HK40077860A - Methods for treating cln2 disease in pediatric subjects - Google Patents

Description

Methods of treating CLN2 disease in pediatric subjects

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority of us provisional patent application No. 62/893,535, filed 2019, 8, 29, which is incorporated herein by reference, is claimed.

Incorporation by reference of electronically submitted material

A list of computer readable nucleotide/amino acid sequences filed concurrently with the present application and identified as follows is incorporated herein by reference in its entirety: an 8,177 byte ASCII (text) file named "54735 _ seqlistingtxt"; created on 8 months and 27 days of 2020.

Technical Field

The present disclosure relates to methods of treating neuropathic ceroid lipofuscinosis (CLN2) and methods of delaying the onset of CLN2 or its associated physiological symptoms in an individual less than 3 years of age.

Background

Neuropathic lipofuscinoid (CLN2) is a rare genetic disease characterized by a deficiency of the lysosomal enzyme tripeptidyl peptidase-1 (TPP1) caused by a mutation in the TPP1 gene. CLN2 disease is inherited as an autosomal recessive genetic disease with an estimated incidence of 0.5 per 100,000 surviving newborns. In the absence of TPP1, lysosomal storage substances, which are normally metabolized by enzymes, accumulate in many organs, and accumulation in the central nervous system leads to the neurodegenerative symptoms typical of CLN2 disease. As indicated by natural history data from independent patient populations in north america and europe, the untreated disease progression of CLN2 disease has been well characterized, and the natural history of the disease is clearly consistent and predictable.

CLN2 disease has a predominantly 'classical' late infant expression pattern. Children with CLN2 disease typically develop to about 3 years of age until the first symptoms appear. Most commonly, CLN2 patients will first develop unproductive seizures and begin to lag in achieving normal speech milestones. By the age of 3, all patients present with one or more signs of disease, including, for example, seizures, dementia, loss of movement, dyskinesias, blindness, clumsiness, ataxia and cognitive decline. Starting from the onset of clinical symptoms, the course of the disease is rapid and aggressive, typically resulting in complete loss of speech, cognition, gait, fine motor, medullary function and vision in 2 to 4 years, leaving the patient immobile, silent and blind. Patients maintain a vegetative state until death, which usually occurs between the ages of 6 and 12.

In assessing the severity of CLN2 disease, expert clinicians have developed two quantitative rating scales and they are used in natural history studies: (1) hamburg scale (Hamburg scale) (Steinfeld et al, J. American J. Med. Genet 2002; 112(4): 347-54); and (2) the Weill Cornell Medical College (WCMC) scale (Worball et al, Neurology 2007; 69(6): 521-35). The structure and the assessment method of the two gauges are similar. Both scales measure the loss of significant neural milestones that CLN2 patients have previously achieved, with each unit loss in the disease rating scale representing a basic milestone of progressive decline.

Analysis of the disease course of untreated CLN 2-affected children showed that after onset of disease, they predictably lost all language and gait within 3 years, with an average loss of 2.1 milestone events per year (i.e. a loss of 2.1 points on the disease rating scale). Language decline usually precedes gait, so that the first year is characterized by loss of clear speech and progression to gait ataxia, the second year is characterized by loss of walking ability and clear language, and the third year is characterized by loss of any locomotor or communication ability.

Recombinant human tripeptidyl peptidase-1 (rhTPP1) is being developed as a possible treatment for CLN2 disease. The rhTPP1 protein is produced in cell culture as a proenzyme (proenzyme), which has no enzymatic activity. Upon uptake to lysosomes, the proenzyme is self-activated at acidic pH (and with lysosomal proteases). The mature native TPP1 protein is a lysosomal serine protease and is the only known mammalian member of the serine-carboxypeptidase (sedolisin) family, characterized by a highly conserved Ser-Glu-Asp (SED) catalytic triad. The catalytic triad on rhTPP1 is formed by S456, E253, and D341. The main activity of the enzyme is a tripeptidyl exopeptidase with broad substrate specificity. Enzyme activity on its substrate leads to the sequential release of tripeptides from the N-terminus of the protein substrate (Oyama et al, J Biochem 2005; 138(2): 127-34). Secondary, significantly weaker endoproteolytic activities have also been reported with an optimum pH of 3 (Lin et al, J. Biochem. 2001; 276(3): 2249-55).

The only commercially available treatments for CLN2 are symptomatic and supportive care; there are currently no approved treatment options to slow or halt The progression of CLN2, which is not to mention reversing The deleterious effects of The Disease (Mole, s.e., and Williams, r.e.,2010, gene reviews (GeneReviews; Chang et al, in Neuronal cerofic lipofusosis (pasteurellosis)), (The neural center Lipofuscinoses) (bat diseases)), (2011, Oxford Univ Press). The ability to preserve motion, language, and/or vision for these children has clinically meaningful benefits to both the patient and the parent/caregiver. Therefore, new therapies for CLN2 are needed that reduce or prevent the deterioration of physiological functions associated with disease.

Disclosure of Invention

The present disclosure relates to methods of treating CLN2 disease or one or more symptoms associated with CLN2 disease in an individual. In an exemplary embodiment, the individual is less than 3 years old. In an exemplary embodiment, the method includes administering to the subject a formulation including recombinant human tripeptidyl peptidase-1 (rhTPP1) in an amount effective to treat CLN2 disease in the subject. The present disclosure also relates to methods of delaying the onset of CLN2 disease or symptoms thereof in an individual. In an exemplary embodiment, the individual is less than 3 years old. In exemplary embodiments, the methods comprise administering to the subject a formulation comprising rhTPP 1in an amount effective to delay the onset of CLN2 disease or symptoms thereof in the subject. In exemplary aspects, the formulation is administered to the subject via intraventricular, intrathecal, or intraocular administration. In an exemplary case, the formulation is administered once every 2 weeks. In various aspects, the formulation is administered at a rate of about 2.5 ml/hour with infusion. In each instance of the presently disclosed methods, a dose of about 300mg or less is administered to the individual. In exemplary aspects, the subject is greater than or about 2 years old, and optionally, a dose of about 300mg of rhTPP1 is administered to the subject. In various aspects, the subject is greater than or about 1 year old and less than 2 years old, and optionally, a dose of about 200mg of rhTPP1 is administered to the subject. In exemplary aspects, each of the 1 st, 2 nd, 3 rd, and 4 th doses administered to an individual (e.g., an individual older than or about 1 year and younger than 2 years) is about 200mg of rhTPP1, and each of the 5 th and subsequent doses administered to an individual is greater than about 200mg of rhTPP 1. In an exemplary case, each of the 5 th and subsequent doses administered to the subject is about 300mg of rhTPP 1. In various aspects, the subject is greater than or about 6 months of age and less than 1 year of age, and optionally, a dose of about 150mg of rhTPP1 is administered to the subject. In various aspects, the individual is less than 6 months of age, and optionally, a dose of about 100mg of rhTPP1 is administered to the individual. In exemplary aspects, based on blood testing, the subject exhibits a decrease in TPP1 enzyme activity. In an illustrative example, the subject is a sibling of the subject diagnosed with CLN2. In an exemplary aspect, the total score of the individual on the athletic and linguistic subscales is from about 3 to about 6. In exemplary aspects, the subject has not been previously treated with stem cell therapy, gene therapy, or enzyme supplementation therapy. In exemplary cases, the method comprises administering to the subject an antihistamine with or without an antipyretic prior to administration of rhTPP1, optionally about 30 to about 60 minutes prior to administration of rhTPP 1. In various aspects, the formulation includes rhTPP1 and at least one pharmaceutically acceptable carrier, diluent, or excipient. In various instances, the formulation includes disodium phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof. In various instances, the method comprises administering a rinse solution to the subject after administering the formulation. In various aspects, the flush solution comprises disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof. In various aspects, the treatment period is at least 10 weeks, at least 20 weeks, at least 40 weeks, at least 80 weeks, or at least 96 weeks.

The foregoing summary is not intended to limit the invention in every aspect thereof, and other features and advantages of the present disclosure will become apparent from the following detailed description, which includes the accompanying drawings. The present disclosure is intended to be related as a unified document, and it should be understood that all combinations of features described herein can be encompassed even if no combination of features is found in the same sentence, paragraph or section of the disclosure. In addition, as an additional aspect, the present disclosure includes all embodiments of the invention, the scope of which is in any way narrower than the variations specifically mentioned above. In connection with aspects of the disclosure described or claimed in "a" or "an," it is to be understood that these terms are intended to mean "one or more" unless the context clearly requires a stricter meaning. With respect to elements described as one or more within a group, it is to be understood that all combinations within the group are contemplated. If an aspect of the present disclosure is described as "comprising" a feature, embodiments "consisting of or" consisting essentially of the feature may also be encompassed. Additional features and variations of the present disclosure will be apparent to those skilled in the art from the entirety of the present application, and all such features are intended as aspects of the present disclosure.

Drawings

Figure 1 depicts the amino acid sequence of rhTPP1 zymogen lacking the relevant signal peptide. The enzyme is preceded by the first 176 amino acid residues and the mature enzyme is 368 amino acids in length starting at position 177.

Fig. 2 depicts the clinical progression of untreated individuals with CLN2 disease in a natural history study and shows a hamburger movement and language composite score of 0 to 6 versus patient age. Also shown are median, quartile and 10%/90% distribution; and mean and 95% confidence interval.

Fig. 3A to 3F depict the clinical assessments of 24 patients cumulatively during treatment and show 0 to 6 hamburger movement and language composite scores. The open circles represent the CLN2 score obtained after or before the first infusion of 300mg rhTPP1, while the open circles represent the CLN2 score obtained after the first infusion of 300mg rhTPP 1. The total score (circle) is shown as well as the contributions of motion/gait (square) and language (triangle) to the total score. Day 1 of the analysis is the date of the first infusion.

Figures 4A to 4I compare the disease score (indicated by the prefix "HAM") of the 0 to 6 hamburger motor and language ensemble scale, with the change in CLN2 score for 9 patients receiving rhTPP1 treatment and untreated natural history patients matched to the individual receiving treatment. The solid line in each group shows the results for the treated patients compared to the results for untreated natural history patients for which the solid line matches, while the results for untreated natural history patients are shown as dashed lines.

FIG. 5 depicts the distribution of clinical changes from baseline in matched untreated natural history patients (circles) during patient matched treatment compared to study individuals (squares).

Fig. 6A to 6I depict the change in CLN2 scores from disease scores on 0 to 9 hamburger movement/language/vision gross scale and matched 9 patients treated with rhTPP1 to untreated natural history patients. The solid line in each group shows the results for the treated patients compared to the results for untreated natural history patients for which the solid line matches, while the results for untreated natural history patients are shown as dashed lines.

Figure 7 depicts the volume (upper panel) and scale (lower panel) of cerebrospinal fluid for all 24 patients measured during the treatment period. Each line represents one patient.

Fig. 8A to 8L depict the brain volume of 24 treated patients. The volume (upper panel) and scale (lower panel) of white matter are shown as the difference between the whole brain volume (dotted line) and CSF and gray matter (dotted line), while gray matter is shown as the difference between CSF and gray matter (dotted line) and CSF (solid line).

Fig. 9A and 9B depict the mean change in CLN2 scores for patients treated with rhTPP1 and untreated natural history patients. Fig. 9A depicts CLN2 scores for 23 patients treated with 300mg rhTPP1 for 48 weeks (dashed line) and an untreated physical history cohort of 41 individuals (solid line). Figure 9B depicts the change from baseline in CLN2 score of 23 patients treated with 300mg of rhTPP1 for 48 weeks.

Fig. 10A to 10L depict clinical assessments of 24 patients who accumulated during treatment and show WCMC (right) gait (square), language (triangle), myoclonus (cross) and eating (diamond) composite scores of 0 to 12 combined hamburger (left panel) movement (square), language (triangle), seizure (cross) and visual composite score (diamond) 0 to 12. The open circles represent the total CLN2 score obtained after or before the first infusion of rhTPP 1300 mg, while the open circles represent the total CLN2 score obtained after the first infusion of rhTPP 1300 mg.

Figure 11 shows a table of clinical laboratory assessments and events tested in the study described in examples 4-10.

Fig. 12 shows a table listing the hamburger LINCL scale.

Figure 13 shows the median (range) PK parameters for cellular lybonase alpha (cerlipenase alfa) after single doses of 30, 100 and 300mg using ICV infusion.

FIG. 14 shows the median (range) PK parameters for cellular Liboninase α following ICV infusion of 300mg QOW.

FIG. 15 shows the mean concentration-time curves of cellular Libornase α in CSF and plasma after infusion of 300mg QOW by ICV. Time 0 represents the start of infusion. SD ═ stable dose phase.

FIG. 16 shows plasma exposure of patient and access matched CSF to cellular Libonase α at 300mg ICV QOW.

Figures 17A-17D show patient characteristics and cellular lybonase α PK in CSF and plasma. Individual patient data is shown as circles. Fig. 17A, gender; fig. 17B, baseline age; fig. 17C, baseline body weight; fig. 17D, baseline CLN2 score. Boxes represent the interquartile spacing (IQR) between the first (Q1) and third (Q3) quartiles, bars within boxes represent median, and whiskers represent the minimum and maximum values that do not contain outliers (i.e., values outside the standard data range are defined as ranging from Q1-1.5 IQR to Q3+1.5 IQR; open circles).

FIGS. 18A-18B show individually access matched PK and ADA status for cellular Ribose. Fig. 18a, CSF; figure 18b, plasma/serum. Open circles for patients who were ADA negative at study visit and open circles for patients who were ADA positive at study visit.

Fig. 19 shows the clinical assessment of CLN2 in CSF and the change from baseline in motor language function composite score for cellular lybonase α PK. Individual patient data is shown as circles. Boxes represent the interquartile spacing (IQR) between the first (Q1) and third (Q3) quartiles, bars within boxes represent median, and whiskers represent the minimum and maximum values that do not contain outliers (i.e., values outside the standard data range are defined as ranging from Q1-1.5 IQR to Q3+1.5 IQR; open circles).

Detailed Description

The following definitions may be useful to aid one skilled in the art in understanding the present disclosure. Unless defined otherwise herein, the scientific and technical terms used in connection with the present disclosure have the meanings that are commonly understood by one of ordinary skill in the art. Where a range of values is provided, it is understood that the invention encompasses each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any particular exclusive limitation within the stated ranges.

The term "family history" refers to an individual who is a blood relative, e.g., siblings, parents, grandparents, great grandparents, etc., diagnosed with CLN2 disease.

In one aspect, the term "fragment" refers to a recombinant protein that includes a portion of the amino acid sequence of rhTPP1 proenzyme as set forth in SEQ ID NO:1 and FIG. 1. For example, a fragment may contain at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the amino acid sequence set forth in SEQ ID NO. 1. In another aspect, a fragment may comprise the full length (368 amino acids long; amino acids 177-544 of SEQ ID NO: 1) mature TPP1 enzyme amino acid sequence set forth in SEQ ID NO:2, a portion thereof and/or at least the catalytic triad formed by amino acid residues S456, E253 and D341. The fragments retain catalytic activity. For example, the fragments exhibit tripeptidyl exopeptidase activity and/or exhibit catalytic activity which results in the sequential release of tripeptides from the N-terminus of the protein matrix. In certain aspects, a "fragment" of rhTPP1 proenzyme comprises at least 500 consecutive amino acids of SEQ ID NO. 1, at least 450 consecutive amino acids of SEQ ID NO. 1, at least 400 consecutive amino acids of SEQ ID NO. 1, at least 368 amino acids of SEQ ID NO. 1, at least 350 amino acids of SEQ ID NO. 1, or at least 300 consecutive amino acids of SEQ ID NO. 1. In other aspects, a "fragment" of rhTPP1 proenzyme includes at least 350 consecutive amino acids of SEQ ID NO. 2, at least 325 consecutive amino acids of SEQ ID NO. 2, at least 300 consecutive amino acids of SEQ ID NO. 2, at least 275 consecutive amino acids of SEQ ID NO. 2, at least 250 consecutive amino acids of SEQ ID NO. 2, or at least 200 consecutive amino acids of SEQ ID NO. 2.

The term "intracerebroventricular" refers to administration of a composition into the ventricular system, e.g., via injection, infusion, or implantation (e.g., into the ventricle).

The term "intraocular" refers to the administration of a composition into the region of the eye, for example via injection, infusion or implantation (e.g. into the eye globe) or topical/ocular administration (e.g. using creams, ointments, gels or droplets).

The term "intrathecal" refers to administration of a composition into the lumbar region, e.g., via injection, infusion, or implantation (e.g., into the subarachnoid space of the spinal cord).

The term "therapeutically effective" refers to any therapeutic benefit resulting from the therapeutic methods of the present invention. For example, such an effect may be a beneficial effect expressed in an appropriate target tissue or organ, which is compared to the measured physiological parameter in the absence of enzyme replacement therapy in that tissue or organ. Such a therapeutic effect may be any reduction or elimination of one or more clinical or subclinical expressions of CLN2 disease. For example, a therapeutically effective treatment ameliorates, reverses, delays, prevents, or reduces the worsening of one or more physiological functions and/or neurological symptoms of CLN2 as described herein.

The term "stable" or "stabilization" refers to a protein-containing formulation in which the protein component thereof substantially retains its physical, functional, and/or chemical stability over time. Stability can be measured at a selected temperature for a selected period of time. Preferably, the formulation is stable at room temperature (about 30 ℃) or at about 40 ℃ for at least 1 month and/or at about 2 ℃ to about 8 ℃ for at least 1 year, and preferably at least 2 years. For example, the extent of protein degradation or aggregation during storage can be used as an indicator of protein stability. Thus, a "stable" formulation may be one in which less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the protein component is present in the formulation in degraded or aggregated form after storage. A "stable" formulation retains essentially the same functional or therapeutic characteristics of a freshly prepared formulation. Various analytical techniques for measuring Protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery (Peptide and Protein Drug Delivery), 247-.

The term "preventing" or "reducing" or grammatical equivalents thereof, when used in reference to preventing or reducing one or more symptoms or physiological consequences of CLN2 disease in an individual, means that the rate of decline of that/those symptoms in a treated CLN2 individual is slower than the rate of decline observed in an untreated CLN2 individual. In this regard, untreated CLN2 may be the same individual subsequently treated with the composition of the invention, or may be the average rate of decline in the symptoms of interest observed as a result of the natural history study disclosed herein.

In the right to forbid patenting of methods of human practice, the meaning of "administering" rhTPP1 or a formulation thereof to a human subject refers to the medical use of rhTPP1 or a formulation thereof, e.g., rhTPP1 or a formulation thereof for treating CLN2 disease as described herein, or the use of rhTPP1 for the manufacture of a medicament for treating CLN2 disease as described herein. Compliance with the broadest reasonable interpretation of the law or regulation defining patentable subject matter is deemed necessary. In the right to not forbid patenting of methods of human practice, "administering" rhTPP1 or formulations thereof includes methods of human practice as well as the aforementioned activities.

The present disclosure provides formulations and kits comprising rhTPP1 and methods of using the same for treating CLN2 disease. Administration of rhTPP1 allowed cells that utilized the cation-independent mannose-6-phosphate receptor (CI-MPR) to take up the protein and localize to lysosomes in the cells throughout the central nervous system. Uptake of the enzyme into the lysosome and subsequent activation promotes increased breakdown of the stored substance in the affected tissue, reduces the progressive accumulation of lysosomal stored substance, and suppresses disease regression. The formulations and methods of the present disclosure provide therapeutic benefits over currently approved therapies.

Formulations

With respect to the present disclosure, the formulation includes an amount of rhTPP1, and in exemplary aspects, the formulation is suitable for intraventricular, intrathecal, and/or intraocular administration. In one aspect, rhTPP1 includes SEQ ID NO 1 or a fragment thereof. rhTPP1 protein and methods of obtaining rhTPP1 protein suitable for use in the formulations and methods described herein are described in U.S. patent nos. 6,302,685 and 8,277,800, which are incorporated herein by reference in their entirety.

