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WO1996039125A1 - Composition et procede d'administration de catalyseurs bio-influants - Google Patents

Composition et procede d'administration de catalyseurs bio-influants Download PDF

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Publication number
WO1996039125A1
WO1996039125A1 PCT/US1996/009262 US9609262W WO9639125A1 WO 1996039125 A1 WO1996039125 A1 WO 1996039125A1 US 9609262 W US9609262 W US 9609262W WO 9639125 A1 WO9639125 A1 WO 9639125A1
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composition
phase
bio
catalyst
matrix
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PCT/US1996/009262
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English (en)
Inventor
Sandeep Kumar
Kenneth J. Himmelstein
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University Of Nebraska Board Of Regents
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Priority to AU62577/96A priority Critical patent/AU6257796A/en
Publication of WO1996039125A1 publication Critical patent/WO1996039125A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases or cochleates; Sponge phases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres

Definitions

  • the present invention relates to treatment of diseases and other pathological conditions associated with enzyme defects or imbalances.
  • the invention provides compositions and methods for supplying enzymes or other bio-affecting catalysts, for long-term enzyme replacement/augmentation therapy.
  • an exogenous enzyme is administered to the patient either intravenously or intramuscularly.
  • enzymes are proteins and are therefore susceptible to rapid proteolytic degradation as well as elimination from the patient's body.
  • the exogenous enzyme may elicit undesirable immunological responses upon being introduced into the body of the patient.
  • the patient's own defenses may actually attack and destroy the enzyme, as well as eliciting detrimental effects to the patient associated with over-stimulation of the patient's immune system and other defense responses.
  • a further disadvantage of conventional enzyme treatment arises due to the frequent requirement for the missing enzyme in localized regions of the body.
  • the exogenous enzyme can fail to provide adequate therapeutic concentrations of the enzyme in the necessary regions of the body for long periods of time.
  • the exogenous enzyme has a very short half-life in the body and may never reach its target location in appreciatable quantities.
  • administration of large quantities of the enzyme is likely to be required. This requirement is inconvenient for the patient and is also impractical, since purified enzyme may not be available, or may be prohibitively expensive, in large enough quantities for long-term treatment.
  • compositions have been proposed for improving delivery of therapeutic agents to their sites of action and for effecting long-term sustained release of therapeutic agents.
  • a number of semi-solid ointments or gels have been shown to be effective for increasing drug retention times, especially in the case of ophthalmic drug delivery.
  • Other compositions have been developed for administration via injection, which comprise polymeric delivery vehicles that are of low viscosity under preparatory conditions, but form a semi-solid gel when subjected to physiological conditions.
  • Lipophilic and amphiphilic compositions have also been utilized in sustained-released drug delivery systems.
  • U.S. Patent No. 5,230,895 to Czarnecki et al. describes a method and composition for treating periodontal disease with a therapeutic agent released in a sustained manner from a glyceride composition that forms a semi-solid gel when placed in the periodontal pocket.
  • U.S. Patent No. 5,151,272 to Engstrom et al. discloses a controlled-released composition for biologically active materials which comprises a gel-like liquid crystalline phase (e.g., lamellar, hexagonal, cubic and micellar) .
  • a gel-like liquid crystalline phase e.g., lamellar, hexagonal, cubic and micellar
  • the biologically active substance is released into the body as a result of erosion of the liquid crystal phase or diffusion of the substance into the surrounding medium.
  • the above-described gels and liquid crystal matrices are all designed to deliver biologically active agents by releasing the agent into the body fluid or tissue into which the composition has been injected, implanted or otherwise administered.
  • release into the body is detrimental for two reasons: first, it exposes the enzyme to proteolytic and immunological attack; second, it allows the enzyme to diffuse from the precise location in the body where it may be required. What is more, because enzymes are catalysts, their release into the body from a gel or liquid crystal matrix is unnecessary. It is necessary only that the enzyme be accessible to its appropriate substrates in the body, and that it be capable of exerting its required enzymatic function and releasing the enzymatic products back to the body where they are required.
  • the compositions should be administrable by syringe and thereafter capable of forming a protective matrix surrounding the enzyme.
  • the compositions should be slowly erodible, so as to eliminate the need for removal after the effective life of the enzyme is over.
  • the present invention provides compositions and methods for administration of bio-affecting catalysts.
  • the term "catalyst” retains its common definition as a substance that influences the speed of a chemical reaction without itself undergoing a permanent change.
