WO1998007442A1 - Preparation a liberation lente renfermant de l'erythropoietine et du polylactide coglycolide - Google Patents

Preparation a liberation lente renfermant de l'erythropoietine et du polylactide coglycolide Download PDF

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WO1998007442A1
WO1998007442A1 PCT/EP1997/004491 EP9704491W WO9807442A1 WO 1998007442 A1 WO1998007442 A1 WO 1998007442A1 EP 9704491 W EP9704491 W EP 9704491W WO 9807442 A1 WO9807442 A1 WO 9807442A1
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epo
preparation according
microspheres
preparation
plg
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PCT/EP1997/004491
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WO1998007442A9 (fr
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Francisco Baralle
Sergio TISMINETSKY
Eduardo Scodeller
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International Centre For Genetic Engineering And Biotechnology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]

Definitions

  • the present invention relates to a sustained-release preparation for the delivery of erythropoietin (EPO) to mammalian subjects, typically humans.
  • EPO erythropoietin
  • EPO stimulates the proliferation and differentiation of erythroid cells, leading to an increase in the number of re iculocytes in the blood, which consequently increases the blood's haematocrit and haemoglobin concentration.
  • Erythropoietin is a sialo-glycoprotein of molecular weight 34,000 Daltons, of which about 40% is accounted for by glycosidic chains. EPO was purified for the first time in sufficient amounts for biochemical studies in 1977. In 1984 its cDNA was isolated and EPO was produced in bacterial cells. This material, although biologically active in vi tro, was not efficient in vivo because the lack of postranslational modification (mainly glycosylation) dramatically shortens EPO's half -life in vivo. EPO produced in baculovirus/insect cell systems is also available.
  • This EPO is glycosylated according to the scheme characteristic of the insect cells and, like bacterial EPO, has a shortened half life in vivo owing to the lack of mammalian-style glycosylation. Hence, neither bacterial EPO nor baculovirus-derived EPO is suitable for therapeutic use in isolation.
  • the dosage can be reduced but both initial therapy and maintenance involve repeated injections that produce peaks of high EPO concentration in the bloodstream followed by a rapid decrease and eventual disappearance from circulation after 5-10 hours of the injection.
  • Chugai describes the microencapsulation of EPO into particles comprised of a biocompatible polymer (polylactide or polylactide coglycolide) .
  • Chugai 's particles have an average particle diameter of from 5 to 100 ⁇ m, a maximum particle diameter of 150 ⁇ m or less and a coefficient of variation of 50% or less (this is a measure of the degree of diversity in the size of the particles. It is defined as (standard deviation of size) + (average particle diameter) x 100) . Therefore, Chugai describes sustained-release preparations comprising particles having a particular range of sizes and a low degree of variation in size, i.e. uniformly-sized particles .
  • preparations of particles having maximum particle sizes of from about 15 ⁇ m to about 80 ⁇ m are exemplified. Small particles having a maximum diameter of 15 ⁇ m and an average diameter of 8 ⁇ m are shown to release EPO at an approximately constant rate over a period of 1 month, whilst a preparation comprising larger particles, more heterogeneous in size and having a maximum diameter of about 80 ⁇ m are unsatisfactory as they burst easily.
  • an effective sustained-release preparation for the delivery of EPO can be formulated using preparations of polylactide coglycolide microspheres whose size distribution is bimodal, such that there are two peaks, an upper and a lower peak, in particle size.
  • the preparations of the invention have been found to release EPO heterogenously in time. After an initial rapid burst, EPO is released at a constant rate that suggests that they will be effective over as much as 1 year or longer. Thus, the preparations of the invention release EPO in two phases , a fast phase and a slow phase .
  • microspheres with heterogenoeous release characteristics have been obtained.