In one aspect, rhTPP1 includes the amino acid sequence of SEQ ID NO:1 (amino acids 1-544 of the amino acid sequence shown in FIG. 1) or a fragment thereof having catalytic activity. In another aspect, rhTPP1 includes the amino acid sequence of SEQ ID NO:2 (amino acids 177-544 of the amino acid sequence shown in FIG. 1) or a fragment thereof having catalytic activity. In yet another aspect, rhTPP1 has detectable enzymatic activity, or is processed in vivo into an enzyme form having detectable enzymatic activity (i.e., "functionality"), and has at least about 70% sequence identity to SEQ ID NO:1 or SEQ ID NO: 2. For example, functional rhTPP1 is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 97% identical to SEQ ID NO 1 or SEQ ID NO 2. In one aspect, the formulation is a liquid formulation comprising rhTPP1 at a concentration of about 1mg/mL to about 100mg/mL, such as about 10mg/mL to about 50mg/mL, about 25mg/mL to about 40mg/mL, or about 30mg/mL to about 60 mg/mL. In various aspects, the formulation comprises rhTPP1 at a concentration of about 1mg/mL to about 100mg/mL, about 5mg/mL to about 80mg/mL, about 10mg/mL to about 50mg/mL, about 20mg/mL to about 40mg/mL, about 25mg/mL to about 35mg/mL, more specifically about 1mg/mL, about 10mg/mL, about 20mg/mL, about 30mg/mL, about 40mg/mL, about 50mg/mL, about 60mg/mL, about 70mg/mL, about 80mg/mL, about 90mg/mL, or about 100 mg/mL. In one aspect, the pH of the formulation is about 5.5 to about 7.5, or about 6.0 to about 7.0, such as about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5.

In one aspect, formulations including rhTPP1 of the present disclosure further include one or more excipients that maintain a critical electrolyte content in cerebrospinal fluid (CSF) or ocular fluid. For example, in one aspect, the formulation further comprises potassium chloride at a concentration of about 0.01mg/mL to about 1mg/mL, such as about 0.1mg/mL to about 0.5mg/mL, about 0.2mg/mL to about 0.8mg/mL, about 0.2mg/mL to about 0.4mg/mL, about 0.15mg/mL to about 0.25mg/mL, or about 0.05mg/mL to about 0.3mg/mL, in addition to rhTPP1 or a fragment thereof. In another aspect, the formulation further comprises magnesium chloride hexahydrate at a concentration of about 0.01mg/mL to about 1mg/mL, such as about 0.1mg/mL to about 0.5mg/mL, about 0.1mg/mL to about 0.8mg/mL, about 0.1mg/mL to about 0.3mg/mL, about 0.15mg/mL to about 0.25mg/mL, or about 0.05mg/mL to about 0.3 mg/mL. In another aspect, the formulation further comprises calcium chloride dihydrate in a concentration of about 0.01mg/mL to about 1mg/mL, such as about 0.1mg/mL to about 0.5mg/mL, about 0.2mg/mL to about 0.8mg/mL, about 0.15mg/mL to about 0.25mg/mL, about 0.1mg/mL to about 0.3mg/mL, or about 0.05mg/mL to about 0.3 mg/mL. In yet another aspect, the formulation includes a combination of all or any of the foregoing.

In another aspect, a formulation comprising rhTPP1 further comprises one or more buffering agents. For example, in various aspects, the formulation further comprises disodium phosphate heptahydrate at a concentration of about 0.01mg/mL to about 1mg/mL, such as about 0.1mg/mL to about 0.5mg/mL, about 0.05mg/mL to about 0.4mg/mL, or about 0.1mg/mL to about 0.3 mg/mL; and/or sodium phosphate monobasic monohydrate at a concentration of about 0.01mg/mL to about 1mg/mL, for example about 0.01mg/mL to about 0.2mg/mL, about 0.05mg/mL to about 0.3mg/mL, or about 0.08mg/mL to about 0.4 mg/mL.

In another aspect, the formulation further comprises an isotonic agent, such as sodium chloride, at a concentration of about 1mg/mL to about 20mg/mL, for example about 1mg/mL to about 10mg/mL, about 5mg/mL to about 15mg/mL, or about 8mg/mL to about 20 mg/mL. Other buffers and isotonic agents known in the art are suitable and may be conventionally suitable for use in the formulations of the present disclosure.

In one aspect, a formulation comprising about 30mg/mL of rhTPP1 further comprises disodium phosphate heptahydrate at a concentration of about 0.11mg/mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08mg/mL, sodium chloride at a concentration of about 8.77mg/mL, potassium chloride at a concentration of about 0.22mg/mL, magnesium chloride hexahydrate at a concentration of about 0.16mg/mL, calcium chloride dihydrate at a concentration of about 0.21mg/mL, and a diluent, such as water for injection.

The rhTPP1 formulations of the present disclosure are stable and can be stored for extended periods of time without unacceptable changes in quality, performance, or purity. In one aspect, the formulation is stable at a temperature of about 5 ℃ (e.g., 2 ℃ to 8 ℃) for at least 1 month, e.g., at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or more. In another aspect, the formulation is stable at a temperature of less than or equal to about-20 ℃ for at least 6 months, such as at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another aspect, the formulation is stable at a temperature of less than or equal to about-40 ℃ for at least 6 months, such as at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another aspect, the formulation is stable at a temperature of less than or equal to about-60 ℃ for at least 6 months, such as at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more.

In one aspect, the formulations of the present disclosure are preservative-free and/or stabilizer-free, and thus do not contain any of thimerosal, phenylmercuric salts, chlorhexidine, phenols, benzoic acid, sorbic acid, parabens, alcohols, or other preservatives commonly found in parenteral or ophthalmic formulations.

In another aspect, the formulations of the present invention may include one or more preservatives, stabilizers, or excipients. In this regard, a variety of well-known and conventionally employed preservatives, stabilizers, and excipients suitable for intrathecal or ICV delivery of protein-containing formulations are known in the art. More specifically, examples of such additives into enzyme-containing formulations suitable for intrathecal or ICV delivery are described in WO2013/096899, which is incorporated herein by reference.

Method

The present disclosure provides methods of treating CLN2 disease comprising administering to an individual in need thereof a therapeutically effective amount of a formulation comprising rhTPP1 described herein. The present disclosure also provides compositions comprising rhTPP1 and the use of rhTPP 1in the manufacture of a medicament for treating the CLN2 disease described herein for treating the CLN2 disease described herein. In one aspect, the severity and progression of CLN2 disease and the therapeutic benefit of administering rhTPP 1in a patient can be measured using the hamburger or WCMC clinical disease rating scale. Both the hamburger and WCMC scales consist of four disease-related domains scored from 0 to 3 according to the subscale, so 3 is normal, 2 is abnormal but functional, 1 is abnormal and apparently dysfunctional, and 0 is no residual function. Two of the four domains, gait/motion and language, are shared between the two scales, and have high internal content effectiveness. Each scale searched for changes that occurred as a function of both disease progression and disease management. Gait, language and visual acuity scales search for disease progression. Seizure frequency, dyskinesias, and feeding depend on care decisions, specifically anticonvulsant drugs and feeding tube management. Clinical progression is typically assessed using an aggregated language and gait subscale, so a score of 6 indicates normal based on age and a score of 0 indicates complete loss of function. Table 1 depicts the WCMC and hamburger CLN2 disease scales.

TABLE 1

In various aspects, the present disclosure provides methods of treating CLN2 disease or one or more clinical symptoms of CLN2 disease, the use of rhTPP 1in the manufacture of a medicament to treat CLN2 disease or rhTPP 1in an individual for treating CLN2 disease in the individual, the method comprising administering to the individual in need thereof a composition comprising a therapeutically effective amount of rhTPP 1.

The present disclosure also provides methods of preventing one or more clinical symptoms of CLN2 disease, comprising administering to an individual in need thereof a formulation comprising rhTPP1 described herein, optionally wherein the individual has a family history of CLN2 disease. In various aspects, the present disclosure provides a method of preventing one or more clinical symptoms of CLN2 disease, the use of rhTPP 1in the manufacture of a medicament for preventing one or more clinical symptoms of CLN2 disease or rhTPP 1in a subject, for preventing one or more clinical symptoms of CLN2 disease in a subject, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of rhTPP1, optionally wherein the subject has a family history of CLN2 disease.

The present disclosure further provides a method of treating CLN2 disease comprising administering to an individual rhTPP 1in a dose effective to maintain physiological function or slow or reduce deterioration of physiological function in an individual in need thereof, wherein the physiological function is speech function, motor function, vision, or eating function. The present disclosure also provides the use of rhTPP 1in the manufacture of a medicament for maintaining physiological function or slowing or reducing the worsening of physiological function in an individual with CLN2 and rhTPP1 for maintaining physiological function or slowing or reducing the worsening of physiological function in an individual with CLN 2; wherein the physiological function is a speech function, a motor function, a vision function or a feeding function.

In one aspect, a method of treating an individual with CLN2 disease or a family history of CLN2 disease comprises administering to the individual rhTPP 1in an amount effective to maintain language function or slow or reduce the worsening of language function. In one aspect, language function deterioration is reduced by at least one point as compared to a previous assessment determined prior to or during treatment as measured using the WCMC or hamburger disease rating scale. In both the WCMC and hamburger scales, a rating of 3 indicates language normal; a score of 2 (identifiably) indicates a language anomaly; 1 indicates that the language is rarely/hardly understandable; and a score of 0 indicates that the language is not understood or cannot speak. In one aspect, the dose of rhTPP1 is effective to maintain the linguistic assessment of the individual at the same level as a previous assessment determined prior to or during treatment, e.g., 3 points, 2 points, or 1 point. In another aspect, the dose of rhTPP1 is effective to slow or reduce the language function deterioration associated with CLN2 in an individual as evidenced by a longer period of time to maintain the language assessment at the same level or a less reduced language function assessment than would be expected in view of the natural progression of the disease.

In another aspect, a method of treating an individual with a family history of CLN2 or CLN2 comprises administering to the individual rhTPP 1in an amount effective to maintain motor function or slow or reduce worsening motor function. In one aspect, deterioration in motor function is reduced by at least one point as compared to a previous assessment determined prior to or during treatment as measured using the WCMC or hamburger disease rating scale. A gait in WCMC scale or a clinical rating scale for locomotion in hamburger scale can be used to assess locomotor function. In both the WCMC and hamburger scales, a rating of 3 indicates normal walking; a score of 2 indicates an abnormal gait but without dependency, such as frequent falls or significant clumsiness; a score of 1 indicates walking abnormalities, requiring assistance, such as walking failure without assistance or only crawling; and 0 indicates immobility/immobility, e.g. most bedridden individuals. In one aspect, the dose of rhTPP1 is effective to maintain the motor function assessment of the individual at the same level as the previous assessment determined prior to or during treatment, e.g., 3 points, 2 points, or 1 point. In another aspect, the dose of rhTPP1 is effective to slow or reduce motor function deterioration associated with CLN2 in an individual, as evidenced by maintaining a motor assessment at the same level for a longer period of time or a reduced motor function assessment, as compared to that expected in view of the natural progression of the disease.

In yet another aspect, a method of treating an individual with a family history of CLN2 or CLN2 comprises administering to the individual rhTPP 1in a dose effective to maintain vision or slow or reduce the progression of vision. In one aspect, the deterioration in vision is reduced by at least one point as compared to a previous assessment determined before or during treatment, as measured using the hamburger disease rating scale. A rating of 3 according to the hamburger scale indicates that the individual recognizes the desired object and grasps the object; 2 points out that the object cannot be grasped in harmony; a score of 1 indicates that the individual responded to light, and a score of 0 indicates that the individual did not respond to visual stimuli. In one aspect, the dose of rhTPP1 is effective to maintain the individual's vision assessment at the same level as the previous assessment determined before or during treatment, e.g., 3 points, 2 points, or 1 point. In another aspect, the dose of rhTPP1 is effective to slow or reduce the individual's vision deterioration associated with CLN2, as evidenced by maintaining the vision assessment at the same level for a longer period of time or a less reduced vision assessment than would be expected in view of the natural progression of the disease.

In another aspect, a method of treating an individual with a family history of CLN2 or CLN2 comprises administering to the individual rhTPP 1in a dose effective to maintain eating function or slow or reduce worsening eating function. In one aspect, the eating function is reduced by at least one minute as compared to a previous assessment determined prior to or during treatment as measured using the WCMC rating scale. A rating of 3 points according to the WCMC scale indicates no swallowing dysfunction; score 2 indicates mild swallowing dysfunction; score 1 indicates moderate swallowing dysfunction and score 0 indicates that the individual has dependency on the gastric tube. In one aspect, the dose of rhTPP1 is effective to maintain the fed function of the individual at the same level as the previous assessment determined before or during treatment, e.g., 3 points, 2 points, or 1 point. In another aspect, the dose of rhTPP1 is effective to slow or reduce the worsening of eating function associated with CLN2 in an individual, as evidenced by maintaining eating function at the same level for a longer period of time or a decreased eating assessment than would be expected in view of the natural progression of the disease.

The present disclosure further provides methods of treating CLN2 disease comprising administering rhTPP1 to an individual in need thereof in an amount effective to improve a physiological function, wherein the physiological function is speech function, motor function, vision, or eating function. The present disclosure also provides the use of rhTPP 1in the manufacture of a medicament for improving physiological function in an individual with CLN2 or rhTPP1 for improving physiological function in an individual with CLN 2; wherein the physiological function is a speech function, a motor function, a vision function or a feeding function. Given the progressive, degenerative nature of the disease, improvements in language function, motor function, vision, and/or eating function are particularly desirable, indicating that the individual has recovered lost function, but are difficult to achieve with current treatment options.

In one aspect, a method of treating an individual having CLN2 disease comprises administering to the individual rhTPP 1in an amount effective to improve language function. In one aspect, the improvement in language function is increased by at least one point as compared to a previous assessment determined prior to or during treatment, as measured using the WCMC or hamburger disease rating scale. For example, an individual may be scored 1 or 2 to score 3, indicating a return to normal language, or scored 1 to score 2.

In another aspect, a method of treating an individual having CLN2 disease comprises administering to the individual rhTPP 1in an amount effective to improve motor function. In one aspect, the improvement in motor function is increased by at least one-point compared to a previous assessment determined prior to or during treatment, as measured using the WCMC or hamburger disease rating scale. For example, an individual may rate from 1 or 2 to 3, indicating a return to normal walking, or from 1 to 2.

In one aspect, a method of treating an individual having CLN2 disease comprises administering to the individual rhTPP 1in an amount effective to improve vision. In one aspect, the improvement in vision is increased by at least one point as compared to a previous assessment determined before or during treatment, as measured using the hamburger disease rating scale. For example, an individual may be rated 1 or 2 to 3, or 1 to 2.

In another aspect, a method of treating an individual having CLN2 disease comprises administering to the individual rhTPP 1in a dose effective to improve fed function. In one aspect, the improvement in eating function is increased by at least one point as compared to a previous assessment determined prior to or during treatment as measured using the WCMC disease rating scale. For example, an individual may increase from a score of 1 or 2 to a score of 3, indicating a return to normal swallowing, or increase from a score of 1 to a score of 2 or 3.

The present disclosure further provides a method of treating CLN2 disease comprising administering rhTPP1 to an individual in need thereof at a dose effective to prevent or treat a neurological symptom of the disease, wherein the neurological symptom is a seizure, a decrease in brain volume, a decrease in gray matter, or an increase in Craniocerebral Spinal Fluid (CSF). The present disclosure also provides the use of rhTPP 1in the manufacture of a medicament for preventing or treating neurological symptoms in an individual with a family history of CLN2 or CLN2 and rhTPP1 for preventing or treating neurological symptoms in an individual with a family history of CLN2 or CLN 2; wherein the neurological condition is a seizure, a decrease in brain volume, a decrease in brain gray matter, or an increase in cranial CSF.

In one aspect, a method of treating an individual with a family history of CLN2 or CLN2 comprises administering to the individual rhTPP 1in a dose effective to maintain or reduce the number of seizures. In one aspect, the dose is effective to reduce the number of monthly seizures experienced by the individual. In another aspect, the dose is effective to increase the seizure rating by at least one-point compared to a previous rating determined prior to or during treatment, as measured using the hamburger disease rating scale. A rating of 3 points according to the hamburger scale indicates no seizures within 3 months; score 2 indicates 1 to 2 seizures within 3 months; score 1 indicates 1 seizure per month; and 0 score indicates more than 1 seizure per month. In one aspect, the dose of rhTPP1 is effective to maintain the individual's seizure assessment at the same level as the previous assessment determined before or during treatment, e.g., 3 points, 2 points, or 1 point. In another aspect, the dose of rhTPP1 may be effective to maintain or reduce seizures in an individual when this is evidenced by maintaining the number of seizures per month for a longer period of time or a lower rate of seizure than would be expected in view of the natural progression of the disease.

In another aspect, a method of treating an individual with a family history of CLN2 or CLN2 comprises administering to the individual a dose of rhTPP1 effective to maintain brain volume or slow or reduce brain volume reduction. Brain atrophy increases as the disease progresses, causing a correlated increase in the volume loss of brain volume and the volume and relative proportion of intracranial CSF. Brain volume can be measured using methods known in the art, including imaging techniques such as Magnetic Resonance Imaging (MRI), computed tomography (CT/CAT), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), electroencephalography (EEG), Magnetoencephalography (MEG), and near infrared spectroscopy (NIRS). In one aspect, the dose of rhTPP1 can be effective to slow or reduce brain volume reduction associated with CLN2 in an individual, as evidenced by maintenance of brain volume for a longer period of time or less brain volume reduction than would be expected in view of the natural progression of the disease.

In another aspect, a method of treating an individual with a family history of CLN2 or CLN2 comprises administering to the individual rhTPP 1in a dose effective to maintain gray brain matter or slow or reduce the reduction in gray brain matter. Loss of gray matter due to brain atrophy occurs as the disease progresses, such that the percentage of gray matter in brain volume decreases. The amount of gray matter in the brain can be assessed using methods known in the art, such as imaging techniques, e.g., MRI, CT/CAT, PET, SPECT, EEG, MEG, and NIRS. In one aspect, the dose of rhTPP1 is effective to slow or reduce gray matter reduction in an individual, as evidenced by maintenance of gray matter granulometry for a longer period of time or less gray matter reduction than would be expected in view of the natural progression of the disease.

In another aspect, a method of treating an individual with a family history of CLN2 or CLN2 comprises administering to the individual a dose of rhTPP1 effective to maintain a volume of cranial CSF or slow down an increase in volume of cranial CSF. Due to brain atrophy, the volume and proportion of total CSF of cranial CSF increases. The amount and proportion of cranial CSF can be assessed using methods known in the art, such as imaging techniques, e.g., MRI and CT/CAT. In one aspect, the dose of rhTPP1 is effective to slow or reduce an increase in cranial CSF in an individual, as evidenced by a volume of cranial CSF maintained for a longer period of time or a lesser increase in the percentage of cranial CSF to total CSF than would be expected in view of the natural progression of the disease.

The aforementioned methods, compositions and uses may further comprise any of the following features, alone and in combination.

In one aspect, the methods, compositions for use, or uses of the present disclosure include administering a formulation, composition, or dose comprising rhTPP1 to an individual continuously or continually over a period of at least about 1 hour, such as at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, or more. In another aspect, the methods or uses of the present disclosure comprise administering to an individual in need thereof a formulation, composition, or dose comprising about 20mg to about 500mg, about 30mg to about 500mg, about 50mg to about 500mg, about 100mg to about 500mg, about 200mg to about 400mg, about 250mg to about 350mg, or about 275mg to about 325mg of rhTPP1, for example about 20mg, about 30mg, about 50mg, about 100mg, about 200mg, about 300mg, about 400mg, or about 500mg of rhTPP 1. In one aspect, the methods or uses of the disclosure comprise administering a formulation, composition, or dose that is about 20mL or less, about 15mL or less, about 10mL or less, about 7.5mL or less, or about 5mL or less, for example about 20mL, about 15mL, about 10mL, about 9mL, about 8mL, about 7mL, about 6mL, about 5mL, about 4mL, about 3mL, about 2mL, about 1mL, or about 0.5mL, per volume of dose or administration event.