  • bio-affecting chemical reactions that occur in, or affect, biological systems
  • bio-affecting catalysts are those that catalyze “bio- affecting” reactions.
  • Bio-affecting catalysts may themselves be biological molecules, such as enzymes, but they need not be.
  • a composition is provided that comprises at least one bio-affecting catalyst retained within a matrix.
  • the matrix functions to substantially prevent release of the bio-affecting catalyst therefrom for a pre-determined time period, preferably calculated relative to the effective lifespan of the catalyst.
  • Catalysts retained within the matrix are in fluid communication with the external medium in which the composition is disposed, thereby enabling accessibility of the catalyst to substrates in the external medium, and release into the external medium of products formed by the catalyst.
  • the catalyst erodes substantially after expiration of the pre-determined time period.
  • the matrix is a high viscosity gel or liquid crystalline phase that not only functions to retain the bio-affecting catalyst, but also functions to protect the bio-affecting catalyst from external proteolytic or other degradative enzymes and/or cellular defense mechanisms present in the physiological environment.
  • High viscosity gel matrices comprise amorphous networks of polymeric molecules, the spaces therebetween providing channels for the fluid communication.
  • High viscosity liquid crystalline matrices comprise semi-regular or regular arrays of amphiphilic molecules, separated by pores or channels for the fluid communication.
  • Liquid crystalline phase high viscosity matrices are preferred in the present invention, with cubic phases being particularly preferred.
  • the above-described composition preferably is prepared as a low viscosity phase mixture comprising the bio-affecting catalyst and at least one matrix-forming compound, and can be induced to undergo a phase transition to a high viscosity phase to produce the matrix with the catalyst retained therein.
  • the phase transition is inducible by exposing the low viscosity phase to changes in one or more conditions, including temperature, pH or solvent composition. In a preferred embodiment, the phase transition is inducible by introduction into the body of a patient.
  • a composition that comprises at least one bio-affecting catalyst retained within a high viscosity cubic phase matrix.
  • the composition is prepared as a low viscosity phase, comprising by weight between about 60% and about 99.99% of glyceryl monooelate, up to about 40% (more precisely, 39.99%) of an aqueous phase, and between about 0.01% and about 40% of one or more bio-affecting substances.
  • a method is provided for supplying a bio- affecting catalyst to a patient.
  • the method comprises administering to a patient a composition comprising at least one bio-affecting catalyst retained within a matrix, which (1) substantially prevents release of the catalyst therefrom for a pre-determined time period, and (2) enables fluid communication between the catalyst and an external medium in which the composition is disposed, resulting in accessibility of the catalyst to substrates in the external medium and release of products into the medium.
  • the above method preferably comprises formulating the composition as a low viscosity phase mixture, preferably injectable, which comprises the bio- affecting catalyst and one or more matrix-forming compounds. The mixture is then induced to undergo a phase transition to a high viscosity phase to produce the matrix with the catalyst retained therein.
  • the low viscosity phase mixture is injected into the patient at a selected location (e.g., intramuscularly, subcutaneously or into a desired tissue or organ) , and the phase transition is induced by exposure of the mixture to the physiological conditions present at the injection site.
  • the composition is administered by removing a component (e.g., body fluid or tissue) from a patient's body, exposing the component to the composition, then returning the component to the patient's body.
  • Fig. 1 is a plot of the fraction of FITC- labelled horseradish peroxidase, FITC-labelled insulin, or methylene blue that has escaped from a cubic phase of glyceryl monooleate as a function of time
  • Fig. 2 is a plot of the percent of horseradish peroxidase (HRP) activity as a function of time in the presence of lipase and/or trypsin; and
  • Fig. 3 is a plot of the percent of catalase activity as a function of time.
  • the composition of the present invention comprises a matrix in which is disposed a bio-affecting catalyst, such as an enzyme.
  • the composition is initially formed as a mixture of the matrix-forming compound(s) and bio-affecting catalyst(s) wherein the mixture exists as a low viscosity phase.
  • the mixture is capable of undergoing a phase transition to a high viscosity phase, such as a liquid crystal or gel, with the bio-affecting catalyst entrapped within.
  • the high viscosity phase matrix comprises pores or channels that allow for the ingress and egress of substrates and products to and from the catalyst disposed therein.
  • the size of the pores or channels is sufficiently small to impede or prevent diffusion of the catalyst from the matrix, and to protect the catalyst from proteolytic enzymes and cells of the immunological response system.
