SE517610C2 - New parenterally administrable microparticle preparation useful for controlled-release of biologically active substances e.g. drug - Google Patents

Abstract

Parenterally administrable microparticle preparation comprises a biologically active substance, which is sensitive to or unstable in organic solvents. The preparation, during the first 24 hours after injection, exhibits a release of the active substance less than 25% of the total release determined from a concentration-time curve in the form of ratio between the area under curve during the 24 hours and total area. Independent claims are included for: (1) a parenteral preparation as above that exhibits a release of active substance in which the maximum plasma concentration is less than 300% of the maximum concentration of the biologically active substance at any time in excess of 48 hours after administration; (2) a preparation as above, which has an amylopectin content in excess of 85% weight, of which at least 80% has an average molecular weight in the range of 10-10000 kDa; (3) a preparation as above which exhibits a release of the active component characterized in that in the release that takes place during any continuos 7 day period the quotient of the active substance divided by the mean concentration is less than five (not including the first 24 hours); and (4) a preparation as above where the mean residence time for an active substance is at least 4 days.

Description

»- v v ~ o-u- u-». In addition, the harmless nature of the PLGA can be exemplified by the fact that several sustained release parenteral preparations based on these polymers have been approved by authorities, including the US Food and Drug Administration.

Parenterally administrable products for sustained release on the market today based on PLGA include Decapeptylm (Ibsen Biotech), Prostap SRW (Lederle), Decapeptyl® Depot (Ferring) and Zoladex® (Zeneca). The drugs in these preparations are all peptides. In other words, they consist of amino acids fused to a polymer with a relatively low degree of polymerization and they do not have a well-defined three-dimensional structure. This in turn usually allows the use of relatively strict conditions during the manufacture of these products. For example, extrusion and subsequent size reduction can be used, which techniques should not be allowed in connection with proteins, as these generally do not withstand such severe conditions.

Consequently, there is also a need for formulations with regulated release of proteins. Proteins are similar to peptides in that they also consist of amino acids, but the molecules are larger and most proteins depend on a well-defined three-dimensional structure in terms of many of their properties, including biological activity and immunogenicity. Their three-dimensional structure can be destroyed relatively easily, e.g. of high temperatures, surface-induced denaturation and in many cases exposure to organic solvents. A very serious disadvantage associated with the use of PLGA, which is an excellent material per se, for delayed release of proteins is therefore the need to use organic solvents to dissolve said PLGA, with the attendant risk that the protein stability is adversely affected and that conformational changes in the protein lead to an immunological reaction in the patient, which can result in both a loss of the therapeutic effect through the formation of inhibitory antibodies and toxic side effects. As it is extremely difficult to determine with certainty whether a complex protein in all parts has retained its three-dimensional structure, it is of great importance to avoid exposing the protein to conditions which may induce conformational changes. .

Despite major efforts to modify PLGA technology to avoid this inherent problem of protein instability during the manufacturing process, developments in this field have been very slow, and the main reason for this is probably that the three-dimensional structures of most proteins are too sensitive to withstand the conditions of manufacture used and the chemically acidic environment formed during the degradation of PLGA matrices. The scientific literature contains a number of descriptions of stability problems in the fabrication of PLGA microspheres due to exposure to organic solvents. As an example of the acidic environment formed during the degradation of PLGA matrices, it has recently been shown that the pH of a PLGA microsphere with a diameter of about 40 microns has been shown to drop to 1.5, which is quite sufficient to denature. or otherwise damage, many therapeutically useful proteins (Fu et al., Visual Evidence of Acidic Environment Within Degrading Poly (lactic-co-glycolic acid) (PLGA) Microspheres. Pharmaceutical Research, Vol. 17, No 1, 2000, 100-106). If the microspheres have a larger diameter, the pH value can be expected to fall further due to the fact that the acidic decomposition products then have a harder time diffusing away and that the autocatalytic reaction is intensified.

The currently most commonly used technique for entrapping water-soluble substances, such as proteins and peptides, is the use of multiple emulsion systems.

The drug substance is dissolved in an aqueous or buffer solution and then mixed with a water-immiscible organic solvent containing the dissolved polymer. An emulsion is formed which has the aqueous phase as internal: nyn- 10 15 20 25 30 35,. ,. »O '" .u n. .H-' __ ,,. N. -: ..-. - ",. .- I ° 'I .. ~', .I f '.- ,, na "_ .o I ...» vu I, ,, n 4',. .-- __ _, ... v- of 4 "phase. Different types of emulsifiers and strong mixtures are often used to create this first emulsion. This emulsion is then transferred, with stirring, to another liquid, usually water, containing another polymer, e.g. polyvinyl alcohol, which gives a water / oil / water triple emulsion.The microspheres are then cured or cured in some way.The most common way is to use a low boiling organic solvent, typically dichloromethane, and to evaporate the solvent. If the organic solvent is not completely immiscible with water, a continuous extraction procedure can be used by adding more water to the triple emulsion.A number of variations of this general procedure are also described in the literature.In some cases the primary emulsion is mixed with a non-water - based phase, eg silicone oil Solid solids in place t for dissolved ones can also be used.

PLGA microspheres containing proteins are described in WO-A1-9013780, where the main feature is the use of very low temperatures during the production of the microspheres in order to preserve high biological activity of the proteins. The activity of encapsulated superoxide dismutase was measured but only on the part released from the particles.

This method has been used to prepare PLGA microspheres containing human growth hormone in WO-A1-9412158, dispersing human growth hormone in methylene chloride containing PLGA, spraying the resulting dispersion in a container with frozen ethanol with a layer of liquid nitrogen over it. freeze the fine droplets and allow them to settle in the nitrogen of the ethanol. The ethanol is then thawed and the microspheres begin to sink into the ethanol, where the methylene chloride is extracted into the ethanol and the microspheres are hardened. Using this methodology, protein stability can be maintained better than in most other processes for entrapping proteins in PLGA microspheres, and recently a product has also been approved by the registration authorities in win 10 15 20 25 30 35 517 610 5 USA. However, it remains to be unequivocally demonstrated for other proteins as well, and the problem of exposing the entrapped biologically active substance to a very low pH during the degradation of the PLGA matrix remains.

However, in the previously mentioned methods based on encapsulation with PLGA, the active substances are exposed to an organic solvent, and this is generally detrimental to the stability of a protein. In addition, the mentioned emulsion processes are complicated and probably problematic in an attempt to scale up to an industrial scale. Furthermore, many of the organic solvents used in many of these processes are associated with environmental problems, and their high affinity for the PLGA polymer makes removal difficult.

A number of attempts to solve the problems described above caused by exposure of the biologically active substance to a chemically acidic environment during the biodegradation of the microsphere matrix and organic solvents in the manufacturing process have been described. In order to avoid acidic environment during decomposition, attempts have been made to replace PLGA as a matrix for the microspheres with a polymer which gives chemically neutral decomposition products and to avoid exposing the biologically active substance to organic solvents, attempts have been made to either manufacture the microspheres in advance and first after working up and drying, tried to charge them with the biologically active substance, or tried to exclude the organic solvent completely during the manufacture of the microspheres.