In various aspects, the methods, compositions for use, or uses of the present disclosure include administering a formulation, composition, or dose comprising rhTPP1 to an individual at a rate of less than or equal to about 2.5mL of formulation, composition, or dose per hour, less than or equal to about 75mg of rhTPP1 per hour, or less than or equal to about 75mg of rhTPP1 per 2.5mL of formulation or composition per hour. The formulation, composition or dose is optionally administered continuously or continually over a period of at least about 4 hours.

In one aspect, the methods, compositions used, or uses of the present disclosure include administering a formulation, composition, or dose comprising rhTPP1 weekly or less frequently, e.g., weekly, every other week, or monthly. More specifically, the methods, compositions, or uses of the present disclosure include administering a formulation, composition, or dose comprising rhTPP1 once every 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In one aspect, the formulation, composition or dose is administered intracerebroventricularly. In another aspect, the formulation, composition or dose is administered intrathecally. In yet another aspect, the formulation, composition or dose is administered intraocularly. In one aspect, the formulation, composition or dose is administered intracerebroventricularly or intrathecally as well as intraocularly. Intraventricular delivery allows penetration into the deep gray matter structures of the brain, such as the thalamus, striatum and midbrain, due to the physiology of CSF flow, in which it allows flow into the third and fourth ventricles, and also penetrates from the ventricles into the subarachnoid space along a slight pressure gradient via the nerve fiber network of the cerebral hemisphere. Intrathecal and intracerebroventricular administration of recombinant enzymes to treat lysosomal storage diseases is described in U.S. patent No. 7,442,372, which is incorporated herein by reference in its entirety.

The formulations, compositions, or doses of rhTPP1 of the present disclosure may be administered as a single bolus injection or as a series of injections (e.g., into the brain, lumbar region, or eye), or as a continuous or sustained infusion, e.g., using an infusion pump or other implant device. In one aspect, a formulation, composition, or dose of rhTPP1 is administered using an infusion system that includes tubing, an in-line filter (e.g., about 0.2 μm), a reservoir (e.g., intrathecal or intracerebroventricular), and a catheter. Typically, when administering a composition intrathecally or intracerebroventricularly, in order to prevent adverse side effects caused by artificially increased intracerebral or intrathecal pressure, a volume of CSF comparable to the volume of the composition to be administered is first removed from the individual prior to administration of the composition. However, as described in example 3, it is demonstrated herein that the formulation, composition, or dose of rhTPP1 of the present disclosure can be administered just prior to administration of the formulation, composition, or dose of rhTPP1 without removing any volume of CSF from the individual.

In one aspect, the methods or uses of the present disclosure comprise intracerebroventricularly administering to an individual with CLN2, at a time period of about 4 hours every week, about 10mL of a formulation, composition, or dose comprising about 300mg of rhTPP 1.

The formulations and compositions of the invention may be administered directly to an individual in need thereof (i.e., non-isocratic), or may be administered after removal of a defined volume of CSF from a previous individual, wherein the defined volume is approximately the same as the volume of the subsequently administered composition (i.e., isocratic).

In one aspect, the methods, compositions for use, or uses of the present disclosure further comprise administering a rinse solution to the subject after administering rhTPP 1. The rinse solution was administered via the same route as rhTPP1 and using the same delivery system (e.g., infusion system) to remove any rhTPP1 remaining in the delivery system and to ensure that the individual received the full intended dose of rhTPP 1. In one aspect, the flush solution is administered to the subject in an amount between about 0.5mL and about 5mL, e.g., about 0.5mL, about 1mL, about 2mL, about 3mL, or about 5mL (e.g., using the same catheter previously used to administer the composition comprising rhTPP 1). In one aspect, the rinse solution includes the same components as a formulation or composition including rhTPP1, but without rhTPP 1. In one aspect, the flush solution includes disodium phosphate heptahydrate at a concentration of about 0.11mg/mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08mg/mL, sodium chloride at a concentration of about 8.77mg/mL, potassium chloride at a concentration of about 0.22mg/mL, magnesium chloride hexahydrate at a concentration of about 0.16mg/mL, calcium chloride dihydrate at a concentration of about 0.21mg/mL, and a diluent, such as water for injection.

In various embodiments, the maximum concentration of rhTPP 1in cerebrospinal fluid (T) after the end of the infusion _max ) Between 4 hours and 10 hours. In various embodiments, the elimination half-life (t) of rhTPP 1in cerebrospinal fluid _1/2 ) Between 5 hours and 20 hours.

Children individual

The disclosure also relates to methods of treating CLN2 disease or one or more symptoms associated with CLN2 disease in a subject and methods of delaying the onset of CLN2 disease or symptoms thereof in a subject, wherein an exemplary subject is a pediatric subject, e.g., less than about 18 years of age. In various aspects, the individual is less than 3 years old. In various aspects, the individual is less than 2 years old. In various aspects, the individual is less than 1 year of age. In various aspects, the individual is greater than or from about 1 month old to about 3 months old, or greater than or from about 1 month old to about 6 months old, or greater than or from about 1 month old to about 9 months old, or greater than or from about 1 month old to about 12 months old. In various aspects, the individual is less than or about 12 months old, but at least or about 2 weeks old, 4 weeks old, 6 weeks old, 12 weeks old, or 16 weeks old. In each case, the individual is less than 12 months of age, but at least or about 5, 6, 7,8, 9, 10, or 11 months of age. In each instance, the individual is at least 12 months old, but less than or about 24 months old, or less than or about 23 months old, or less than or about 22 months old, or less than or about 21 months old, or less than or about 20 months old, or less than or about 19 months old, or less than or about 18 months old, or less than or about 17 months old, or less than or about 16 months old, or less than or about 15 months old, or less than or about 14 months old, or less than or about 13 months old. In each case, the individual is less than 24 months old, and at least or about 13 months old, or at least or about 14 months old, or at least or about 15 months old, or at least or about 16 months old, or at least or about 17 months old, or at least or about 18 months old, or at least or about 19 months old, or at least or about 20 months old, or at least or about 21 months old, or at least or about 22 months old, or at least or about 23 months old. In each instance, the individual is greater than or about 24 months old, and less than or about 36 months old, or less than or about 35 months old, or less than or about 34 months old, or less than or about 33 months old, or less than or about 32 months old, or less than or about 31 months old, or less than or about 30 months old, or less than or about 29 months old, or less than or about 28 months old, or less than or about 27 months old, or less than or about 26 months old, or less than or about 25 months old.

In an exemplary embodiment, the method includes administering to the subject a formulation including recombinant human tripeptidyl peptidase-1 (rhTPP1) in an amount effective to achieve a desired result, e.g., treating CLN2 disease or symptoms thereof or delaying the onset of CLN2 disease or symptoms thereof. In each instance of the methods disclosed herein, a dose of about 300mg or less of rhTPP1 is administered to the individual. In exemplary aspects, the subject is greater than or about 2 years old, and optionally, a dose of about 300mg of rhTPP1 is administered to the subject. In various aspects, the subject is greater than or about 1 year old and less than 2 years old, and optionally, a dose of about 200mg of rhTPP1 is administered to the subject. In exemplary aspects, multiple doses of rhTPP1 are administered to an individual. In exemplary aspects, the methods comprise administering an initial dose followed by administration of a subsequent dose. In various aspects, the initial dose is lower than the subsequent dose. In various aspects, the initial dose is higher than the subsequent dose. In various aspects, the initial dose is about 200mg of rhTPP1, and subsequent doses are higher. In exemplary aspects, the initial dose is about 200mg of rhTPP1, and subsequent doses are at least 50% higher, such as at least or about 300mg of rhTPP 1. In exemplary aspects, each of the 1 st, 2 nd, 3 rd, and 4 th doses administered to an individual (e.g., an individual older than or about 1 year and younger than 2 years) is about 200mg of rhTPP1, and each of the 5 th and subsequent doses administered to an individual is greater than about 200mg of rhTPP 1. In an exemplary case, each of the 5 th and subsequent doses administered to the subject is about 300mg of rhTPP 1. In various aspects, the subject is greater than or about 6 months of age and less than 1 year of age, and optionally, a dose of about 150mg of rhTPP1 is administered to the subject. In various aspects, the individual is less than 6 months of age, and optionally, a dose of about 100mg of rhTPP1 is administered to the individual. In various aspects, the formulation is administered once every 2 weeks, once every two weeks, once every other week, or about once every 14 days (± 3 days). In various aspects, the formulation is administered with infusion at a rate of about 2.5mL per hour or at a lower rate.

In exemplary aspects, based on blood testing, individuals, e.g., pediatric individuals, exhibit a decrease in TPP1 enzyme activity. In an exemplary case, an individual, such as a pediatric individual, is a sibling of an individual diagnosed with CLN2. In an exemplary case, an individual, such as a pediatric individual, is not a sibling of the individual diagnosed with CLN2. In an exemplary aspect, an individual, e.g., a pediatric individual, is scored from about 3 to about 6 (e.g., about 3, about 4, about 5, about 6) based on the overall score of the athletic and linguistic subscales. In an exemplary aspect, the score of the individual after rhTPP1 has been administered is closer to the score associated with "healthy" or "normal" according to the scores recited in table 1.

In exemplary aspects, an individual, e.g., a pediatric individual, has not been previously treated with stem cell therapy, gene therapy, or enzyme supplementation therapy. In exemplary cases, the method comprises administering to the subject an antihistamine with or without an antipyretic prior to administration of rhTPP1, optionally about 30 to about 60 minutes prior to administration of rhTPP 1. In various aspects, the formulation includes rhTPP1 and at least one pharmaceutically acceptable carrier, diluent, or excipient. In various instances, the formulation is any of the aforementioned formulations, including but not limited to formulations comprising disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or combinations thereof. In various instances, the method comprises administering a rinse solution to the subject after administering the formulation. In various aspects, the rinse solution is any of those described herein. In various aspects, the flush solution comprises disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof. In various aspects, the treatment period is at least 10 weeks, at least 20 weeks, at least 40 weeks, at least 80 weeks, or at least 96 weeks. In various aspects, the treatment period is longer than 96 weeks. In various aspects, the methods treat CLN2 disease or delay the onset of CLN2 disease or symptoms thereof without causing a Serious Adverse Event (SAE). In various aspects, the individual has an ICV device. In various aspects, the method comprises implanting an ICV device in a subject.

Reagent kit

The present disclosure further provides kits comprising the formulations of rhTPP1 described herein in dosages and forms suitable for administration to a patient. In one aspect, a kit includes a formulation including rhTPP1 at a concentration of about 30mg/mL, disodium phosphate heptahydrate at a concentration of about 0.11mg/mL, sodium phosphate monobasic monohydrate at a concentration of about 0.08mg/mL, sodium chloride at a concentration of about 8.77mg/mL, potassium chloride at a concentration of about 0.22mg/mL, magnesium chloride hexahydrate at a concentration of about 0.16mg/mL, calcium chloride dihydrate at a concentration of about 0.21mg/mL, and a diluent, such as water for injection. In one aspect, the kit further comprises instructions for intracerebroventricular, intrathecal and/or intraocular administration of a therapeutic composition of the invention in addition to the therapeutic formulation. In another aspect, the kit further comprises a wash solution as described herein. In yet another aspect, the kit further comprises a system for administering the formulation comprising any or all of the following components: tubing, inline filters, reservoirs for implantation, and catheters. In one aspect, a kit may include a catheter, reservoir, or other device preloaded with a therapeutic formulation of the present disclosure. For example, catheters pre-loaded with about 100mg of rhTPP1, about 200mg of rhTPP1, about 300mg of rhTPP1, about 400mg of rhTPP1, or about 500mg of rhTPP 1in a pharmaceutically acceptable formulation are particularly contemplated. Alternatively, the kit may comprise a refillable catheter, reservoir or other device and an appropriate amount of enzyme for refilling the device.

In certain embodiments, a kit of the invention may comprise one or more of the following components: extension lines (e.g. product number 536040, Smiths Medical, Dublin OH), in-line filters (e.g. product number FS116, Smiths Medical), port needles (e.g. product number 21-2737-24, Smiths Medical), a syringe or two or more syringes (e.g. product number 309604, Becton Dickinson, Franklin Lakes, NJ) or a syringe needle or two or more syringe needles (e.g. product number 305196, Becton Dickinson).

The disclosure will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

Examples of the invention

The following examples describe formulations including rhTPP1 for Intracerebroventricular (ICV) administration and the results of administering the formulations to human patients compared to matched untreated natural history patients.

Example 1

Formulation of rhTPP1 for intraventricular administration

rhTPP1 was produced in a genetically engineered CHO host cell line and purified using standard chromatography methods, as described in U.S. Pat. No. 6,302,685 and Sleat et al 1997, Science 277: 1802-. rhTPP1 is produced in the form of an inactive pro-enzyme that self-activates at acidic pH upon uptake into lysosomes. The calculated isotopic average molecular weight of the enzyme form prior to rhTPP1 was about 59 kDa. The apparent molecular weight of the mature enzyme is approximately 46 kDa. The amino acid sequence of the rhTPP1 proenzyme is set forth in SEQ ID NO. 1 and shown in FIG. 1. The enzyme is preceded by the first 176 amino acid residues and the mature enzyme is 368 amino acids in length starting at position 177 and is set forth in SEQ ID NO 2.

The rhTPP1 formulation used in the examples was a sterile solution for ICV infusion. It is a clear and colorless to pale yellow liquid containing rhTPP1 protein formulated at a concentration of 30 mg/mL. The formulation was packaged in a container closure system consisting of a type 1 clear borosilicate glass vial closed with a fluoropolymer coated butyl rubber stopper and capped with an aluminum seal. The formulations were stored and served frozen at a temperature of-40 ℃ ± 10 ℃. The target pH of the formulation was pH 6.5.

The composition of the rhTPP1 formulation used in the examples is provided in table 2.

TABLE 2

The rhTPP1 formulation is carefully designed to mimic the characteristics of human CSF, e.g., the concentration of critical electrolytes is similar to that found in vivo in human CSF, and the formulation does not contain any conventional preservatives or stabilizers as excipients. No safety issues, i.e., severe adverse reactions, were reported or observed following administration of rhTPP1 formulation, which may not have been previously predictable.

Stability studies were conducted to monitor time-temperature stability under long-term (. ltoreq. -60 ℃) and accelerated conditions (5. + -. 3 ℃) in accordance with the ICH guidelines and following the protocol. The stability samples were stored in small size vials made of the same material as the full size package. Stability data collected on supportive and clinical batches demonstrate that rhTPP1 formulations are stable at ≦ -60 ℃ for at least 36 months, and at 5 ± 3 ℃ for at least 6 months, which was unexpected given that the formulations lack preservatives and stabilizers commonly found in pharmaceutical products. Table 3 shows the results of the stability tests.

TABLE 3

Example 2

Study of natural disease history

Quantitative assessments of disease progression were analyzed in a cohort of 41 untreated CLN2 patients for the natural history of CLN2. The hamburger clinical scale was used to assess age-appropriate nerves and functional domains affected by disease.

A quantitative description of clinical decline in untreated natural history CLN2 individuals is shown in figure 2. Natural history analysis confirms a clear and predictable relationship of age to disease severity. After the onset of motor and linguistic symptoms, there is a substantial tendency for rapid linear decay, with the average child losing about 2 milestone events per year (linear rate of decay of 2.1 points per year). There are largely predictable courses of disease, but there are still some 'late-onset' cases that account for less than 20% of the population in the cohort. These patients tend to have late onset symptoms and mild disease for a longer period of time, but then die with rapid and active decline, usually 2 to 3 years later than typical forms.

Quantitative clinical progression from the hamburger cohort was confirmed by overlaying clinical grade assessments from independent (patient and scorer) cohorts of WCMC (n ═ 49). Although the clinical description of the independent CLN2 cohort was similar, this was the first confirmation of a robust quantitative relationship in disease progression in different patient groups. Both cohorts of CLN2 patients had the vast majority of typical late stage infant onset and progression, and a smaller proportion of children with 'late-onset' expression, often with early disease expression at the age of 5 rather than 3 years. Neural deterioration of CLN2 patients was reproducibly retrieved using a scale of motor (gait) and language function. Based on the foregoing analysis, a cohort of natural study individuals was determined to be an appropriate non-treated control population, and the average rate of decline of symptoms of CLN2 disease in this untreated natural history population can be used as an effective and informative comparative example for any prevention or reduction in the rate of decline of symptoms in individuals afflicted with CLN2 disease caused by administration of the compositions of the present invention.

Example 3

Phase 1/phase 2 open marker dose escalation study of CLN2 patients

The study was an open label therapeutic clinical trial to evaluate the safety, tolerability, and efficacy of rhTPP1 formulations of the present disclosure delivered via ICV catheters to children with CLN2 disease at doses of 300mg (10mL total volume) every other week. The study was designed to evaluate safety and tolerability starting at low doses (30mg and 100mg), but when the independent data monitoring committee indicated that lower doses were safe, all patients were escalated to the high expected therapeutic dose (300 mg). The study duration for all enrolled patients was treated for 48 weeks at a stable expected therapeutic dose of 300mg ICV every other week. The primary study objective was to assess the safety and tolerability of rhTPP1 formulations of the present disclosure administered to individuals with CLN2 disease using implanted ICV reservoirs and catheters, and to assess efficacy using the CLN2 disease-specific rating scale score, compared to natural medical history data after 12 months of treatment. The secondary study objective was to evaluate the effect of treatment on the measure of brain atrophy, compared to CLN2 disease natural history data after 12 months of treatment.

The primary inclusion criteria was CLN2 diagnosis and a registration age of at least 3 years. Patients with baseline disease rating scores less than 3 (using the hamburger 0 to 6 aggregate motor/language scale) at screening were excluded from the study. Patients under 3 years of age are likely to be progressing due to age rather than treatment, as depicted by the horizontal line on the progression curve. Patients with a score of 2 or less at screening are also less linear, more variable, and may be considered more difficult to treat due to the disease stage. Thus, the treatment group is defined by age and score only to encompass early and highly predictable decline.

The mean age at enrollment was 4.0 years with slightly more girls than boys, mainly caucasians. Clinical CLN2 scores at screening and baseline are shown in table 4 below, which shows the hamburger motor/language score and the total score of screening and baseline for each study cohort.

TABLE 4

Overall, the pre-treatment CLN2 score biased towards more advanced disease. The expectation score will be low in view of the rapid development of the disease and the difficulty of identification. In addition, there was a decline in scores at screening as well as during baseline assessment (just prior to placement of ICV reservoir) (up to two weeks). There were 4 patients in the screening group who scored 3 who lost one point at baseline, and two patients in the screening group who scored 4 lost 3 points at baseline. Two patients who entered at 6 years old (i.e., roughly normal) were siblings of the affected child. The deployment, demographics, and characteristics of the population of individuals are summarized in table 5 below.

TABLE 5

^a Enrolled patient 1287-.

^b Similar to the distribution of natural medical history population

^* General genotype: c.622C>T and c.509-1G>C

All enrolled patients received a steady dose of 300mg ICV every other week. Group 1 was exposed to 30mg ICV for 1 month or more every other week, then rose to 100mg ICV for 4 weeks or more every other week, while group 2 began receiving 100mg ICV for 4 weeks or more every other week. Both cohort 1 and cohort 2 were raised to 300mg ICV every other week, and all subsequent patients, including cohort 3, were started to be dosed at a stable dose regimen of 300mg ICV every other week for ≧ 48 weeks. The 300mg dose was administered via an ICV catheter over a period of about 4 hours in the form of a 10mL infusion. A volume of CSF, e.g., equal to the amount of rhTPP1 formulation to be administered, was not removed just prior to the start of infusion, which is atypical, but surprisingly did not cause any adverse effects. An amount of about 2mL of flush solution was administered to the individual via the same ICV catheter immediately after administration of the 300mg dose. The rinse solution was consistent with the formulation in table 2, but without rhTPP 1. A single dose of 300mg of enzyme per administration event is significantly higher than the enzyme replacement therapy previously administered intrathecally or ICV, and thus the safety and efficacy profile observed after administration of such high doses of drug may not have been previously predictable. More specifically, it may not have been previously possible to predict the administration of a single dose of 300mg of rhTPP1 without the associated serious, unmanageable adverse events.