  • the present invention can be practiced with a variety of bio-affecting catalysts. These catalysts may be naturally-occurring enzymes or enzyme derivatives, or they may be any other catalyst (e.g., catalytic peptides or peptide derivatives) useful for producing biological products normally supplied by a naturally-occurring enzyme in a patient's body.
  • Enzyme classes that may be utilized in the present invention include, but are not limited to, oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases (or synthetases) . Examples of enzymes suitable for use in the present invention, include but are not limited to, the enzymes set forth in Table "A" below. - 8 -
  • L-asparaginase Acute Substrate lymphoblastic Asparagine leukemia Mechanism: deprivation of substrate needed by tumor
  • Urease Uremia Substrate Urea ⁇ -galactosidase Fabry's disease or Substrates: gal-
  • Hexosaminidases A Sandoff's disease Substrates: GM2 and B ganglioside, asilo-GM2 ganglioside, and globoside ⁇ -N-acetyl Tay-Sachs disease Substrates: GM2 hexosaminidases ganglioside and
  • Glucosyl Gaucher's disease Substrates cerebramidase and Glucosyl- ⁇ - ceramide, glucocerebrosidas glucosyl- e sphingosive, and sialyl lactosylceramide
  • one or more bio-affecting catalyst can be simultaneously entrapped within the matrix. If more than one catalyst is entrapped, they can act independently or as co-catalysts in a complex system.
  • the matrix may further comprise coenzymes and other cofactors required to accomplish complex bio-catalytic functions.
  • the matrix possesses pores or channels that allow for the movement of substrates and products to and from the enzyme and the external environment. The pores or channels are sufficiently narrow to prevent or impede the bio-affecting catalyst from diffusing out of the matrix. Retention may also be facilitated by partitioning of the catalyst with the lipid phase of the matrix (preferred matrices of the invention comprise a lipid phase) .
  • anchor molecules include, but are not limited to: 1-palmitic acid and other fatty acids, peptides, proteins, polyethylene glycol and other polymers, or a combination of such molecules.
  • the bio-affecting catalyst is disposed within a matrix comprising pores or channels of a size that enable entry/exit of substrates/products, while concomitantly preventing exit of the bio-affecting catalyst during its functional lifetime and substantially preventing entry of proteolytic or other enzymes or immunological defense cells of the body.
  • the pore or channel size may vary, but is preferably within the range of 0.1 to 100 nm, more preferably between 1 and 20 nm, and most preferably on the average of 5 nm.
  • the matrix preferably comprises a relatively uniform channel structure, that may be adjusted by varying the composition and relative concentrations of components used to prepare the matrix. Optimum size ranges are formulated depending on the size of the bio- affecting catalyst, as well as sizes of substrates and product molecules.
  • the matrix should be made from one or more matrix-forming compounds that can be mixed with the bio-affecting catalyst as a low viscosity phase, to enable even distribution of the bio-affecting catalyst in the matrix to be formed.
  • the mixture then should be capable of undergoing a phase transition to a high viscosity phase, thereby forming the matrix in which the bio-affecting catalyst is disposed.
  • the transition from the low viscosity phase to the high viscosity phase is effectuated, for example, by a change in pH, temperature, and/or solvent composition (e.g., by contacting an alcoholic or lipid mixture with water, ionic amphiphiles or salts) .
  • the phase transition can be induced ex vivo, prior to administration into the body of a patient, and thereafter be introduced into the body by implantation.
  • the composition is injected as a low-viscosity phase mixture through a syringe, so that it can be easily placed virtually anywhere within the patient's body, and thereafter undergoes a phase transition to form the high viscosity matrix in which the bio-affecting catalyst is entrapped.
  • the high viscosity phase formed by these reversibly-gelling compositions are gels comprising amorphorous polymer networks in a substantially aqueous environment.
  • Preferred gels comprise low molecular weight polymers (e.g., mw ⁇ 50,000), which will slowly dissolve in the body, or polymers that are otherwise degradable in the body, to eliminate the need for surgical removal.
  • gels can be used, it has been discovered in accordance with the present invention that matrices having well structured pores or channels provide better accessibility of the bio-affecting catalysts to substrates (and better release of products) than do gel matrices with amorphous polymer networks, or other matrices having poorly formed channel structures. Accordingly, preferred for use in the present invention are compounds capable of undergoing a phase transition to a liquid crystal structure, such as a cubic, hexagonal or reverse hexagonal phase. Of these, cubic phases are most preferred because of their uniform pore/channel structure.