For example, highly branched starch of relatively low molecular weight (maltodextin, average molecular weight about 5000 Da) has been covalently modified with acrylic groups to transfer this starch in a form which can be solidified into microspheres and the resulting polyacrylic starch has been converted to particulate form by emulsion polymerization toluene / chloroform (4: 1) as outer phase (Characterization of Polyacrylic Starch Microparticles as Carries for Prote- = »» ~ ø 10 15 20 25 30 35 517 e 1 o šïï * 2fï = 6 ins and Drugs. Artursson et al, J Pharm Sci, 73, 1507-1513). Proteins could be trapped in these microspheres, but the manufacturing conditions expose the biologically active substance to both organic solvents and high shear forces in the manufacture of the emulsion. The resulting microspheres dissolve enzymatically and the pH can be expected to remain neutral. The resulting microspheres are not suitable for parenteral administration, especially repeated ones, for several reasons. Most important is the incomplete and very slow biodegradability of both the starch matrix (Biodegrable Microspheres IV. Factors Affecting the Distribution and Degradation of Polyacrylic Starch Microparticles. Laakso et al., J. Pharms Sci, 75, 962-967, 1986). synthetic polymer chain that crosslinks the starch molecules. In addition, these microspheres are too small, with a diameter <2 μm, to be suitable for injection into the tissues for prolonged release, as tissue macrophages can easily phagocytose them. Attempts to increase the rate of degradation and the degree of degradation by introducing a potentially biodegradable ester group to bind the acrylic groups to the highly branched starch did not lead to the intended result and even these polyacrylic starch microspheres were biodegradable too slowly and incompletely. of Polyacrylic Starch Microparticles Prepared from Acrylic acid Esterified Starch. Laakso and Sjöholm. 1987 (76) pp 935-939. J Pharm Sci.).

Polyacrylic dextran microspheres have been fabricated in two-phase aqueous systems (Stenekes et al., The Preparation of Dextran Microspheres in an All-Aqueous System: Effect of the Formulation Parameters on Particle Characteristics).

Pharmaceutical Reserach, Vol 15, No 4, 1998, 557-561 and Franssen and Hennink, A novel preperation method for polymeric microparticles without using organic solvents, Int J Pharm 168, 1-7, 1998). This approach avoids exposing the biologically active substance to organic solvents, but otherwise the microspheres acquire properties similar to those described for polyacrylic starch microspheres above, making them unsuitable for repeated parenteral use. administration. Given that humans do not have specific dextran-degrading enzymes, the rate of degradation should be even lower than for polyacrylic starch microspheres. The use of dextran is also associated with a certain risk of severe allergic reactions.

Manufacture of starch microspheres using non-chemically modified starch using an outer phase oil has been described (US 4,713,249; Schröder, U., Crystallized carbohydrate spheres for slow release and targeting. Methods Enzymol. 1985 ( 112) 116-128; Schröder, U., Crystallized carbohydrate spheres as a slow release matrix for biologically activated substances. Bio- materials 5: 100-104, 1984). The microspheres are solidified in these cases by precipitation in acetone, which leads both to the biologically active substance being exposed to an organic solvent and to the fact that the starch's natural tendency to solidify by physical crosslinking is not utilized during the manufacturing process. This in turn leads to microspheres with an inherent instability as the starch after resuspension in water and upon exposure to the body fluids will strive to form such crosslinks. In order to obtain a water-in-oil emulsion, high shear forces are required and the microspheres formed are too small to be suitable for parenteral prolonged release.

EP 213303 A2 describes the production of microspheres of, among other things, chemically unmodified starch in two-phase aqueous systems, utilizing the starch's natural ability to solidify by forming physical crosslinks, and immobilizing a substance in these microspheres in order to avoid exposing it to biological. the active substance for organic solvents. The methodology described, in combination with the starch quality nun- 0 »vpøu lO 15 20 25 30 35 u uno I '517 610 8 defined, does not give rise to completely biodegradable particles. The resulting particles are also not suitable for injection, especially for repeated injections over a longer period of time, as the described starch quality contains excessive amounts of foreign plant protein. Contrary to what is taught by this patent, it has now also surprisingly been discovered that significantly better yield and higher charge of the biologically active molecule can be obtained if significantly higher concentrations of the polymers are used than are required to form two phase. the water system and this also leads to advantages in terms of the conditions for obtaining stable, non-aggregated, microspheres and their size distribution. The temperature treatments described are not useful for sensitive macromolecules and the same applies to reprocessing which involves drying with either ethanol or acetone.

Alternative methods for producing microspheres in two-phase water systems have been described. In US 5,981,719 microparticles are manufactured by mixing the biologically active macromolecule with a polymer at a pH near the isoelectric point of the macromolecule and stabilizing the microspheres by supplying energy, preferably heat. The lowest proportion of macromolecule, i.e. the biologically active substance, in the preparation is 40% and this is too high for most applications and leads to great uncertainty in the amount of active substance injected as the dose of the microparticles becomes too low. Although the manufacturing method is described as mild and capable of maintaining the biological activity of the captured biologically active substance, the microparticles are stabilized by heating and in the examples given, heating takes place to at least 58 ° C for 30 minutes and in many cases to 70-90 °. C during the corresponding time, which sensitive proteins, whose biological activity is dependent on a three-dimensional structure, can not be expected to tolerate, and even in cases where the protein has apparently tolerated manufacturing prolapse »10 15 20 25 30 35 5 17 6 10 9 cess there is a risk of small, but still not negligible, changes in the protein conformation. The outer phase always uses a combination of two polymers, usually polyvinylpyrrolidone and PEG, which complicates the manufacturing process by washing both of these substances away from the microspheres in a reproducible and safe manner. The microparticles formed are too small (in the examples values below 0.1 μm are given in diameter) to be suitable for parenteral prolonged release after e.g. subcutaneous injection, when macrophages, which are cells specialized in phagocytic particles and present in the tissues, are easily able to phagocytose microspheres up to 5-10, possibly 20 μm, and the phagocytic particles are located intracellularly in the lysosomes, where both the particles and the biologically active substance is broken down, thereby losing the therapeutic effect.

The very small particle size also makes the processing of the microspheres more complicated, as desirable methods, such as filtration, cannot be used. The same applies to US 5,849,884.

US 5,578,509 and EP 0 688 429 B1 describe the use of two-phase water systems for the manufacture of macromolecular microparticle solutions and the chemical or thermal crosslinking of the dehydrated macromolecules to form microparticles. It is highly undesirable to chemically crosslink the biologically active macromolecule, either by itself or with the microparticle matrix, as such chemical modifications have several serious disadvantages, such as reducing the bioactivity of a sensitive protein and risking induction of an immune response to the new antigenic determinants on the protein, leading to a need for extensive toxicological studies to investigate the safety of the product.