  • Yan e_t a_l Vaccine vol. 13, No. 7 pp 645-651; 1995
  • the preparations of the invention comprise EPO, not anti-ricin IgG, and do not release EPO in bursts. Indeed, this would be undesirable, as it would lead to undesirable peaks in EPO levels in the patient.
  • the preparations of the invention allow long term maintenance of EPO at physiological levels in the bloodstream of the subject, thus eliminating the need for frequent administration of EPO and reducing the risk of thrombosis and hypertension. Thus they are clinically advantageous over preparations in which EPO is not associated with a sustained release mechanism and over known sustained-release preparations, which are not effective over long periods.
  • the invention provides:
  • a sustained release preparation comprising polylactide coglycolide (PLG) microspheres which comprise erythropoietin (EPO) ; said microspheres having a size distribution which is bimodal, having an upper peak and a lower peak in particle size;
  • PLG polylactide coglycolide
  • EPO erythropoietin
  • composition comprising a preparation according to the invention and a pharmaceutically acceptable carrier;
  • PLG polylactide coglycolide
  • EPO erythropoietin
  • the plasmid pKG5 comprises: a) the EcoRI-PvuII fragment of pBR322 (2394 bp) at positions 3693 to 176; b) the PvuII-Hindlll fragment from SV40 (322 bp, 5191..5243, 1..270) at positions 176 to 498 containing the replication origin core region and the early TTIRNA promoter including the tandemly repeated 72bp segments; c) the BamHI-BcII fragment from SV40 (237 bp, 2533..2770) at positions 556 to 797, containing splice and polyadenylation signals from the SV40 early region; and d) a marker gene (neo) , coding for the aminoglycoside 3' phosphotranferase, from transposon Tn5 confers resistance to the antibiotic G418.
  • the neo gene (positions at pKG5 : 1606 to 2400) is preceded by the promoter (fragment Clal-Bglll, positions at pkG5 : 795 to 1571) and followed by the polyadenylation signal (Smal-EcoRI fragment, positions at pKGF : 2571 to 3693) of the thymidine kinase (tk) gene from herpes simplex virus.
  • the sustained release preparations of the invention comprise microspheres (also known as microcapsules) . These are small particles prepared by encapsulating a pharmaceutically active agent, in this case EPO, in an outer layer consisting of the polymer polylactide coglycolide (PLG) .
  • a pharmaceutically active agent in this case EPO
  • PLG polymer polylactide coglycolide
  • the microspheres of the invention comprise a "core" of active material (EPO) surrounded by a layer of PLG.
  • microspheres release the EPO at a rate determined by their size and chemical composition. In particular, the smaller the microspheres, the greater the rate of release.
  • the releasing mechanism is a combination of bulk and/or surface erosion and diffusion through the pores that exist in the polymer layer, it is possible to vary some factors (drug loading polymer molecular weight, composition, particle size and porosity) in order to vary the release characteristics of the EPO.
  • the preparations of the invention comprise microspheres of widely varying sizes, and these sizes are in a bimodal distribution. It is probable that this accounts for their ability to release EPO at relatively constant rates over long periods of time, with the larger microspheres releasing EPO slowly and the smaller ones doing so more rapidly.
  • the distribution of particle sizes is bimodal. This means that there are two peaks in particle size, an upper peak and a lower peak. These peaks are peaks in terms of the frequency of particles of a given size. Thus there are two sizes or size ranges in which high preparations of the total number of particles in the preparations are found.
  • the upper peak occurs at from 30 to 150 ⁇ m, more preferably at from 50 to 130 ⁇ m, 50 to 100 ⁇ m or 100 to 130 ⁇ m. Most preferably, the upper peak occurs at from 50 to 70 ⁇ m.
  • the two peaks typically contain a high proportion of the total number of microspheres in the preparation. For example, each peak may contain up to 10, up to 20, up to 30 or up to 40% of the total number of microspheres.
  • the two peaks may be of any size relative to one another.
  • the upper peaks may contain a higher proportion of the total number of microspheres that the lower one, or the lower peak may contain a higher proportion of the microspheres.