  • Liquid crystalline phases are common in amphiphilic lipid systems, including compositions comprising simple surfactants, straight- or branched- chain lipids and/or complex biological lipid mixtures.
  • Some amphiphilic lipid systems are capable of undergoing a phase transition, e.g., upon temperature increase from a low viscosity lamellar phase (i.e., a phase formed by lipid-water systems in which lipid bilayers alternate with water layers) to a high viscosity liquid crystalline phase.
  • the liquid crystalline phase may be a cubic phase (i.e., an isotropic phase with a three-dimensional structure in which the lipid bilayer forms a continuous structure that separates two identical networks of water channels) , a hexagonal phase (lipids forming tubular micelles with hydrophobic tails facing internally) or reverse hexagonal phase (tubular micelles with hydrophilic heads facing internally) .
  • compositions were formulated for the purpose of releasing drugs or other bio-active compounds into the body, not for retention of a bio-affecting catalyst, for purposes of protecting the catalyst while enabling entry and exit of substrates and products, as disclosed herein in accordance with the present invention.
  • compositions of the invention are preferably formulated from the following general classes of compounds at the indicated preferred concentration ranges:
  • aqueous phase water, salt solution, buffer, etc.
  • a formulation in which the mixture contains no initial aqueous phase may be prepared. Such a formulation will form the liquid crystal phase upon administration into an aqueous environment, such as the body, the transition induced by absorption of water and/or temperature increase from room temperature to body temperature.
  • bio-affecting catalysts, co-catalysts and/or co-factors in appropriate effective concentrations, preferably about 0.01% - 50% (but could be less or greater depending on the specific catalysts and other substances used) ;
  • Amphiphilic molecules that may be utilized in preparing compositions of the invention include, but are not limited to: lipids, surfactants and soaps capable of forming high viscosity liquid crystalline phases. These components may be used alone or in combination.
  • Nonlimiting examples of lipids that may be utilized in the compositions of the invention include glycerides, phospholipids, fatty acids and sphingolipids.
  • Glycerides include, but are not limited to, monoglycerides, diglycerides and triglycerides.
  • the monoglyceride, glyceryl mono(cis-9)oleate also referred to as glyceryl monooleate
  • glyceryl monooleate is used, as described in greater detail below.
  • Nonlimiting examples of soaps that may be incorporated into compositions of the invention include anionic and cationic soaps.
  • examples of surfactants include, but are not limited to, nonionic and zwitterionic surfactants.
  • the aqueous medium may contain a biologically acceptable alcohol, such as ethanol, for purposes of improving the solubility of polymers in the low viscosity phase (see, e.g., U.S. Patent Application No. 08/261,731, the disclosure of which is incorporated herein by reference) .
  • bio-affecting catalysts have been discussed in detail hereinabove.
  • Compositions of the invention may contain one or more such bio-affecting catalysts, along with co-catalysts and co-factors, as required.
  • compositions of the invention may also be included in compositions of the invention for various purposes, such as to modify the structure of the high viscosity phase and/or to accelerate or decelerate degradation of the matrix.
  • ingredients include, but are not limited to, glycerol and other similar solvents, lipids (e.g., glycerides, phospholipids, sphingolipids, cholesterol, fatty acids, etc.), oils, proteins and other biologically compatible molecules.
  • lipids e.g., glycerides, phospholipids, sphingolipids, cholesterol, fatty acids, etc.
  • oils proteins and other biologically compatible molecules.
  • compositions of the invention include, e.g., preservatives, protein stabilizers, enzyme inhibitors (e.g., lipase inhibitors, esterase inhibitors, protease inhibitors) , as well as ingredients for stabilizing enzymes, reducing escape of enzymes from the matrix, agents that modify the structure of the matrix (e.g., by modifying the pore or channel size or by modifying the rate of degradation of the matrix) .
  • enzyme inhibitors e.g., lipase inhibitors, esterase inhibitors, protease inhibitors
  • agents that modify the structure of the matrix e.g., by modifying the pore or channel size or by modifying the rate of degradation of the matrix.
  • the matrix in its high viscosity phase should be slowly erodible in a physiological environment.
  • the term "erodible” means that (1) the matrix is susceptible to degradation by enzymes and/or other agents naturally occurring in a physiological environment such as a patient's body, or (2) the matrix slowly dissolves, or (3) the matrix erodes by a combination of dissolution, degradation and/or other means.
