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WO1997006817A1 - Haematopoietic recovery - Google Patents

Haematopoietic recovery Download PDF

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Publication number
WO1997006817A1
WO1997006817A1 PCT/GB1996/002006 GB9602006W WO9706817A1 WO 1997006817 A1 WO1997006817 A1 WO 1997006817A1 GB 9602006 W GB9602006 W GB 9602006W WO 9706817 A1 WO9706817 A1 WO 9706817A1
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WO
WIPO (PCT)
Prior art keywords
stem cell
recovery
haematopoietic
cfu
myelosuppressive
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PCT/GB1996/002006
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French (fr)
Inventor
Brian Iles Lord
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British Biotech Pharmaceuticals Limited
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Priority to AU67501/96A priority Critical patent/AU6750196A/en
Publication of WO1997006817A1 publication Critical patent/WO1997006817A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19Cytokines; Lymphokines; Interferons
    • A61K38/195Chemokines, e.g. RANTES
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19Cytokines; Lymphokines; Interferons
    • A61K38/20Interleukins [IL]
    • A61K38/2053IL-8

Definitions

  • This invention relates to the use of proteinaceous molecules which are stem cell chemokines (SCCs), e.g. LD78, to regulate the recovery of haematopoiesis after myelosuppressive insult or therapy.
  • SCCs stem cell chemokines
  • continuous administration of an SCC enhances the rate and quality of the recovery of haematopoiesis following cytotoxic myelosuppressive insult or therapy.
  • haematopoietic stem cells are both pluripotential - that is they can give rise to all cell types - and capable of self-renewal. This is defined by their ability to repopulate animals whose haematopoietic system has been destroyed by radiation. Stem cells represent a very small percentage of bone marrow cells, and are normally quiescent. When stimulated to divide, they give rise to more committed, differentiated daughter cells with greater proliferative potential. The term stem cell is often also applied to these so-called early progenitor cells. Sequential rounds of division and differentiation give rise to an enormous amplification of cell numbers, necessary for the production of mature blood ceils.
  • CSFs Colony Stimulating Factors
  • G-CSF granulocyte-colony stimulating factor
  • EPO erythropoietin
  • Leukocytic haematopoietic cells are important in maintaining the body's defence against disease.
  • macrophages and lymphocytes are involved in potentiating the body's response to infection and tumours; granulocytes (neutrophils, eosinophils and basophils) are involved in overcoming infection, parasites and tumours.
  • Other cell types derived from haematopoietic stem cells include platelets and erythrocytes. Platelets form an important element in the haemostatic mechanism through initiating thrombus formation by their adhesion to each other and to damaged surfaces, and by the release of factors which assist in the formation of the fibrin clot. Erythrocytes are mainly involved in the transport of oxygen.
  • neutrophils along with other granulocytes, are an essential component of the body's cellular defences against infection is illustrated by the fact that individuals with a leukocyte dysfunction such as LAD (leukocyte adhesion deficiency) are very prone to infection.
  • LAD leukocyte adhesion deficiency
  • Neutrophils are continuously produced in large numbers from myeloid precursors in the bone marrow. Neutrophils are released into the circulation from where they can enter the tissues in response to chemotactic signals released locally during infection or tissue damage. The activated neutrophil can then attack the infective agent by release of enzymes and free-radicals, as well as by phagocytosis. Circulating and tissue neutrophils have a short half-life of about 2hr.
  • the neutropaenia resulting from chemotherapy or radiotherapy occurs within days of treatment, and leaves the patient vulnerable to infection until the haematopoietic system has recovered sufficiently for neutrophil counts to recover.
  • CSFs such as G-CSF and GM-CSF to enhance the neutrophil recovery rate by stimulating the division and differentiation of neutrophil precursors.
  • G-CSF G-CSF
  • GM-CSF GM-CSF
  • SCIs stem cell inhibitor proteins
  • a known stem cell inhibitor protein known as MIP-1 ⁇ (macrophage inflammatory protein), or huMIP-1 ⁇ or LD78 (for the human form), is a peptide of about 69 amino acids and is a member of a growing family of molecules with homologous structure - the chemokine or intercrine family. Other notable members of this family include IL-8 and platelet factor 4.
  • MIP-1 ⁇ A clinical use for MIP-1 ⁇ emerged when it was discovered that it was the same molecule as a factor purified from bone marrow some years earlier (3).
  • This factor stem cell inhibitor protein, was defined by its ability to put early haematopoietic progenitor cells (stem cells) out of cycle. Because stem cells are needed for repopulation of the bone marrow, there is a great deal of interest in the use of this protein (also known as LD78) as a marrow protective agent during cancer chemotherapy.
  • LD78 haematopoietic progenitor cells
  • a number of routes for the production of the wild-type molecule as well as engineered variants with improved physico-chemical properties are described in Patent Application WO-A- 93/13206.
  • Macrophage Inflammatory Protein-1 has been recognised as a haemopoietic stem cell proliferation inhibitor by its capacity to protect multipotent progenitor cells from cytotoxic agents which are effective against cells in DNA synthesis, both in vitro and in vivo (1-7).
  • MIP-1 ⁇ was given in vivo to protect haemopoietic spleen colony-forming units (CFU-S) (8) from the effects of hydroxyurea (HU)
  • CFU-S haemopoietic spleen colony-forming units
  • stem cell inhibitor molecules such as MIP-1 ⁇ can be used to protect haematopoietic stem cells from cycle specific cytotoxic agents.
  • the present invention is based on the findings that continuous (as opposed to multiple bolus injections) administration of the SCI starting (a) before commencement of a course of myelosuppressive therapy or (b) during a course of myelosuppressive therapy or (c) immediately after a course of myelosuppressive therapy has ceased, and proceeding after the therapy has finished, has unexpected beneficial effects on the recovery of haematopoiesis.
  • continuous (as opposed to multiple bolus injections) administration of the SCI starting (a) before commencement of a course of myelosuppressive therapy or (b) during a course of myelosuppressive therapy or (c) immediately after a course of myelosuppressive therapy has ceased, and proceeding after the therapy has finished has unexpected beneficial effects on the recovery of haematopoiesis.
  • similar benefits are implied for the use of other SCCs in the same way as SCIs.
  • a method for enhancing haematopoietic recovery in a subject in need thereof which comprises commencing continuous administration of a stem cell chemokine to the subject
  • stem cell chemokine or “SCC” as used throughout this specification refers to any chemokine molecule or analogue or variant thereof to which haematopoietic stem and progenitor cells or their progeny respond. That definition includes any chemokine which has demonstrated biological activity on a haematopoietic stem cell, progenitor cell, maturing lineage specific cell, mature cell of either myelomonocytic or lymphoid lineages including but not limited to monocytes, macrophages, neutrophils, platelets, basophils, eosinophils, dendritic cells etc.
  • Chemokines or their analogues encompassed in this definition possess significant amino acid identity (>20% homology) or structural similarity to all or part of the family of chemotactic cytokines called chemokines, or if produced via recombinant DNA expression, the nucleic acid encoding the chemokine or analogue would hybridise under stringent conditions to nucleic acid encoding a known chemokine, such as LD78, or would do so but for the redundancy of the genetic code.
  • LD78 belongs to a super-family of related chemotactic cytokines (19). which have recently been called Chemokines. All of the chemokines have four conserved cysteines and are grouped into two sub-families according to their chromosomal location and the position of the first two cysteines, which are either adjacent (CC proteins or ⁇ subfamily; Iocated on human chromosome 4) or separated by one amino acid (CXC proteins or ⁇ subfamily; Iocated on human chromosome 17). Both ⁇ and ⁇ subfamilies of the chemokine superfamily are included in this definition.
  • chemokines share amino acid homology (the amino acid sequences of the chemokines can be found in various sequence databases such as EMBL or SwissProt) and have very similar tertiary structures. This means that information obtained for one is likely to be applicable to others. Baggiolini et al. (20) review the various members of the ⁇ and ⁇ subfamilies of the chemokine superfamily.
  • stem cell chemokines include but are not limited to: LD78 (huMIP-1 ⁇ ), muMIP-1 ⁇ , MIP-1 ⁇ (ACT- 2), Rantes, IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ , neutrophil activating protein (NAP-2), monocyte chem-attractant and activating protein (MCAF), epithelial cell-derived neutrophil activating protein (ENA78), platelet factor 4 (PF4), interferon-gamma inducible protein ( ⁇ lP10), granulocyte chemotactic protein 2 (GCP-2), MCP-1 , MCP-2 and MCP -3.
  • SCC molecules are known stem cell inhibitors, including: IL-8, PF4, MIP-1 ⁇ , Rantes, I N PRO L etc.
  • variant (or its synonym for present purposes “analogue”) is used, broadly, in a functional sense. Variants may possess amino acid deletions, substitutions and/or insertions. As a practical matter, though, most variants will have a high degree of homology with the prototype molecule if biological activity is to be substantially preserved. Variants or analogues may have improved biophysical properties and include those proteins capable of being more easily expressed or purified. Variants may also possess less toxicity when administered to the patient. It will be realised that the nature of changes from the prototype molecule is more important than the number of them.
  • nucleic acid coding for an analogue may for example hybridise under stringent conditions (such as at approximately 35°C to 65°C in a salt solution of approximately 0.9 molar) to nucleic acid coding for the prototype molecule, or would do so but for the degeneracy of the genetic code.
  • the stem cell chemokine for use in the invention is huMIP-1 ⁇ , in a more preferred embodiment it is BB-10010 a demultimerised LD78(huMIP-1 ⁇ ) analogue (as described in Example 7 of Patent Application No. WO- A-93/13206).
  • Myelosuppressive (also termed myeloablative) therapy refers to treatments that cause marrow and haematopoietic cells to be destroyed, but does not include their complete annihilation. Such treatments include chemotherapy and radiotherapy. After myelosuppressive therapy, the haematopoietic system is damaged and the levels of circulating mature blood cells, maturing lineage specific cells in haematopoietic tissues and stem cells are reduced.
  • haematopoietic recovery as used herein is meant the process of renormalisation of the haematopoietic system after such damage. The system may be said to have recovered completely when the levels of cells measured have risen to be within the range normally expected for each cell type.
  • CD34+ 1800-16200/ml blood CFU- GM 17-430/ml blood
  • BFU-E 20-190/ml blood white blood cells 3-11 x 10 9 /L
  • red blood cells 3.8 - 6 x 10 12 /L platelets 140 - 400 x 10 L
  • neutrophils 1.7 - 7.5 x 10 9 /L lymphocytes 1 - 3.5 x 10 9 /L
  • procedures for estimating cell numbers are interoperator and interlaboratory specific, with interassay variation often resulting in different estimations of cell numbers.