  • the upper peak contains a higher proportion of the microspheres than the lower one.
  • each peak and hence the proportion of the mcrospheres contained in it, can be determined by standard statistical techniques known in the art.
  • Preparations of the invention preferably have a minimum particle diameter of from 1 to lO ⁇ m, more preferably from 3 to 5 ⁇ m; and a maximum particle diameter of greater than 150 ⁇ m, preferably from 150 to 250 ⁇ m, more preferably from 150 to 220 ⁇ m and most preferably around 200 ⁇ m, for example 180 to 190 ⁇ m, 190 to 200 ⁇ m, 200 to 210 ⁇ m or 210 to 220 ⁇ m.
  • a minimum particle diameter preferably from 1 to lO ⁇ m, more preferably from 3 to 5 ⁇ m; and a maximum particle diameter of greater than 150 ⁇ m, preferably from 150 to 250 ⁇ m, more preferably from 150 to 220 ⁇ m and most preferably around 200 ⁇ m, for example 180 to 190 ⁇ m, 190 to 200 ⁇ m, 200 to 210 ⁇ m or 210 to 220 ⁇ m.
  • the microspheres of the invention comprise, as an active ingredient, Erythropoietin (EPO) .
  • EPO Erythropoietin
  • This EPO may be obtained from any suitable source. For example, it may be extracted from animals that produce it or it may be produced recombinantly. Recombinantly produced human EPO is preferred, and this is commercially available. Non-human EPO may also be used.
  • Recombinantly produced EPO may be prepared by any suitable method, for example biosynthesis in transformed bacterial, e.g. E. coli cells, which generates unglycosylated EPO; biosynthesis in transformed mammalian, e.g. CHO or BHK, cells, which produces fully glycosylated EPO; or biosynthesis in baculovirus-infected insect cells.
  • EPO from this source is extremely short lived although very active in vivo; for this reason, this baculovirus-derived EPO may be particularly suitable for delivery in vivo after entrapment in biodegradable microspheres according to the invention. It is envisaged that continuous EPO release according to the invention will compensate for the short half-life and give a good activity of clinical value.
  • EPO electroactive polymer
  • the industrial production of EPO is carried out on rollers at 37°C without C0 2 .
  • the culture is harvested every 48 hours for up to 10 days (5 harvests) before replacing it with fresh cells.
  • the media is DMEM plus antibiotic but lacking foetal calf serum, geneticin (G418) and methotrexate .
  • the EPO obtained is of high purity with a specific activity of about 100000 u/mg.
  • the yield of the process is about 50%.
  • Ultrapure recombinant human EPO suitable for use according to the invention is available from (Amersham) under Catalogue No. 286-EP as a sterile-filtered solution in 50% glycerol in 25 mM Hepes buffer, pH 7.2 and has a purity of >97%, as determined by SDS-PAGE visualized by silver stain, and a specific activity of greater than 100,00 units/A 2 ⁇ 0 .
  • the in vi tro biological activity of this preparation measured in a cell proliferation assay using a factor-dependent human erythroleukemic cell line, TF-l (Kitamura, T. et al . , 1989, J. Cell. Physiol. 140:323, gives an ED50 of 0.1 units/ml.
  • tissue-culture grade EPO is available from (Amersham) under Catalogue No. 287-TC as a sterile-filtered solution of PBS with carrier, having a greater than 100,000 units/A 280 .
  • the in vi tro biological activity of this preparation measured in a cell proliferation assay using a factor-dependent human erythroleukemic cell line, TF-l (Kitamura, T. e ⁇ al . , 1989, J. Cell. Physiol. 140:323) gives an EP50 of 0.1 units/ml.
  • EPO for use according to the invention amy be of any suitable purity. Preferably, it is substantially pure. EPO of at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% purity is preferred.
  • the EPO may have any suitable specific activity.