  • An erodible matrix eliminates the need for removal of the composition upon completion of the treatment. If an erodible matrix is utilized, the rate of erosion should be adjusted so that the matrix erodes substantially after the effective life span of the bio-affecting catalyst is completed.
  • the rate of lipid degradation can be delayed by adding lipase inhibitors to the matrix- forming mixture in its low viscosity phase.
  • the inclusion of lipase inhibitors functions to reduce to activity of lipases that may attack the lipid-containing matrix of the composition. As a result, the useful lifetime of the compositions are increased.
  • An exemplary composition of the invention comprises, as a matrix-forming compound, the polar lipid, glyceryl monooleate (hereinafter GMO) , also known as monoolein. At 0-40% (preferably 10-20%) (by weight) water in GMO, the mixture forms a low viscosity, opaque lamellar phase in which bio-affecting catalysts and other ingredients may be solubilized.
  • GMO glyceryl monooleate
  • the viscosity of the lamellar phase is low enough that the GMO-catalyst mixture can be passed through a syringe. Upon injection into a patient's body, the mixture undergoes a phase transition to a high viscosity, clear cubic phase
  • phase transition is induced by the absorption of water and increase in temperature (from room temperature to body temperature) .
  • the cubic phase gradually erodes in the body, eliminating the need for surgical removal.
  • the GMO cubic phase comprises aqueous channels with average diameters of about 5 nm, which allow substrates and product to migrate to and from the bio- affecting catalyst (which, in this preferred embodiment, is a naturally-occurring enzyme or combination of enzymes) .
  • the cubic phase stabilizes the enzyme and protects the enzyme from proteases, macrophages and other macromolecules and cells involved in the patient's immune or defense systems.
  • the channels are sufficiently narrow so that enzymes are entrapped in the cubic phase, and are substantially prevented from exiting the cubic phase. As discussed above, under certain circumstances it may be desirable to further reduce the rate at which the enzyme (or other co-factors or bio-affecting catalysts) escape from the cubic phase.
  • the present invention also provides methods of using the compositions described above. In one method, the compositions are used for replacing or supplementing at least one enzymatic function in a patient.
  • the term "patient" refers to both humans and animals.
  • the enzyme supplementation therapy may be accomplished by providing the deficient naturally occurring enzyme; alternatively, an enzyme derivative or synthetic substitute catalyst may be utilized to supply the enzymatic function deficient in the patient.
  • a mixture is prepared of the enzyme or other bio-affecting catalyst and the matrix-forming compound(s) , along with other desired ingredients.
  • the mixture is prepared under conditions whereby the mixture forms a low viscosity phase with the enzyme solubilized within the phase.
  • the mixture is then subjected to conditions that induce a phase transition to a high viscosity phase, preferably an ordered liquid crystalline phase.
  • the phase transition is induced ex vivo , and the resultant matrix-enzyme composition is administered to the patient by implantation into the desired location of the patient's body.
  • the mixture is administered to the patient by placing the mixture in a syringe and injecting it into the patient intramuscularly, subcutaneously or into a selected tissue.
  • the phase transition is induced by the physiological conditions within the patient's body, including increased temperature and exposure to water.
  • the mixture also may be injected into a predetermined region of the body, so as to provide increased concentrations of the enzyme or other bio- affecting catalyst in that area.
  • the general method described above is applicable not only to enzyme replacement/augmentation therapy, but may also be used for treatment of substrate-dependent tumors.
  • a substrate-dependent tumor is human acute lymphoblastic leukemia. Lymphoblastic leukemia cells are deficient in L- asparagine synthetase and depend on exogenous supplies of L-asparagine formed in surrounding normal cells.
  • Current therapy for lymphoblastic leukemia involves intravenous administration of the enzyme, L-asparaginase. L- asparaginase degrades L-asparagine, thereby depleting the body of L-asparagine. This causes the malignant cells to die of starvation due to lack of adequate supply of L- asparagine, which is an essential amino acid.
  • compositions of the invention may provide a more effective treatment for substrate-dependent tumors.
  • injection of a composition comprising L- asparaginase into a tissue or organ having high blood flow e.g. spleen, liver, subcutaneous injection
  • a composition comprising L- asparaginase into a tissue or organ having high blood flow (e.g. spleen, liver, subcutaneous injection) will result in an increased half-life of the enzyme as well as sequestering it from recognition by the immune response system.
  • removal of L-asparagine from the patient's body may be effected over a longer term, with administration of less L-asparaginase.