  • the rate of haematopoietic recovery (which is faster when the method of the invention is used than with the methods of the prior art) can be estimated by use of assays known in the art of murine haematopoiesis. These include the marrow repopulating ability (MRA) assay (also known as the pre-CFU-S assay), the CFU-S assay, nucleated cell counts from femoral cell suspensions or from blood and red cell counts from these tissues. These assays are described in: Haematopoiesis, A Practical Approach, Testa NG, Molineux G (eds):Oxford,New York,Tokyo, IRL Press at Oxford University Press, 1993.
  • MRA marrow repopulating ability
  • assays In humans, not all of these assays are possible. However, a plethora of appropriate assays are known which can be used to estimate the state of haematopoiesis at steady state and after myeloabiative insult or therapy. Any of these methods could be used singly or in combination to estimate the state of haematopoietic recovery.
  • These assays are clonogenic in vitro assays eg. CFU-GM, BFU-E, CFU-GEMM etc, LTCIC and CAFC assays (see ref:18 and 21-23), marrow cellularity and mature cell number counts including neutrophils, platelets or lymphocytes, taken from marrow biopsies or other haematopoietic tissues or from blood.
  • the benefit of the invention is observed if a faster recovery to a certain value (eg. time to neutrophil count >500/mm3) or greater number at a certain time (eg. number of CFU-GM or LTCIC at 10 days post myelosuppressive therapy) of any of the haematopoietic cell types is measured compared to control value without any stem cell chemokine treatment or by treatment using the regimes of the prior art.
  • the benefit of the invention is also observed if no changes in the rate or extent of recovery were seen but if individuals which received stem cell chemokine treatment in accordance with the invention are better able to withstand subsequent rounds of myelosuppressive insult or therapy. This is seen as faster or greater recovery in subsequent rounds of therapy, or by a reduction in the level of toxicity in subsequent rounds of repeated cycles of chemotherapy.
  • Faster mature cell recovery may be an indication of the ability to withstand subsequent rounds of chemotherapy or radiotherapy, however it should not be the sole guide.
  • the state of the marrow is a far better guide. Greater numbers of early progenitors or stem cells gives a better indication of the "quality" of the marrow recovery. If these levels approach the numbers found in normal individuals then the marrow is more fully recovered and is more likely to be able to sustain haematopoiesis after subsequent rounds of therapy.
  • This invention calls for continuous stem cell chemokine treatment over a prolonged period. This means that the prolonged nature of its use may indeed inhibit the recovery of the mature cells whilst improving the quality of the marrow stem and progenitor cell recovery.
  • stem cell effects may occur at the expense of mature cell recovery.
  • the positive effects of prolonged continuous administration of the stem cell chemokine on stem cell recovery may be at the price of slower mature cell recovery, and this may therefore require therapeutic intervention with growth factors or by transfusions of neutrophils, platelets or blood to sustain the patient through the nadir of myelosuppression.
  • the beneficial effects of the stem cell chemokine may become apparent in subsequent rounds of myelosuppressive therapy when the greater number of stem cells will allow the marrow to withstand the myelosuppression even better.
  • the clinician may choose to balance the optimum effects of the stem cell chemokine on recovery with the potential prolonged cytopenia.
  • the ability to undergo more rounds of myelosuppressive therapy is of great clinical significance. Often patients do not recover from the first few rounds of therapy and cannot proceed to the later rounds in a multiple cycle regimen. This reduces the rate of remission and the immunocompromised patients are more susceptible to opportunistic infection. It would be much better if a greater proportion of patients could continue all of the planned rounds of therapy and if restoration of the immune system was speeded up.
  • the use of this invention increases the ability of patients to withstand additionai rounds of therapy and/or higher dosages of the myelosuppressive insult than currently possible.
  • the invention is applicable following cycle specific and non-cycle specific chemotherapies and radiotherapy.
  • the invention provides for enhanced haematopoietic recovery after myeloabiative therapy by prolonged continuous administration of a stem cell chemokine
  • a pharmaceutical composition for continuous administration comprising a stem cell chemokine.
  • continuous administration means a mode of administration whereby the agent is administered to the subject such that the agent is maintained in the body, preferably the blood, at substantially the same concentration over time, as opposed to administrations that result in a peak in the concentration of the agent followed by a gradual or rapid decrease in concentration, eg. following bolus intra venous injection.
  • Such administration may comprise continuous infusion.
  • continuous does not imply that uninterrupted administration, or administration for 24 hours per day, is essential.
  • Continuous administration of the stem cell chemokine is generally via a device which ensures delivery for a substantial proportion of each day of treatment.
  • the continuous administration should be for more than 4hr, 8hr, 12hr, 16hr, 20hr or 23hr.
  • the SCC is administered continuously throughout each day without interruption.
  • delivery devices may need to be replenished and that there may be periods when administration is discontinuous as described above.
  • patients may require other therapies during the course of their treatment which may be related to the use of stem cell chemokines, the patients underlying disease state or for entirely different reasons which are incompatible with stem cell chemokine administration.
  • the stem cell chemokine administration could be suspended until it was appropriate to resume administration.
  • Continuous administration can be via i.v., i.p., i.m. or s.c. routes. Other routes such as transdermal may also be possible.
  • the ablation of the haematopoietic system caused by myelosuppressive therapies is not instantaneous but takes several days for the nadir of cytopenia to develop.
  • Cytotoxic chemotherapeutic myelosuppressive agents once injected have a clearance ⁇ -half-life from the body which is agent specific eg. 5-FU approx 10min, AraC 2hrs, Cyclophosphamide 10hrs.
  • agent specific eg. 5-FU approx 10min, AraC 2hrs, Cyclophosphamide 10hrs.
  • the clinician may decide to begin administration for a period of up to 7 days before the myelosuppressive treatment is initiated.
  • stem cell chemokine or stem cell inhibitor
  • the stem cell chemokine (or stem cell inhibitor) treatment would normally be continued throughout the duration of the myelosuppressive treatment.
  • the myeloabiative treatment would be completed within 24hrs although longer treatment regimens would be considered.
  • the clinician would then seek to continue the protection with stem cell chemokine until the body had cleared sufficient of the cytotoxic chemotherapeutic myelosuppressive agent to render any remaining agent ineffective. A clinician may decide that 50%, 25%, 12.5% or 6.25% of remain drug might be an ineffective dose.
  • the stem cell chemokine should not be administered throughout the duration of the multiple cycles of chemotherapy. There should preferably be a break in treatment with stem cell chemokine before the next cycle of myelosuppressive treatment begins. This break should preferably be >25% and not less than 10% of the cycle duration.
  • the stem ceil chemokine may be administered for an undefined period of time before myelosuppressive treatment begins, throughout the day of the treatment (day 0)and preferably for at least 8 ⁇ half-lives of the myelosuppressive agent after administration of the agent ceases. If a cocktail of myelosuppressive agents has been used then the longest ⁇ half-life should be used in the calculation. For AraC this period would be 64hr after AraC treatment ceased or through days 1 and 2 and part way through day 3 of the clinical protocol. If beneficial the duration of administration of stem cell chemokine could be even longer. The maximum duration of administration is for 90% of cycle time but preferably 75% or less of cycle time.
  • this invention calls for continuous administration of stem cell chemokine for a minimum of days 0, 1 and 2 and part of day 3 of the experimental protocol and not longer than from day 0 through to day 26 and preferably no longer than day 22 of the clinical protocol.
  • Clinical regimens which administer a stem cell chemokine for a shorter period that 8 ⁇ half-lives of the myelosuppressive agent after the agent has ceased to be administered are not the subject of this invention.
  • stem cell chemokine dosage regimens seek only to use the stem cell inhibitory and therefore protective effects of some stem cell chemokines such as LD78 and do not attempt to make use of the utility of prolonged use of stem cell chemokines on self-renewal or regeneration of the haematopoiesis system.
  • the invention aiso includes the use of a stem cell chemokine in the preparation of a medicament for use in continuous administration for:
  • a method for enhancing haematopoietic recovery in a subject having undergone, or undergoing myelosuppressive therapy comprises the continuous administration of an effective amount of a stem cell chemokine.
  • An element to haematopoietic recovery arising from the effects of continuous administration of a SCC is the enhanced mobilisation of haematopoietic cells to the blood.
  • stem cell chemokine for use in continuous administration is LD78 or MIP-1 ⁇ or analogues thereof.
  • the de-multimerised LD78 analogue BB-10010 (as described in Patent Application No. WO-A-93/13206) is used in continuous administration.
  • Molecules useful in the present invention can be prepared from natural or recombinant sources. Methods for expression and purification of stem cell chemokines by recombinant techniques are known in the art.
  • the preferred form of the natural MIP-1 ⁇ molecule is a 69 amino acid form of LD78 described by Obaru e al. (25).
  • the active ingredient may be administered parenterally in a sterile medium.
  • the drug can either be suspended or dissolved in the vehicle.
  • the stem cell chemokine will be administered in the form of a sterile composition comprising the purified protein in conjunction with physiologically acceptable carriers, excipients or diluent.
  • adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
  • suitable pharmacological compositions is routine to those skilled in the art, as exemplified by "Remington's Pharmaceutical Sciences” 15th Edition, incorporated herein by reference.
  • Dosage of stem cell chemokines in accordance with any aspect of the invention will be such as to be effective and will be under the control of the physician or clinician considering various factors such as the condition, sex, body weight and diet of the patient and the severity of the myelosuppressive treatment administered.
  • doses may be in the range of from 0.1 ⁇ g/kg/day and 10mg/kg/day, preferably a dose between 1 ⁇ g/kg/day and 1 mg/kg/day more preferably from 10 ⁇ g/kg/day to 0.2 mg/kg/day.
  • the most effective dose of stem cell chemokine given throughout the treatment can be determined by clinicians following normal clinical research practice in dose-response studies.
  • the optimal dose may be constant throughout the duration of dosing or may change. For instance a high dose initial induction might be appropriate.
  • the invention will be described in the following non-limiting examples and by the following figures in which:
  • Figures 1 and 2 diagrammatically represents the data of Tables 3 and 4 of eight and twelve day CFU-S in the bone marrow of mice subjected to repeated 14 d cycles of 4.5 Gy y -rays.
  • the graphs refer to mini-osmotic pump dispensing BB-10010 (•) or PBS (O).
  • Figures 3 and 4 diagrammatically represents the data of Tables 3 and 4 of eight and twelve day CFU-S in the spleen of mice subjected to repeated 14 d cycles of 4.5 Gy y -rays.
  • the graphs refer to mini-osmotic pump dispensing BB-10010 (•) or PBS (O).
  • Figures 5 and 6 diagrammatically represents the data of Tables 3 and 4 of the 1 day post irradiation nadirs in bone marrow CFU-S following sequential 2- weekly doses of 4.5 Gy ⁇ -rays.
  • O Irradiation only;
  • Figures 7 and 8 diagrammatically represents the data Tables 3 and 4 of the 14 day post irradiation recovery values for bone marrow CFU-S following sequential 2-weekly doses of 4.5 Gy ⁇ -rays.