Results

Effect of treatment on clinical assessment of gait and language: the primary assessment tool to quantitatively assess clinical severity was a 0 to 6 point aggregation of gait and language sub-tables shared by the two hamburger and WCMC disease rating scales. This scale retrieves a predictable, rapid and progressive clinical decline in matched untreated natural history patients, which is used as a comparative example for the primary efficacy analysis.

Gait/language disease assessment scores for 23 patients with treatment duration exceeding 42 weeks are shown in figures 3A to 3F. Of the 23 patients, 3 patients (1244-1001, 1244-1002 and 1244-1003) were from group 1(C1), 3 patients (1244-1004, 1244-1006 and 1287-1005) were from group 2(C2), 3 patients (1244-1008, 1244-1009, 1244-1010) were from group 3(C3), and 14 patients (0119-1020, 0146-1021, 0146-0141022, 0146-1023, 1244-1023, 1011 4-1012, 1244-1017, 1244-1024, 1321013, 1323-1014, 1323-1015, 1323-1016, 1323-1018 and 1323-9) were from 300mg stable drug administration (SBO) group. As expected, language deficits are generally more advanced than gait deficits. Entry scores are not randomly distributed; disease progression was significant for 12 patients with a combination entry score of 3 and a combination entry score of 6 for 2 patients. Given rapid progression and disease confirmation, children often express significant decline or siblings that express those that are significant decline.

After treatment with the rhTPP1 formulation of the present disclosure (shown in table 2 above), the CLN2 gait/language disorder assessment score was stable as shown in figures 3A to 3F. Eleven of the 23 patients did not show irreversible regression during the treatment period. Four patients experienced single unit regression early in treatment, but no irreversible regression thereafter. Two patients (1244 1008 and 1323 1013) dropped by one unit from 3 to 2 points between screening and baseline, but did not experience any additional loss in the assessment during treatment. Based on the results, significant therapeutic benefit occurred in all patients regardless of cohort (initial dose) or entry score. In many patients, a reversal in decline was assessed. For example, the patient's 1287-1005 (fig. 3B) assessment declined by 2 units within the first month of treatment, which represents a loss of function in both gait and language. However, this patient recovered one unit on the 60 th treatment day, and there was no net change thereafter until the 440 th analysis day. The score for recovery is the gain in language, emphasizing the clinical importance of single unit changes.

None of the 2 patients with a score of 6 at entry lost one unit of assessment. There were 7 of 12 patients with an entry score of 3 who did not show irreversible decline, and 2 stable after the initial single unit decline. Thus, therapeutic benefit is evident in patients with significant deficits and disease progression.

The median rate of decline in the untreated natural history population was estimated to be 2.1 units per year as demonstrated by the CLN2 natural history study. Thus, all patients in the treatment group had an improved assessment compared to the expected outcome for the untreated natural history population.

To establish a clearer disease course relationship between treated patients and matched untreated natural history patients, each study patient was matched to untreated natural history patients using parameters of baseline CLN2 score, age, and genotype. Although there are no clear subgroups or factors predicting the progression of CLN2 disease, these parameters are most often used to define disease severity. Individual treated patients were compared to members of a natural history cohort with similar gait/language assessment scores at baseline, as shown in figures 4A-4I. Patients in the study were matched using the baseline CLN2 score as follows: for a given study patient with a given baseline score, all natural history patients reported one or more CLN2 assessments with the same CLN2 score were identified. If the baseline CLN2 score for the study patient is 2,3, 4, or 5, then the CLN2 versus time profile for each natural history patient is shifted left or right such that it overlaps with the baseline score for the study patient. If a natural history patient has multiple assessments equal to the baseline CLN2 score of the study patient, then the intermediate time points of the multiple assessments are used for time-shifting. If the baseline CLN2 score for the study patient was 6 points, then the final score for the 6 natural history patient was used for time-shifting. Sensitivity analysis was performed using other matching criteria, and the results from these analyses were consistent with the analysis of score matching.

Fig. 4A-4I show results from individuals treated with the rhTPP1 formulation of the present disclosure plotted against matched untreated natural history patients. Treated individuals and untreated natural history patients were matched using disease assessment scores using 0 to 6 units of gait and language subscales as an aggregate. The assessments were compared over a one year treatment period. Individuals receiving rhTPP1 were therapeutically beneficial compared to all members of the matched untreated natural history patient group. The assessment of subject 1244-. The assessment of subjects 1244-1002 (fig. 4B) increased from 3 units to 4 units, decreased from 4 units to 2 units, and increased from 2 units to 3 units, so that the overall disease assessment remained unchanged compared to day 1 at the end of the study. The individuals 1244 + 1003 (FIG. 4C) and 1244 + 1010 (FIG. 4I) maintained 6 units of assessment, i.e., normal motor and language function, throughout the study. Individuals 1244-1004 (FIG. 4D) and 1244-1009 (FIG. 4H) maintained 3 units of assessment throughout the study. The assessment of the individuals 1244-1006 (FIG. 4E) was first reduced from 3 units to 2 units, but recovered one unit before being reduced from 3 units to 2 units again, with no net change thereafter. The assessment of individuals 1244-1008 (fig. 4G) first decreased from 3 units to 2 units, after which there was no net change.

In contrast to all treated individuals, prior to the end of the comparison period, the irreversible assessments in the majority of their matched untreated natural history patients decreased from 3 units to 0 units, indicating complete loss of combined gait and language function. Analysis of the match confirms the therapeutic benefit of those patients who maintain their disease rating score and also have an initial rating decline, but then stabilize.

The most complex reaction (individuals 1287-1005) is shown in figure 4F. Although this patient dropped rapidly by 2 units within the first month of the study, from a baseline score of 3 points to a study score of 1 point, the patient was able to recover one unit and stabilize at 2 points. Interpretation of this course is elucidated by comparison of the score matched untreated natural history patients. Clinical course was worse in 15 of 18 score-matched untreated natural history patients and was identical in only 2 score-matched untreated natural history patients (single untreated natural history match was not evaluable). Clinical course in untreated patients is always worsening, often with little time between lost milestones. The lost function and subsequent stability has never been re-established. Thus, matching with the most complex treated profile also indicates significant therapeutic benefit.

FIG. 5 shows the distribution of clinical changes from baseline for patient-matched treatment durations in matched untreated natural history patients compared to study individuals. As previously described, there was no change in the baseline disease rating scale for 7 out of 9 patients (> 75%). For this 7 patients during treatment, all matched untreated natural history patients declined by at least one unit, but more commonly by multiple units or 2 to 4 units. As an example, patient 1244-. Thus, treated patients did not change over the same time period, but 14 of the 18 matched untreated natural history patients (> 75%) lost all gait and language functions. There was a lower effect of a baseline entry score of 3, where many matched untreated natural history patients lost all available units of assessment, but 2 patients with an entry score of 6 (patients 1244 + 1003 and 1244 + 1010) showed importantly that these matches were also actively decreasing, some of which decreased 4 and 5 points over the treatment period. This observation clearly demonstrates substantial therapeutic efficacy clinically; most treated children remain in clinical assessment with an untreated natural history match, with active loss of speech and independent gait over the same period of time, with many people completely losing function. The remaining 2 treated patients with loss of a single fraction (individuals 1287-. Overall, 97% of untreated natural history patients with matching scores scored worse than treated individuals.

Using multiple matching criteria (e.g., baseline, age, and genotype), nearly 100% comparison showed favorable therapeutic effects compared to matched untreated natural history patients. The mean treatment difference for the matched criteria in all treated patients compared to natural history patients ranged from 1.9 to 2.1 points depending on the matched criteria used.

FIG. 9A shows the mean change in motor-language assessments for patients treated for > 48 weeks (n-21; dashed line) and natural history cohorts (n-41; solid line). The mean decrease in disease rating for the treated patients was 0.43 (standard deviation 0.839), with a median decrease of 0.00 units over 48 weeks. By contrast, the mean decrease in disease rating for the natural history cohort was 2.09 (standard deviation 0.966), with a median decrease of 1.87 units over 48 weeks. Overall, the expected clinical decline in treated patients was significantly reduced (p <0.0001) 79%. Figure 9B shows the change in motor-language scores for patients (n ═ 23) measured from the first 300mg dose (baseline) to the last before the last 300mg dose at ≧ 48 weeks. Overall, 65% (15 of 23) of the patients improved or had no clinical disease progression during treatment, and 87% (20 of 23) of the patients expressed more preferentially (i.e., changed score by-1 or more) during treatment than untreated individuals from natural history studies. These analyses consistently support the following conclusions: there was significant and clinically meaningful stability of CLN2 scores in treated patients compared to matched members of the untreated natural history group that declined rapidly and predictably.

Effect of treatment on clinical assessment of vision: in untreated CLN2 patients, vision loss occurs later than the decline in language and gait, but once symptoms appear, the course of the disease is predictably rapid and progresses to blindness. Therefore, preserving vision is an important therapeutic outcome. Visual loss can be retrieved on a 0 to 3 point subscale in a similar manner to other subscales, where 3 is normal and 0 is functional blindness. During the treatment period, most of the treated patients did not experience irreversible loss in the visual component range. When matching treated patients to untreated natural history individuals by score, age and genotype using a combination of scales of gait, language and vision subscales (0 to 9 units), it is evident that untreated matched natural history patients lose additional points compared to the treated group.

Fig. 6A-6I show results from nine individuals treated with the rhTPP1 formulation of the present disclosure plotted against matched untreated natural history patients on a disease assessment score using 0 to 9 units of gait, language and vision subscales as an aggregate. The assessment of the individual 1244-1001 (fig. 6A) was decreased by one unit from score 6 to 5, but immediately thereafter restored to a score of 6, with no net change thereafter. The assessment of subjects 1244-1002 (fig. 6B) increased from score 5 to score 6, then decreased from score 6 to score 4, then increased from score 4 to score 5, so that the overall disease assessment at the end of the study remained unchanged compared to day 1. The individuals 1244 + 1003 (FIG. 6C) and 1244-1010 (FIG. 6I) maintained a 9 point assessment throughout the study, indicating gait; the language function and the vision are normal; individuals 1244-1004 (FIG. 6D) and 1244-1009 (FIG. 6H) maintained a 6 point assessment throughout the study, and individuals 1244-1008 (FIG. 6G) maintained a 5 point assessment throughout the study. The initial assessment of the individuals 1244-1006 (fig. 6E) is reduced by one unit from score 6 to 5 and then again to 4, but is restored by one unit to an assessment of 5, after which there is no net change. The individual 1287-1005 (FIG. 6F) was rated as decreasing from 6 to 4 points, then increasing from 4 to 5 points and decreasing to 4 points, but again returned to one unit to a final rating of 5 points.

During the treatment period, the addition of the visual subscale did not change nine treated patients. However, the visual loss of patients with untreated natural history who match the score contributes significantly to the aggregate score. There were multiple untreated natural history matches compared to treated patients, with differences greater than 3 points, indicating a contribution of visual deterioration to the aggregate score during the study. Thus, adding a vision sub-chart increases the difference between treated patients and matched untreated natural history patients. Since there is no irreversible loss of disease assessment units in matched untreated natural history patients, the observation of the therapeutic efficacy of rhTPP 1in connection with arresting disease progression and stabilization function can be extended from exercise/gait and language to the clinical domain encompassing vision.

Effect of treatment on overall disease assessment: patients were also evaluated during the study using a 12-part table containing combinations of complete hamburger or WCMC scores. The 12-part scale score is the sum of the individual scores for (1) locomotion/gait, (2) speech, (3) seizures/myoclonus, and (4) vision/eating by the patient. Figures 10A to 10L show the results of individuals treated with rhTPP1 formulations of the present disclosure using a combination hamburger (left panel) and WCMC scale (right panel) of 0 to 12 units. Sixteen of the 23 patients had no irreversible decline according to at least one scale and 8 had increased scores according to at least one scale at the end of the treatment period compared to baseline, confirming the overall therapeutic benefit of patients receiving rhTPP 1.

Effect of treatment on brain volume: MRI was used to assess the secondary endpoint of treated patients. The disease process is characterized by atrophy, cell loss, and abnormal signaling. These parameters are correlated to patient age and disease rating score either individually or as a composite score. Thus, disease progression is generally considered to be associated with an atrophic MRI index, and various MRI parameters have been shown to be associated with the age and disease severity of CLN2 disease (Dyke et al, J. neuroradiology (AJNR Am J. Neuroadiol.) -2013; 34(4): 884-9); (Paniagua et al, clinic neuroradiology (Clin neuroadiol.) 2013; 23(3): 189-96). The imaging database supporting these conclusions is based on cross-sectional correlations of a large number of patients, however, there is no longitudinal acquisition of MRI images within the patient. Thus, longitudinal study-derived MRI analysis cannot be matched equally to similarly derived natural medical history databases.

For analysis of treated patients, MRI acquisition parameters were standardized across the entire hardware platform at the study site. The data is compiled identifying information acquired locally and has been passed to a central imaging core laboratory. The images were randomized so that the independent radiologist did not know the patient or the temporal relationship to the baseline. Changes in brain volume were reconstructed from the random independent central readings. Data was analyzed to longitudinally compare the study to the baseline of the treated population. Figure 7 shows an overview of MRI measured brain volume of treated patients. Brain atrophy results in a greater volume and proportion of intracranial CSF. These increases in atrophy measurements correlated with the age and severity of CLN2 patients. Longitudinal plots of CSF volume and proportion of treated patients indicate that the measurement of CSF parameters also appears to be almost unchanged, if at all. MRI volumetric determination for all patients appeared constant and consistent with the stability of the assessment.

Fig. 8A to 8L show longitudinal MRI assessment of brain volume in treated patients. Active neurodegeneration in CLN2 patients is characterized by a large loss of gray matter and a compensatory increase in CSF. However, during the evaluation period, the brain volume of the treated patients was very stable and there was no evidence of a neurodegenerative process. The differences between CSF and gray matter curves (dashed lines) and CSF curves (solid lines) in each of the upper and lower panels in fig. 8A-8L show that gray matter granulometry of each of the treated patients remained stable throughout the study. The change in cortical gray matter particle volume as a percentage of total brain volume from the last measurement prior to the first 300mg infusion (baseline) compared to the last observation after treatment at > 48 weeks is shown in table 6 below.

TABLE 6

Longitudinal changes in cortical granules were-1% per year in normal children between 4-12 years of age, but-12.5% per year for untreated CLN2 patients. During treatment with rhTPP1, the volumes of CSF, gray matter, and white matter remained relatively constant, reducing 89% of the disease-associated cortical granule loss.

Adverse events: one patient dropped out of the study due to failure to comply with the protocol. The remaining 23 patients remained in the study and were resistant to treatment with rhTPP1 drug via the ICV pathway. There were no deaths, treatment-related withdrawals, or study discontinuations for safety-related reasons. All patients were dosed intra-operatively for one week, consistent with the minimal impact of device implantation. Of the total 325 infusions, only 5 (1.5%) were discontinued for any reason, with only 2 (0.6%) discontinued for reasons associated with adverse events. The most common non-CLN 2 disease-related adverse events observed in the study were fever, allergy, and upper respiratory tract infection (each accounting for 25% of the total population of individuals). Typically, these events are mild, self-limiting and medically regulated. Investigator-defined allergic events were associated with minimal peripheral expression and were medically managed with a combination of antipyretics, antihistamines, and/or steroids. Laboratory data confirmed the lack of clinically relevant changes in the surrounding laboratory. In cerebrospinal fluid, some patients develop mild, transient cerebrospinal fluid cytosis, with no change in CSF glucose or protein. Overall, evaluation of safety parameters demonstrated that treatment with the rhTPP1 formulation of the present disclosure via ICV infusion could be tolerated in all patients.

Conclusion

Clinical studies demonstrated significant clinical benefit in patients with each treatment exposure over 36 weeks, characterized by complete arrest of progression of CLN2 disease, which constitutes the greatest therapeutic benefit, since function acquisition was not expected over a time frame for patients with moderate progression and mobility deterioration.

This finding is even more convincing when patients are matched to members of the natural medical history database based on a number of parameters, including baseline disease assessment, age, and genotype. This match discloses that matched untreated natural history patients experienced significant loss of function during the same time period that the treated individuals experienced stopped disease progression when treated with rhTPP1 drug. Thus, all treated patients showed a suppression of disease progression compared to active disease progression in matched untreated natural history patients. Based on the available natural medical history data, the median rate of decline for the untreated natural medical history population was estimated to be 2.1 points per year, with each declining unit representing a significant milestone loss of physiological function. For most patients entering the study, keeping 2 units means continuing to walk independently with meaningful communication.

Overall, the results demonstrate that the rhTPP1 formulations and methods of treatment of the present disclosure have an acceptable safety/tolerability profile. No individual discontinued study or treatment due to adverse events. One individual, due to failure to follow the protocol, exited the study after one treatment dose. Analysis of PK and immunogenicity discloses high CNS delivery and no antibody formation in CSF.

The foregoing examples demonstrate that the formulations and methods described herein comprising rhTPP1 are effective in preventing or treating one or more clinical symptoms of CLN2 disease and/or CLN2. In diseases where the clinical course is characterized by rapid, unalterable and irreversible neurodegenerative disease progression, especially in each treated patient, cessation of disease progression is a significant and unexpected clinical benefit.

Example 4

This example describes a phase 2 open label study to evaluate the safety, tolerability, and efficacy of formulations including rhTPP1 for intraventricular administration to pediatric patients less than 18 years of age who have CLN2 disease.

Object of study

The main goals of this study include the following: (1) evaluating the safety and tolerability of TPP1 administered via an Intracerebroventricular (ICV) device, and (2) evaluating the efficacy of treatment as a delay in progression of the CLN2 motor-language clinical scale.

Secondary goals of this study included the following: (1) assessing immunogenicity in CSF and serum, (2) assessing MRI parameters of disease progression, and (3) assessing the effect of treatment on the total hamburger clinical rating scale.

The exploratory goals of this study included the following: (1) assessing developmental achievement, (2) assessing abnormal involuntary movements, (3) assessing retinal anatomy using Optical Coherence Tomography (OCT), (4) determining seizure, type and frequency, (5) assessing quality of life metrics, and (6) analyzing putative molecular biomarkers from CSF and plasma.

Study plan: overall research design and planning

This would be a phase 2 open-label study for CLN2 patients who have demonstrated CLN2 gene mutations and impaired TPP1 activity. Formulations including rhTPP1 (referred to herein as "TPP 1") will be administered biweekly using ICV infusion. Since both actual and ethical issues preclude concurrent or untreated control individuals, findings can be compared to prior history data for CLN2 registration and/or data from siblings (as needed). An overview of events is provided by the access in FIG. 11.

Study suitability will be determined prior to admission of the individual to surgery to implant the ICV access device (< 21 days). Baseline visits to collect clinical scores and clinical laboratory parameters will be completed no more than two days prior to the first infusion.

The planned enrollment for this study was at most 5 individuals. Patients eligible to enroll in this study must have a sibling diagnosed with CLN2 registered in the previous study described in example 3. All subjects were administered a dose of TPP1(300mg) every 14 days for at least 96 weeks from the day of the first infusion (+3 days). Administration may be tailored to the investigator's judgment of adverse events during the course of the trial by decreasing to 150mg and/or by decreasing the infusion rate.

Patients in this study will be required to surgically implant an ICV reservoir for administration of TPP 1. MRI will be performed prior to the procedure to ensure proper planning and placement of the ICV access device. Patients will be closely monitored in a care-intensive environment 48 hours post-operatively. Upon placement of the ICV access device, the individual and their caregiver will be provided with written instructions that provide detailed information about the signs and symptoms of device complications, as well as instructions as to when to return to the point of study for device evaluation. The hospitalized patient will have another follow-up phone call within 48 hours of discharge.

The first infusion will be performed at least 14 days from surgery to implant the ICV reservoir and no more than 28 days post surgery. Thereafter, study visits were made every two weeks ± 3 days from the day of the first infusion (+3 days). Generally, functional and QOL assessment should precede MRI and sampling, which should precede infusion; when a subject is sedated for MRI, sample collection can be performed. If the individual aborts the study prematurely, the visit should be scheduled to terminate prematurely within 3 days.