Microparticles made by chemical crosslinking with glutaraldehyde are already known and are generally considered unsuitable for repeated parenteral administration in humans. The microparticles described in US 5,578,509 generally suffer from the same disadvantages as described in US 5,981,719, with unsuitable manufacturing conditions for sensitive proteins, either by subjecting them to chemical modification or for harmful temperatures, and a microparticle size distribution that is too narrow for parenteral, prolonged release and that complicates reprocessing of the microspheres after manufacture.

WO 97/14408 describes the use of air suspension technology for the preparation of microparticles for prolonged release after parenteral administration without exposing the biologically active substance to organic solvents. However, the publication does not provide guidance on the process according to the invention or on the new microparticles that can be obtained thereby.

US 5,470,582 produces a microsphere consisting of PLGA and containing a macromolecule by a two-step process, where the microsphere itself is first made using organic solvents and charged with the macromolecule at a later stage, where the organic solvent has already been removed. This procedure leads to too low a content of the biologically active substance, usually 1-2%, and to a very large proportion being released immediately after injection, which is usually extremely inappropriate. This too rapid initial release is very high even at a charge of 1% and becomes even more pronounced at higher contents of the active substance in the microspheres. Upon degradation of the PLGA matrix, the pH drops to levels that are usually not acceptable for sensitive macromolecules.

That starch is theoretically a very suitable, perhaps even ideal, matrix material for microparticles has been known for a long time because starch does not need to be dissolved in organic solvents and has a natural tendency to solidify and because there are body enzymes that can break down starch into bodily and neutral substances, ultimately glucose, as well as that starch, probably due to the resemblance to uu: - _ nvu wow. av, u ... I: ~ ||| 1017 20 25 30 35 517 610 11 body glycogen, has been shown to be non-immunogenic.

However, despite great efforts, starch with properties that allow the production of microparticles suitable for parenteral use and conditions that allow the production of completely biodegradable microparticles under mild conditions that allow the capture of sensitive biologically active substances, such as proteins, have not been described previously.

Starch granules naturally contain impurities, such as starch proteins, which make them unsuitable for parenteral injection. Accidental deposition of insufficiently purified starch, as can occur in operations where many types of surgical gloves are powdered with stabilized starch granules, can cause very serious side effects. Starch granules per se are also not suitable for repeated parenteral administration for the reason that they are not completely biodegradable within acceptable periods of time.

Starch microspheres made from acid hydrolyzed and purified starch have been used for parenteral administration in humans. The microspheres were made by chemical crosslinking with epichlorohydrin under strongly alkaline conditions. The chemical modification then obtained of the starch leads to a decrease in biodegradability so that the microspheres can be completely dissolved by the body's enzymes, such as α-amylase, but not completely converted to glucose as the end product. Neither the manufacturing method nor the resulting microspheres are suitable for the immobilization of sensitive proteins, and as the control experiments show, acid hydrolyzed starch is also not suitable for the production of either completely biodegradable starch microspheres or starch microspheres containing a high-density microsphere. biologically active substance, such as a protein.

Hydroxyethyl starch (HES) is administered parenterally in high doses to humans as a plasma replacement. HES is prepared by starch granules from starch consisting largely exclusively of highly branched amylopectin, so-called is rolysed for a reduction in the molecular weight distribution, and 'waxy maize', or waxy maize starch, acid hydride - then hydroxyethylated under alkaline conditions and acid hydrolyzed once more to achieve an average molecular weight of around 200,000 Da. Then filtration, extraction with acetone and spray drying are performed. The purpose of the hydroxyethylation is to prolong the duration of the effect, as unmodified amylopectin is degraded very rapidly by α-amylase and its residence time in circulation is about 10 minutes. HES is not suitable for the production of fully biodegradable microspheres containing a biologically active substance, as the chemical modification leads to a significant reduction in the rate and completeness of biodegradation and to the natural tendency of starch to be solidified by the formation of non-covalent crosslinks. eliminated. In addition, highly concentrated solutions of HES become too viscous to be useful for the production of microparticles. The use of HES in these high doses shows that parenterally useful starch can be produced, even if HES is not useful for the manufacture of microspheres without chemical crosslinking or precipitation with organic solvents.

WO 99/00425 describes the use of heat-resistant proteolytic enzymes with a broad pH optimum to purify starch granules from surface-associated proteins. The resulting granules are not suitable for parenteral administration as they still contain the starch proteins contained within the granules and there is a risk that residues of the added proteolytic enzymes remain in the granules. The granules are also not suitable for the production of parenterally administrable starch microspheres in two-phase aqueous systems, as they have the wrong molecular weight distribution to be able to be used in a sufficiently high concentration, even after dissolution, and in those cases micro-10 15 20 25 30 35 517 610 13 spheres can be obtained, they are probably not completely biodegradable.

The use of shear to modify molecular weights in order to obtain better starch for the manufacture of tablets is described in the U.S. distribution of starch, 5,455,342. The starch obtained is not suitable for parenteral administration due to the high content of starch proteins, which may be present in denatured form after shear, and the starch obtained is also not suitable for the production of biodegradable starch microspheres for parenteral administration or for use in two-phase water systems for the production of such starch microspheres.

Thus, a process for the preparation of parenterally administrable starch formulations having the following properties would be highly desirable: a process which makes it possible to capture sensitive biologically active substances in microparticles while retaining their biological activity; A process by which biologically active substances can be trapped under conditions which do not expose them to organic solvents, high temperatures or high shear forces and which allow them to retain their biological activity; A process which enables high loading of a parenterally administrable preparation with also sensitive biologically active substances; A process by which a substantially completely biodegradable and biocompatible preparation can be prepared which is suitable for injection parenterally and in the degradation of which chemically neutral substances are formed; A process by which a parenterally injectable preparation having a size exceeding 20 μm and more preferably exceeding 30 μm can be prepared in order to avoid phagocytosis of tissue macrophages and to simplify the reprocessing thereof during manufacture; In a process for the preparation of microparticles containing a biologically active substance, which can be used as an intermediate in the preparation of a preparation for controlled, prolonged or delayed release and enables rigorous quality control of the chemical stability of the captured biological substance and biological activity; A process utilizing a parenterally acceptable starch suitable for the production of substantially completely biodegradable starch microparticles; A substantially completely biodegradable and biocompatible microparticulate formulation which is suitable for injection parenterally and in the degradation of which chemically neutral substances are formed; A microparticulate preparation containing a biologically active substance and having a particle size distribution suitable for coating by means of air suspension technology and sufficient mechanical strength for this purpose; A coated microparticulate formulation containing a biologically active substance, which formulation provides a controlled release after parenteral administration; Such and other objects are achieved by means of the invention defined below.

DESCRIPTION OF THE INVENTION The present invention relates to a novel microparticle preparation. More specifically, it is a microparticle preparation which is biodegradable, which contains a biologically active substance and which is intended for parenteral administration of this substance to a mammal, especially a human. Primarily, the parenteral administration means that the microparticles are intended for injection.