  • EPO having an activity of from 50,000 to 150,000 ⁇ /mg is preferred, with EPO having an activity of from 80,000 to 120,000 ⁇ /mg, such as about 100,000 ⁇ /mg being particularly preferred.
  • the microspheres of the invention may comprise any effective amount of EPO, and the amount of EPO contained in the microspheres will vary, for example with the size of the microspheres and with the therapeutic application.
  • the microspheres of the invention comprise a polyactide coglycolide (PLG) capsule.
  • PLG is a suitable material because it is biocompatible and biodegradable. This PLG may have any suitable characteristics. For example, it may have any average molecular mass, for example from 1000 to 20000 with PLG having an average molecular mass of 5000 to 10000 being preferred.
  • the PLG may comprise any suitable proportions of lactide and glycolide moieties.
  • the ratio of lactide to glycolide may be from 90:10 to 10:90 with ratios of from 20:80 to 80:20 being preferred and ratios of from 40:60 to 60:40 being more preferred, and a ratio of 50:50 being most preferred.
  • Lactide is optically active and any proportions of D and L isomers may be present, ranging from pure D-Lactide to pure L-lactide, with racemates comprising 50% D-lactide and 50% L-lactide being preferred.
  • microspheres of the invention may be prepared by any means which results in a suitable distribution of particle sizes and other characteristics, as described herein.
  • EPO- containing microspheres may be prepared by a water-in-oil-in water emulsion process similar to that by the process of Eldridge et . ai, (1991: Infection and Immunity pp 2978-2986), but incorporating the modifications described in the Examples.
  • the bimodal distribution of particle sizes according to the invention which is atypical and surprising, is different from that obtained by Eldridge e_t al.
  • water-in-oil-in- water emulsion processes of the invention involve two emulsification steps, and this distribution is believed to be, at least to some extent, a function of the stirring speed profile during the formation of the second emulsion.
  • this stirring was not performed at constant speed but rather at two speeds, 200 rpm for the first half of the process and 500 rpm for the second half.
  • This bimodal profile is believed to be responsible for the bimodal EPO release profile of the microspheres of the invention.
  • suitable speeds for the first period may be from 500 to 10000 rpm, preferably from 1000 to 10000 rpm, more preferably from 1500 to 2500 rpm or from 1800 to 2200 rpm.
  • a particularly preferred speed for the first period is 2000 rpm.
  • suitable speeds may be from 100 to 5000 rpm, preferably from 100 to 1000 rpm, more preferably from 200 to 800 rpm or from 400 to 600 rpm.
  • a particularly preferred speed is 500 rpm.
  • the two agitation periods at different speeds may be of any suitable length, and of any suitable length in relation to each other.
  • the two periods may be equal or unequal in length.
  • the two periods may occupy, respectively, from 90% and 10% to 10% and 90% of the total emulsification period. It is preferred that the two periods occupy from 80% and 20 to 20% and 80% of the emulsification period, preferably from 60% and 40% to 40% and 60% of the period. In a preferred embodiment, the two periods occupy 50%, or around 50% each of the total emulsification period.
  • the transition between the two different stirring speeds may take place at any speed, e.g. gradually or instantaneously.
  • preparation procedures will vary slightly depending on the nature of the microspheres to be produced; for example, preparative procedures may be tailored to give microspheres with a particular size distribution, or maximum or minimum particle size (diameter) for which they are intended.
  • the invention provides icrosphere preparations obtained, or obtainable, by the processes of the invention.
  • the "core load" of EPO in the microspheres may vary between preparations or within a single preparation.
  • the core load of EPO may be from 0.1 to 10% of the total mass of the microspheres, with core loads of 0.5 to 5% being preferred and EPO core loads of 1 to 2% being particularly preferred.