For all infusions, the individual will be monitored in an inpatient setting for at least 24 hours from the start of the infusion. For the first infusion only, the individual will also return to the clinic for a follow-up visit 72 hours after the infusion begins. After all visits, the parent or legal guardian will be called up within 48 hours to determine health status.

Vital signs will be measured within 30 (+ -5) minutes prior to each infusion, every 60 (+ -5) minutes during each infusion, and 1 and 4 hours (+ -5 minutes) after each infusion. For the first dose, vital signs will be measured within 30 (+ -5) minutes before infusion begins (or resumes), every 30 (+ -5) minutes during infusion, 0.5 and 1 hour (+ -5 minutes) after the end of infusion, and every 4 hours (+ -15 minutes) until discharge.

Efficacy will be measured using the CLN2 motor-language clinical rating scale. Hamburger 0 to 12 points CLN2 total scale will be collected. Other secondary efficacy measures would include developmental status, seizure frequency, involuntary movements, and retinal anatomy and quality of life metrics. The safety and tolerability of treatment will be assessed by collecting Adverse Events (AEs), body findings, vital signs, ECGs, EEG, and clinical laboratory tests. Researchers will assess the severity, and relationship of AEs to study drug and/or ICV access devices.

Since allergic reactions may be associated with ERT administration, subjects should be pre-treated with an age-appropriate dose of antihistamine (and if appropriate an antipyretic) about 30 minutes prior to infusion. According to standard practice in the institution, the investigator may pre-treat the subject with an age-appropriate sedative drug, as appropriate, about 30 minutes prior to infusion of TPP 1.

The time relationship of drug infusion will be studied to define TRE. This should be distinguished from clinical assessment of infusion response. Therefore, adverse events occurring within 24 hours of the start or restart of infusion of TPP1 are defined as TRE.

Certain events occurring within a 24 hour period after infusion may be allergy-mediated. Allergic reactions are characterized by the occurrence of an adverse local or systemic reaction upon exposure to an allergen. Symptoms of anaphylaxis may include fever, chills, skin symptoms (urticaria, angioedema, rash), respiratory symptoms (dyspnea, wheezing), gastrointestinal symptoms (nausea, vomiting, abdominal pain) and/or cardiovascular changes (hypotension/hypertension).

In severe cases, severe allergies (systemic anaphylaxis) may also occur. Severe allergy is the most severe form of anaphylaxis in which symptoms may appear during infusion or within hours; if severe allergies are left untreated, death may result.

Symptoms of severe allergy may include skin and/or mucosal tissue involvement (e.g., systemic urticaria, itching or flushing, swelling of the lips-tongue-palate), dyspnea (e.g., dyspnea, wheezing-bronchospasm, wheezing, peak expiratory flow reduction, hypoxemia), and reduced blood pressure or peripheral organ dysfunction (e.g., hypotonia, syncope, incontinence).

To date, no severe allergy or severe allergy-like reactions have been developed by the TPP1 study. However, if a severe allergic reaction, severe allergic event or severe allergy (defined as a grade 3 or higher allergic event) is suspected, blood samples will be collected within 1 hour of the event to assess C4, serum tryptase and total IgE; to assess drug-specific IgE, blood samples will be collected no more than 8 hours after the event occurs (or before the next infusion).

Another possible AE is infection from indwelling ICV catheters. If infection is suspected, blood and CSF samples will be drawn for evaluation. Subsequent treatment procedures may include antibiotic therapy and catheter replacement or withdrawal. If TPP1 treatment is suspended, TPP1 may be resumed if the dose missed consecutively after the last given dose is no more than 4.

The evaluation schedule for the study plan will be provided in fig. 11.

Screening

Prior to performing any study-related procedures, the parent or legal guardian will provide informed consent to the eligible patients for the study. The patient will be evaluated to determine if they meet the study entry criteria (example 5) and are candidates suitable for implantation of an ICV access device. Diagnosis of CLN2 disease as determined by TPP1 enzyme activity (dried blood spots) should be available at the time of screening. Whether or not this genotype information is available, blood will be collected at the screening visit for CLN2 gene analysis. In addition, blood for TPP1 enzyme activity (dried blood spots) will be collected and subjected to a centralized analysis at the screening visit. The screening procedure should be completed no more than 21 days prior to ICV reservoir implantation surgery.

The CLN2 rating scale will be performed globally as appropriate for the individual. The assessment guide provides detailed instructions on assessment of the assessment Scale, and Imaging Charter (see denver Development Scale ii) provides instructions on MRI assessment with a centralized facility. In addition, a full physical examination will be performed.

Surgery and first dose

The study individual will be sent to a hospital for surgical implantation of an MRI compatible ICV access device in the lateral ventricle of the right hemisphere; surgery and anesthesia will be performed under the guidance of the neurosurgeon (see handbook of research Pharmacy (), for compatible ICV reservoirs and catheters). MRI will be performed preoperatively to ensure proper planning and placement of the ICV access device. Generally, surgery and post-operative care will be determined by the standard of care of the research center and the specific clinical needs of the individual. Individuals will be monitored post-operatively for at least 48 hours in a care-intensive environment. Upon placement of the ICV access device, the individual and their caregiver will be provided with written instructions that provide detailed information about the signs and symptoms of device complications, as well as instructions as to when to return to the point of study for device evaluation. Follow-up phone calls will be made again within 48 hours after the hospitalized patient is discharged.

The first infusion will be performed at least 14 days from surgery to implant the ICV reservoir and no more than 28 days post surgery. At the first (and each subsequent) study drug administration, the investigator will assess the patency, location, and skin integrity of the reservoir. Device access is performed using strictly sterile techniques. The skin covering the reservoir is inspected for the appropriate needle insertion site. The needle insertion site must be intact with no signs of rupture, wound, infection or rash. The needle used is a small gauge non-apertured tip. Once the reservoir is accessed, the needle is fixed to ensure minimal risk of removal or removal. Guidelines for evaluating reservoir devices for leakage and replacement are provided in the research pharmacy handbook. The reservoir may be replaced during the clinical study at the discretion of the researcher and/or neurosurgeon.

Discussing study design, including selection of control groups

This study must be conducted in patients with CLN2 disease, as the potential development of ICV access device implantation or TPP1 autoimmunity, and its unknown long-term health consequences, are acceptable risks for healthy volunteers. However, within a few years it is almost certain that serious disability and death can balance these risks in a patient population.

Diagnosis of CLN2 disease is typically based on analysis of enzyme activity, which would be required to participate in this study. Diagnosis of CLN2 disease as determined by TPP1 enzyme activity (dried blood spots) should be available at the time of screening. Blood will be collected at the screening visit for CLN2 gene analysis, whether or not genotype information is available.

Since CLN2 disease is fatal during childhood, the study population must be children. Since therapeutic benefit is unlikely to be obtained in advanced CLN2 disease, given the massive depletion of cortical neurons, participation in the study requires a lack of massive CLN2 disease progression, resulting in a study population comparable to that of TPP1 gene therapy studies (worgalil, 2008, human gene therapy (hum. Safety assessments will also be limited in patients with advanced stages that are essentially in a nutritional state. The study was limited to affected children 1 year of age or older.

Clinical rating scale was developed: hamburger scale to record the natural history of CLN2 disease (Steinfeld,2002, journal of american medical genetics).

Although all four fields of the hamburger rating scale would be complete (fig. 12), the motion and language fields are the most useful for this study. Language and motion are the earliest loss of function domains, and therefore, the aggregate CLN2 motion-language scale is a suitable measure for efficacy. Efficacy endpoints will compare the motor and language subscale scores of treated study individuals with untreated patients, obtained from natural history data of CLN2 registration and/or data from siblings as required.

The remaining two domains in the rating scale (fig. 12) are unlikely to provide information for this study. To search for seizure and dyskinesia expression in CLN2 Disease in more detail, the adaptation domain from the uniform pasteurisation (battlen Disease) rating scale (UBDRS) was incorporated into the protocol. The UBDRS field records involuntary movements and seizures by type, frequency and severity (Kwon,2011, Neurology).

Due to practical (limited number of patients available) and ethical (neurosurgical treatment of children with fatal neurological disease) concerns, this study design cannot involve simultaneous, matched, randomized, blind or untreated control individuals (Arkin,2005, human gene therapy); (Crystal,2004, "human Gene therapy"). The motor and language subscales of the hamburger CLN2 disease scale and the total score will be compared with the natural medical history data in the registry of the medical centre of the university of hamburger, germany and/or data from siblings (as required).

Example 5

This example describes the selection of study populations for the phase 2 open label study described in example 4.

Study plan: selection of study population

Patients confirmed the progression of CLN2 disease may be eligible for participation in this study. Additional criteria for reference in this study are detailed below. Individuals had to meet the following criteria to participate in the study (table 7):

TABLE 7

Example 6

This example describes the duration of time that the individual participated in the study described in examples 4 and 5.

Individual participation would involve surgical implantation of an ICV access device, followed by recovery 14 to 28 days post-surgery, and all individuals would last at least 96 weeks of treatment. Treatment will continue until all procedures are completed, the subject withdraws consent and discontinues the study, the investigator discontinues the study or terminates the study. A security follow-up visit will be made 6 months after the final TPP 1infusion, but if the individual participates in an extended study, enrolls or otherwise may continue to have access to TPP1 within 6 months after the last infusion, then no security follow-up visit is required (for AE/SAE report indications, see section 10.2.1).

Example 7

This example describes the treatments administered in the studies described in examples 4 to 6.

TPP1 was administered to all study individuals every two weeks, preferably in the morning after at least a 2 hour fast using ICV infusion. When using a feeding tube, the tube should be closed 2 hours prior to infusion. All individuals will be infused with 300mg of TPP1, but for safety reasons the dose can be reduced to 150mg if necessary.

Administration guidance

Surgical implantation of ICV reservoirs will be performed prior to study drug administration. Researchers will assess the patency, location, and skin integrity of the reservoir at each study drug administration (see handbook of study Agents). At each administration, the investigator will aspirate 1-2ml of CSF into the device cannula prior to administration of study drug to check patency. Device access is performed using strictly sterile techniques. The skin covering the reservoir is inspected for the appropriate needle insertion site. The needle insertion site must be intact with no signs of rupture, wound, infection or rash. The needle used is a small gauge non-apertured tip. Once the reservoir is accessed, the needle is fixed to ensure minimal risk of removal or removal. The reservoir may be replaced during the clinical study at the discretion of the researcher and/or neurosurgeon.

TPP1 was administered to all study individuals every two weeks, preferably in the morning after at least a 2 hour fast using ICV infusion. When using a feeding tube, the tube should be closed 2 hours prior to infusion, and unless otherwise specified, the study procedure for each study visit should be prior to study drug infusion. The date, time, volume and concentration of each dose of study drug administered to each individual will be recorded in the pharmacy log provided for the study and on the appropriate CRF. A research Agents Manual provides further instructions regarding the preparation and administration of research drugs.

The subject will be admitted for each infusion of TPP 1. For all infusions, the individual will be monitored in an inpatient setting for at least 24 hours from the start of the infusion. For the first infusion only, the individual will also return to the clinic for a follow-up visit 72 hours after the infusion begins. After all visits, the parent or legal guardian will be called up within 48 hours to determine health status.

Since allergic reactions may be associated with ERT administration, subjects should be pre-treated with age-appropriate antihistamines (and antipyretics (if appropriate)) about 30 minutes prior to infusion of TPP 1. According to standard practice in the institution, the investigator may pre-treat the subject with an age-appropriate sedative drug, as appropriate, about 30 minutes prior to infusion of TPP 1. Prior to pre-operative administration for infusion, clinical, developmental and QOL assessments should be performed.

For 300mg administration, TPP1 will be infused at 2.5 ml/hour ICV to deliver the full volume in approximately 4(± 1) hours. A syringe pump should be used to ensure a uniform infusion rate, with proper delivery range, delivery rate accuracy, and warnings of incorrect delivery or occlusion. If termination of the dose is required for safety or other reasons, the dose can be restarted and completed at the same rate as long as the total dose is administered within 10 hours of preparing the dosage syringe.

Normal infusions will be 300mg administered over 4(± 1) hours. If an individual experiences an AE, the researcher may reduce the dose or infusion rate after negotiating with the medical guardian for future infusions. The change in infusion should be done in the following order:

(I) reduce the infusion rate and administer drug over a total of 6 hours

(II) the dose administered over a standard time of 4 (. + -. 1) hours was reduced to 150mg

(III) decreasing the rate of infusion and administering 150mg over a total of 6 hours

Safety monitoring

Upon placement of the ICV access device, the individual and their caregiver will be provided with written instructions that provide detailed information about the signs and symptoms of device complications, as well as instructions as to when to return to the point of study for device evaluation. The subject will be admitted for each infusion of TPP 1. For all infusions, the individual will be monitored in an inpatient setting for at least 24 hours from the start of the infusion. For the first infusion only, the individual will also return to the clinic for a follow-up visit 72 hours after the infusion begins. After all visits, the parent or legal guardian will be called up within 48 hours to determine health status.

For the first infusion, Vital Signs will be measured within 30 (+ -5) minutes before infusion begins or resumes, every 30 (+ -5) minutes during infusion, 0.5 and 1 hour (+ -5 minutes) after infusion ends, and every 4 hours (+ -15 minutes) until discharge (see Vital Signs).

For each subsequent infusion, vital signs will be measured within 30 (+ -5) minutes prior to the start of the infusion, every 60 (+ -5) minutes during the infusion, and 1 and 4 hours (+ -5 minutes) after the end of the infusion.

Individuals need to be monitored regularly throughout the duration of the infusion for adverse events and epileptic seizures by appropriately trained personnel. If epileptic seizures occur, infusion may be discontinued by the investigator as appropriate. During study drug infusion, the vicinity of the bed requires suitably trained personnel and equipment for available emergency resuscitation (including epinephrine) due to the possibility of allergic reactions (severe allergy or general allergies). All individuals should be endovenous during the infusion period if urgent treatment is required.

Symptoms of anaphylaxis may include fever, chills, skin symptoms (urticaria, angioedema, rash), respiratory symptoms (dyspnea, wheezing), gastrointestinal symptoms (nausea, vomiting, abdominal pain) and/or cardiovascular changes (hypotension/hypertension). If more severe symptoms occur, such as angioedema (swollen tongue or throat) or wheezing, the infusion should be terminated.

To date, no severe allergy or severe allergy-like reactions have been developed by the TPP1 study. However, if severe allergy is suspected, local guidelines should be followed. If severe anaphylaxis, severe TRE or severe TRE (defined as a TRE of grade 3 or higher) is suspected, blood samples will be collected within 1 hour of the event to assess C4, serum tryptase and total IgE; to assess drug-specific IgE, blood samples should be collected no more than 8 hours after the event (or before the next infusion).

Safety assessments were performed during and after each infusion. At the discretion of the investigator, the individual may need to maintain a longer observation period. If an AE consistent with TRE is observed (section 7.4.1), appropriate intervention may include discontinuation of infusion, reduction in infusion rate, or administration of antihistamines, oxygen, liquids, or steroids. If the infusion is resumed after the interruption, the initial rate should be about half the rate at which the reaction occurs. More details of infusion improvements are provided in the research pharmacy handbook.

The parent or legal guardian is instructed to contact the researcher to discuss any AEs after discharge.

The use of ICV access devices can lead to infection, prolonged use of the reservoir leading to cerebral hemorrhage, reservoir leakage and seizures (Karavelis,1996, Neurosurgery (Neurosurgery) (Kronenberg,1998, Pain (Pain)). Additional surgery may be required to secure or replace the device. Throughout the study, patients will be monitored for potential infections (hyperthermia, coughing, rash, headache, swelling or drainage of the incision area) and signs of leakage or failure of the ICV reservoir (see the research and pharmaceutical handbook).

Due to the inherent safety concerns around implanted devices,

the individual should be scheduled to remove the ICV access device no more than 4 weeks after completion of the study visit or premature termination of the visit. If the individual intends to continue to receive TPP1 (e.g., via a commercially available product, registered use, or other TPP1 study) after participating in this study, the device need not be removed. After removal of the device, the individual should return to the study site for a security follow-up visit after 4 weeks (+ -3 days).

Method for assigning individuals to treatment groups

Individuals will participate in the situation where they are available without concern for any entry criteria or other patient characteristics.

Dose selection

The planned dose was 300 mg.

The recommended dose level was derived from the dose content used in TPP1 jejunal dog studies (Vuillemenot,2011, molecular genetic metabolism (mol. Pharmacological effects, including improved function and increased longevity, have been well documented in TPP1 jejunal dogs at dose levels of 4mg and 16 mg. Since TPP1 activity in brain tissue is proximally related to CNS lysosomal storage substances, a determination of scaling by brain mass can predict therapeutic doses in humans. The human equivalent dose is calculated by utilizing the brain mass to adjust proportionally. Human brain reaches an average of about 75% of adult body weight by age 2 and 100% by age 5 (Giedd,1996, cortex (Cereb. cortex)). If the adult brain mass is 1400g, healthy children 2 to 7 years old range from 1050 to 1400 g. Given the progressive brain atrophy in CLN2 patients, assuming a mean mass of 1000g, a scaling factor of 20-fold was generated based on a 50g dachshund mean brain mass.

The non-observed adverse effect content (NOAEL) of clinical disease, which also received the benefit of non-clinical studies in dachshunds, was 48mg, which would correspond to 960mg in humans.

Preliminary analysis of the study in example 3 demonstrated that administration of 300mg every 2 weeks via an Intracerebroventricular (ICV) device of all children participating in the study had a positive benefit risk profile. Furthermore, clinical scores stabilized in those children receiving TPP1 for more than 36 weeks, as opposed to matched untreated controls (where in most matches, the decline was rapid and profound).

Time-sequential selection of doses for individual subjects

The mean CNS half-life was about 2 weeks, indicating that therapeutic TPP1 levels in the CNS can be maintained with every two week dosing. TPP1 concentration in CSF will remain above lysosomal Kuptake for approximately 48 hours following single ICV or IT-L infusion in species with CSF kinetics similar to that of humans (Vuillemenot,2014, "-toxicology applications pharmacology" (toxicol. appl.,) "," "Vuillemenot, 2011," "molecular genetics metabolism"; 0190-09-071). In these same species (dogs and monkeys), the CNS distribution of TPP1 is widespread in many brain regions.

Selection of infusion volume and rate

Non-clinical studies in dachshunds and cynomolgus monkeys used infusion rates of about 5% of the total CSF volume per hour. This is expected to represent a safe infusion rate that minimizes changes in total CSF volume and intracranial pressure. Dachshunds received ICV infusion at a rate of 0.6 ml/hr for 2-4 hours, while monkeys received 0.88 ml/hr for 3.6 hours. No effect was observed in these studies, indicating safety issues due to infusion rate. In the CLN2 patient population, the estimated CSF volume was about 100 mL. For the proposed clinical trial, infusion of 10mL volumes over a 4 hour period represents an infusion rate of 2.5% of the total CSF volume per hour, which is about half the rate of no safety effect in non-clinical studies. Therefore, it is expected that infusion of 10mL over 4 hours will be safe in CLN2 patients.

Are not aware of

This is a one-armed, open label study. A point of study assessment of safety and clinical severity will be made, blinding to treatments and individuals that will participate after meeting the conditions.

Supervision of MRI assessment will be performed by the independent radiologist by the central Imaging facility, as defined in Imaging Charter. Interpretation radiologists and software analysis will be blinded to the individual and time of study. All individual identification information will be re-edited before the endpoint is evaluated.

Prior and concomitant medications

Medications (prescription, over-the-counter, and herbal) and nutritional supplements taken during the 30 day period before informed consent will be recorded in the CRF at screening. In each subsequent visit (or within one week of prematurely terminating the visit), changes in any medication (dose, frequency, new drug, or cessation) due to the previous visit will be recorded in the CRF.

Any concomitant medications added or discontinued during the study should be recorded in the CRF (or within one week of early termination of the visit).

The subject may be taking anticonvulsant agents and drugs for myoclonus, tremors, agitation and pain. Researchers will be required to keep these protocols unchanged throughout the study unless changes are required due to lack of efficacy or toxicity.