  • Compositions of the invention should be particularly effective in the case of substrate-dependent solid tumors, where they can be injected near or in the tumor to deplete malignant cells of a required substrate.
  • compositions of the invention may also be utilized for detoxification of various body fluids and tissues, both in vivo and ex- vivo.
  • the bio-affecting catalyst e.g., comprising glutamine synthetase
  • a toxic substance e.g., ammonia
  • a non-toxic substance e.g., amino acids such as glutamine
  • Such compositions may also be used in extracorporeal shunting or filtering devices, such as dialysis devices or circulation devices in which a patient's blood is directed out of the body, through external tubing and back into the body.
  • compositions of the invention may be placed in such devices so that a selected body fluid comes in contact with the composition during its flow through the extracorporeal device.
  • Compositions formulated to comprise bio-affecting catalyst having detoxification functions may be used to advantage to purge these body fluids of an unwanted or toxic substance during such procedures.
  • compositions and methods of the present invention mixtures of a matrix and various enzymes or complex enzyme systems were prepared.
  • MyverolTM 18-99 glyceryl monooleate (GMO) obtained from Eastman Chemical Company
  • GMO glyceryl monooleate
  • the enzyme systems tested include horseradish peroxidase, catalase, and a combination of human cytochrome P450-2A6, cytochrome P450 reductase, and glucose-6-phosphate dehydrogenase.
  • HRP horseradish peroxidase
  • ABTS 2,2'-azino-bis-[3- ethylbenzthiazoline-6-sulfonic acid]
  • HRP concentrations in solution can be determined using fluorescein isothiocyanate-labelled HRP (hereinafter, HRP-FITC) , which can be detected fluorimetrically at an excitation wavelength of 490 nm and an emission wavelength of 515 nm.
  • HRP-FITC fluorescein isothiocyanate-labelled HRP
  • the samples of lamellar and cubic phase GMO containing HRP (or HRP-FITC) used in the examples were prepared as follows. A 10 mM solution of phosphate buffer (pH 7.4) containing HRP was mixed with GMO such that the final composition was, in weight percent, 85.00% GMO, 14.87% buffer, and 0.13% HRP. The mixture was stored at room temperature for 24 hours to form the lamellar phase. The lamellar phase was then exposed to a 10 mM solution of phosphate buffer (pH 7.4) at 37°C, in order to simulate physiological conditions.
  • Each gram of the lamellar phase absorbed about 0.3 grams of buffer and formed the cubic phase with a composition, in weight percent, of about 65.0% GMO, 34.9% buffer, and 0.1% HRP.
  • a composition, in weight percent, of about 65.0% GMO, 34.9% buffer, and 0.1% HRP did not affect the formation of the lamellar phase or the transition to the cubic phase upon exposure to simulated physiological conditions.
  • the cubic phase was then removed from the beaker and thoroughly rinsed.
  • the outer layers of the cubic phase were removed from all sides and a small portion of the core of the cubic phase weighing about 0.1 grams was placed in a beaker.
  • the cubic phase was then treated with a substrate solution in the same manner described in the previous paragraph.
  • the color of the substrate solution changed to a bluish-green, again indicating the formation of product.
  • the activity demonstrated by the core removed from the interior of the cubic phase indicates that the enzyme is homogeneously distributed throughout the cubic phase and that the enzyme is not denatured within the cubic phase.
  • the remaining buffer solution was removed and 4 mL of a substrate solution containing 0.25 mM ABTS and 0.05 mM H 2 0 2 was added to the vial.
  • the HRP containing layer turned deep bluish-green while the upper cubic phase layer remained colorless.
  • the substrate solution on top of the upper cubic phase layer also acquired a bluish-green color.
  • the immobilized HRP assay involved placing 1 gram of a cubic phase containing HRP at the bottom of a glass vial (12 mm inner diameter) .
  • the cubic phase was thoroughly rinsed with buffer solution to remove any surface associated HRP.
  • a 2 mL aliquot of a substrate solution containing 0.25 mM ABTS and 0.05 mM H 2 0 2 was poured on top of the cubic phase in the vial and allowed to react for 2 minutes with continuous shaking. At the end of the 2 minutes, the reaction was stopped by decanting the substrate solution into another vial containing 2 mL of a solution containing 2% dodecyl sulfate and 0.1% sodium azide.
  • the concentration of the products was measured spectrophotometrically by determining the absorbance of the solution at 410 nm following appropriate dilution.
  • a similar assay procedure was used to test the activity of HRP in free solution.