Since the microparticles are primarily intended for injection, it is preferably a matter of producing particles with an average diameter in the range 10-200 μm, usually 20-100 μm and especially 20-80 μm.

The term "microparticles" is used in connection with the invention as a general term for particles of a certain size in accordance with per se known technology. One type of microparticle is thus microspheres, which have a substantially spherical shape, while the term microparticle may generally include deviation from such an ideal spherical shape. The per se known concept of microcapsule also falls within the term microparticle in accordance with known technology.

According to one aspect of the present invention, there is provided a parenterally administrable, biodegradable microparticle preparation containing a biologically active substance which, during the first 24 hours after injection, exhibits a release of the active substance which is less than 30% of the total the release, determined from a concentration-time curve in the form of the ratio between area during the curve during said first 24 hours and total area during the curve in question.

Preferably, the release during the first 24 hours after the injection is less than 20%, preferably less than 15%, more preferably less than 10% and most preferably less than 5%, of the total release.

According to another aspect of the present invention, it provides a microparticle preparation of the above-mentioned type, which during the first 48 hours after injection shows a release of the active substance where the maximum concentration in plasma or serum is less than 300% of the maximum concentration. of the biologically active substance at any time exceeding 48 hours after injection.

Anno »10 15 20 25 30 35 5 17 6 1 0 gi: 16 Said maximum concentration is preferably less than 200% and more preferably less than 100% of the maximum concentration in question.

A further aspect of the invention is represented by a microparticle preparation of the above-mentioned kind, which shows a release of the biologically active substance where the bioavailability of said substance is at least 35% of the bioavailability obtained when the substance is injected intravenously in soluble form.

Said bioavailability is preferably at least 45%, more preferably at least 50%, of the bioavailability obtained when the biologically active substance is injected intravenously.

Another aspect of the present invention is represented by a microparticle preparation of said kind, which exhibits a release of the active substance characterized in that in the release which takes place during any continuous seven-day period, the ratio between the highest concentration of the biologically active the substance in serum or plasma divided by the mean concentration during the said seven-day period less than 5, provided that the selected seven-day period does not include the first 24 hours after injection.

Said release is preferably less than 4 times, more preferably less than 3 times and most preferably less than 2 times.

According to a further aspect, the invention provides a microparticle preparation of the type indicated, which exhibits a release of the biologically active substance where the average residence time of the substance in question is at least 4 days.

Preferably, said average residence time is at least 7 days, more preferably at least 9 days, e.g. at least 11 days or especially at least 13 days.

The features which have been set forth for the aspects of the invention presented above may be combined in any suitable combinations. 1: --- 10 15 20 25 30 35 17 For any of these aspects of the invention, it is preferred that the biologically active substance is a protein, a peptide or a polynucleotide. Most preferably it is a protein, e.g. a recombinantly produced protein.

Interesting groups of substances in question are hormones, especially human growth hormone, or a growth factor.

More specifically, according to a preferred embodiment of the invention, the preparation contains a substance selected from growth hormone, insulin, erythropoietin, interferon α, interferon β, interferon γ, Factor VII, Factor VIII, glucagon-like peptide 1 or 2 and C-peptide. .

For the various characteristics that have been stated for the microparticle preparation according to the invention, it is more specific that these are primarily the concepts of MRI, burst and bioavailability.

These can be defined according to the following MRI One goal of preparations for controlled release is to obtain an extended release of the active material. One measure that can be used to quantify the release time is mean residence time (MRT), which is the accepted concept in pharmacokinetics.

MRI is the average time that the molecules introduced into the body stay in the body. (Clinical Pharmacokinetics. Concepts and Applications. Malcolm Rowland and Thomas N. Tozer. 2 “ed. Lea & Febiger, Philadelphia London) The MRI value can be calculated from plasma concentration data by the following formula. .frun 10 15 20 25 30 517 610 -a saw 18 00 J: Cdr MRT = ° OO j Cd: o where C is the plasma concentration and t is the time Bëšâš A common problem with controlled release preparations for parenteral use is that immediately after administration in the body releases a large part of the drug during the early phase. This is referred to in the professional literature as a “burst effect”. This is usually due to the drug being on the surface of the formulation or the formulation (which may consist of microparticles) cracking. A low brush effect is very desirable because a high concentration of the drug can be toxic and in addition the part that disappears quickly in the first time is poorly utilized, which means that more drugs are required to maintain a therapeutic level of the drug during the intended treatment time.

Burst is defined as the proportion of the drug that is absorbed during the first 24 hours of the total proportion that is absorbed.

Mathematically, it can be defined using "area under curve" calculations from plasma concentration curves. 24h j Cd: Burst = 0 0 100% w I Cd: o Bioavailability Bioavailability is a measure of how much of the administered drug is absorbed from the site of administration to the blood in active form. Bioavailability is often compared with data from intravenous drug delivery; 10 15 20 25 30 35 517 610 19 let, where one thus has no absorption barriers, and is then called absolute bioavailability, Absolute bioavailability is defined according to the following formula: __A (Å: x- [hv _kDx-AÉKQV where AUC xär area-under -curve the value of the formulation tested, AUCiv is the area-under-curve value of an intravenous administration of the drug, DX is the dose of the drug in the formulation and Dh is the intravenous dose.

The determination of the release profile and the pharmacokinetic parameters is preferably performed by animal experiments. The most relevant species, due to its resemblance to humans, is the pig. In cases where the biologically active substance can induce an immune response during the test that risks affecting the determination of the pharmacokinetic parameters of the biologically active substance, inhibition of the immune response should be used, e.g. by drug treatment, which is known in the art, and details can be obtained from the scientific literature, for example Agersö et al, (J. Pharmacol Toxicol 41 (1999) 1-8). The microparticle preparation according to the invention can be prepared by a method, which comprises a) preparing an aqueous starch solution comprising starch having an amylopectin content exceeding 85% by weight, wherein the molecular weight of said amylopectin is reduced, preferably by shear, so that at least 80% by weight of the material is in the range of 10-10000 kDa, and having an amino acid nitrogen content of less than 50 pg per g dry weight of starch, the starch concentration of the solution being at least 20% by weight, b) combining the biologically active substance with the starch solution under such conditions that a composition is formed in the form of a solution, emulsion or suspension of said substance in the starch solution, ~ w | zn 10 15 20 25 30 35 5 17 6 1 0 gi: 20 c) mixes the composition obtained in step b) with an aqueous solution of a polymer capable of forming a two-phase aqueous system, so as to form an emulsion of starch droplets, which contain the biologically active substance, as the inner phase in an outer phase of said poly additional solution, where the starch droplets suitably have the desired size for the microparticles; d) cause the starch droplets obtained in step c) to gel to starch particles via the starch's natural ability to solidify, e) dry the starch particles, and f) optionally apply a bioregulation. biodegradable polymer, preferably by air suspension technology, on the dried starch particles.