Treatment compliance

Study medication will be administered to individuals at the point of study by qualified professionals. The date, time, volume, and concentration of each dose must be recorded in the dispensing log and on the appropriate CRF. In the event that the dose of study treatment is missed or incomplete, the investigator should record the cause and any other relevant information on the CRF as appropriate.

Dietary or other regimen restrictions

There were no dietary or other regimen restrictions for this study. The study will be continued for individuals who require a PEG (percutaneous endoscopic gastrostomy) tube as a standard of care during the course of the study. When using a feeding tube, the tube should be closed 2 hours prior to infusion.

Example 8

This example describes the efficacy and safety variables of the studies of examples 4-7.

Variable of efficacy

Although this study was designed to primarily assess safety and tolerability, efficacy will be measured using the CLN2 motor-language clinical rating scale. CLN2 quantitative tables and MRI measurements of disease progression were collected from 0 to 12 points of total hamburgers as secondary endpoints. Other exploratory efficacy measures would include developmental status, seizure frequency, involuntary movements, retinal anatomy, and quality of life metrics.

CLN2 clinical rating Scale

Disease severity will be assessed using the CLN2 clinical rating scale (Steinfeld,2002, journal of american medical genetics); (Worgalil, 2008, human Gene therapy). This table consists of four fields with inherent content validity. In each domain, a score of 0 to 3 is assigned, and the total score is calculated by adding the four domain scores to give a final score of 0 (severely impaired) to 12 (normal).

Since the motor and language domains are most relevant to CLN2 disease progression (section 9.2), it will be used to assess efficacy (appendix 1).

The scorers will be identified as qualified practitioners who have been trained in the definition and examples of CLN2 disease assessment scales. All scorers at all study points will be required to pass training courses designed to standardize definitions and gauge anchor points throughout the study before conducting a study assessment. The duration of treatment for each of the participating patients should be assessed by a scorer whenever possible. In addition, patient scoring should be performed at the same time as the study visit, preferably in the morning prior to the procedure and/or infusion. Before pretreatment for TPP 1infusion, CLN2 scale assessments should be made.

Danfo development Scale II

Denver II is a revision and renewal of the denver development screening test.

Both tests were designed to monitor the development of infants and preschool children. The test covers four basic functions: personal socialization (e.g., smiling), well-adapted movements (e.g., grasping and drawing), language (e.g., combining words), and gross movement (e.g., walking). The test covers ages from birth to 6 years of age.

Modified unified pap rating scale non-voluntary exercise scale

The modified unified papanicolaou disease rating scale (mbudrs) involuntary movement scale is a rating scale that measures the type, frequency and severity of common involuntary movements associated with CLN2 disorders such as myoclonus and dystonia.

Magnetic resonance imaging

All image data will be acquired on a 1.5Tesla MRI platform. Study MRI will contain localizers specified in Imaging Charter, 3D T1 weighted sagittal, T2 weighted gradient echo, diffusion weighted axial and FLAIR axial acquisitions. The total scanner time is less than 60 minutes and is expected to be done in most sedated individuals.

Volumetric analysis of the image will be done by estimating the volume of total cortical gray matter and the proportion of cranial CSF.

All patients will receive MRI, without any contrast media, prior to implantation of the ICV access device to ensure proper planning and placement. In conducting this MRI, no study assessment related to MRI is conducted, and any readings obtained during MRI are not included in the analysis of study data.

When the investigator suspects an infection or shunt dysfunction, an MRI scan should also be performed. In this case, according to Imaging Charter, MRI should be performed with or without intravenous contrast, at least with T1 weighted axial and sagittal aspects. For suspected meningitis, the brain should be MRI with or without contrast agent according to the imaging manual.

Optical coherence tomography

Optical Coherence Tomography (OCT) is a non-invasive imaging test that uses light waves to take a picture of the cross-section of each layer of the retina to measure its thickness. These measurements may aid in the early detection and treatment of retinal diseases. OCT will be performed locally and should be performed prior to infusion.

Modified unified pap rating scale seizure scale

The mUBDRS seizure Scale measures the type and frequency of seizures in CLN2 patients within the first 3-month interval. The scale is completed with a review of the caregiver/family members during the time period between study visits.

PedsQL

The PedsQLTM universal core scale is designed to measure the quality of life of children and adolescents. The assessment is simple, practical and suitable for development. The instrument responds to clinical changes over time (Msall,2005, "review of mental retardation and developmental disorders research (ment. record. dev disclosure. res Rev)). Four parental reports cover ages of 1-12 months, 13-24 months, 2-4 years, and 5-7 years, and contain questions about physical, emotional, and social functions, as well as school functions where applicable. Patients older than 7 years of age will not be evaluated using this tool during the study.

EQ-5D-5L

EQ-5D-5L instruments are self-reported questionnaires designed to measure general Health status (EuroQol Group,1990, Health Policy (Health Policy), Brooks,1996, Health Policy). EQ-5D-5L consists of 2 parts: a system is described that assesses overall health from 5 dimensions and 5 degrees of sensory problems (movement, self care, daily activity, pain/discomfort and anxiety/depression) in an EQ visual analog scale (EQ VAS).

CLN 2-specific QoL questionnaire

CLN 2-specific QoL questionnaire was a disease-specific complement to PedsQL using the same format and quantification. The questionnaire is a new instrument designed in collaboration with the patient's family and advocate community to search for basic care and quality of life issues for advanced infant CLN2 disease.

Questionnaire for infant quality of life

The infant quality of life questionnaire (ITQOL) is used to assess the health of children between the ages of 2 months and 5 years. This tool requires parents of preschool children to dislike the physical and psychosocial domains, such as development, pain, mood, and the effects of child health on parents.

Immunogenicity

Immunogenicity testing will be performed at a central laboratory using validated immunogenicity assays on serum and CSF samples. Blood samples (serum) will be collected for TAb testing, and CSF samples will be collected for TAb and NAb testing before the first infusion (baseline or week 1 before infusion), every 12 weeks thereafter, and at the safety follow-up (or within one week of early termination of visits). Collection must precede infusion. Nabs will be tested in CSF at baseline and at subsequent time points only when TAb is positive in CSF.

If additional laboratory work is required for the development of an allergic reaction at a later time, baseline total IgE and drug-specific IgE levels will be obtained using the baseline samples.

To date, no severe allergy or severe allergy-like reactions have been developed by the TPP1 study. However, if a severe allergic reaction, severe allergic event or severe allergy (defined as a grade 3 or higher allergic event) is suspected, blood samples will be collected within 1 hour of the event to assess C4, serum tryptase and total IgE; to assess drug-specific IgE, blood samples will be collected no more than 8 hours after the event occurs (or before the next infusion).

Clinical laboratory evaluation

Blood and urine samples will be collected for routine clinical laboratory evaluation (hematology, urinalysis) and subjected to centralized analysis. The collection should be prior to infusion.

Any abnormal test result determined by the researcher to be clinically significant should be repeated until the cause is determined, the value returned to baseline or within normal limits, or the researcher determines that the abnormal value is no longer clinically significant.

All abnormal clinical laboratory pages should be initialized and dated by the investigator and accompanied by comments about clinical significance. Each clinically significant laboratory result will be recorded as a medical history at the time of screening and subsequently as an AE.

If known, a diagnosis associated with an abnormality in clinical laboratory results considered clinically significant by the investigator should be recorded on the AE CRF.

Table of clinical laboratory tests performed as described below (table 8):

TABLE 8

Blood chemistry	Hematology	Urine analysis
			Albumin	Heme	Appearance of the product
Alkaline phosphatase	Blood volume ratio	Colour(s)
			ALT(SGPT)	WBC count	pH
AST(SGOT)	RBC counting	Specific gravity of
			Direct bilirubin	Platelet count	Ketones
Total bilirubin	Differential cell count	Protein
			Blood urea nitrogen		Glucose
Calcium carbonate		Bilirubin
			Carbon dioxide		Nitrite salt
Chloride compound		Urobilinogen
			Total Cholesterol		Heme
C reactive protein
			Creatinine
Creatine kinase
			Glucose
GGT
			LDH
Phosphorus (P)
			Potassium salt
Total protein
			Sodium salt
Uric acid

Cerebrospinal fluid monitoring

Within 30 (+ -5) minutes prior to each infusion, standard clinical laboratory CSF samples (differential cell count, protein and glucose) will be collected for routine monitoring. Small volumes of CSF will be collected from ICV reservoirs and analyzed locally. Collection should be prior to study drug infusion.

Biomarkers

Samples of plasma and CSF will be collected to analyze putative molecular/biochemical biomarkers. Collection should be prior to study drug infusion. The test samples will be pooled.

Other laboratory evaluation

Individuals experiencing SAE that may be associated with TPP1 or other related AEs may need to draw additional blood samples to assess immunogenicity or safety parameters.

Vital signs, physical examination and other observations

Vital signs

For the first infusion, vital signs (SBP, DBP, heart rate, respiratory rate, and body temperature) will be measured within 30 (+ -5) minutes before the infusion begins or resumes, every 30 (+ -5) minutes during the infusion, 0.5 and 1 hour (+ -5 minutes) after the end of the infusion, and every 4 hours (+ -15 minutes) until discharge.

For each subsequent infusion, vital signs will be measured within 30 (+ -5) minutes prior to the start of the infusion, every 60 (+ -5) minutes during the infusion, and 1 and 4 hours (+ -5 minutes) after the end of the infusion.

Physical examination

A full physical examination will include the general appearance (head, eyes, ears, nose and throat), cardiovascular, dermatological, lymphatic, respiratory, gastrointestinal, genitourinary, musculoskeletal, and weight and height.

Simple physical examinations would include gross appearance, cardiovascular, respiratory, neurological, and gastrointestinal assessments.

The use of an ICV reservoir device for intraventricular drug administration requires monitoring of the patient throughout the study for potential infections (high body temperature, coughing, rash, headache, mental state changes, swelling or drainage of the incision area) and signs of leakage or failure of the ICV reservoir (swelling of the skin around the reservoir site, difficulty in CSF extraction, erythema of the scalp, swelling of the reservoir device or fluid exudation upon infusion).

The investigator will assess the patency, location, and skin integrity of the reservoir at each study drug administration. Prior to infusion, the investigator will examine the site of the fluid reservoir for scalp edema, erythema or skin breakdown. Patency was again assessed during pre-infusion sampling and at the time of infusion. Difficulty in obtaining the desired CSF volume required for the sample prior to infusion or evidence of leakage of the ICV reservoir (skin swelling around the reservoir site, scalp erythema, reservoir device swelling, or fluid seepage) would facilitate further assessment of reservoir failure prior to continuing infusion. Other surgical consultation may be required, including surgery, to secure or replace the device.

Clinically significant abnormalities will be recorded under or after "medical history" at screening as AEs.

Neurological examination

Complete neurology will include level of consciousness, speech, language, cranial nerves, motor intensity, motor tension, abnormal motion, reflexes, upper limb sensations, lower limb sensations, gait, humper (Romberg), nystagmus, and coordination abilities.

Electrocardiogram

Standard 12-lead electrocardiograms contain heart rate, heart rhythm, time intervals, axes, conduction defects and anatomical abnormalities. ECG will be performed within 15 (+ -5) minutes after the end of the infusion.

If a clinically significant abnormality is found, the researcher or prescribing personnel will assess whether study participation or continuation is appropriate; clinically significant abnormalities will be recorded in the medical history during screening and later as AEs.

Electroencephalogram

The recording criteria awaken the EEG. If a clinically significant abnormality is found, the researcher or prescribing personnel will assess whether it is appropriate to conduct study participation or continue the study; clinically significant abnormalities will be recorded under a "history" during the screening.

Pregnancy test

At any time during the screening and study period, female individuals who will be judged by the investigator to be fertile (as defined by menstruation) will be tested for pregnancy using the urine pregnancy test; whenever pregnancy is problematic, additional urine tests will be performed. If the urine test result is positive or ambiguous, then a serum pregnancy test will be performed.

Example 9

This example describes the study procedure for the study of examples 4-8.

After explaining the nature of the study, written informed consent of the parent or legal guardian must be obtained prior to any study-related procedures. Within 21 days of hospitalization for implantation of the ICV access device, the following procedures will be performed:

informed consent

Confirmation of diagnosis of CLN2 disease by TPP1 enzyme Activity (dried blood spots)

Blood for CLN2 Gene analysis

Hamburger CLN2 disease rating Scale with video (FIG. 12)

Validation criteria for the study entrance

Cranial MRI

Complete physical examination including medical history

CLN2 specific QoL questionnaire

Pregnancy test (female with fertility)

Evaluation of SAE in relation to protocol-imposed interventions and seizure history after informed consent

Concomitant medication

Surgical access

Eligible study candidates will be admitted for surgical implantation of an ICV access device into the right ventricle. MRI will be performed preoperatively to ensure proper planning and placement of the ICV access device. The individual should be observed in a care-intensive environment for at least 48 hours post-operatively. The following procedure will also be followed:

AE evaluation, including history of persistent epileptic seizures

Concomitant medication

Upon placement of the ICV access device, the individual and their caregiver will be provided with written instructions that provide detailed information about the signs and symptoms of device complications, as well as instructions as to when to return to the point of study for device evaluation. The hospitalized patient will have another follow-up phone call within 48 hours of discharge.

Baseline access and first infusion

Baseline access

The following baseline values will be recorded within 2 days prior to the first infusion (which will occur at least 14 days post-surgery and no more than 28 days post-surgery):

hamburger CLN2 disease rating Scale with video (appendix 1)

ECG (12 leads) (heart rate, time interval, axis, conduction defect and anatomical abnormality)

EEG (Standard Wake-up)

Cranial MRI

CSF (TAb and NAb in all individuals) and serum (TAb and drug-specific IgE in all individuals) for immunogenicity

Complete physical examination

Examination of nerves

Clinical laboratory tests (hematology, blood chemistry and urinalysis)

Modified unified pap rating Scale non-voluntary exercise Scale (mUBDRS-sports)

Modified unified papanicolaou disease assessment Scale seizure Scale (mUBDRS-seizure)

Optical coherence tomography

QOL questionnaire for infants

·PedsQL

·EQ-5D-5L

Danfo II developmental Scale

CLN2 specific QoL questionnaire

Ophthalmic evaluation

AE evaluation, including history of persistent epileptic seizures

Concomitant medication

First infusion

Generally, for all infusions, assessment of function and QOL should be done prior to MRI and blood sampling; blood samples may be collected while the individual is sedated for MRI examination.

First infusion-day 1

The following procedure will be performed on day 1 of the first infusion:

CSF monitoring (cell count, protein, glucose)

Patency/infection of the device

Study drug infusion

Cerebrospinal fluid and plasma for biomarkers

Vital signs within 30 (+ -5) minutes before the start of infusion, every 30 (+ -5) minutes during infusion, 0.5 and 1 hour (+ -5) after the end of infusion and every 4 hours (+ -15 minutes) until discharge

Simple physical examination

AE assessment (additional blood samples may be collected by the investigator for safety or immunogenicity testing of any AE of interest) and history of persistent seizures

Concomitant drug assessment

Post infusion monitoring for at least 24 hours in an inpatient setting

First infusion-days 2-6

The following procedure will be performed 2-6 days after the first infusion:

vital signs (day 2) (every 4 to 20 hours after infusion)

Clinical laboratory tests (hematology, blood chemistry and urinalysis) (day 2)

CSF monitoring (cell count, protein, glucose) (day 6)

Simple physical examination (day 6)

Unobstructed/infected equipment (day 6)

AE assessment (additional blood samples may be collected by the investigator for safety or immunogenicity testing of any AE of interest) (daily) and history of persistent seizures

Concomitant drug assessment (daily)

After the discharge of the hospitalized patient since the first infusion, a follow-up phone call will be made with the parent/guardian within 48 hours.

Every 2 weeks

The following evaluations and procedures should be performed every 2 weeks during the study. All study visits should be made every 2 weeks from the day of the first infusion (+ -3 days). Unless otherwise specified, all assessments and procedures should be completed prior to conducting study drug infusions. Generally, assessment of function and QOL should be done prior to MRI and blood sampling; blood samples may be collected while sedating an individual for MRI.

For all visits, if no security issues are observed, the individual may be discharged after 24 hours, with medical stability. Approximately 48 hours after discharge, a follow-up phone call to the parent or legal guardian was made to determine health.

Simple physical examination

CSF monitoring (differential cell count, protein, glucose)

Device patency/infection assessment

Study drug administration

Vital signs within 30 (+ -5) minutes before the start of infusion, every 60 (+ -5) minutes during infusion, 1 and 4 hours (+ -5 minutes) after the end of infusion

AE assessment (additional blood samples may be collected by the investigator for safety or immunogenicity testing of any AE of interest) and history of persistent seizures

Concomitant drug assessment

Call parents/guardians by phone within 48 hours after access

Every 4 weeks

During the study, the following evaluations and procedures should be performed every 4 weeks. Where applicable, blood sampling may be performed while MRI sedation is being performed.

Routine clinical laboratory tests (hematology, blood chemistry and urinalysis)

Every 12 weeks

During the study, the following evaluations and procedures should be performed every 12 weeks. Unless otherwise specified, all assessments and procedures should be completed prior to conducting study drug infusions. Generally, assessment of function and QOL should be done prior to MRI and blood sampling; blood samples may be collected while sedating an individual for MRI.

Record hamburger CLN2 disease rating Scale (FIG. 12)

Examination of nerves

CSF and serum sampling for immunogenicity

mUBDRS-locomotion

mUBDRS-seizure

Optical coherence tomography

Quality of life questionnaire for infants

·PedsQL

Danfo II developmental Scale

CLN2 specific QoL questionnaire

·EQ-5D-5L

Every 24 weeks

During the study, the following evaluations and procedures should be performed every 24 weeks:

ECG (12 leads) (heart rate, heart rhythm, time interval, axis, conduction defect and anatomical abnormality) within 15 (+ -5) minutes after the end of infusion

EEG (Standard Wake-up)

Cranial MRI

CSF and plasma for biomarker analysis

Complete physical examination

Every 48 weeks

During the study, the following evaluations and procedures should be performed every 48 weeks:

ophthalmic evaluation

Completion of study or early termination of access

At study completion or early termination, subjects will return to the study site within 3 days. The following process will be completed:

recorded hamburger CLN2 disease rating Scale (appendix 1)

ECG (12 lead) heart rate, heart rhythm, time interval, axis, conduction defects and anatomical abnormalities) [ if infused, 15 (+ -5) minutes after infusion ended ]

Cranial MRI

CSF monitoring (cell count, protein, glucose)

CSF and plasma for biomarker analysis

CSF and serum sampling for immunogenicity

Vital signs (SBP, DBP, heart rate, oral body temperature and respiratory rate)

Complete physical examination

Examination of nerves

Routine clinical laboratory tests (hematology, blood chemistry and urinalysis)

Danfo II developmental Scale

CLN2 specific QoL questionnaire

Ophthalmic evaluation

AE assessment (additional blood samples may be collected by the investigator for safety or immunogenicity testing of any AE of interest) and history of persistent seizures

Concomitant drug assessment

An ICV access device for an individual who will not continue to receive TPP1 under other circumstances (e.g., commercial use, participation in registration, participation in another TPP1 clinical study, etc.) should be removed after the study is completed or access is terminated early. Device removal should be performed no more than 4 weeks after study completion visit or ETV.

Device and security follow-up

After completion of the following steps, subjects will return to the study site 4 weeks (± 3 days) after removal of the ICV access device:

vital signs (SBP, DBP, heart rate, oral body temperature and breathing rate)

Simple physical examination (including careful examination of a device site prior to inspection for signs of infection, etc.)

Serum sampling for immunogenicity

Examination of nerves

Routine clinical laboratory tests (hematology, blood chemistry and urinalysis)

AE evaluation (the investigator may collect additional blood samples for safety or immunogenicity testing, for any AE of interest)

Concomitant drug assessment

For individuals who do not have device removal, they will be exempted from their 4-week device security follow-up visit as they will continue to receive TPP 1in other circumstances (e.g., commercial use, enrolment participation, participation in another TPP1 clinical study, etc.).