  • the free HRP assay involved preparing a solution of HRP in a 10 mM phosphate buffer solution (pH 7.4) and a substrate solution of 0.25 mM ABTS and 0.05 mM H 2 0 2 . Aliquots of each solution were mixed to yield a final volume of 4 mL.
  • the activity of HRP was measured for fixed ABTS and H 2 0 2 concentrations with different HRP concentrations, as well as at fixed HRP and H 2 0 2 concentrations with different ABTS concentrations.
  • the reaction was allowed to proceed for 2 minutes. At the end of the 2 minutes, 4 mL of a solution containing 2% sodium dodecyl sulfate and 0.1% sodium azide was added to stop the reaction. The concentration of products formed was determined spectrophotometrically by determining the absorbance of the solution at 410 nm following appropriate dilution.
  • the stability of HRP immobilized in the cubic phase was compared to the stability of HRP in free solution. Aliquots of 1 gram pieces of cubic phase containing HRP were submerged in a 10 mM phosphate buffer solution and stored at 37°C. For comparison, a 4 ⁇ g/mL solution of HRP in a 10 mM phosphate buffer solution (pH 7.4) was also stored at 37°C. The HRP entrapped in the cubic phase and the HRP in free solution were tested using the assay procedures described above. Each measurement was performed in triplicate. The averages of the three measurements are given in Table 2.
  • Example 7 Effect of Lipase and Protease Enzymes on Immobilized Enzyme Activity and Matrix Structure
  • protease and esterase lipolytic enzymes circulating in the body.
  • protease and esterase lipase enzymes
  • trypsin a protease enzyme
  • lipase an esterase enzyme
  • a first group of three aliquots were then exposed to a 10 mM phosphate buffer solution (pH 7.4) as a control.
  • a second group of three aliquots were exposed to a solution of 0.03 mg/mL trypsin in phosphate buffer.
  • a third group of three aliquots were exposed to a solution of 0.03 mg/mL lipase in phosphate buffer.
  • a fourth group of three aliquots were exposed to a solution of 0.03 mg/mL trypsin and 0.03 mg/mL lipase in phosphate buffer. All of the aliquots were placed in an incubator at 37°C. The solutions in the vials were removed and replaced with fresh solutions every 8 hours.
  • Catalase catalyzes the breakdown of hydrogen peroxide (H 2 0 2 ) to water and oxygen.
  • H 2 0 2 absorbs light at a wavelength of 240 nm and, therefore, catalase activity can be measured spectrophotometrically by monitoring the decrease in absorption at that wavelength.
  • catalase immobilized in the cubic phase of GMO retains its enzymatic activity
  • catalase in a 50 mM phosphate buffer solution (pH 7.0) was mixed with GMO and allowed to form a cubic phase such that 1 gram of the cubic phase contained 100 units of catalase.
  • Three aliquots, each containing 1 gram of the cubic phase containing catalase, were placed in three separate glass vials along with 1 mL of a 50 mM phosphate buffer solution (pH 7.0) and stored at 37°C.
  • the activity of the immobilized catalase was determined on days 0, 1, 3, 7, 10, and 14 by the following procedure.
  • the cubic phases in the vials were thoroughly rinsed with a 50 mM phosphate buffer solution to remove any surface associated catalase.
  • the cubic phases were then exposed to 3 mL of a 50 mM H 2 0 2 and 50 mM phosphate buffer solution (pH 7.0).
  • a 50 mM H 2 0 2 and 50 mM phosphate buffer solution pH 7.0
  • 0.5 mL aliquots were withdrawn from the vials and replaced with fresh 0.5 mL aliquots of the 50 mM H 2 0 2 and 50 mM phosphate buffer solution (pH 7.0).
  • the cytochrome P450 family consists of membrane bound enzymes that metabolize many chemicals in the body.
  • CYP-2A6 is a coumarin 7-hydroxylase enzyme: it converts coumarin (substrate) to 7-hydroxycoumarin (product) .
  • the rate of formation of the product can be measured fluoro etrically with excitation at 390 nm and emission at 440 nm.
  • G6PDase glucose-6-phosphate dehydrogenase
  • Enzyme solutions in 50 mM Tris were added to GMO to form the cubic phase such that 1 gram of matrix contained 2 mg CYP-2A6/CYP-r protein and 20 units of G6PDase.