  • mice Male B6D2F1 (C57BI? x DBA2 ) mice aged 10 wks at the start of experiments were used throughout and all procedures were carried out under licence from the Home Office, Animals (Scientific Procedures) Act, 1986. BB-10010/MIP-1 ⁇
  • BB-10010 is a non-aggregating, genetically engineered variant of human MIP-1 ⁇ (or LD78) comprising a single amino acid substitution of Asp26>Ala.
  • the construction of BB-10010 is described in example 7 in WO-A-9313206.
  • mice (groups of 20) were irradiated with 15.25 Gy 60Co ⁇ -rays (0.95 Gy/hr). They were then injected intravenously with 0.2 ml of a freshly prepared suspension of bone marrow or spleen cells from mice treated as described below. Eight days (10 mice) and 12 days (10 mice) later the recipient mice were killed. Their spleens were excised, fixed and the colonies counted.
  • MRA Marrow Repopulating Ability Assays
  • the MRA was measured as the generation of 12 d CFU-S during 13 days' growth in the marrow by an extension of the CFU-S assay (see ref. 13 for details), on day 14 of the 3rd and 4th treatment cycles.
  • An extra 5 irradiated (primary) recipients were injected with the bone marrow suspension. After 13 days their femora were removed. Bone marrow suspensions were made and assayed for CFU-S12 in secondary groups of 10 irradiated recipients.
  • MRA was calculated as (c x p x q)/N per femur or c x p x q x IO 5 per IO 5 cells:
  • 1/q fraction of donor marrow cells injected into primary recipient
  • 1/p fraction of primary recipient marrow injected into secondary recipient.
  • mice Groups of 3 mice were implanted subcutaneously with mini-osmotic pumps delivering BB-10010 (40 ⁇ g/24 hr period for 7d) or Phosphate Buffered Saline (PBS). Three to four hours later the mice were exposed to 4.5 Gy ⁇ -rays from a caesium-137 source (dose rate 2.5 Gy/min) and after 7 days the spent pumps were removed. Groups of mice were killed at 1 , 7 and 14 days after irradiation and their femora and spleens removed. Cell suspensions were made in Fischer's medium from the bone marrow and spleen (see ref.
  • BM bone marrow
  • Iabje_2 Cellularity of Bone Marrow and Spleen in Mice Subjected to Repeated Cycles of Sub-Lethal Irradiation. With or Without
  • mice were exposed to 4.5 Gy ⁇ -rays on day 0 and at 14 day intervals thereafter. Data are for 3 to 4 experiments ⁇ standard error. Data for day 0 are standardized norms for these mice and are presented simply as approximate reference points.
  • T able 3 8 d_CFU ⁇ S in Bone Marrow and Spleen of Mice Subjected to Repeated Cycles of Sub-Lethal lnadia.tjojL.Wjth gr .Without BB-10010.
  • mice were exposed to 4 5 Gy ⁇ -rays on day 0 and at 14 day intervals thereafter. Data are for 3 to 4 experiments 1 standard error. Data for day 0 are standardized norms for these mice and are presented simply as approximate reference points.
  • mice were exposed to 4.5 Gy ⁇ -rays on day 0 and at 14 day intervals thereafter. Data are for 3 to 4 experiments 1 standard error. Data for day 0 are standardized norms for these mice and are presented simply as approximate reference points.
  • e 3_ M A of Progenitor Cells in Bone Marrow of Mice Subjected to Repeated Cycles of Sublethal Irradiation, With or Without
  • Marrow repopulating ability is normally recorded for these mice at about 10 s per femur or 500 per 10 5 marrow cells (14).
  • Fourteen day recovery-marrow in the 3rd and 4th cycles of sublethal irradiation yielded only 6500-7300 per femur which is less than 10% of normal (Table 5).
  • Treatment with BB-10010 yielded an MRA of 26600 after the third cycle and 11206 after the fourth cycle of irradiation (Table 5).
  • the cellular concentration of MRA cells was increase by 1.3-3 times following continuous BB-10010 administration treatment.
  • BB-10010 gave little, protection from the first dose of irradiation but the better recovery characteristics, particularly in respect of the highly enriched MRA, ensured that the second and subsequent irradiations caused progressively less initial damage and that the recovery patterns did not significantly deteriorate.
  • Figures 1 to 4, 7 and 8 show that CFU-S recoveries are generally much better with BB-10010 treatment.
  • Example 1 shows the ability of BB-10010 to improve haematopoietic recovery when it is continuously administered via minipump for 7 days. This example shows that a shorter period of continuous administration is much less effective in inducing this recovery.
  • Prolonged continuous administration of BB--10010 is more efficacious than multiple injections.
  • Example 1 and comparative example 1 show that prolonged continuous administration of BB-10010 for 7 days significantly improves haematopoietic recovery through multiple cycles of myelosuppressive therapy and that it is advisable to administer the BB-10010 for more than 3 days in this model system.
  • Comparative example 2 illustrates the value of continuous minipump administration of BB-10010 relative to repeated injections over the same period.
  • the methods were as in example 1 and comparative example 1 , using 2 cycles of radiation treatment together with BB-10010 treatment over the first 7 days of each cycle.
  • BB-10010 was administered by (a) 7 day minipump (40 ⁇ g/d), (b) by daily subcutaneous injections of 40 ⁇ g or (c) by twice daily injections of 20 ⁇ g.
  • the pumps were implanted 3 to 4hr and the injections started 2 hrs before irradiation.
  • CFU-S were assayed after 14 days recovery in the second treatment cycle. Table 7 show that only continuous administration by pump provided significant enhancement in CFU-S recovery and illustrates the advantage of this form of administration relative to a repeated bolus injection regimen.
  • BB-10010 administration was started 3 to 4 hours prior to the radiation treatment. It is possible that BB-10010 protected stem cells from the myelosuppressive therapy as well as, or instead of, enhancing their recovery. Examples 1 and comparative example 1 provide evidence which argues that much of the haematopoietic enhancing effect of prolonged continuous BB-10010 administration may occur after several days of BB-10010 administration post-irradiation.
  • the 3 and 7 day minipump experiments had the same pre-irradiation dosing of BB-10010 so their direct protective effects should be identical; but the 7 day minipumps were considerably more effective than the 3 day minipumps. This suggests that the additional 4 days of administration are important to the ultimate enhancing effects of BB-10010.
  • this example describes the activity of BB-10010 dosed continuously for 7 days via minipumps inserted after the cessation of radiation. In this study there can be no direct protective contribution of BB-10010.
  • Minipumps implanted before irradiation (0-7d) generated good enhancement of recovery giving twice as many CFU-S/f. Pumps fitted 1 day after irradiation showed less efficacy (-1.4 fold enhancement) while those present in the second phase of the recovery cycles were ineffective.
  • BB-10010 can protect the progenitor CFU- S population from a generalised myelosuppressive agent (eg radiation) while its subsequent, but not delayed, continuous availability is separately instrumental in promoting the recovery rate of the surviving CFU-S.
  • a generalised myelosuppressive agent eg radiation
  • BB-10010 Continuous administration of BB-10010 enhances the peripheral blood mobilisation of progenitor cells from the bone marrow.
  • BB-10010 can enhance the efficacy of progenitor cell mobilisation from marrow to peripheral blood induced by G-CSF (24).
  • Prolonged continuous administration of BB-10010 improves the efficacy of progenitor cell mobilisation bv G-CSF.
  • Hendry JH, Lajtha LG The response of hemopoietic colony-forming units to repeated doses of X-rays. Radiat Res 52: 309, 1972.
  • Ploemacher RE, van Os RP, van Buerden CAJ, Down JD Murine hemopoietic stem cells with long-term engraftment and marrow repopulating ability are less radiosensitive to gamma irradiation than are spleen colony forming cells. Int J Radiat Biol 61 : 489, 1992.

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Abstract

Enhanced haematopoietic recovery following myelosuppressive therapy by continuous administration of a stem cell chemokine.

Description

Haematopoietic recovery
Field of the Invention.
This invention relates to the use of proteinaceous molecules which are stem cell chemokines (SCCs), e.g. LD78, to regulate the recovery of haematopoiesis after myelosuppressive insult or therapy. According to the invention continuous administration of an SCC enhances the rate and quality of the recovery of haematopoiesis following cytotoxic myelosuppressive insult or therapy.
Background to the Invention
The various mature blood cell types are all ultimately derived from a single class of progenitor cell known as haematopoietic stem cells. True stem cells are both pluripotential - that is they can give rise to all cell types - and capable of self-renewal. This is defined by their ability to repopulate animals whose haematopoietic system has been destroyed by radiation. Stem cells represent a very small percentage of bone marrow cells, and are normally quiescent. When stimulated to divide, they give rise to more committed, differentiated daughter cells with greater proliferative potential. The term stem cell is often also applied to these so-called early progenitor cells. Sequential rounds of division and differentiation give rise to an enormous amplification of cell numbers, necessary for the production of mature blood ceils. This process of division and differentiation is subject to regulation at many levels to control cell production. Positive factors such as the Colony Stimulating Factors (CSFs) act to promote division of early progenitors and differentiation down particular lineages, for example granulocyte-colony stimulating factor (G-CSF) drives neutrophil production whilst erythropoietin (EPO) promotes formation of erythrocytes.
Leukocytic haematopoietic cells are important in maintaining the body's defence against disease. For example, macrophages and lymphocytes are involved in potentiating the body's response to infection and tumours; granulocytes (neutrophils, eosinophils and basophils) are involved in overcoming infection, parasites and tumours. Other cell types derived from haematopoietic stem cells include platelets and erythrocytes. Platelets form an important element in the haemostatic mechanism through initiating thrombus formation by their adhesion to each other and to damaged surfaces, and by the release of factors which assist in the formation of the fibrin clot. Erythrocytes are mainly involved in the transport of oxygen.
That neutrophils, along with other granulocytes, are an essential component of the body's cellular defences against infection is illustrated by the fact that individuals with a leukocyte dysfunction such as LAD (leukocyte adhesion deficiency) are very prone to infection. Neutrophils are continuously produced in large numbers from myeloid precursors in the bone marrow. Neutrophils are released into the circulation from where they can enter the tissues in response to chemotactic signals released locally during infection or tissue damage. The activated neutrophil can then attack the infective agent by release of enzymes and free-radicals, as well as by phagocytosis. Circulating and tissue neutrophils have a short half-life of about 2hr.
An important consequence of this high turnover rate is that neutrophil numbers drop very rapidly when the bone marrow is damaged. This can occur during some viral infections, but clinically the most important cause is chemotherapy or radiotherapy used to treat malignant disease. Such treatments destroy dividing cells within the tumour, but also devastate other highly proliferative cell populations such as the bone marrow and gut epithelial cells. The bone marrow toxicity kills haematopoietic precursors indiscriminately, but the major impact on mature cell numbers is seen with neutrophils and to a lesser extent platelets because of the short half-life of these cells. The neutropaenia resulting from chemotherapy or radiotherapy occurs within days of treatment, and leaves the patient vulnerable to infection until the haematopoietic system has recovered sufficiently for neutrophil counts to recover.