Safety follow-up

If an individual is involved in an extended study or registry or may continue to use TPP1 within 6 months after the final infusion, then no security follow-up access is required. If necessary, subjects will return to the study site 6 months after the last study treatment, after completion of the following procedures:

ECG (12 leads) (heart rate, heart rhythm, time interval, axis, conduction defects and anatomical abnormalities) [ if infused, within 15 (+ -5) minutes after infusion ended ]

Serum sampling for immunogenicity

Vital signs (SBP, DBP, heart rate, oral body temperature and breathing rate)

Complete physical examination

Routine clinical laboratory tests (hematology, blood chemistry and urinalysis)

AE assessment (additional blood samples may be collected by the investigator for safety or immunogenicity testing of any AE of interest) and history of persistent seizures

Concomitant drug assessment

Termination of the study

The study will end after the last individual completes the last security follow-up visit. BioMarin retains the right to discontinue the study at any time for clinical or administrative reasons, including but not limited to participation in poor or non-compliance programs or programs for GCP, and to abort individual researchers or research sites for clinical or administrative reasons. In addition, if BioMarin believes that the safety of the study individual may be compromised, the study may be terminated.

Example 10

This example describes the results, modifications, and discussion of the studies described in examples 4-9.

Drug and rinse solution, component and formulation development

The drug product studied as described in examples 4-9 was a lyophilized injectable formulation containing 150mg of cellular lybonase α per 5mL of solution in a single 10mL glass vial. The drug product contained the following excipients: disodium hydrogen phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate and water for injection. The flush solution is supplied for the purpose of completely administering the drug remaining in the administration line to maintain patency of the line after intracerebroventricular administration of the drug. The composition of the rinse solution was the same as the drug, except that the rinse solution contained no active and was supplied as a 5mL solution in a single 10mL glass vial.

Mechanism of action of cellular Ribonaccase alpha

The cellular libonnase alpha is a recombinant hTPP1 precursor consisting of 544 amino acids. The amino acid sequence is identical to that of the precursor of hTPP 1in vivo. N-linked oligosaccharides, such as bisphosphorylated high mannose chains, bind to aspartic acid residues (Asn191, Asn203, Asn267, Asn294 and Asn424) in the five N-linked oligosaccharide spectra and are taken up by CI-M6PR through the oligosaccharide into target cells or lysosomes (J Biol Chem 2001; 276: 2249-55). Thereafter, the propeptide fragment is cleaved in an acidic environment by in vivo proteases to produce active enzymes (e.g., journal of biochemistry 2004; 279,31058-67; journal of biochemistry 2009; 284:3985-97), and it cleaves tripeptides from polypeptides accumulated in lysosomes, preventing the increase in storage mass and, thus, the progression of the CLN2 disease.

Pharmacokinetics

In this study, Brineuro was indicated for patients other than the Japanese CLN2 (target sample size: 5), and Brineuro was administered to the patients. The following table (table 9) shows the CSF and plasma pharmacokinetic parameters for individual patients receiving a 200 or 300mg intracerebroventricular dose.

TABLE 9

Pharmacokinetic parameters of CSF and plasma of Individual patients receiving intracerebroventricular doses of 200 or 300mg ^a)

C _max : maximum concentration in CSF or plasma

AUC _0-t : area under CSF or plasma concentration-time curve from administration to last quantification time point t

a) In this study, pharmacokinetic assessments were performed after the date of data expiration; the data for the pharmacokinetic parameters for cellular Ribose alpha are shown for 5 patients who received Brineura between 2 days.

b) Dose 49; c) dose 13

Discussion of this study

The key inclusion criteria in this study were a blood test based decrease in TPP1 enzyme activity, age 1 year or older, a 3 to 6 point overall score for siblings, sports and linguistic subscales participating in the study of example 3, ¹⁹ and have not been previously treated with stem cell therapy, gene therapy, or enzyme supplementation therapy.

This study consisted of: a post-operative recovery period of 14 to 28 days and a study drug treatment period of 96 weeks after the procedure of implanting the device for intracerebroventricular administration.

Dosage and administration were 300mg of TPP1 (Brineuro) per 2 weeks over a period of about 4 hours (infusion rate: 2.5mL/h)Intraventricular dose of (a). The treatment period was 96 weeks. ²⁰

In addition, the study protocol was revised a little later after the data expiration so that study participation was no longer limited to siblings of CLN2 patients participating in the study of example 3. The eligible ages for study participation were changed to be between birth and <18 years of age, and the dose and administration for patients less than 2 years of age were changed to be as follows: 100mg for patients born to <0.5 years of age; 150mg for patients aged 0.5 or more and <1 year old; and ≧ 1 year and <2 years, the first 4 doses of 200mg, and then 300mg every 2 weeks thereafter. All doses were administered intracerebroventricularly at an infusion rate of 2.5 mL/h.

The safety analysis group and efficacy analysis group each contained four treated patients (baseline age:2 to 5 years old). For efficacy, table 10 shows the clinical score scale from CLN2 ²¹ And their sum (ML score) and the change from baseline to the last evaluation time point. No baseline drop was found on any clinical rating scale; all were unchanged.

Watch 10

Baseline score and change therein from baseline to last evaluation time point for each clinical rating Scale (study 109-203)

Number of patients (%)

a) Of the 4 patients enrolled by the date of data expiration, 1 baseline ML subscale scored 1, and because this patient had autism at the same time, this patient was excluded from language and ML subscale assessments.

AE 22 (upper respiratory tract infection, malaise, seizures, partial seizures, constipation and abdominal pain; fever, sleep disturbance and fever; influenza, fever and gastroenteritis; and vomiting, allergy, dropletization, bronchitis, generalized tonic-spastic seizures, upper respiratory tract infection, generalized tonic-spastic seizures and gastroenteritis) was reported in all 4 patients with respect to safety. Of these events, 3 (2 patients with fever and 1 patient with allergy) were considered ADRs.

No mortality was reported. It was reported that 3 patients had severe AE (2 patients were febrile and 1 patient was allergic) and all these events were considered ADR. No AEs leading to discontinuation of treatment were reported.

Anti-cell lybonase α antibodies were positive in the sera of all 4 patients up to 13 weeks after the start of treatment. No anti-cellular lybonase α antibodies were detected in CSF in any of the patients until 37 weeks after initiation of treatment.

No clinically significant changes were reported in vital signs or 12 lead ECG.

Efficacy of

In this study, the study protocol was revised after data cutoff as it relates to the efficacy of Brineuro in CLN2 patients younger than 3 years old, so that eligible study population patients were aged from birth to birth<Age 18. Based on the latest data obtained from the data acquisition, ²⁷ brineurora has been administered to 11 patients, 5 of which were under 3 years of age (3 were 2 years of age and 2 were 1 year of age). Table 11 shows ML scores at baseline age. From baseline to the one-time assessment time point, the ML score was unchanged in 8 patients (4 patients maintained score 6,3 patients maintained score 4, and 1 patient maintained score 2), the score was improved in 2 patients (1 patient increased from 5 to 6, and 1 patient increased from 1 to 2), and 1 patient was scored at 1(the patient's score was from 4 down to 3). Of the 3 2-year-old patients, 2 maintained an ML score of 6, and the score of the other patients increased by 1 (from 5 to 6). Two patients younger than 2 years maintained an ML score of 6.

TABLE 11

Distribution of ML scores at baseline and final assessment time points

Number of patients (%)

a) Both patients under 2 years of age had a baseline age of 1 year.

b) This data contained the MS scores of the autistic patients described in table 29, a).

In terms of efficacy in patients involving less than 3 years of age, the results demonstrate that there is no worsening of symptoms during the Brineura therapy.

In view of the above, it is believed that the use of Brineura may be expected to be effective in preventing disease progression of CLN2 in younger patients.

Safety and serious adverse events

About<Safety of CLN2 patient 3 years old: in this study, Brineuro was administered in 11 patients (6 patients ≧ 3 years, 3 patients 2 years, and 2 patients 1 year), and all 11 patients reported AEs, based on the most recent data. Among them, in the case of the above-mentioned,3 patients aged 3 years ≧ 3 (1 to 3 episodes of fever per patient) and 3<Events in a 3 year old patient piece (2 patients with fever and allergies at 2 years old; 1 patient with drug allergies and drug allergies at 1 year old) were evaluated as ADRs and the results of all these events were "restored". 6 patients (Escherichia coli urinary tract infection), gastrointestinal fistula and pneumonia, Propionibacterium (Propionibacterium) tested positive, fever, adenoid hypertrophy and rhinitis, device insertion complications, periorbital hematoma, and fever, caries and fever) aged 3 years or more and 3 patients<SAE was reported in patients aged 3 years (persistent epilepsy and infections in patients aged 2 years; fever, influenza and flu; and hemomas in allergy and medical device sites). Of these, 2 events (fever and fever; and fever) in patients aged 3 years and 2<Events in patients aged 3 years (fever and allergies in patients aged 2 years) were assessed as ADRs, and the results of all these events were "recovered". Based on the above, in this study<No new clinically significant AEs were observed in patients aged 3 years.

The safety of the Brineura infusion was considered acceptable based on the incidence of AEs in clinical studies and overseas port marketing experience.

Overall, the efficacy of a certain level of Brineura can be expected and the safety of Brineura appears to be acceptable when administered intracerebroventricularly based on previous studies in CLN2 patients aged 3 and this study in CLN2 patients comprising <3 years old.

Dosage and administration

The intraventricular route of administration of Brineura was decided to bypass the blood-brain barrier by using surgically placed implantable intraventricular devices to deliver enzymes directly and sufficiently to the central nervous system to achieve efficacy against neurological symptoms.

In a major pharmacodynamic study, 16mg of cellular Ribonase alpha was intracerebroventricularly administered every 2 weeks in dogs (dachshunds) deficient in TPP1, ³⁾ prolonged onset of neurological symptoms is accompanied by a tendency to survive for a long period of time. Based on the fact that TPP1 activity in brain tissue is more closely related to accumulated lysosomal material in the central nervous system, clinical doses were studied based on human dose scaling factors according to brain weight. Considering that the human brain reaches 75% of the weight of an adult brain before the age of 2 years and almost 100% before the age of 5 years (Cereb Cortex 1996; 6:551-60), the weight of a brain from the age of 2 to 7 years may be in the range of 1050 to 1400g by an average adult brain weight of 1400 g. In view of the potential for progressive brain atrophy in CLN2 patients, the applicant postulated that the brain weight of CLN2 patients in this age group may be about 1000 g. Since the mean Brain weight of dachshunds was estimated to be 50g (Peptide Drug Delivery to the Brain), Raven Press,1991:112), the scaling factor that determines human dose was 20-fold, and the dose of 16mg of cellular Ribonase α administered to a TPP 1-deficient dog (dachshund) at > 2 years old was considered to be 320mg based on the Brain weight ratio between species. Subsequently, the study described in example 3 in CLN2 patients aged 3 or more was started, with the cohort starting at 30mg, for example, and titrated to 300mg for safety. After confirmation of tolerability, 300mg was evaluated for efficacy and safety, including fixed dose time periods. For this study, the protocol was revised (after data expiration). Based on<Brain weight selection dose for 2 year old patients ³⁹ To study the efficacy and safety of Brineuro: patients born to 0.5 years of age are<100mg, 0.5 to<150mg for patients aged 1 year, 1 year to<The first four doses in a 2 year old patient were 200mg, and the subsequent doses were 300 mg. At age 1 to<In patients 2 years of age, the dose is estimated to be 300mg based on their brain weight. However, the initial dose was set at 200mg as 0.5 to the start of treatment with Brineuro<An intermediate dose for a patient of 1 year of age, and increasing the dose from 150mg to 300mg depending on its age based on safety considerations. For dosing intervals, Brineuro was administered in clinical studies at a dose interval of once every two weeks, sinceResults of tissue distribution in non-clinical pharmacokinetic studies etc. indicate that exposure to pharmaceutically active substances in the CNS is expected to maintain a dose interval once every two weeks.

For children younger than 2 years of age, the doses were reduced with reference to the following table (table 12).

TABLE 12

Typically, this drug is infused at a rate of 2.5 ml/hour by using an infusion pump, but the infusion rate is reduced depending on the patient.

Allergies that include severe allergies may occur when such a drug is administered. To alleviate symptoms, it is contemplated to pre-treat the patient with antihistamines for 30 to 60 minutes, with or without an antipyretic before the infusion begins.

For infusion rates in clinical studies, Brineura was administered intracerebroventricularly at infusion rates of 2.5 ml/hour at all ages. If the infusion rate is estimated to be about 100mL CSF in patients 2 to 7 years old and in<Approximately 50mL CSF in a 1 year old patient (peptide drug delivery to the brain, Raven Press 1991:112), then the infusion volume of Brineura is estimated to be less than about 10% of CSF. CSF production rates in humans are typically about 20 ml/hr (1962, Am J Physiol), about 12% of the Brineuro infusion rate (2.5 ml/hr), and at 0.5 years of age CSF production rates (estimated at about 2.5-4.7 ml/hr) ⁴⁰ It was also assumed to be greater than the Brineuro infusion rate (2.5 ml/hour). Based on these data, the intraventricular infusion rate of Brineuro appears to have no significant effect on the rate of CSF or CSF production in humans.

In view of the above, it was demonstrated that Brineura can effectively inhibit disease progression without causing any significant safety problems based on the results of a previous study in which 300mg was intracerebroventricularly administered to CLN2 patients aged at > 3 years every 2 weeks and 300mg or less of this study was intracerebroventricularly administered to CLN2 patients aged at <3 years every 2 weeks.

References cited in examples 4-9

The following references are cited herein in accordance with the following numbering, and/or are incorporated by reference herein.

Arkin, LM, Sondhi, D, worgali, S, Suh, LH et al "human Gene therapy (Hum Gene Ther)' 16[9],1028-1036.2005 in the face of problems of therapeutic misunderstanding, decision participation and personal motivation in clinical studies based on Gene-drugs for fatal disorders" (stabilizing the issues of therapeutic approaches, outcome, and personal mobility in genetic media-based clinical research results for human disorders).

Awano, T, Katz, ML, O' Brien, DP, Sohar, I et al, frame shift mutations in canine TPP1 (ortholog of human CLN2) in young dachshunds with neuropathic lipofuscinoid disease (A frame shift mutation in canine TPP1(the ortholog of human CLN2) in a jungle Dachshot with neural peptide lipofectin @) _ molecular Gene metabolism (Mol Genet Metab) 89[3], 254-260.2006.

Brooks r. "EuroQol: current participation status (EuroQol: the current state of play), "health policy" 37[ 1]],53-72.1996。

Chang, M.CLN2, New York, Oxford Univ Press,2011:

crystal, RG, Sondhi, D, Hackett, NR, Kaminsky, SM et al, Clinical protocols (Clinical protocols), Administration of replication-deficient adeno-associated virus gene transfer vectors expressing human CLN2 cDNA to the brain of children with advanced stage infant neuropathic ceroid (Administration of a replication-specific infection-associated virus gene transfer vector expressing the human CLN2 cDNA to the brain of the child with disease of human tissue injury neurological disease, human gene therapy 15[11], 1-1154.2004.

Dierenfeld, AD, McEntee, MF, Vogler, CA, Vite, CH et al, < replacement enzyme at birth,. alpha-L-iduroniside improves symptoms in the brain and periphery of dogs with type I mucopolysaccharidosis (tying the enzyme alpha-L-iduronidase at birth, animals and systems in the brain and period of teeth with mucopolysaccharidosis type I > ] < scientific transformation medicine (Sci Transl Med) < 2[60],60ra 89.2010.

Dyke, JP, Sondhi, D, Voss, HU, Shungu, DC et al, Assessment of Disease Severity in advanced infant neuropathic Ceroid Lipofuscinosis Using Multiparametric MR Imaging (Assessment of Disease Severity in Late infant neurological fashion in lipid Lipofuscinosis Using Multiparametric MR Imaging), J.USA.J.Neuroradiology 2012.

EuroQol group, "EuroQol-a new facility for the measurement of health-related quality of life (EuroQol-a new facility for the measurement of health-related quality of life)," health policy "16 [3], 199-208.1990.

Quantitative magnetic resonance imaging of human brain development, Giedd, JN, Snell, JW, Lange, N, Rajapakse, JC et al: 4-18 years old (Quantitative magnetic resonance imaging of human brain damage: agents 4-18.) "cortex of brain 6[4], 551-560.1996.

Karavelis A, Foroglou G, Selviaridis P et al, [ Intraventricular administration of morphine in 90patients to control intractable cancer pain (Intra viral administration of morphine for control of intraspecific cancer pain in 90patients ] ], neurosurgery [ 39[1], 57-62.1996.

Kronenberg MF, Lamier I, Rifici C et al, "seizures associated with intraventricular and intrathecal morphine boluses" (Epilestic sectional associated with intranuclear branched and intragastric morphine bolus.) "pain" 75[2], 383-387.1998.

Kurachi, Y, Oka, A, Mizuguchi, M, Ohkoshi, Y et al, "neurology" 54[8],1676-1680.2000, "Rapid immunodiagnosis of classical advanced infant neuropathic lipofuscinoses (Rapid immunological diagnosis of classic late neurological disease).

Kwon JM, Adams H, Rothberg PG, et al, quantitative physiological decline in juvenile neuropathic lens in cervical lipofuscinosis (Batten disease), neurology 77[20], 1801-1807.2011.

Lishner M, Perrin RG, Feld R et al, complications associated with omaya reservoir in patients with cancer: margaret principals Hospital Experience and Literature reviews (compartment associates with Ommaya Reservoirs in Patients with Cancer: The Princess Margaret Hospital Experience and a Review of The residence.) medical year of medicine (Arch Intern Med) 150[1], 173-176.1990.

Msall ME. "Measuring functional skills in pre-school children at risk of neurologic developmental disabilities" (Measuring functional bones in preschool children at risk for neurological developmental disorders "), overview of mental retardation and developmental disorders research (reviewed in 11,263-273.2005).

Seitz, D, Grodd, W, Schwab, A, Seeger, U et al, J.S. neuroradiology 19[7],1373-1377.1998, MR imaging and localized proton MR spectroscopy in advanced infant neuropathic ceroid lipofuscinosis (MR imaging and localized proton MR spectroscopy in membrane induced neurological lipofectinosis).

Sleat, DE, El-Banna, M, Sohar, I, Kim, KH et al, Residual levels of tripeptidyl-peptidase I activity significantly ameliorates the disease in advanced infant neuropathic ceroid lipofuscinoses (molecular levels of tripeptidyl-peptidase I activity in enzyme-induced myocardial lipofectamine metabolism.) (molecular Gene metabolism 94,222-233.2008).

Sleat, DE, Wiseman, JA, El-Banna, M, Kim, KH et al, A mouse model of classical advanced infantile neuropathic ceroid lipofuscinoses based on targeted disruption of the CLN2 gene resulted in loss of tripeptidyl-peptidase I activity and progressive neurodegeneration (amino model of classical neurological cortical basal on targeted disruption of the CLN2 gene responses in a low of tertiary-peptidyl-peptidase I activity and progressive neurodegeneration.) -J Neurosis (J Neurosci) 24[41], 9117-9126.2004.

Steinfeld, R, Heim, P, von Gregory, H, Meyer, K et al, "advanced infant neuropathic ceroid lipofuscinosis: quantitative description of the clinical course in patients with CLN2 mutation (Late amino neurological cancer lipofectinosis: quantitative description of the clinical course in patients with CLN2 mutations.) journal of medical genetics 112[4], 347-354.2002.

Vuillemotot, BR, Katz, ML, Coates, JR, Kennedy, D et al, "Intrathecal tripeptidyl-peptidase 1 reduction of lysosomal storage in canine models of advanced infant neuropathic lipofuscinoid disease (Integrated tripeptidyl-peptide 1 reduction in a canine model of late infant neurological waxy lipofuscinosis)," molecular Gene metabolism "104 [3], 325-337.2011.

Vuillemoot, BR, Kennedy, D, Reed, RP, Boyd, RB, et al, & recombinant human tripeptidyl peptidase-1infusion into the monkey CNS: safety, pharmacokinetics and distribution (Recombinant human tripeptidyl peptide enzyme-1 infusion to the monkey CNS: safety, pharmacokinetics, and distribution), "toxicology applied pharmacology (Toxicol Appl Pharmacol)" 277[1], 49-57.2014.