  • HSA human serum albumin
  • the sustained-release preparations of the invention typically have certain characteristics as far as their release of EPO in vivo or in vi tro is concerned. Typically they release EPO at an approximately constant rate over a long period, typically greater than one month, and preferably for 1 to 6 months, from 6 months to one year or more .
  • the microsphere preparations of the invention release EPO in a biphasic manner. It is believed that this is, at least partly, caused by the bimodal size distribution of the particles, and that this, in turn, is at least partly caused by the bimodal stirring regime which may be used in preparing the microspheres .
  • microspheres of the invention may be administered in any suitable amount and by any suitable route in order to deliver EPO to human or animal, preferably human, subjects.
  • each dose of the preparation will contain from 1000 to 100000 units of EPO, preferably 5000 to 50000 units, more preferably 10000 to 20000 or 20000 to 30000 units, for an adult human of average size. For larger or smaller humans, or for animals, these doses can be adjusted in relation to body mass.
  • a typical dose for research uses in mice may comprise from 10 to 100 units of EPO, preferably from 20 to 50 units of EPO, for example 20 to 25 units.
  • the preparations of the invention are administered to subjects deficient in EPO and the dose size and release characteristics of the preparation will be adjusted to increase the level of EPO in the subject's bloodstream towards the level found in normal subjects.
  • the release characteristics of the preparation will be such that a normal or near-normal level is maintained over a long period of time, 95 mentioned above.
  • Any suitable dosage regime that maintains a suitable blood level of EPO may be used.
  • a dose may be administered from a 1 to 50 times per year, within monthly and bimonthly dosages being preferred.
  • sustained release preparations of the invention may be administered by any suitable route, with injection being preferred, parenteral injection being particularly preferred and subcutaneous injection being most particularly preferred.
  • the erythropoietin (EPO) gene was isolated from human genomic DNA using standard PCR and cloning techniques .
  • the EPO gene (approx 2.1 kb) was isolated from genomic DNA prepared from the white blood cells of healthy individuals. PCR techniques using primers designed in the fashion shown in the enclosed sequence .
  • the cloning and sequencing manipulations were done following standard genetic engineering methods (Sambrook et al , 1987. Molecular Cloning: a laboratory manual ed. C.S.H. University Cold Spring Harbor) .
  • the gene sequence was modified without changing the amino acid sequence to allow easier genetic manipulation and.
  • the amino acid and the nucleotide sequence were checked in the final construct .
  • the gene was cloned into expression vector pKG5 (that confers geneticin (G418) resistance) and was introduced into CHO DHFR " cells together with the plasmid pSV2 DHFR (that complements the DHFR (dihydrofolate reductase) insufficiency.
  • the vector map of the pKG5 plasmid is given in Fig. 6, this vector having been constructed over 12 years ago using fragments from known vectors.
  • the SV40 expression unit segment was derived from the plasmid pSVl described by Krieg et al . , J. Mol . Biol . 180, 615-643 (1984) . It was modified for the cloning site using synthetic oligonucleotides .
  • the TK neo gene has the promoter and poly A sites of Herpes TK (Colbere-Gerapin et al . , J. Molec. Biol. 150 1-14 (1981).
  • the EPO sequence inserted includes 2 nucleotides preceding the ATG and 15 nucleotides following the stop codon.
  • the cells were selected for G418 resistance and checked for EPO production.
  • the EPO producing clones were subjected to gene amplification by selection with methotrexate (Mtx) .
  • the end product was a cell line that produces 10 7 units of EPO/litre of culture per 24 hours.
  • the cells can be grown in orMEM medium lacking ribo-and deoxyribonucleotides (GIBCO 072- 02000) complemented with gentamicin 50 mg/1, L-glutamine 2 mM, geneticin (G418) 500 mg/1, Fetal Calf Serum (FCS) (dialysed) 10%, Methotrexate 10 "7 M.
  • GEBCO 072- 02000 ribo-and deoxyribonucleotides
  • FCS Fetal Calf Serum
  • the mRNA and amino acid sequences of this EPO were compared to these of the industrial product.