  • One gram matrix (in duplicate) was placed in a glass vial along with 1 ml 50 mM Tris (pH 7.4) solution. At selected intervals, the matrix was rinsed thoroughly with fresh Tris solution to remove surface associated enzymes, if present.

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Abstract

L'invention concerne des compositions et des procédés d'administration d'un catalyseur bio-influant, tel qu'une enzyme. Les compositions comprennent au moins une enzyme ou un autre catalyseur bio-influant, contenus à l'intérieur de la matrice. La matrice présente une barrière entre l'enzyme piégée et des enzymes de décompositions extérieures ou des systèmes de défenses cellulaires du corps d'un patient, et empêche également la libération de l'enzyme. La matrice comprend des pores ou des voies permettant l'entrée et la sortie de substrats et de produits. La matrice est de préférence une phase cubique à haute viscosité d'un ou de plusieurs composés amphiphiles adaptés, tels que le monooléate de glycéryle. Des procédés préférés de l'invention consistent à formuler la composition sous la forme d'une phase injectable à faible viscosité, laquelle subit une transition de phase lors d'une exposition à des conditions physiologiques.
PCT/US1996/009262 1995-06-06 1996-06-06 Composition et procede d'administration de catalyseurs bio-influants WO1996039125A1 (fr)

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AU62577/96A AU6257796A (en) 1995-06-06 1996-06-06 Composition and method for administration of bio-affecting c atalysts

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US65262395A 1995-06-06 1995-06-06
US08/652,623 1995-06-06

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010411A3 (fr) * 1999-08-06 2001-06-21 Max Delbrueck Centrum Depot de principe actif implantable
US6773703B1 (en) 1999-01-29 2004-08-10 Beiersdorf Ag Protein-containing hydrogels
EP1322422A4 (fr) * 2000-07-18 2004-11-17 Univ California Procede et appareil de preparation de mesophases lipidiques
WO2007070561A3 (fr) * 2005-12-13 2008-03-20 Southeastern Medical Technolog Agents pour controler des fluides biologiques et leurs procedes d’utilisation
JP2008523149A (ja) * 2004-12-13 2008-07-03 サウスイースタン メディカル テクノロジーズ 体液を調節する薬剤及びその使用方法
US20090325171A1 (en) * 2008-05-13 2009-12-31 Thomas Hirt Vesicles for use in biosensors
KR101340586B1 (ko) 2011-12-12 2013-12-11 강원대학교산학협력단 광 응답성을 나타내는 큐빅상 조성물 및 그 제조방법

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5162430A (en) * 1988-11-21 1992-11-10 Collagen Corporation Collagen-polymer conjugates

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5162430A (en) * 1988-11-21 1992-11-10 Collagen Corporation Collagen-polymer conjugates

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6773703B1 (en) 1999-01-29 2004-08-10 Beiersdorf Ag Protein-containing hydrogels
WO2001010411A3 (fr) * 1999-08-06 2001-06-21 Max Delbrueck Centrum Depot de principe actif implantable
JP2003506397A (ja) * 1999-08-06 2003-02-18 マックス−デルブルック−セントルム フュア モレキュラー メディツィン 埋め込み可能な有効成分デポ剤
US7556827B1 (en) 1999-08-06 2009-07-07 Max-Delbrück-Centrum-für Molekulare Medizin Implantable active ingredient depot
EP1322422A4 (fr) * 2000-07-18 2004-11-17 Univ California Procede et appareil de preparation de mesophases lipidiques
US7410803B2 (en) 2000-07-18 2008-08-12 The Regents Of The University Of California Method and apparatus for preparing lipidic mesophase material
US7482166B2 (en) 2000-07-18 2009-01-27 The Regents Of The University Of California Method and apparatus for preparing lipidic mesophase material
JP2008523149A (ja) * 2004-12-13 2008-07-03 サウスイースタン メディカル テクノロジーズ 体液を調節する薬剤及びその使用方法
US8535709B2 (en) 2004-12-13 2013-09-17 Southeastern Medical Technologies, Llc Agents for controlling biological fluids and methods of use thereof
WO2007070561A3 (fr) * 2005-12-13 2008-03-20 Southeastern Medical Technolog Agents pour controler des fluides biologiques et leurs procedes d’utilisation
US20090325171A1 (en) * 2008-05-13 2009-12-31 Thomas Hirt Vesicles for use in biosensors
KR101340586B1 (ko) 2011-12-12 2013-12-11 강원대학교산학협력단 광 응답성을 나타내는 큐빅상 조성물 및 그 제조방법

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