One known way to minimise the impact of this neutropaenia has been to use CSFs such as G-CSF and GM-CSF to enhance the neutrophil recovery rate by stimulating the division and differentiation of neutrophil precursors. Such an approach can shorten the period of neutropenia but not abolish it. *»
An alternative and complementary approach is to use negative regulators of haematopoiesis such as stem cell inhibitor proteins ("SCIs") to protect early progenitors by causing them to go out of cell-cycle during the period of exposure to the cytotoxic agent. Such cells, when held out of cycle, are more resistant to the toxic effects of chemotherapy.
A known stem cell inhibitor protein, known as MIP-1α (macrophage inflammatory protein), or huMIP-1α or LD78 (for the human form), is a peptide of about 69 amino acids and is a member of a growing family of molecules with homologous structure - the chemokine or intercrine family. Other notable members of this family include IL-8 and platelet factor 4.
A clinical use for MIP-1α emerged when it was discovered that it was the same molecule as a factor purified from bone marrow some years earlier (3). This factor, stem cell inhibitor protein, was defined by its ability to put early haematopoietic progenitor cells (stem cells) out of cycle. Because stem cells are needed for repopulation of the bone marrow, there is a great deal of interest in the use of this protein (also known as LD78) as a marrow protective agent during cancer chemotherapy. A number of routes for the production of the wild-type molecule as well as engineered variants with improved physico-chemical properties are described in Patent Application WO-A- 93/13206.
Macrophage Inflammatory Protein-1 (MIP-1α) has been recognised as a haemopoietic stem cell proliferation inhibitor by its capacity to protect multipotent progenitor cells from cytotoxic agents which are effective against cells in DNA synthesis, both in vitro and in vivo (1-7). In one series of experiments in which MIP-1α was given in vivo to protect haemopoietic spleen colony-forming units (CFU-S) (8) from the effects of hydroxyurea (HU), the subsequent recovery rate of the (partially) protected population appeared to be higher than in the control (HU treated) population (5). This was complementary to earlier observations that the partially purified inhibitor enhanced the generation of haemopoietic ceils in long-term bone marrow cultures (9). Furthermore, it led to the suggestion that, in addition to its capacity to block the progression of progenitor cells into DNA-synthesis, MIP-1α might also have effects on the self-renewal and differentiation capacity of the surviving muitipotent progenitor cell population (5). This enhanced rate of recovery of the CFU-S population has recently been confirmed and direct measurements showed the self-renewal capacity of the CFU-S population surviving HU treatment to be enhanced by MIP-1α given in vivo (10). This switch from differentiation potential, in favour of self-renewal potential may not necessarily adversely affect the production of differentiated cells. Indeed, the increased reserves of "stem" cells can provide a pool that is more than adequate to compensate the reduced differentiation rate.
The art therefore teaches that stem cell inhibitor molecules such as MIP-1α can be used to protect haematopoietic stem cells from cycle specific cytotoxic agents.
It is therefore known that in animal models of cytotoxic cancer therapy, administration of MIP-1α/LD78 or a variant thereof which is engineered to be less prone to multimerisation can protect a proportion of haematopoietic cells from the destructive effects of such therapy by blocking cell cycling, and therefore accelerate the recovery of haematopoiesis after cessation of the therapy. In the published studies, the SCC (i.e. MIP-1α/LD78 or their variants) was administered in bolus doses by single or multiple injections, shortly before the start of cytotoxic therapy to ensure that the protective effect has time to develop, or in a few studies before and after the cytotoxic therapy was started.
It is also known from animal models, that molecules such as MIP-1α/LD78, IL-8 etc. can cause a rapid release (mobilisation) of haematopoietic stem, progenitor and mature cells into the blood stream from whence they can be purified and used for example in peripheral blood stem cell transplantation treatments (PBSCT). This mobilisation phenomenon merely reflects the movement of the haematopoietic cells into the circulating blood pool and does not represent an increase in the total number of haematopoietic cells. The art also teaches that MIP-1 α/LD78 or their variants, may act to enhance the rate of recovery following progenitor cell depletion. Brief description of the invention.
In contrast to the published use of SCIs summarised above, the present invention is based on the findings that continuous (as opposed to multiple bolus injections) administration of the SCI starting (a) before commencement of a course of myelosuppressive therapy or (b) during a course of myelosuppressive therapy or (c) immediately after a course of myelosuppressive therapy has ceased, and proceeding after the therapy has finished, has unexpected beneficial effects on the recovery of haematopoiesis. In addition, based on the known properties of the other members of the SCC family, similar benefits are implied for the use of other SCCs in the same way as SCIs.
Detailed description of the invention.
According to the invention there is provided a method for enhancing haematopoietic recovery in a subject in need thereof, which comprises commencing continuous administration of a stem cell chemokine to the subject
(a) before commencement of a course of myelosuppressive therapy;
(b) during a course of myelosuppressive therapy;
(c) immediately after a course of myelosuppressive therapy has ceased; and continuing administration until in cases (a) and (b) at least the myelosuppressive therapy has ceased, and in all cases (a), (b) and (c) until at least a desired degree of haematopoietic recovery has taken place.
The term "stem cell chemokine" or "SCC" as used throughout this specification refers to any chemokine molecule or analogue or variant thereof to which haematopoietic stem and progenitor cells or their progeny respond. That definition includes any chemokine which has demonstrated biological activity on a haematopoietic stem cell, progenitor cell, maturing lineage specific cell, mature cell of either myelomonocytic or lymphoid lineages including but not limited to monocytes, macrophages, neutrophils, platelets, basophils, eosinophils, dendritic cells etc. Chemokines or their analogues encompassed in this definition possess significant amino acid identity (>20% homology) or structural similarity to all or part of the family of chemotactic cytokines called chemokines, or if produced via recombinant DNA expression, the nucleic acid encoding the chemokine or analogue would hybridise under stringent conditions to nucleic acid encoding a known chemokine, such as LD78, or would do so but for the redundancy of the genetic code.
It is recognised in the art that LD78 belongs to a super-family of related chemotactic cytokines (19). which have recently been called Chemokines. All of the chemokines have four conserved cysteines and are grouped into two sub-families according to their chromosomal location and the position of the first two cysteines, which are either adjacent (CC proteins or β subfamily; Iocated on human chromosome 4) or separated by one amino acid (CXC proteins or α subfamily; Iocated on human chromosome 17). Both α and β subfamilies of the chemokine superfamily are included in this definition. These chemokines share amino acid homology (the amino acid sequences of the chemokines can be found in various sequence databases such as EMBL or SwissProt) and have very similar tertiary structures. This means that information obtained for one is likely to be applicable to others. Baggiolini et al. (20) review the various members of the α and β subfamilies of the chemokine superfamily. Examples of such stem cell chemokines include but are not limited to: LD78 (huMIP-1α), muMIP-1α , MIP-1β(ACT- 2), Rantes, IL-8, GROα, GROβ, GROγ, neutrophil activating protein (NAP-2), monocyte chem-attractant and activating protein (MCAF), epithelial cell-derived neutrophil activating protein (ENA78), platelet factor 4 (PF4), interferon-gamma inducible protein (γlP10), granulocyte chemotactic protein 2 (GCP-2), MCP-1 , MCP-2 and MCP -3. A number of these SCC molecules are known stem cell inhibitors, including: IL-8, PF4, MIP-1α, Rantes, I N PRO L etc.
In addition to MIP-1α/LD78, Broxmeyer et al. (18) demonstrate that the following chemokines : MIP-2α, PF4, IL-8 and MCAF, also have similar myelosuppressive effects.
The term "variant" (or its synonym for present purposes "analogue") is used, broadly, in a functional sense. Variants may possess amino acid deletions, substitutions and/or insertions. As a practical matter, though, most variants will have a high degree of homology with the prototype molecule if biological activity is to be substantially preserved. Variants or analogues may have improved biophysical properties and include those proteins capable of being more easily expressed or purified. Variants may also possess less toxicity when administered to the patient. It will be realised that the nature of changes from the prototype molecule is more important than the number of them. As guidance, though, at the amino acid level, and taking MIP-1α as an example, it may be that (in increasing order of preference) at least 20, 30, 40, 50, 60, 65, 67 or 68 of the residues will be the same as the naturally occurring prototype molecule; at the nucleic acid level, nucleic acid coding for an analogue may for example hybridise under stringent conditions (such as at approximately 35°C to 65°C in a salt solution of approximately 0.9 molar) to nucleic acid coding for the prototype molecule, or would do so but for the degeneracy of the genetic code.
In a preferred embodiment of the invention the stem cell chemokine for use in the invention is huMIP-1α, in a more preferred embodiment it is BB-10010 a demultimerised LD78(huMIP-1α) analogue (as described in Example 7 of Patent Application No. WO- A-93/13206).
Myelosuppressive (also termed myeloablative) therapy as used throughout this specification refers to treatments that cause marrow and haematopoietic cells to be destroyed, but does not include their complete annihilation. Such treatments include chemotherapy and radiotherapy. After myelosuppressive therapy, the haematopoietic system is damaged and the levels of circulating mature blood cells, maturing lineage specific cells in haematopoietic tissues and stem cells are reduced. By the term "haematopoietic recovery" as used herein is meant the process of renormalisation of the haematopoietic system after such damage. The system may be said to have recovered completely when the levels of cells measured have risen to be within the range normally expected for each cell type. These levels have been established and are well known for mature blood cells and would be known for those skilled in specialist haematology for the various identifiable progenitors. For instance, estimation of the numbers of each cell type in 20 or even 10 random healthy individuals would provide a range to describe the 'normal' level of each cell type. Complete recovery of the haematopoietic system is not essential for continuing a course of multiple cycle myelosuppressive therapy. The haematopoietic system is deemed to have recovered sufficiently for further rounds of myelosuppressive treatment when the levels of neutrophils, and/or platelets and/or progenitor cells (as measured by CD34+ or CFU- GM assays) have recovered to greater than 25% of the Iower normal range value. The following values for the normal range of the various cell types are by way of example and are in no way designed to limit the invention: CD34+ 1800-16200/ml blood; CFU- GM 17-430/ml blood; BFU-E 20-190/ml blood; white blood cells 3-11 x 109/L; red blood cells 3.8 - 6 x 1012/L; platelets 140 - 400 x 10 L; neutrophils 1.7 - 7.5 x 109/L; lymphocytes 1 - 3.5 x 109/L; monocytes 0.2 - 0.6 x 109/L; eosinophils 0.03 - 0.5 x 109/L; basophils 0.01 - 0.1 x 109/L It is recognised in the art that procedures for estimating cell numbers are interoperator and interlaboratory specific, with interassay variation often resulting in different estimations of cell numbers.