  • Amino acid sequences were determined on the purified EPO using a model 477A protein microsequencer from Applied Biosystems. According to the method of Hewick et al . (J. Biol. Che . , 256, 7990-7997, 1981) .
  • mRNA sequences were determined by cDNA amplification with the specific primers from total RNA prepared from the producer cell line.
  • the techniques for amplification and sequence were the standard ones (Maniatis, Fritsch and Sambrook, 1982.
  • EPO EPO-EPO
  • EPO activity calibrated against the second international reference preparation of erythropoietin (Annable, L. et al . , 1972, Bull. Wld. Hlth. Org 47: 99) using an in vivo bioassay which measures the incorporation of 56 Fe into red blood cells of exhypoxic polycythemia mice (Cotes and Bangham, 1961, Nature 191:1065).
  • 5mg of purified EPO corresponds to 10 6 international units.
  • EPO cDNA was prepared from RNA extracted from the cell line described in Example 1. The cDNA was placed under the control of the polyhdrin promoter (Quelle et al (1992) Protein Expression and purification 3, pp461-469) and a recombinant baculovirus system producing EPO was constructed.
  • the amount of protein in the microparticles was determined by a bicinchoninic acid protein assay (Pierce Chemical Co, Rockford, Illinois) after disruption of the microparticles and extraction of the entrapped protein, with the microparticles being disrupted and the protein extracted as follows: 20-25 mg of microspheres were dissolved in 2 ml of C1 2 CH 2 (methylene chloride) and the protein extracted 3 times with 0.5 ml of 0. IN NaOH. The aqueous layer was subjected to protein determination by the bichinconic assay method (Pierce, see above) .
  • the emulsification technique of Eldridge e_t al was modified to obtain a bimodal distribution of the microspheres' diameter (see Fig. 7) .
  • the stirring speed was initially 2000 rpm and half way through the process it was switched to 500 rpm.
  • the amount of protein found to be present in the microspheres (the "core load") is a function of the protein concentration to be entrapped in the aqueous solution used for the first emulsion. 12,000 units of recombinant EPO and 15 mg of human serum albumin were dissolved and entrapped in 1 g of
  • Figure 1 shows the appearance of this material analysed by (part a) light microscopy and by (part b) scanning electron microscopy.
  • Ames tests for mutagenesis can be performed on the final preparation (Ames, B.N., McCann J. and Yamasaki E., Methods for detecting carcinogens and utagens with the
  • Salmonella/mammalian-microsome mutagenicity test Muta t . Res . , 31, 347-363, 1975). These also revealed low toxicity.
  • the EPO preparation of the invention was tested for toxicity, in vi tro and in vivo activity.
  • EPO release from PLG microspheres was examined by incubation of 10 g of microspheres in 1 ml of receiving fluid. (Phosphate buffered saline-sodium azide 0.02% maintained at 37°C withdrawn periodically and replaced with the same amount of fresh fluid) . As can be seen in Figure 2, approximately 35% of the encapsulated EPO is available for quick release during the first and second days followed by a slower release for a period of 30 days. The amount of EPO released per day from the PLG after the initial burst varies between 0.5 and 1%, based on the total amount initially present .
  • the first phase is characterised by an initial burst of EPO release (rapid release from surface bound and incompletely encapsulated protein) .
  • EPO release rapid release from surface bound and incompletely encapsulated protein
  • the second phase the release occurs by diffusion through aqueous pores and channels as the molecular weight of the polymer decreases.
  • the integrity of this protein after being kept at room temperature for long periods of time, was checked by polyacrylamide gel electrophoresis (PAGE) .
  • PAGE polyacrylamide gel electrophoresis
  • the gel migration of the material taken at different times during the course of the experiment corresponds to the known molecular weight of EPO (34kDa) and has the electrophoretic mobility of the original material taken before encapsulation.