An important aspect in connection with this process is, in other words, that a certain kind of starch is used as the microparticle matrix. A starch which is particularly suitable, as well as a process for its preparation, is carefully described in the Swedish patent application which has the same filing date as the present patent application and which has the title Pharmaceutically acceptable starch. Details regarding this can in other words be obtained from the said Swedish patent application, the content of which in this respect is hereby incorporated into the present text via reference.

However, the main features of such starch will be described below. In order for completely biodegradable microparticles with a high yield of the active substance to be formed in a two-phase aqueous system and for the obtained starch microparticles to have the properties which will be described below, the starch must generally consist predominantly of highly branched starch. in the natural state of the starch granule is called amylopectin. It must also have a molecular weight distribution that makes it possible to achieve the desired concentrations and gelation rates. other 10 15 20 25 30 35 21 In this context, it can be added that the term biodegradable means that the microparticles, after parenteral administration, dissolve in the body into the body's own substances, ultimately glucose. The biodegradability can be determined or examined by incubation with a suitable enzyme, e.g. alpha-amylase, in vitro. It is advisable to add the enzyme several times during the incubation period, so as to ensure that there is active enzyme present in the incubation mixture at all times. The biodegradability can also be examined by parenteral injection of the microparticles, e.g. subcutaneous or intramuscular, and histological examination of the tissue as a function of time. Biodegradable starch microparticles normally disappear from the tissue within a few weeks and usually within a week. In cases where the starch microparticles are coated with a release-regulating coating, e.g. coated, it is usually this envelope that determines the rate of biodegradability, which in turn determines when alpha-amylase becomes available to the starch matrix.

Biocompatibility can also be investigated by parenteral administration of the microparticles, e.g. subcutaneously or intramuscularly, and histological evaluation of the tissue, it being important to note that the biologically active substance, which is often a protein, itself has the ability to induce e.g. an immune system in case it is administered in another species. Thus, for example, many recombinantly produced human proteins can elicit immune responses in experimental animals.

The starch must also have a purity that is acceptable for the manufacture of a parenterally administrable preparation. It must also be able to form sufficiently stable solutions at a sufficiently high concentration to enable admixture of the biologically active substance under conditions which allow the bioactivity of the substance to be maintained, while at the same time it must be able to spontaneously solidify in a controlled manner to co- s. v ~ u .. 10 15 20 25 30 35 517 610 22 early biodegradable, microparticles must be obtained. High concentration is also important to prevent the biologically active substance from being distributed unacceptably to the outer phase or to the interface between the inner and outer phases.

A number of different embodiments with respect to the nature of the starch are as follows.

The starch preferably has a purity of at most 20 pg, more preferably at most 10 pg and most preferably at most 5 pg, amino acid nitrogen per g of dry weight starch.

The molecular weight of the above-mentioned amylopectin is preferably reduced so that at least 80% by weight of the material is in the range 100-4000 kDa, more preferably 200-1000 kDa and most preferably 300-600 kDa.

The starch further preferably has a content of amylopectin with the reduced molecular weight in excess of 95% by weight, more preferably in excess of 98% by weight. In addition, it can of course consist of 100% by weight of such amylopectin.

According to another variant, the starch is of such a nature that it can be dissolved in water in a concentration exceeding 25% by weight. This generally means the ability to dissolve in water in accordance with the technique known per se, ie usually dissolution at elevated temperature, for example up to approximately 80 ° C.

According to another variant, the starch lacks covalently bonded extra chemical groups of the kind found in hydroxyethyl starch. By this is generally meant that the starch contains only those groups which are present in natural starch and have not been modified in any other way, such as in hydroxyethyl starch, for example.

Another variant means that the starch has an endotoxin content of less than 25 EU / g.

Another variant means that the starch contains less than 100 microorganisms per gram, often even less than 10 microorganisms per gram. The starch can be further defined as being purified from surface localized proteins, lipids and endotoxins by washing with aqueous alkali solution, molecular weight reduced by shear and purified from internal proteins by ion exchange chromatography, preferably anion exchange chromatography.

The fact that amylopectin constitutes the main constituent of the starch used generally means that its proportion is 60-100% by weight, calculated on dry weight starch.

In some cases, it may be beneficial to use a smaller proportion, e.g. 2-15% by weight, short chain amylose to modify the gelation rate in step d). The average molecular weight of said amylose is preferably in the range 2.5-70 kDa, especially 5-45 kDa. Other details regarding short-chain amylose can be found in U.S. Patent No. 3,881.99l.

In the formation of the starch solution in step a), heating is generally used according to the technique known per se for dissolving the starch. A particularly preferred variant here also means that the starch is dissolved during autoclaving, whereby it is also preferably sterilized. This autoclaving is carried out in aqueous solutions, e.g. water for injection or suitable buffer.

If the biologically active substance is a sensitive protein or other temperature sensitive substance, the starch solution must cool to a suitable temperature before it is combined with the substance in question. Which temperature is suitable is determined primarily by the thermal stability of the biologically active substance, but in general a temperature below about 60 ° C, even more preferably below 55 ° C, is suitable.

According to an alternative, the active substance is therefore combined with the starch solution at a temperature of at most 60 ° C, more preferably at most 55 ° C, and preferably in the range 20-45 ° C, especially 30-37 ° C. For the mixing operation in step b) it is furthermore appropriate to use a weight-ratio starch biologically active substance in the range from 3: 1 to 100: 1.

For the mixing operation, it also applies that the active substance is mixed with the starch solution before a two-phase aqueous system is formed in step c). The active substance may then be in loose form, e.g. in a buffer solution, or in solid, amorphous or crystalline form and at a suitable temperature, which is usually between room temperature (20 ° C) and 45 ° C, preferably not more than 37 ° C.

It is possible to add the starch solution to the biologically active substance or vice versa. Then the biologically active substances that are suitable for use in this system, e.g. proteins, usually macromolecules, when a solution of a dissolved macromolecule with starch is also mixed, for example, an emulsion can be formed, where the macromolecule usually represents the inner phase, or a precipitate. This is perfectly acceptable, provided that the biologically active substance retains or does not significantly lose its bioactivity.

Thereafter, a homogeneous solution, emulsion or suspension is created, by stirring, which can be done by suitable techniques. Such a technique is well known in the art, for example magnetic agitation, propeller agitation or the use of one or more static mixers. An especially preferred embodiment of the invention is represented in this case by the use of at least one static mixer.

Thus, in the preparation of the starch microparticles, the concentration of starch in the solution to be converted into solid form, in which the biologically active substance is to be incorporated, must be at least 20% by weight in order to form starch microparticles having the proper properties. Exactly which starch concentration works best in each individual case can be easily titrated out for each individual biologically active substance at the charge in the microparticles desired in the individual case. It should be noted in this context that the biologically active substance to be incorporated into the microparticles can affect the two-phase system and the gelling properties of the starch, which also means that customary experiments are carried out in order to determine the optimal conditions in the individual case. Most often, experiments show that the starch concentration should advantageously be at least 30% by weight and in some special cases at least 40% by weight. especially not more than 45% by weight.