In the murine preclinical models described in the non-limiting examples herein, the rate of haematopoietic recovery (which is faster when the method of the invention is used than with the methods of the prior art) can be estimated by use of assays known in the art of murine haematopoiesis. These include the marrow repopulating ability (MRA) assay (also known as the pre-CFU-S assay), the CFU-S assay, nucleated cell counts from femoral cell suspensions or from blood and red cell counts from these tissues. These assays are described in: Haematopoiesis, A Practical Approach, Testa NG, Molineux G (eds):Oxford,New York,Tokyo, IRL Press at Oxford University Press, 1993.
In humans, not all of these assays are possible. However, a plethora of appropriate assays are known which can be used to estimate the state of haematopoiesis at steady state and after myeloabiative insult or therapy. Any of these methods could be used singly or in combination to estimate the state of haematopoietic recovery. These assays are clonogenic in vitro assays eg. CFU-GM, BFU-E, CFU-GEMM etc, LTCIC and CAFC assays (see ref:18 and 21-23), marrow cellularity and mature cell number counts including neutrophils, platelets or lymphocytes, taken from marrow biopsies or other haematopoietic tissues or from blood. The benefit of the invention is observed if a faster recovery to a certain value (eg. time to neutrophil count >500/mm3) or greater number at a certain time (eg. number of CFU-GM or LTCIC at 10 days post myelosuppressive therapy) of any of the haematopoietic cell types is measured compared to control value without any stem cell chemokine treatment or by treatment using the regimes of the prior art. The benefit of the invention is also observed if no changes in the rate or extent of recovery were seen but if individuals which received stem cell chemokine treatment in accordance with the invention are better able to withstand subsequent rounds of myelosuppressive insult or therapy. This is seen as faster or greater recovery in subsequent rounds of therapy, or by a reduction in the level of toxicity in subsequent rounds of repeated cycles of chemotherapy.
Faster mature cell recovery may be an indication of the ability to withstand subsequent rounds of chemotherapy or radiotherapy, however it should not be the sole guide. The state of the marrow is a far better guide. Greater numbers of early progenitors or stem cells gives a better indication of the "quality" of the marrow recovery. If these levels approach the numbers found in normal individuals then the marrow is more fully recovered and is more likely to be able to sustain haematopoiesis after subsequent rounds of therapy. This invention calls for continuous stem cell chemokine treatment over a prolonged period. This means that the prolonged nature of its use may indeed inhibit the recovery of the mature cells whilst improving the quality of the marrow stem and progenitor cell recovery. One skilled in the art will appreciate that the desirable stem cell effects may occur at the expense of mature cell recovery. Thus it is to be appreciated that the positive effects of prolonged continuous administration of the stem cell chemokine on stem cell recovery may be at the price of slower mature cell recovery, and this may therefore require therapeutic intervention with growth factors or by transfusions of neutrophils, platelets or blood to sustain the patient through the nadir of myelosuppression. The beneficial effects of the stem cell chemokine may become apparent in subsequent rounds of myelosuppressive therapy when the greater number of stem cells will allow the marrow to withstand the myelosuppression even better. In practice it is to be recognised that the clinician may choose to balance the optimum effects of the stem cell chemokine on recovery with the potential prolonged cytopenia. In murine models, repeated sub-lethal doses of irradiation result in a long-term accumulation of damage such that recovery of the CFU-S population is successively more impaired (12). Such damage is equivalent to that frequently encountered in chemotherapy programmes where successive cycles of treatment have to be delayed or terminated after 4 or 5 cycles because of deteriorating marrow recovery. Experimentally, it is known that repeated doses of sub-lethal irradiation aiso result in accumulating damage to the spleen colony forming units (15). It is envisaged that the continuous administration of a stem cell chemokine in accordance with the invention will enhance the recovery of the stem cell population and will allow the subject to receive more cycles of myelosuppressive therapy or even higher dosages of myelosuppressive agent.
The ability to undergo more rounds of myelosuppressive therapy is of great clinical significance. Often patients do not recover from the first few rounds of therapy and cannot proceed to the later rounds in a multiple cycle regimen. This reduces the rate of remission and the immunocompromised patients are more susceptible to opportunistic infection. It would be much better if a greater proportion of patients could continue all of the planned rounds of therapy and if restoration of the immune system was speeded up. The use of this invention increases the ability of patients to withstand additionai rounds of therapy and/or higher dosages of the myelosuppressive insult than currently possible.
The invention is applicable following cycle specific and non-cycle specific chemotherapies and radiotherapy.
The invention provides for enhanced haematopoietic recovery after myeloabiative therapy by prolonged continuous administration of a stem cell chemokine
According to one aspect of the invention there is provided a pharmaceutical composition for continuous administration comprising a stem cell chemokine. As used herein the term "continuous administration" means a mode of administration whereby the agent is administered to the subject such that the agent is maintained in the body, preferably the blood, at substantially the same concentration over time, as opposed to administrations that result in a peak in the concentration of the agent followed by a gradual or rapid decrease in concentration, eg. following bolus intra venous injection. Such administration may comprise continuous infusion. Specifically, for the reasons appearing below "continuous" does not imply that uninterrupted administration, or administration for 24 hours per day, is essential. Continuous administration of the stem cell chemokine is generally via a device which ensures delivery for a substantial proportion of each day of treatment. Such devices are well known in the art. In increasing order of preference, the continuous administration should be for more than 4hr, 8hr, 12hr, 16hr, 20hr or 23hr. Preferably, the SCC is administered continuously throughout each day without interruption. However, it is recognised that delivery devices may need to be replenished and that there may be periods when administration is discontinuous as described above. In addition, patients may require other therapies during the course of their treatment which may be related to the use of stem cell chemokines, the patients underlying disease state or for entirely different reasons which are incompatible with stem cell chemokine administration. At the clinicians discretion the stem cell chemokine administration could be suspended until it was appropriate to resume administration. Continuous administration can be via i.v., i.p., i.m. or s.c. routes. Other routes such as transdermal may also be possible.
Continuous administration of the SSC in accordance with the invention continues in cases (a) and (b) until myelosuppressive therapy has ceased, and in all cases until at least the desired degree of haematopoietic recovery is attained. The reasons for these requirements appear below:
The ablation of the haematopoietic system caused by myelosuppressive therapies is not instantaneous but takes several days for the nadir of cytopenia to develop. Cytotoxic chemotherapeutic myelosuppressive agents once injected have a clearance β-half-life from the body which is agent specific eg. 5-FU approx 10min, AraC 2hrs, Cyclophosphamide 10hrs. Thus it would take a variable amount of time for the clearance of the drug to bring the agent to an ineffective level. Currently, in order to protect the marrow progenitors during myeloabiative treatment the clinician may decide to begin administration for a period of up to 7 days before the myelosuppressive treatment is initiated. This would give time to develop the protective effect of stem cell chemokine administration. More preferably a shorter time might be chosen of 3, 2 or 1 day prior to myelosuppressive treatment. The stem cell chemokine (or stem cell inhibitor) treatment would normally be continued throughout the duration of the myelosuppressive treatment. Preferably the myeloabiative treatment would be completed within 24hrs although longer treatment regimens would be considered. The clinician would then seek to continue the protection with stem cell chemokine until the body had cleared sufficient of the cytotoxic chemotherapeutic myelosuppressive agent to render any remaining agent ineffective. A clinician may decide that 50%, 25%, 12.5% or 6.25% of remain drug might be an ineffective dose. These doses, by way of example oniy, indicate that the duration one must continue treatment is dependent upon which agent is used. If it is assumed for illustration that 6.25% of the initial drug dose is a safe dose to relieve stem cell chemokine protection, then the clinician would continue stem cell chemokine treatment for 4β-half-lives for example, 5-FU for 40min, AraC for 8hr and cyclophosphamide for 40hrs after the myelosuppressive administration ceased.
With radiotherapy, although the treatment is usually performed over a short period of time, most of the cellular damage inflicted by the radiation, such as tumour and normal cell death, occurs during normal biological processes after the radiation dosing, and the damage caused by it, has ceased.
The above calculations provide guidance on what the expected duration of stem cell chemokine administration might be to obtain the myeioprotective effects of a stem cell chemokine such as LD78. This invention relates to the surprising degree of effectiveness that prolonged administration has, particularly on haematopoietic recovery. Thus we would define prolonged administration as significantly longer (and arbitrarily 2x) than these times. At its minimum, "prolonged" has therefore been defined as 2x the period necessary to reach the ineffective dose of the cytotoxic chemotherapeutic myelosuppressive agent based on its β-half-life clearance rate, and fcr radiotherapy, arbitrarily as 48 hours. However, to achieve the beneficial effects of prolonged stem cell chemokine administration it is likely that even longer treatment would be required. More preferably prolonged treatment means 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more of continuous stem cell chemokine administration after the cessation of myelosuppressive treatment.
Preferably the stem cell chemokine should not be administered throughout the duration of the multiple cycles of chemotherapy. There should preferably be a break in treatment with stem cell chemokine before the next cycle of myelosuppressive treatment begins. This break should preferably be >25% and not less than 10% of the cycle duration.
The stem ceil chemokine may be administered for an undefined period of time before myelosuppressive treatment begins, throughout the day of the treatment (day 0)and preferably for at least 8 β half-lives of the myelosuppressive agent after administration of the agent ceases. If a cocktail of myelosuppressive agents has been used then the longest β half-life should be used in the calculation. For AraC this period would be 64hr after AraC treatment ceased or through days 1 and 2 and part way through day 3 of the clinical protocol. If beneficial the duration of administration of stem cell chemokine could be even longer. The maximum duration of administration is for 90% of cycle time but preferably 75% or less of cycle time.
Using AraC as an example, and assuming a 4 week cycle time with AraC being given on day 0, this invention calls for continuous administration of stem cell chemokine for a minimum of days 0, 1 and 2 and part of day 3 of the experimental protocol and not longer than from day 0 through to day 26 and preferably no longer than day 22 of the clinical protocol. Clinical regimens which administer a stem cell chemokine for a shorter period that 8 β half-lives of the myelosuppressive agent after the agent has ceased to be administered are not the subject of this invention. These shorter clinical stem cell chemokine dosage regimens seek only to use the stem cell inhibitory and therefore protective effects of some stem cell chemokines such as LD78 and do not attempt to make use of the utility of prolonged use of stem cell chemokines on self-renewal or regeneration of the haematopoiesis system.
For the reasons discussed above, it will be appreciated that the invention aiso includes the use of a stem cell chemokine in the preparation of a medicament for use in continuous administration for:
(a) enhancing haematopoietic recovery following myelosuppressive therapy; and/or
(b) reducing the normal haematopoietic stem cell regeneration period following myelosuppressive therapy; and/or
(c) enabling the subject to receive more cycles of myelosuppressive therapy, compared to subjects not receiving stem cell chemokine.