  • the molecular integrity as measured in a PAGE is not enough to assure physiological activity, the in vivo effect of the release material was also evaluated.
  • PLG-EPO microspheres were incubated for 30 days at 37°C in PBS. Subsequently, an aliquot of the receiving fluid was injected in posthypoxic polycytemic mice, a standard EPO measurement (Cotes and Bangham (1961) : Nature 191 ppl065-1067 ; Cotes (1988) : British Medical Journal pp296-806) . The released EPO stimulated the uptake of 59 Fe in mice, indicating that its physiological activity remained unaltered after the whole procedure .
  • mice were subcutaneously injected for in vivo studies with the same batch of 125 I-EPO. Animals were sacrificed at different times (1 to 21 days) after injection. The radioactivity was measured in samples collected from liver, thyroid gland, blood and urine. From these data it can be seen that, as in the case of the in vi tro release, there is an initial burst followed by a steady release at least until day
  • Physiological activity was measured in posthypoxic polycytemic mice as described in (a) above using EPO produced by the cell line described in Example 1. This method is limited by its short duration as the production of endogenous EPO by posthypoxic mice restarts after a few days. Nevertheless a definitive prolonged effect was observed for up to 5 days after injection of microencapsulated EPO.
  • EPO in microspheres of the invention allows a biologically active form to be kept for long periods at room temperature avoiding stabilizers and/or cold chain, (c) Biological activity in vivo in the 5/6 nephrecto ised rat model.
  • Haematocrit Hct
  • This anaemia can be corrected to a Hct of 50% by treatment with recombinant EPO, 25 units intraperitioneal, twice weekly (Garcia, et al (1988) : PNAS 85 pp6142-6146) .
  • Fig. 4 shows the values of the Hct measured in tail blood of conscious rats after renal ablation without treatment (inverted triangles ⁇ ) , treated with a single dose of rEPO (open circles 0) , twice weekly does of rEPO for 4 weeks open triangles or with the EPO preparation of the invention (full circles •) .
  • Figure 5 compares the in vi tro release characteristics of the two preparations, in terms of the percentage of EPO remaining in the microspheres at a series of time-points (as a fraction of the total EPO originally encapsulated).
  • Chugai' s preparation releases EPO at an approximately constant rate over the course of one month
  • the EPO preparation of the present invention after an initial rapid burst, releases a far smaller fraction of its encapsulated EPO (approximately 40%) in the same time.
  • the rate of release is approximately constant at around 3% per week. At this rate of release, EPO could be maintained of physiological concentrations for several months, or even as much as one year or more.
  • the preparation of the invention has very different release characteristics than those known in the art, as represented by the Chugai preparation. Accordingly, it is clear that the preparation of the invention will provide a sustained release of EPO over a far longer period than previously known preparations, as represented by that of Chugai .

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Abstract

L'invention concerne une préparation à libération lente. Cette préparation comprend des microsphères de polylactide coglycolide (PLG) qui comprennent de l'érythropoïétine. Ces microsphères présentent une granulométrie qui est bimodale, avec un pic supérieur et un pic inférieur de granulométrie. L'invention concerne aussi des compositions pharmaceutiques comprenant ces préparations. L'invention concerne également un procédé d'émulsion d'eau dans l'huile dans l'eau pour produire une préparation à libération lente.
PCT/EP1997/004491 1996-08-23 1997-08-18 Preparation a liberation lente renfermant de l'erythropoietine et du polylactide coglycolide WO1998007442A1 (fr)

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GB9617732.4 1996-08-23
GB9617732A GB2316316A (en) 1996-08-23 1996-08-23 Sustained release of erythropoietin from microspheres

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WO1998007442A1 true WO1998007442A1 (fr) 1998-02-26
WO1998007442A9 WO1998007442A9 (fr) 1998-07-23

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GB2316316A (en) 1998-02-25

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