The maximum limit is usually 50% by weight. It is not normally possible to obtain these high starch concentrations without the use of molecular weight-reduced, highly branched starch.

With regard to the polymer used in step c) for the purpose of forming a two-phase water system, information on a large number of polymers capable of forming two-phase systems with starch as internal phase is published in this particular field of technology. All such polymers may be considered useful in the present context. However, a particularly suitable polymer is polyethylene glycol. Preferably, this polyethylene glycol has an average molecular weight of 5-35 kDa, more preferably 15-25 kDa and especially about 20 kDa.

The polymer is dissolved in a suitable concentration in aqueous or aqueous solution, which term also includes buffer solution, and tempered to a suitable temperature. This temperature is preferably in the range 4-50 ° C, more preferably 10-40 ° C and usually 10-37 ° C.

The concentration of the polymer in the aqueous solution is suitably at least 20% by weight and preferably at least 30% by weight, and preferably at most 45% by weight. An especially preferred range is 30-40% by weight.

The mixing operation in step c) can be performed in many different ways, e.g. by using propeller agitator or at least one static mixer. The mixing is normally carried out in the temperature range 4-50 ° C, preferably 20-40 ° C, often about 37 ° C. In a batch process, the starch solution may be added to the polymer solution or vice versa. In case static mixers or mixers are used, the operation is conveniently carried out by pumping the two solutions in two separate pipelines into a common pipeline containing the mixers.

The emulsion can be formed using low shear forces, when there is no high surface tension unlike oil / water or water / oil emulsions, and then between the phases in water / water emulsions, it is primarily the viscosity of the starch solution that must be overcome for that the drops should reach a certain size- In most cases, it is sufficient to stomach- e.g. when the amount of microparticles to be produced exceeds the distribution. net or propeller agitation. On a larger scale, 50 g, it is convenient to use so-called baffles to obtain an even more efficient agitation in the used container. An alternative way of forming the water / water emulsion is to use at least one static mixer, the starch solution being suitably pumped at a controlled rate into a tube in which the static mixers have been placed. The pumping can be done with any suitable pump, provided that it provides a uniform flow rate under these conditions, does not subject the mixture to unnecessarily high shear forces and is acceptable for the manufacture of parenteral preparations in terms of purity and absence of unwanted substances. Even in cases where static mixers are used to create the emulsion, it is often advantageous to allow the solidification to microparticles to take place in a vessel with suitable stirring.

A preferred embodiment of the process according to the invention means that in step c) the polymer solution is added to the composition in at least two steps, where an addition takes place after the emulsion has been created or has begun to be created.

Of course, it is also within the scope of the present invention to add the polymer solutions in several steps and thereby, for example, to change the average molecule 10 15 20 25 30 35 1 noo ~ 1 c nun n coon Q couuu .no no un.- 517 610 f The weight and / or concentration of the polymer used, e.g. to increase the concentration of starch in the internal phase, where this is desired.

Important for the preparation of the microparticles according to the present invention is that the solidification takes place via the starch's natural tendency or ability to gel, and not by, for example, precipitation with organic solvents, such as acetone. The latter procedure can thus lead to the biologically active substance being exposed to organic solvent, which in many cases is unacceptable, and to the natural formation of the physical crosslinks required for a regulated ways to obtain stable microparticles are absent.

It is also important in connection with the solidification of the microparticles that this takes place under conditions that are mild for the constituent biologically active substance, or the substances. In other words, it is primarily a question of using a temperature that is not harmful to the substance present. It has surprisingly been found that the criteria for this and for the formation of stable microparticles with a suitable size distribution can be more easily met if more than one temperature level is used during the solidification. It is especially advantageous if the solidification process is initiated in the two-phase system at a lower temperature than the temperature used in the final phase of the solidification. A preferred embodiment involves starting the solidification in the range 1-20 ° C, preferably 1-10 ° C, especially around 4 ° C, and ending the same in the range 20-55 ° C, preferably 25-40 ° C , A confirmation that the selected conditions are in particular around 37 ° C. direct or suitable can be obtained by stating that the starch microparticles have the desired size distribution, are stable during the subsequent washing and drying operations and dissolve essentially completely enzymatically in vitro and / or that the incorporated substance has been encapsulated in an effective way and have bi- .unc. n q n nn .. _ ... v uu. 10 15 20 25 30 35 517 610 28 retain bioactivity. The latter are usually examined by chromatographic methods or by other methods established in the art, in vitro or in vivo, after dissolving the microparticles enzymatically under mild conditions, and are an important part of ensuring a robust and safe manufacturing process for sensitive biologically active substances. It is a great advantage that the microparticles can be completely dissolved using mild conditions, as this minimizes the risks of preparation-induced artifacts, which are common when, for example, organic solvents are needed to dissolve the microparticles, which is the case, for example, when these consist of a PLGA matrix.

The microparticles formed are preferably washed in a suitable manner, to remove the outer phase and of excess active substance. Such washing is conveniently effected by filtration, which is made possible by the good mechanical stability of the microparticles and the appropriate size distribution. Washing by centrifugation, removal of the supernatant and resuspension in the washing medium can also often be appropriate. Each washing procedure uses one or more suitable washing media, which are usually buffered aqueous solutions. In connection with this, screening can also be used, if necessary, to adjust the size distribution of the microparticles, for example to eliminate the content of too small microparticles and to ensure that no microparticles above a certain size are present in the finished product.

The drying of the microparticles can take place in any suitable way, e.g. by spray drying, freeze drying or vacuum drying. The drying method chosen in the individual case often depends on what is most suitable for maintaining the biological activity of the entrapped biologically active substance. Process considerations also come into play, such as capacity and purity .. .. vv .. w.- ~ -.... 10 15 20 25 30 35 517 610 non now 29 heat aspects. Freeze-drying is often the preferred drying method, as it, when properly designed, is extremely mild with respect to the entrapped biologically active substance. The fact that the incorporated biologically active substance has retained its bioactivity can be determined by means of an analysis suitable for the substance after enzymatic dissolution of the substance under mild conditions. Suitable enzymes for use in connection with starch are alpha-amylase and amyloglucosidase, individually or in combination, in which case they should have been found to be free from any proteases which may degrade proteins. The presence of proteases can be detected by methods known in the art and, for example, by mixing the biologically active substance in control experiments and determining its integrity in the usual way after incubation with the intended enzyme mixture under the conditions which will then be used. inverted to dissolve the microparticles. In cases where the preparation is found to contain contamination of proteases, it can be replaced with a preparation of higher purity or purified from proteases. This can be done with techniques known in the art, for example by chromatography with az macroglobulin bound to the appropriate chromatography material.

The enzymes used may need to be purified from e.g. contaminating proteases to avoid artifactic degradation of sensitive substances incorporated into the microparticles, e.g. recombinant proteins. This can be achieved by techniques known in the art, e.g. by chromatography with fibronectin bound to a suitable chromatography material.