According to a further aspect of the invention there is provided for a method for enhancing haematopoietic recovery in a subject having undergone, or undergoing myelosuppressive therapy, which method comprises the continuous administration of an effective amount of a stem cell chemokine.
An element to haematopoietic recovery arising from the effects of continuous administration of a SCC is the enhanced mobilisation of haematopoietic cells to the blood.
Lord et al (24) demonstrate that a single dose of BB-10010 can cause mobilisation of haematopoietic cells. In accordance with this invention the use of minipumps to provide a prolonged administration of stem cell chemokine can provide substantially improved mobilisation results. In preclinical models the prolonged administration is as effective as multiple doses of G-CSF, a cytokine with demonstrated clinical utility in progenitor and stem cell mobilisation regimens.
In a preferred embodiment of the invention ihe stem cell chemokine for use in continuous administration is LD78 or MIP-1α or analogues thereof. In a more preferred embodiment the de-multimerised LD78 analogue BB-10010 (as described in Patent Application No. WO-A-93/13206) is used in continuous administration. Molecules useful in the present invention can be prepared from natural or recombinant sources. Methods for expression and purification of stem cell chemokines by recombinant techniques are known in the art. The preferred form of the natural MIP-1α molecule is a 69 amino acid form of LD78 described by Obaru e al. (25). Given the low amounts of SCI present in natural sources, its production by a recombinant route is greatly preferred. In view of the tendency of SCI to multimerise to form large macromolecular complexes, it is also preferred that an engineered variant of the molecule that does not associate beyond a tetramer be used. Such variants, and their production, are the subject of International Patent Application No. WO-A-93/13206.
The active ingredient may be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Typically, the stem cell chemokine will be administered in the form of a sterile composition comprising the purified protein in conjunction with physiologically acceptable carriers, excipients or diluent. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. The preparation of suitable pharmacological compositions is routine to those skilled in the art, as exemplified by "Remington's Pharmaceutical Sciences" 15th Edition, incorporated herein by reference.
Dosage of stem cell chemokines in accordance with any aspect of the invention will be such as to be effective and will be under the control of the physician or clinician considering various factors such as the condition, sex, body weight and diet of the patient and the severity of the myelosuppressive treatment administered. As general but not exclusive guidance, though, doses may be in the range of from 0.1 μg/kg/day and 10mg/kg/day, preferably a dose between 1 μg/kg/day and 1 mg/kg/day more preferably from 10μg/kg/day to 0.2 mg/kg/day. The most effective dose of stem cell chemokine given throughout the treatment can be determined by clinicians following normal clinical research practice in dose-response studies. The optimal dose may be constant throughout the duration of dosing or may change. For instance a high dose initial induction might be appropriate. The invention will be described in the following non-limiting examples and by the following figures in which:
Figures 1 and 2 diagrammatically represents the data of Tables 3 and 4 of eight and twelve day CFU-S in the bone marrow of mice subjected to repeated 14 d cycles of 4.5 Gy y -rays. The graphs refer to mini-osmotic pump dispensing BB-10010 (•) or PBS (O).
Figures 3 and 4 diagrammatically represents the data of Tables 3 and 4 of eight and twelve day CFU-S in the spleen of mice subjected to repeated 14 d cycles of 4.5 Gy y -rays. The graphs refer to mini-osmotic pump dispensing BB-10010 (•) or PBS (O).
Figures 5 and 6 diagrammatically represents the data of Tables 3 and 4 of the 1 day post irradiation nadirs in bone marrow CFU-S following sequential 2- weekly doses of 4.5 Gy γ-rays. (O) Irradiation only; (•) Irradiation + BB-10010.
Figures 7 and 8 diagrammatically represents the data Tables 3 and 4 of the 14 day post irradiation recovery values for bone marrow CFU-S following sequential 2-weekly doses of 4.5 Gy γ-rays. (O) Irradiation only; (•) Irradiation + BB-10010.
Materials and Methods
The methods used in the following examples are well known to those skilled in the art of murine haematology. A more detailed description of the assays can be found in "Haemopoiesis: A Practicai Approach", IRL Press. Eds. Testa and Molineux, Oxford University Press, Walton Street, Oxford.
Mice
Male B6D2F1 (C57BI? x DBA2 ) mice aged 10 wks at the start of experiments were used throughout and all procedures were carried out under licence from the Home Office, Animals (Scientific Procedures) Act, 1986. BB-10010/MIP-1α
BB-10010 is a non-aggregating, genetically engineered variant of human MIP-1α(or LD78) comprising a single amino acid substitution of Asp26>Ala. The construction of BB-10010 is described in example 7 in WO-A-9313206.
CFU-S Assays
Haemopoietic spleen colony-forming units (CFU-S) were assayed as previously described (13). Briefly, mice (groups of 20) were irradiated with 15.25 Gy 60Co γ-rays (0.95 Gy/hr). They were then injected intravenously with 0.2 ml of a freshly prepared suspension of bone marrow or spleen cells from mice treated as described below. Eight days (10 mice) and 12 days (10 mice) later the recipient mice were killed. Their spleens were excised, fixed and the colonies counted.
Marrow Repopulating Ability Assays (MRA)
The MRA was measured as the generation of 12 d CFU-S during 13 days' growth in the marrow by an extension of the CFU-S assay (see ref. 13 for details), on day 14 of the 3rd and 4th treatment cycles. An extra 5 irradiated (primary) recipients were injected with the bone marrow suspension. After 13 days their femora were removed. Bone marrow suspensions were made and assayed for CFU-S12 in secondary groups of 10 irradiated recipients. MRA was calculated as (c x p x q)/N per femur or c x p x q x IO5 per IO5 cells:
Where N = number of donor marrow cells per femur c = number of CFU-S12 colonies per secondary spleen
1/q = fraction of donor marrow cells injected into primary recipient
1/p = fraction of primary recipient marrow injected into secondary recipient.
Three secondary assays were carried out for each primary recipient bone marrow pool for which p = 10, 20 and 40. In these experiments q = 50 or 100. Example 1
Enhanced haematopoietic recovery following prolonged (7d continuous administration of BB-10010.
Repeated sub-lethal doses of irradiation result in a long-term accumulation of damage such that recovery of the CFU-S population is successively more impaired (12). Such damage is equivalent to that frequently encountered in chemotherapy programmes where successive cycles of treatment have to be delayed or terminated after 4 or 5 cycles because of deteriorating marrow recovery. Experimentally, it is known that repeated doses of sub-lethal irradiation also result in accumulating damage to the spleen colony forming units (15). We chose, therefore, to use irradiation as a convenient model to test the potential of BB-10010 to maintain recovery of the stem cell populations through multiple cycles of myelosuppressive therapy.
The 4 x 4.5 Gy irradiation model described by Hendry et al (15) was adopted though the repeat interval was shortened to 14 days in order to ensure suboptimally recovered haemopoietic tissue.
Groups of 3 mice were implanted subcutaneously with mini-osmotic pumps delivering BB-10010 (40 μg/24 hr period for 7d) or Phosphate Buffered Saline (PBS). Three to four hours later the mice were exposed to 4.5 Gy γ-rays from a caesium-137 source (dose rate 2.5 Gy/min) and after 7 days the spent pumps were removed. Groups of mice were killed at 1 , 7 and 14 days after irradiation and their femora and spleens removed. Cell suspensions were made in Fischer's medium from the bone marrow and spleen (see ref. 13 for details) counted and assayed for day 8 (CFU-S8) and day 12 (CFU-S 12) colony-forming units. The cellular concentrations of the inoculation suspensions were adjusted so that 0.2 ml injected contained the fraction of a femur or spleen indicated in Table 1. These figures are approximate values. Throughout the course of the four experiments reported below, they were continuously refined to optimize the average spleen colony counts, where possible, at about 10. The two week cycle of pump, irradiation and assays was repeated 3 more times in a total observation period of 56 days. On days 42 and 56 (ends of the 3rd and 4th treatment cycles) marrow cells were additionally assayed for MRA (Table 5).
Table 1 : Fractions of One Femur or Spleen Injected for CFU-S Assay
Assay Day' BM Spleen MIP-BM MlP-Spieen
1 1/3 1/6 1/3 1/6
7 1/10 1/10 1/40 1/40
14 1/100 1/100 1/100 1/100
Repeated for each 14 d cycle of treatment and assay. BM = bone marrow
The repeated irradiation treatment regimen was conducted in four separate experiments and the overall results are shown in Tables 2-4 and Figures 1 and 2. For each cycle of treatment, the cellularities of bone marrow and spleen fell to about 20-30% of normal within 24 hrs but recovered to near normal levels within 14 days (Table 2). Bone marrow CFU-S were reduced to less than 1% of their normal levels and in 14 d, CFU- S8 and CFU-S12 recovered to 40 and 20% respectively (see Fig. 1).
Iabje_2: Cellularity of Bone Marrow and Spleen in Mice Subjected to Repeated Cycles of Sub-Lethal Irradiation. With or Without
BB-10010
Tiιne (d) Femur (xϊθ 6) Spleen (x106) Femur (x106) Spleen (xϊθ 6) with BB-10010 with BB-10010
._ __ __ _ _
1 5.1±0.5 32± 6 4.2± 0.4 41± 8
7 15.8±2.0 35± 6 20.0± 2.5 44±11
14 17.0±1.7 115±12 18.1± 2.3 86±18
15 7.2±1.6 39± 5 8.2± 5.6 34± 1
21 23.2 97 20.0 45
28 16 5±2 1 84±12 23.3± 2.7 140± 2 — to
29 4.0±1.4 40±11 4.1± 0.7 35± 6 ° 35 13.4 52 24.5 52
42 16.0±2.4 113±26 18.9±16 113±27
43 4.1±0.3 25± 8 5.0± 0.4 18± 6 49 20.8 60 14 57
56 14.7±2.5 131±25 22.3± 3.1 185±56
Mice were exposed to 4.5 Gy γ-rays on day 0 and at 14 day intervals thereafter. Data are for 3 to 4 experiments ± standard error. Data for day 0 are standardized norms for these mice and are presented simply as approximate reference points.
Figure imgf000022_0001
T able 3: 8 d_CFU^S in Bone Marrow and Spleen of Mice Subjected to Repeated Cycles of Sub-Lethal lnadia.tjojL.Wjth gr .Without BB-10010.
Time (d) Femur Spleen Femur Spleen with BB-10010 with BB-10010
0 4000 1500 4000 1500
1 14± 4 4± 4 15± 5 6± 6 7 149±30 52±27 308± 92 536±264 14 1513±364 681±523 1833±441 630±372
15 17± 2 0 21± 6 4± 3 21 180±10 237± 9 331±33 1080±20 28 345±152 352±152 1135173 13671286
29 6± 2 1± 1 9± 5 4± 1 35 71± 8 61± 6 400±28 880±48 42 318±61 843±286 1147±236 7841211
43 4± 1 1± 1 11± 3 616 49 12717 116± 9 94±13 0
56 355±144 904±248 14471571 18451565
Mice were exposed to 4 5 Gy γ-rays on day 0 and at 14 day intervals thereafter. Data are for 3 to 4 experiments 1 standard error. Data for day 0 are standardized norms for these mice and are presented simply as approximate reference points.