Worgarl, S, Kekatpure, MV, Heier, L, Ballon, D et al, neurology 69[6],521-535.2007, Neurological deterioration in neuropathic in vitro lipofuscinosis in advanced infants.

Worball, S, Sondhi, D, Hackett, NR, Kosofsky, B et al, "Treatment of advanced infant neuropathic ceroid lipofuscinosis by CNS administration of serotype 2adeno-associated virus expressing CLN2 cDNA (Treatment of late amino neural lipoid lipofuscinosis by CNS administration of a serotype 2 ado-associated virus expressing CLN2 cDNA.)" human Gene therapy "19 [5], 463-474.2008.

Xu, S, Wang, L, El-Banna, M, Sohar, I et al, Large-volume intrathecal enzyme delivery to improve survival in mouse models of advanced stage infant neuropathic lipofusoid disease (Large-volume interventional intervention patients with a mouse model of late animal neurological disease), molecular therapy (Mol Ther) 19[10],2011。

Example 11

This example describes additional pharmacokinetic and pharmacodynamic analyses performed on patient samples from the phase 1/phase 2 study of example 3.

Pharmacokinetic analysis: CSF and blood (plasma) samples for pharmacokinetic analysis were collected after the initial dose, the first dose at each new dose level during the up-dosing phase, and at weeks 5 and 13 of the stable dose phase. Samples were collected pre-dose (within 0.25 hours before infusion started) and at 0.25, 4, 8,20, 72 and 120 hours after infusion ended. Additional CSF and blood (plasma) samples were collected initially prior to dosing and every 4 weeks of the stable dose phase, whenever no continuous samples were collected. CSF samples were obtained from the lateral ventricle using the ICV port.

CSF and plasma samples were analyzed for cellular lipinase alpha concentrations using a validated electrochemiluminescence immunoassay (ECLA) method (biomain Pharmaceutical, Novato, CA, US). The lower limit of quantitation in CSF (LLOQ) was 20ng/mL and in plasma was 16 ng/mL. The inter-assay accuracy (% absolute system of relative error) and precision (% variation is a few%) of the quality control group was 13.2% in CSF and 17.2% in plasma throughout the sample testing procedure.

PK parameters were estimated based on CSF and plasma concentration-time data using non-compartmental analysis (NCA) using Phoenix WinNonlin 6.4(Pharsight, Cary, NC, USA). Maximum concentration (C) was recorded directly from observed data _max ) And maximum concentration time (T) _max ). Other PK parameters estimated were elimination half-life (t) _1/2 ) (ii) a Time 0 to last time canArea under the concentration-time curve (AUC) over time to measure concentration _0-t ) It is estimated using the linear trapezoidal rule; extrapolation of area under infinite concentration-time curve (AUC) _0-∞ ) (ii) a Clearance (CL) of absorbed fraction; end-based volume of distribution (V) _z ) (ii) a And steady state distribution volume (V) _ss )。

Immunogenicity analysis: CSF and blood (serum) samples for immunogenicity were collected at baseline, every 4 weeks during the up-dosing phase and every 4 weeks at the start and after the stable dosing phase. CSF and serum samples were tested against total anti-drug antibodies (TAb) specific for cellular liprenase a using a validated bridge electrochemiluminescence assay (biomain Pharmaceutical, Novato, CA, US). TAb positive samples in CSF were further characterized using a validated cell-based flow cytometry analysis (BioMarin Pharmaceutical, Novato, Calif., US) against neutralizing antibodies (NAb) that prevent cellular Libannase alpha uptake into lysosomes. NAb testing was performed only in CSF samples, since the target site of action was CNS, and TAb positive samples were tested for NAb response. The complete immunogenic approach and the results of this study have been previously reported [ Cherukuri-2018 ].

Statistical analysis: demographic characteristics are summarized for PK populations. PK parameters were summarized in a descriptive manner using biological matrices, dose groups and study visits. PK parameters were evaluated graphically for demographic, immunogenicity, safety, and efficacy parameters, as analysis of PK populations failed to evaluate statistical significance. For the analysis without time as a co-variable, the mean PK parameters for each patient over the course of treatment with 300mg QOW were used as a representative measure of exposure for the individual patients. C was calculated by using 300mg QOW dosing and intensive PK sampling (i.e., 300mg for the first dose, and 5 and 13 weeks for the stable dose phase) during the study visit _max And AUC _0-t Average values of the values to derive the average PK parameter.

Results

PK parameters were estimated in all patients within various dose levels and study visits. At 300mg QOW, 24 patients had evaluable PK data in CSF versus 15 patients in plasma.

Single dose PK data can be obtained from patients receiving initial doses of cellular risporase α at 30 (n-3), 100 (n-3) or 300mg (n-17) (4/4 from dose, and 13/14 directly involved in the stable dose phase) (fig. 13).

In CSF, a peak concentration was observed at the first sampling time point after the end of the 4 hour infusion and it appeared to be biphasic off. CSF exposure increased by a smaller dose proportion, median C, than dose increased 10-fold from 30 to 300mg _max And an increase in AUC of about 5 to 7 fold. One patient in the 100mg group was highly exposed after its initial dose, and therefore, the exposure parameter for the 100mg dose content was highly variable due to the small sample size. C of this patient _max And AUC (shown by the maximum reported for the 100mg group) is higher than the median of the 300mg group. Although it was inconclusive to discover that this outlier exposure was considered publicly, the CSF exposure of this patient after a subsequent infusion of 300mg was lower than its exposure after the initial 100mg dose.

During the initial phase of study performance, plasma PK samples were stored outside the stability range, and thus no data was available in the 30mg group, and only in one patient in the 100mg group. Concentrations in plasma peaked between 8 and 20 hours after the end of the 4 hour ICV infusion, based primarily on a 300mg dose level, and appeared to decline in two phases, remaining above the lower limit of quantitation (LLOQ) over 72 hours.

Multiple dose PK data were evaluated from patients who were directly involved in the stable dose phase (n-14) who received 300mg of cellular liprenase α QOW throughout the study (fig. 14-15).

PK parameters in CSF were similar between day 1, week 5 and week 13 visits. Although variable, plasma T on visit _max 、C _max And AUC _0-t Comparable to no discernible trend. Median C in plasma with 300mg QOW ICV administration _max About 1000-fold lower than in CSF and median AUC in plasma _0-t About 300 to 1000 times lower than in CSF. Contrast of C in plasma in CSF based on patient matched and visit matched PK _max Or AUC _0-t There is no significant correlation between the magnitudes of (A), (B), (C), (D), and D) a)16). In access, C _max And AUC _0-t The individual-to-individual variation was 26% -73% in CSF and 31% -49% versus 54% -89% and 59% -103% in plasma, respectively. In access, C _max And AUC _0-t In-person changes of 33% and 24% in CSF versus 69% and 80% in plasma, respectively.

Pharmacokinetic and patient characteristics: the potential effect of baseline patient characteristics on cellular lybonase α PK was evaluated against a 300mg QOW protocol. C _max And AUC _0-t Is used to represent exposure of individual patients over the course of therapy, and is considered appropriate due to lack of drug accumulation or time-dependent PK with 300mg QOW. Baseline gender, age, weight or CLN2 score had no significant effect on exposure of cellular lybonase alpha to CSF or plasma (fig. 17A-17D). With age, plasma C _max There was a slight trend towards increase, but no plasma AUC exhibited _0-t 。

Pharmacokinetics and immunogenicity: total antibodies against cellular lybonase alpha (TAb) were detected in the CSF of 5/24 (21%) patients and in the serum of 19/24 (79%) patients over the duration of the study. CSF tap responses were first detected at week 13 of the stable dose phase, while serum tap responses were detected at the earliest sampling time point (week 5 of the up-dosing phase). No neutralizing antibody (NAb) was detected in the CSF of any of the 5 patients with CSF tap positive, and therefore was not available for further analysis.

To determine whether cellular Ribose alpha PK is affected by the development of anti-drug antibodies (ADAs), visited C will have a positive TAb response _max And AUC _0-t And compared to visits with negative TAb reactions. The matched exposure parameters and ADA status (i.e., stable dose at day 1, week 5, and stable dose at week 13) were assessed from all patients starting treatment at 300mg and having evaluable PK and ADA data. Eighteen patients with CSF data (4/4 from the dose escalation phase and 14/14 directly involved in the stable dose phase) and 14 patients with plasma/serum data (1/4 from the dose escalation phase and 13/14 from the stable dose phase) were included for analysis.

As shown in figure 18A, CSF C-free in patients using CSF ADA status _max And AUC _0-t A discernible trend of (a). CSF C with positive ADA response visit _max And AUC _0-t The values are well within the distribution of exposure values with negative ADA reactions. CSF AUC of ADA Positive visits for two patients with visit-matched PK and CSF ADA positivity _0-t A reduction of 17-27% compared to ADA negative visits. Plasma C with serum ADA status in and within patients _max And AUC _0-t No correlation was observed between (fig. 18 b). Serum ADA visit-positive plasma exposure spans the exposure range of ADA negative visits within individual patients.

Changes in motor-language scores were used to assess the relationship between cellular lybonase alpha PK and efficacy outcomes from 300mg QOW at the start to the end of the study. Of the 23 patients in this analysis, 2 were increased by one, 13 were unchanged, 5 were lost by one, and 3 were lost by two after 48 weeks of treatment, with a total response rate of 87% (20/23). Change in patient motor-language score at week 48 and individual mean C in CSF _max And AUC _0-t Not relevant (fig. 19). Patients with decreased scores had CSF exposure parameters within the distribution of patients with no change in score or increased scores. Similarly, no correlation was shown when assessed using the maximum reduction in patient score over the 48-week treatment period.

The relationship between PK and adverse events was also analyzed. As noted above, study drug-related events that occurred in at least 10% of the study population were included for analysis: fever 46% (11/24), allergy 33% (8/24), seizure 33% (8/24), epilepsy 17% (4/24), headache 13% (3/24) and vomiting 13% (3/24). There was no significant difference in Cmax and AUC0-t in CSF or plasma between patients with or without fever, allergy, seizures or epilepsy. Headache patients have a slightly higher tendency for CSF exposure than non-event patients, and emesis patients are more likely to be exposed to both CSF and plasma. The exposure of headache or vomiting patients does not generally exceed the highest exposure observed in patients without either of the events.

The above results indicate that the initial ICV infusions were 30, 100 andafter 300mg, cellular lybonase α demonstrated a sub-dose proportional increase in CSF exposure. Single dose range (2.08X 10 respectively) ⁵ 、6.65×10 ⁵ And 1.42X 10 ⁶ ng/mL) CSF C _max The amount of ICV dose generally corresponding to about 100mL of CSF administered into the human brain (3.00X 10, respectively) ⁵ 、1.00×10 ⁶ And 3.00X 10 ⁶ ng/mL) were consistent (Pardridge et al, J.Cereb. blood Flow Metab.) -17, 713-731, 1997). At 300mg QOW, based on comparable C in study visits _max AUC, CL and V _ss No apparent accumulation or no time dependence of CSF or plasma PK. This corresponds to a CSF half-life of 6.2-7.7 hours in the patient and a calculable plasma half-life of 11.8 hours in one patient, taking into account the dosing frequency every two weeks. It should be emphasized that CSF half-life does not directly reflect the target site, since CNS tissue half-life (from evaluation in monkeys) and lysosomal half-life (from ex vivo human fibroblasts) for days to weeks are most relevant to the rationale for therapeutic agents administered every two weeks. In patients, CSF concentrations are greater than lysosomal k _{Absorption of} Up to about 4 days, which is based on animal data, the widespread distribution of surface enzymes to CNS tissues. This is supported by an estimate of the CSF distribution volume that exceeds a typical CSF volume of about 100 mL. Direct administration of cellular ribavirin enzyme α to the internal CSF space of the brain produces approximately three orders of magnitude higher exposure than the periphery, where the C between CSF and plasma _max Or the magnitude of AUC is not correlated; indicating that plasma PK is not a good substitute for CSF PK. With CSF T occurring immediately after the end of infusion _max In contrast, plasma T _max 8 hours after completing the 4 hour ICV infusion. The blood-CSF barrier leaks compared to the BBB, and thus ICV administered drugs are transported out of the brain via the CSF flow track and absorbed into the peripheral blood stream in the spider villi (Pardridge et al, fluid Barriers CNS 8:7,2011).

CSF and plasma PK variations between patients cannot be explained by patient demographics, as intrinsic factors seem to be unrelated to cellular risonase alpha exposure. Design of ICV dose of Liboninase alpha based on brain qualityAnd thus CSF exposure would be expected to have no significant change in this study for the range of age (3-8 years) and body weight (14.5-26.0 kg). Human brain reaches an average of about 75% of adult mass by age 2 and 100% by age 5, with progressively decreasing brain to body weight ratios during development (Giedd et al, cortex (Cereb.) 6:551-560, 1996). The brain weight of the non-affected human is between the ages of 3 and 8-9 years, with an average of 1.09-1.18kg for males and 1.27-1.37kg for females, respectively, compared to a body weight of 14.1-26.0kg and 15.6-27.5kg (Dekaban et al, annual journal of neurology, 4,345-356, 1978). Thus, brain weight change was only 8% in this study over the age range compared to 80% for body weight. Notably, with age, plasma C _max The slight trend of increase is likely due to the disproportionate change in body weight versus brain weight during early childhood. Despite age-matched brain weight, the amount of ICV dose is absorbed into significantly less body weight and correspondingly less blood volume, resulting in more concentrated systemic exposure.

Patient-to-patient variation in CSF exposure may be due to differences in disease severity rather than inherent variations in ICV administered enzymes, as intra-patient variation is much smaller than patient-to-patient variation (CSF C) _max Is 33%, and CSF AUC _0-t 24%). Although no correlation between CSF exposure and baseline CLN2 scores was shown, there may be a pathological effect on the CNS that is translated in CSF PK, but not into clinical assessment score changes. Because systemic absorption thereafter occurs, inter-and intra-patient variation in plasma PK is significantly higher than in CSF, in part because of the insufficient number of plasma samples with quantifiable concentrations.

Based on analysis of patient matches and visit matches at 300mg QOW, the presence of ADA in CSF and serum appeared to have no effect on PK in CSF and plasma, respectively. Most treated patients found ADA in serum, suggesting that plasma exposure to cellular lybonase α would likely cause ADA positivity in serum, consistent with other ERT's, because patients lack endogenous proteins (Long et al, clinical therapy 39: 118;. 129, 2017). The appearance of ADA responses in this study was previously shown to not predict an adverse safety profile or adverse treatment outcome (Cherukuri et al, clinical immunology 197:68-76,2018). For the most common adverse events associated with cellular ribavirin enzyme α, there was no clear correlation between CSF and plasma exposure and the occurrence of fever, allergy, seizure or epilepsy. Considering the small sample size of both events (3/24 patients), the frequency was low, and therefore the explanation for the slight tendency for increased exposure with concomitant headache and vomiting was limited.

The response to treatment appeared to be unrelated to the magnitude of CSF exposure, as measured by change in CLN2 score after 48 weeks at 300mg QOW, indicating that the greatest benefit was obtained over the 300mg QOW exposure range. Notably, CSF exposure in CLN2 patients exceeded that associated with an effective 16mg dose in TPP1 empty dogs, suggesting that these exposures are within the plateau of the exposure-response relationship (Katz et al, J. Neuroscientific research 92,1591-1598, 2014; Vuillemotot et al, molecular Gene metabolism 114,281-293, 2015). Having a reportable CSF AUC at a clinical 300mg dose despite inter-and intra-patient PK variations _0-t Has a value of 91% (62/68) of visits higher than the mean CSF AUC in TPP1 empty treated dogs _0-t (6.45×10 ⁶ ng-hr/mL) (Vuillemenot 2015, supra). Low expression of TPP1 has been shown to significantly ameliorate disease in CLN2 mutant mouse studies, where only 6% of normal TPP1 activity in the brain increases longevity to almost the same as wild-type mice (Sleat 2008, supra). Taken together, these non-clinical and clinical data indicate that cellular libonase promontory oval ICV at 300mg QOW provided sufficient TPP1 exposure to the CNS to obtain meaningful therapeutic benefit. This is the first feature of clinical CSF and plasma pharmacokinetics of ICV administered proteins.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Sequence listing

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<120> method for treating CLN2 disease in pediatric individuals

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Claims (28)

1. A method for treating neurogenic ceroid lipofuscinosis (CLN2) in an individual less than 3 years of age, comprising administering to the individual a formulation comprising recombinant human tripeptidyl peptidase-1 (rhTPP1) in an amount effective to treat CLN2 disease in the individual.

2. A method for delaying the onset of neuropathic lipofusinoid disease (CLN2) or symptoms thereof in an individual less than 3 years of age comprising administering to the individual a formulation comprising recombinant human tripeptidyl peptidase-1 (rhTPP1) for intraventricular, intrathecal or intraocular administration.

3. The method of claim 1 or 2, wherein the formulation is administered to the subject via intraventricular, intrathecal, or intraocular administration.

4. The method of any one of the preceding claims, wherein the formulation is administered once every 2 weeks.

5. The method of any one of the preceding claims, wherein the formulation is administered by infusion at a rate of about 2.5mL per hour.

6. The method of any one of the preceding claims, wherein the individual is administered a dose of about 300mg or less.

7. The method of claim 6, wherein the individual is greater than or about 2 years old.

8. The method of claim 7, wherein the individual is administered a dose of about 300mg of rhTPP 1.

9. The method of claim 6, wherein the individual is greater than or about 1 year old and less than 2 years old.

10. The method of claim 9, wherein the individual is administered a dose of about 200mg of rhTPP 1.

11. The method of claim 10, wherein each of the 1 st, 2 nd, 3 rd, and 4 th doses administered to the individual is about 200mg of rhTPP1, and each of the 5 th and subsequent doses administered to the individual is greater than about 200mg of rhTPP 1.

12. The method of claim 11 wherein each of the 5 th and subsequent doses administered to the individual is about 300mg of rhTPP 1.

13. The method of claim 6, wherein the individual is greater than or about 6 months of age and less than 1 year of age.

14. The method of claim 13, wherein the individual is administered a dose of about 150mg of rhTPP 1.

15. The method of claim 6, wherein the individual is less than 6 months of age.

16. The method of claim 15, wherein the individual is administered a dose of about 100mg of rhTPP 1.

17. The method of any one of the preceding claims, wherein the individual exhibits a decrease in TPP1 enzyme activity based on a blood test.

18. The method of any one of the preceding claims, wherein the individual is a sibling of the individual diagnosed with CLN2.

19. The method of any one of the preceding claims, wherein the individual has a total score of about 3 to about 6 according to the athletic and linguistic subscales.

20. The method of any one of the preceding claims, wherein the individual has not been previously treated with stem cell therapy, gene therapy, or enzyme supplementation therapy.

21. The method of any one of the preceding claims, comprising administering antihistamine to the individual with or without an antipyretic prior to administration of the rhTPP1, optionally about 30 to about 60 minutes prior to administration of the rhTPP 1.

22. The method of any of the preceding claims, wherein the formulation comprises the rhTPP1 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

23. The method of claim 22, wherein the formulation comprises disodium phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof.

24. The method of any one of the preceding claims, comprising administering a rinse solution to the subject after administering the formulation.

25. The method of claim 24, wherein the flush solution comprises disodium phosphate pentahydrate, monosodium phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride hydrate, water for injection, or a combination thereof.

26. The method of any one of the preceding claims, wherein a treatment period is at least 10 weeks, at least 20 weeks, at least 40 weeks, at least 80 weeks, or at least 96 weeks.

27. A composition comprising a formulation comprising recombinant human tripeptidyl peptidase-1 (rhTPP1) for intracerebroventricular, intrathecal or intraocular administration for the treatment of neuropathic lipofuscinoid disease (CLN2) in an individual younger than 3 years of age.

28. Use of a formulation comprising recombinant human tripeptidyl peptidase-1 (rhTPP1) for intracerebroventricular, intrathecal or intraocular administration for the manufacture of a medicament for the treatment of neuropathic lipofuscinoid disease (CLN2) in an individual younger than 3 years of age.