In order to modify the release properties of the microparticles, it is also possible to apply a release-regulating coating of a biocompatible and biodegradable polymer. Examples of suitable polymers in this context can be found in the prior art, and in particular polymers of lactic acid and glycolic acid (PLGA) can be mentioned. The application of the casing in question is preferably carried out with the aid of air suspension technology. A particularly suitable such technique is described in WO97 / 14408, and details in this regard can thus be taken from this document, the contents of which are hereby incorporated by reference into the text. The starch microparticles which are obtained by the method of the present invention are extremely well suited for coating or coating by means of said air suspension techniques, and the obtained coated microparticles are particularly well suited for parenteral administration.

When using the microparticles prepared, whether or not they are coated with a release-regulating outer shell, and especially to enable injection, the dry microparticles are suspended in a suitable medium. Such media and procedures in these respects are well known in the art and need not be described further here. The injection itself can be done through a suitable needle or with a needle-free injector. It is also possible to inject the microparticles with the aid of a dry powder injector, without first resuspending them in an injection medium.

In addition to the advantages mentioned above, the above-mentioned process has the advantage that the yield of the biologically active substance is generally high, that it is possible to obtain a very high content of the active substance in the microparticles while maintaining the bioactivity of the substance. the resulting microparticles have the right size distribution for use in parenteral, controlled (eg delayed or prolonged) release, as they are too large to be phagocytosed by macrophages and small enough to be injected through small needles, e.g. 23G-25G, and that during the decomposition of the microparticles, body-specific and neutral decomposition products are formed, whereby, for example, it is possible to avoid exposing the active substance to an excessively low pH value. In addition, the process itself is particularly well suited for rigorous quality control. ua .. u 10 15 20 25 30 35 31 The preparation according to the invention is of particular interest in connection with proteins, peptides, polypeptides, polynucleotides and polysaccharides or generally other drugs or biologically active substances which are sensitive to or unstable in for example, organic solvents. Recombinantly produced proteins are a very interesting group of biologically active substances. In general, however, the invention is not limited to the presence of such substances, since the idea of the invention is applicable to any biologically active substance which can be used for parenteral administration. Thus, except in connection with sensitivity or instability problems, the invention may also be of particular interest in cases where it would otherwise be difficult to remove solvents or where toxicological or other environmental problems could occur.

Examples of biologically active substances of the type indicated above are growth hormone, erythropoietin, interferon (a, ß, y-type), vaccine, epidermal growth hormone, Factor IV, V, VI, VII, VIII and IX, LHRH analog, insulin , macrophage colony stimulating factor, granulocyte colony stimulating factor and interleukin.

Useful biologically active substances of the non-protein drug type can be selected from the following groups: Antitumor agents, antibiotics, anti-inflammatory agents, antihistamines, sedatives, muscle relaxants, antiepileptic agents, antidepressants, antiallergic agents, bronchodilators, cardiotonic agents, antiarrhythmic agents, vasodilators , antidiabetic agents, anticoagulants, hemostatic agents, narcotics and steroids.

However, the new microparticle preparation according to the invention is not limited to those which can be prepared by the above-mentioned process, but comprises all microparticle preparations of the type in question, regardless of the production methodology. avv- u »nunn 10 15 20 25 30 35 517 610 22"; 32 In addition, microparticles may consist essentially of starch having a content exceeding 85% by weight of amylopectin, for which at least 80% by weight has an average molecular weight within in the range of 10-1,000 kDa, which have an amino acid content of less than 50 pg per dry weight of starch and which lack covalent chemical crosslinking between the starch molecules.

The starch on which the microparticles in question are based is preferably one of the starch varieties defined above in connection with the process.

According to a variant of the microparticles, these are those in which the bioactivity of the biological substance is at least 80%, preferably at least 90%, of the bioactivity which the substance showed before it was incorporated into the starch. Most preferably, said bioactivity is mainly retained or preserved in the microparticles. Yet another variant is represented by microparticles which are biodegradable in vitro in the presence of alpha-amylase and / or amyloglucosidase.

Another variant is represented by those which are biodegradable and eliminated from tissue after subcutaneous or intramuscular administration.

An especially preferred variant of the microparticles is represented by particles having a release-regulating envelope of at least one film-forming, biocompatible and biodegradable polymer.

This polymer is preferably a homo- or copolymer made of alpha-hydroxy acids and / or cyclic dimers of alpha-hydroxy acids, said alpha-hydroxy acid being preferably selected from the group consisting of lactic acid and gluconic acid, and wherein the cyclic dimer is preferably selected from the group consisting of glycolides and lactides.

Such polymers or dimers (for example of the PLGA type) are carefully described per se in the prior art, so further details regarding such can be obtained from it. »In addition 10 15 20 25 30 35 33 A further preferred variant of the microparticles is of course represented by such microparticles which are manufacturable, or produced, by a process as defined above, either generally or in the form of any preferably a preferred embodiment of said method.

With regard to the determination of the biological activity for the microparticles containing active substance, it applies that this may take place in a manner suitable for each individual biological substance. In cases where the determination takes place in the form of animal experiments, a certain amount of the biologically active substance incorporated into the starch microparticles is injected, possibly after dissolution of these microparticles enzymatically under mild conditions in advance and the biological response is compared with the response given. obtained after injection of the corresponding amount of the same biologically active substance in a suitable solution. In cases where the evaluation takes place in vitro, for example in test tubes or in cell culture, the biologically active substance is preferably made fully available before the evaluation by dissolving the starch microparticles enzymatically under mild conditions, after which the activity is determined and compared with the activity of a control solution with the same concentration of the biologically active substance in question. In any case, the evaluation must include any non-specific effects of the degradation products of the starch microparticles.

The invention will now be further illustrated by the following non-limiting examples. For these, as for the rest of the text, the stated percentages refer to% by weight, unless otherwise stated. Examples 1-7 relate to comparative experiments, while Examples 8-15 represent the invention.

EXAMPLES Examples la - le-b Control experiments: procedure for the preparation of starch microspheres according to EP 213303 A2. The purpose of these 10 15 20 25 30 35 517 610 34 Control experiments was to show the current state of the art. The starch concentration was generally 5%, and (average molecular weight 6 kDa). was poured into the PEG solution (5g, the PEG concentration was 6% (log) tempered to 70 ° C) The starch solution and stabilized with stirring at room temperature overnight. The materials were then resuspended in 95% ethanol, as they could not be filtered. During the various steps, the presence of discrete microspheres was observed and, where possible, the biodegradability was examined in vitro with α-amylase after reprocessing. Initially, attempts were made to work up the microspheres by filtration, but when this was not possible, centrifugation (Sorfvall, SS34, 5 min, 10,000 rpm 20 ° C), suction of the supernatant and addition of 10 ml of 95% ethanol were used.

Example la Manufacture of potato starch starch microspheres.

The potato starch (Acros organics, Lot AOl364230l) formed a very highly viscous clear solution already at 5%.

After stabilization overnight, discrete microspheres had not formed without any kind of precipitation. After washing with 95% ethanol, no discrete starch microspheres were found, but a rather hard and tough lump.