Table 4: 12 d CFU-S in Bone Marrow and Spleen of Mice Subjected to Repeated Cycles of Sub-Lethal Irradiation. With or Without
BB-10010.
Time (d) Femur Spleen Femur Spleen with BB- 10010 with BB-10010
4000 1000 4000 1000
1 201 4 61 4 191 6 1 11 6
7 1341 27 251 6 4051194 6911509 14 7411120 4211248 11171192 4461220
15 171 1 51 2 211 5 91 1 21 801 8 681 8 2671 22 4401 26 28 4281 69 2061 71 10771259 7131 69 t to
29 81 1 41 1 121 3 61 1 35 521 4 451 10 2251 17 2691 31 42 3681163 4121194 9531306 5671217
43 61 2 101 5 121 2 171 5 49 911 1 1021 6 1281 22 350 56 2621 88 5961128 12061401 14731578
Mice were exposed to 4.5 Gy γ-rays on day 0 and at 14 day intervals thereafter. Data are for 3 to 4 experiments 1 standard error. Data for day 0 are standardized norms for these mice and are presented simply as approximate reference points.
Figure imgf000024_0001
Iab|e 3_: M A of Progenitor Cells in Bone Marrow of Mice Subjected to Repeated Cycles of Sublethal Irradiation, With or Without
BB-10010.
Day Bone Marrow Cells Per CFU-S,2 MRA Per Donor Donor Femur Primary Femur Donor Femur (x106) (= c x p) (= c x p q)
0 Normal" 20 12 10^ ±ϊ.1.10"
42 Irradiated 13.5 65.31 3.5 65331 352 (n=3) Irradiated + MIP-1α 17.75 266 114 2660011386
56 Irradiated 15.7510.05 61.01 4.6 72661 688 (n=9) Irradiated + MIP-1α 22.0 13.2 1 14 119 1 120611313
3 secondary assays were carried out for each primary recipient bone marrow pool (taken at 13d after transplant) for which p = 10, 20 and 40. In these experiments q = 50 or 100.
Data for normal mice are taken from reference 14.
Current data are mean values 1 standard error.
Figure imgf000025_0001
Repeated cycles of irradiation increased the damage to CFU-S in the marrow. One day post-irradiation, CFU-S survival levels were successively Iower (Figs.1. 2, 5 and 6) as were the 14 d recovery levels (Figs. 1, 2 and 8) CFU-S8 reaching only 10% and CFU- S12, 6% of their starting levels, after the fourth cycle of treatment. Splenic CFU-S recoveries were maintained better throughout the repeated irradiation cycles but CFU- S8 were recovering to only 50% of their initial value and CFU-S12 oniy to 35% (Figs. 3 and 4).
The one day nadirs in cellularity were unaffected by the presence of a pump delivering BB-10010 though in the bone marrow, the 14 day recovery values were somewhat better than in the controls (Table 2). Intrinsic errors in measuring the very low CFU-S numbers one day after irradiation are unavoidably high. Figures 5 and 6, however, indicate that BB-10010 had little effect in the first cycle, but after the fourth cycle of treatment, about twice as many CFU-S survived. The degree of recovery was maintained with each successive treatment cycle (Figs. 1 , 2, 7 and 8) and at the end of the fourth treatment cycle both CFU-S8 and CFU-S12 were about 35% of normal levels in the BB-10010 treated mice compared to less than 10% without BB-10010.
Marrow repopulating ability is normally recorded for these mice at about 10s per femur or 500 per 105 marrow cells (14). Fourteen day recovery-marrow in the 3rd and 4th cycles of sublethal irradiation yielded only 6500-7300 per femur which is less than 10% of normal (Table 5). Treatment with BB-10010 yielded an MRA of 26600 after the third cycle and 11206 after the fourth cycle of irradiation (Table 5). The cellular concentration of MRA cells was increase by 1.3-3 times following continuous BB-10010 administration treatment.
BB-10010 gave little, protection from the first dose of irradiation but the better recovery characteristics, particularly in respect of the highly enriched MRA, ensured that the second and subsequent irradiations caused progressively less initial damage and that the recovery patterns did not significantly deteriorate. Figures 1 to 4, 7 and 8 show that CFU-S recoveries are generally much better with BB-10010 treatment.
This example demonstrates that prolonged continuous administration of BB-10010 causes a significant improvement to the rate of haematopoietic recovery of the animals in each cycle of myelosuppressive therapy and left the animals more able to withstand subsequent rounds of therapy. This discovery is contrary to the accepted art of stem cell inhibitors. Until this discovery it was generally thought that prolonged administration of a stem cell inhibitor might result in an inhibition of haematopoietic recovery.
Comparative Example 1
Prolonged continuous administration to observe the recovery enhancing effects of BB- 10010.
Example 1 shows the ability of BB-10010 to improve haematopoietic recovery when it is continuously administered via minipump for 7 days. This example shows that a shorter period of continuous administration is much less effective in inducing this recovery.
Methods were as described in example 1 with the exception that CFU-S measurements were made at the end of the second cycle of radiation and recovery only, and that minipumps delivering BB-10010 for 7 days were compared with minipumps delivering BB-10010 for 3 days only. CFU-S8 and CFU-S 12 were measured in 2 experiments and CFU-S 10 in 3 further experiments. Results were comparable in all cases and are represented in Table 6 as average CFU-S per femur.
Two cycles of radiation and recovery resulted in 500 CFU-S per femur at 28 days. Pumps delivering 40μg BB-10010 per day for 3 and 7 days increased this to 800 and 1250 CFU-S/femur respectively.
This example demonstrates the efficacy of longer periods of continuous treatment with BB-10010 during the recovery periods following myelosuppressive treatment. Table 6: Effect of Continuous Infusion of BB-10010 on CFU-S Recovery Following
Radiation
Treatment CFU-S Per Femur
4.5 Gy γ-rays 458 ± 51 plus 7d BB-10010 1250 ± 108 pius 3d BB-10010 799 ± 81
No. Experiments 3
Comparative Example 2
Prolonged continuous administration of BB--10010 is more efficacious than multiple injections.
Example 1 and comparative example 1 show that prolonged continuous administration of BB-10010 for 7 days significantly improves haematopoietic recovery through multiple cycles of myelosuppressive therapy and that it is advisable to administer the BB-10010 for more than 3 days in this model system.
Comparative example 2 illustrates the value of continuous minipump administration of BB-10010 relative to repeated injections over the same period. The methods were as in example 1 and comparative example 1 , using 2 cycles of radiation treatment together with BB-10010 treatment over the first 7 days of each cycle. BB-10010 was administered by (a) 7 day minipump (40μg/d), (b) by daily subcutaneous injections of 40μg or (c) by twice daily injections of 20μg. The pumps were implanted 3 to 4hr and the injections started 2 hrs before irradiation. CFU-S were assayed after 14 days recovery in the second treatment cycle. Table 7 show that only continuous administration by pump provided significant enhancement in CFU-S recovery and illustrates the advantage of this form of administration relative to a repeated bolus injection regimen.
Table 7: Comparison of Continuous Infusion with Repeated Injections of BB-10010 on CFU-S Recovery Following Radiation
Treatment CFU-S Per Femur
4.5 Gy γ-rays 544 ± 58 plus 7d continuous BB-10010 at 40μg/d 1220 ± 77 plus 7 daily 40μg bolus injections of BB- 711 ± 67 10010 plus twice daily bolus injections of 20μg 550 ± 56 BB-10010 for 7 days
Example 2
Prolonged continuous administration of BB-10010 commencing after myelosuppressive therapy enhances haematopoietic recovery.
In the above examples. BB-10010 administration was started 3 to 4 hours prior to the radiation treatment. It is possible that BB-10010 protected stem cells from the myelosuppressive therapy as well as, or instead of, enhancing their recovery. Examples 1 and comparative example 1 provide evidence which argues that much of the haematopoietic enhancing effect of prolonged continuous BB-10010 administration may occur after several days of BB-10010 administration post-irradiation. The 3 and 7 day minipump experiments had the same pre-irradiation dosing of BB-10010 so their direct protective effects should be identical; but the 7 day minipumps were considerably more effective than the 3 day minipumps. This suggests that the additional 4 days of administration are important to the ultimate enhancing effects of BB-10010. To investigate the timing of administration further, this example describes the activity of BB-10010 dosed continuously for 7 days via minipumps inserted after the cessation of radiation. In this study there can be no direct protective contribution of BB-10010.
Again, the 2 cycle radiation treatment was employed and in each 14 day cycle, 7 day minipumps were implanted (a) 3 to 4 hrs prior to irradiation (0-7d), (b) 1 day post irradiation (1-8d) or (c) 7 days post irradiation (7-14d). CFU-S were measured on d14 of the second cycle. Table 8 show the results of two such experiments.
Minipumps implanted before irradiation (0-7d) generated good enhancement of recovery giving twice as many CFU-S/f. Pumps fitted 1 day after irradiation showed less efficacy (-1.4 fold enhancement) while those present in the second phase of the recovery cycles were ineffective.
This example illustrates that the presence of BB-10010 can protect the progenitor CFU- S population from a generalised myelosuppressive agent (eg radiation) while its subsequent, but not delayed, continuous availability is separately instrumental in promoting the recovery rate of the surviving CFU-S.
Table 8: Recovery of CFU-S Following Radiation: Dosing of BB-10010 at Various
Times before and after Radiation
BB-10010 Treatment CFU-S Per Femur
4.5 Gy γ-rays only 730 ± 94 days 0-7 1270 ± 127 days 1-8 960 ± 69 days 7-14 690 ± 64 Example 3.1
Continuous administration of BB-10010 enhances the peripheral blood mobilisation of progenitor cells from the bone marrow.
It has been demonstrated that BB-10010 can enhance the efficacy of progenitor cell mobilisation from marrow to peripheral blood induced by G-CSF (24).
Clinical circumstances sometimes require or utilise a combination of myelosuppressive drugs and G-CSF to ensure adequate mobilisation. In this example it is demonstrated that continuous infusion of BB-10010, through the period of myelosuppressive drug administration, up to and through the period of G-CSF treatments can accelerate the mobilisation process.
Three day minipumps dispensing a continuous dose of 100μg/kg/d BB-10010 (or PBS) were inserted 3 to 4 hours before injecting 200mg/kg Cyclophosphamide (Cy). On day 3 100μg/kg G-CSF (G) were injected and peripheral blood (pb) was harvested on day 4 for progenitor cell number assays.
Without BB-10010 or G-CSF intervention no CFU-Sd8 were observed four days after cyclophosphamide therapy (Table 9). The use of G-CSF caused an increase in blood CFU-Sd8 and the combination of BB-10010 and G-CSF led to the highest yield of CFU-Sd8 four days after cyclophosphamide therapy.
Without BB-10010 or G-CSF intervention white blood cell counts recovered 7 days after cyclophosphamide treatment. The combination of BB-10010 and G-CSF resulted in significant mobilisation of white blood cells four days after cyclophosphamide therapy.
This example demonstrates that continuous infusion of BB-10010 may improve the efficacy of peripheral blood stem cell mobilisation. From a practical (clinical) point of view, such an application should allow reduced treatment periods for mobilisation purposes, minimise consumption of G-CSF and improve yield of stem and mature blood cells.
Example 3.2
Prolonged continuous administration of BB-10010 improves the efficacy of progenitor cell mobilisation bv G-CSF.
As a corollary to example 3.1, extension of the period of BB-10010 infusion from 3 to 7 days allowed examination of the optimal duration of BB-10010 treatment (Table 9).
7 day minipumps dispensing a continuous dose of 100μg/kg/d BB-10010 (or PBS) were inserted 3 to 4 hours before injecting 200mg/kg Cyclophosphamide (Cy). On days 3 to 6, 100μg/kg G-CSF (G) were injected and peripheral blood (pb) was harvested on days 4 to 7 for progenitor and cell number assays.
With the continued infusion of BB-10010 beyond 3 days, more CFU-S were available in blood at 5, 6 and 7 days than were available after 4 days. Thus prolonged administration of the stem cell chemokine analogue BB-10010 was beneficial. The combination of G-CSF and BB-10010 for 4 or 5 days was also beneficial with greater numbers of circulation CFU-S in the groups of animals receiving both agents compared to those with G-CSF alone. This observation was confirmed by GM-CFC assays. On days 4, 5, 6, and 7 of the experiment there were greater numbers of circulating GM-CFC in the BB-10010 treated groups. On days 5 and 6 there was a beneficial effect of the G-CSF and BB-10010 treatment compared to G-CSF alone. Table 9: Mobilisation of Peripheral Blood Stem Cells with Cyclophosphamide and G-CSF is Enhanced by Continuous Administration of BB-10010
Treatment Day 4 Day 5 Day 6 Day 7
3 Day MINIPUMP WBC Counts (x10'6/ml)
Cyclophosphamide 0 1.4 3.7 5.2
+G-CSF 0.8 2.8 19.4 17.7 +G-CSF+BB-10010 1.0 8.4 12.3 12.6
CFU-S8/ml
Cyclophosphamide 0 215 310 490
+G-CSF 40 635 755 930 +G-CSF+BB-10010 441 605 575 890
7 Day MINIPUMP WBC Counts (x10"6/ml)
Cyclophosphamide 2.9 1.4 2.9 7.1
+BB-10010 1.5 2.8 7.1 6.1
+G-CSF 1.3 4.4 25.9 30.4
+G-CSF+BB-10010 3.1 12.2 21.3 26.7
CFU-S8/ml
Cyclophosphamide 0 390 400 410
+BB-10010 373 520 600 580
+G-CSF 383 480 1330 2020
+G-CSF+BB-10010 499 960 1290 1740
GM-CFC/ml
Cyclophosphamide 0 4600 2100 460
+BB-10010 4862 6230 13670 4530
+G-CSF 5683 4100 21700 20660
+G-CSF+BB-10010 4518 8900 27700 17700 References
1. Lord Bl, Mori KJ, Wright EG, Lajtha LG: An inhibitor of stem cell proliferation in normal bone marrow. Br J Haematol. 34: 441 , 1976.
2. Wright EG, Lord Bl: Regulation of CFU-S proliferation by locally produced and endogenous factors. Biomed Exp 27: 215, 1977.
3. Graham GJ, Wright EG, Hewick R, Wolpe SD, Wilkie NM, Donaldson D, Lorimore S, Pragnell IB: Identification and characterisation of an inhibitor of haemopoietic stem cell proliferation. Nature 344: 442-444, 1990.
4. Clements JM, Craig S, Gearing AJH, Hunter MG, Heyworth CM, Dexter TM, Lord Bl: Biological and structural properties of Mip-1 expressed in yeast. Cytokine 4: 76, 1992.
5. Lord Bl, Dexter TM, Clements JM, Hunter MG, Gearing AJH: Macrophage inflammatory protein protects multipotent hematopoietic cells from the myelosuppressive effects of hydroxyurea in vivo. Blood 79: 2605, 1992.
6. Dunlop DJ, Wright EG, Lorimore S, Graham GJ, Holyoake T, Kerr DJ, Wolpe SD, Pragnell IB: Demonstration of stem cell inhibition and myeloproliferative effects of SCI/rh MIP-1 in vivo. Blood 79: 2221 , 1992.
7. Cooper S, Mantel C, Broxmeyer HE: Myelosuppressive effects in vivo with very low dosages of monomeric recombinant murine macrophage inflammatory protein- 1 Exp Hematol 22: 186, 1994.
8. Till JE, McCulloch EA: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14: 213, 1961. JJ
9. Lord Bl, Liu FL, Pojda Z, Spooncer E: Inhibitor of haemopoietic CFU-S proliferation: assays, production sources and regulatory mechanisms, in Najman A, Guigon M, Gorin N-C, Mary J-Y (eds): The Inhibitors of Hematopoiesis, vol 162. London, UK, INSERM/John Libbey Eurotext, p227, 1988.
10. Lord Bl: MIP-1 increases the self-renewal capacity of the hemopoietic spleen- colony-forming cell population in vivo. Submitted for publication.
11. Verfaiilie CM, Catanzarro PM, Li WN: Macrophage-inflammatory protein 1 alpha, interleukin-3 and diffusible marrow stromal factors maintain human hematopoietic stem ceils for at least eight weeks in vitro. J Exp Med 179: 643, 1994.
12. Hendry JH, Lajtha LG: The response of hemopoietic colony-forming units to repeated doses of X-rays. Radiat Res 52: 309, 1972.
13. Lord Bl: In vivo assays for multipotential and marrow repopulating cells, in Testa NG, Molineux G (eds): Haemopoiesis, A Practical Approach. Oxford, New York, Tokyo, IRL Press at Oxford University Press, p1, 1993.
14. Lord Bl, Woolford LB: Proliferation of spleen colony forming units (CFU-S8, CFU- S13) and cells with marrow repopulating ability. Stem Cells 11 : 212, 1993.
15. Hendry JH, Testa NG, Lajtha LG: Effect of repeated doses of X-rays or 14 MeV neutrons on mouse bone marrow. Radiat Res 59: 645, 1974.
16. Meijne EIM, van der Winden-Groenewegen RJM, Ploemacher RE, Vos O, David JAG, Huiscamp R. The effects of X-irradiation on hematopoietic stem cell compartments in the mouse. Exp Hematol 19: 617, 1991.
17. Ploemacher RE, van Os RP, van Buerden CAJ, Down JD: Murine hemopoietic stem cells with long-term engraftment and marrow repopulating ability are less radiosensitive to gamma irradiation than are spleen colony forming cells. Int J Radiat Biol 61 : 489, 1992.
18. Broxmeyer HE, Sherry B, Cooper S, Lu L, Maze R, Beckmanπ MP, Cerami A and Ralph P. Comparative analysis of the human macrophage inflammatory protein family of cytokines (chemokines) on proliferation of human myeloid progenitor cells. Interacting effects involving suppression, synergistic suppression, and blocking of suppression. J. Immunology. Vol. 150(8 pt 1), pp3448-3458, 1993.
19. Covell DG, Smythers GW, Gronenborn AM and Clove GM. Analysis of hydrophobicity in the α and β chemokine families and its relevance to dimerisation. Protein Sci. 3:2604-2072, 1994.
20. Baggiolini M, Dewald B, and Moser B. lnterleukin-8 and related Chemotactic Cytokines-CXC and CC Chemokines. Advances in Immunology vol. 55:97-179,1994.
21. Tsukamoto AS et al. Biological characterisation of stem cell present in mobilized peripheral blood of CML patients. Bone Marrow Transplantation. 14 Suppl. 3:S25-32, 1994.
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24. Lord Bl, Woolford LB, Wood LM, Czaplewski LG, McCourt M, Hunter MG and Edwards RM. Mobilization of early hematopoietic progenitor ceils with BB-10010:A genetically engineered variant of human macrophage inflammatory protein-1α. Blood. Vol. 85(No.12):3412-3415, 1995. 25. Obaru K, Fukuda M, Maeda S and Shumada K. A cDNA clone used to study mRNA inducible in human tonsillar lymphocytes by a tumor promoter. J. Biochem. 99: 885-894, 1986.

Claims

Claims:
1. A method for enhancing haematopoietic recovery in a subject, by commencing continuous administration of a stem cell chemokine
(a) before commencement of a course of myelosuppressive therapy;
(b) during a course of myelosuppressive therapy;
(c) immediately after a course of myelosuppressive therapy has ceased; and continuing administration until in cases (a) and (b) at least the myelosuppressive therapy has ceased, and in all cases (a), (b) and (c) until at least a desired degree of haematopoietic recovery has taken place.
2. A method for enhancing haematopoietic recovery in a subject having undergone, or undergoing myelosuppressive therapy, which method comprises the continuous administration of an effective amount of a stem cell chemokine.
3. The use of a stem cell chemokine in the preparation of a medicament for use in continuous administration for:
(a) enhancing haematopoietic recovery following myelosuppressive therapy; and/or
(b) reducing the normal haematopoietic stem cell regeneration period following myelosuppressive therapy; and/or
(c) enabling the subject to receive more cycles of myelosuppressive therapy, compared to subjects not receiving stem cell chemokine.
4. A pharmaceutical composition for continuous administration comprising a stem cell chemokine.
5. A method or use or composition as claimed in any of claims 1 to 4 wherein the stem cell chemokine is selected from the group consisting of: LD78 (huMIP-1α), muM!P-1α, MIP-1 β(ACT-2), Rantes, IL-8, GROα, GROβ, GROy, neutrophil activating protein (NAP-2), monocyte chemo-attractant and activating protein (MCAF), epithelial cell-derived neutrophil activating protein (ENA78), platelet factor 4 (PF4), interferon-gamma inducible protein (γlP10), granulocyte chemotactic protein 2 (GCP-2), INPROL, MCP-1 , MCP-2 and MCP -3, or their analogues.
6. A method or use or composition as claimed in any of claims 1 to 5 wherein the stem cell chemokine is huMIP-1α (LD78) or an analogue thereof.
7. An LD78 analogue as claimed in claim 6 which is BB-10010.
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EP0866806A4 (en) * 1995-10-24 2002-01-09 Smithkline Beecham Corp Method of mobilizing hematopoietic stem cells
EP0848012A1 (en) * 1996-12-13 1998-06-17 Roche Diagnostics GmbH Use of polypeptides for treating thrombocytopenias
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