Examples 1a-b Manufacture of starch microspheres containing BSA.

Starch microspheres were prepared according to Example 1a, with the difference that a protein (bovine serum albumin (BSA), 20%, the creation of the two-phase system. After stabilization over 0.1 ml) was mixed with the starch solution before night, discrete microspheres had not formed but some precipitation and after washing with 95% ethanol, a tough lump was obtained and no discrete microspheres.

Example lb Manufacture of soluble starch microspheres. .av lO 15 20 25 30 35 517 610 z Ia. The soluble starch (Baker BV - Deventer Holland, Lot No M27602) gave a slightly opalescent solution when heated to 95 ° C. No discrete microspheres had formed after stirring overnight without any precipitation. After washing with 95% ethanol, discrete microspheres could not be observed but the starch had precipitated in the form of small gravel-like particles. After drying, about 4 mg of particles of a total of 500 mg of starch were obtained. Upon incubation with α-amylase in vitro, approximately 65% of the starch matrix was resistant and did not dissolve.

Examples lb-b Incorporation of BSA into starch microspheres made from soluble starch.

The procedure of Example 1b was repeated except that BSA (20%, 0.1 ml) was mixed with the starch solution before the creation of the two-phase system. After overnight stabilization, discrete particles had formed, which were worked up, after which the biodegradability was examined in vitro by incubation with α-amylase, with the result that approximately 57% of the matrix was soluble. The yield of the protein was low and could not be quantified as the concentration obtained after the partial dissolution of the microspheres was lower than the lowest standard in the standard curve for the HPLC method.

Example 1c Manufacture of starch microspheres from oxidized soluble starch.

The starch (Perfectamyl A3108, Stadex) formed a clear solution after heating to 95 ° C. No discrete microspheres had formed after stabilization overnight and no solid material could be found in the sample at all.

Example lc-b: | »ø | 10 l5 20 25 30 35 517 610 36 Incorporation of BSA into starch microspheres made from soluble starch.

The procedure of Example 1c was repeated, except that BSA (20%, 0.1 ml) was mixed with the starch solution before the creation of the two-phase system. After stabilization, a precipitate which did not resemble starch was observed and after washing and drying about 2 mg of solid material were obtained.

Example ld Production of starch microspheres from native highly branched starch (amylopectin).

The native amylopectin starch (Cerestar SF 04201) gave a clear and viscous solution when heated to 95 ° C.

After stirring overnight, no discrete microspheres could be observed, but the sample consisted of some kind of precipitate. After washing with 95% ethanol, the sample had become a slimy mass and contained no discrete starch microspheres.

Example ld-b Incorporation of BSA into starch microspheres made from native highly branched starch (amylopectin).

The procedure of Example 1d was repeated, except that BSA (20%, 0.1 ml) was mixed with the starch solution before the creation of the two-phase system. After stabilization, a precipitate that did not resemble starch was observed and after washing and drying, a tough lump was obtained.

Example le Manufacture of starch microspheres from acid hydrolyzed and sheared amylopectin.

The starch originally consisted of acid hydrolyzed waxy maize (Cerestar 06090) and was also mechanically sheared to give a molecular weight distribution which is better suited for the manufacture of starch microspheres in two-phase aqueous systems. After heating to about 95 ° C, a clear solution was obtained. After stirring overnight, no discrete microspheres could be observed but the sample consisted of some kind of precipitate. After washing with 95% ethanol, no discrete microspheres could be observed but the starch had formed small particles.

Example 1e-b Incorporation of BSA into Starch Microspheres Made of Acid Hydrolyzed and Sheared Starch (Amylopectin) The procedure of Example 1e was repeated except that BSA (20%, 0.1 ml) was mixed with the starch solution prior to the formation of two phase phases. the system. After stabilization overnight, no discrete microspheres could be observed. On the other hand, an extremely small precipitation could be observed. After processing, the amount obtained was so small that no exact determination could be made.

Example 2 Preparation of starch microspheres of amylose.

Starch microspheres were made from amylose (from Serva) in order to investigate their biodegradability in vitro and in vivo. Since amylose gels are too fast for manual production to be possible, a machine was used that enables continuous production. The central unit in the machine is a microwave unit that enables rapid heating of the starch to about 150 ° C, followed by cooling to about 45 ° C before mixing with protein or buffer solution. The starch solution was prepared in distilled water as it would otherwise turn brown on heating in the microwave unit. The starch consisted of pregelatinized amylose from pea (4%) and despite repeated homogenization, several lumps remained. The flows were adjusted according to: starch (5 ml / min), Tris buffer, pH 7.8 (1.2 ml / min) and the PEG solution (2.4% by weight PEG with an average molecular weight of 300000 Da) which also contained 6, 9% sodium chloride and 14.3% nitrile. The microparticles were allowed to stabilize at room temperature for a few hours and then in the refrigerator overnight. They were worked up by the following centrifuge washes: three times with 70% ethanol, three times with 5 mm phosphate buffered saline containing 1 mM calcium chloride and 0.02% sodium azide, pH 7.4), and finally 99.5% ethanol three times. The microparticles were dried in vacuo. Since there were some very small particles in the preparation, they were tried to be removed by sedimentation in 99.5% ethanol three times, which did not lead to their complete removal. A total of 9.5 g of microparticles was obtained and after the sedimentation 8.5 g remained.

The particle size distribution was determined with a Malvern Mastersizer and was found to be wide, with the smallest microspheres around 5 μm and the largest over 160 μm. The mean diameter calculated from the volume was 25 μm. Approx. 30-40% of the starch matrix was dissolved when the microspheres were incubated with α-amylase at 37 ° C in vitro for two weeks.

This comparative example shows the difficulties in making microspheres from amylose due to the fact that it is difficult to prepare homogeneous solutions of, requires very high temperatures to dissolve and is sensitive to degradation during exposure to these high temperatures, and gels very quickly. The example also shows that the resulting microspheres have too wide a size distribution, both immediately after manufacture and after attempts to narrow the size distribution, and an excessive content of microspheres with a size of less than 10 μm to be well suited for extended release. after injection subcutaneously or intramuscularly. The example also shows that the biodegradability, examined in vitro, is not complete during the examined period of time.

Example 3 Preparation of starch microspheres containing β-lactoglobulin. 10 15 20 25 30 35,. n .. nu 517 610 39 Starch microspheres were made from amylose (Serva) to investigate the effectiveness of immobilization of a model protein, β-lactoglobulin. As this amylose gels too fast for fully manual manufacture to be possible, a machine with a microwave unit is used which allows rapid starch heating to about 150 ° C, followed by cooling to about 45 ° C before mixing with protein or buffer solution. The starch solution (10%) was prepared in distilled water and the flow was set at 5 g / minute.

The starch solution (2.5 ml) was collected in a plastic beaker and when the temperature dropped to 50-70 ° C (0.6 ml, 8.3% in buffer) was added thereto almost immediately thereafter (10 ml). %, average molecular weight 20,000 3.0 ml) to the starch solution with stirring and the protein solution with magnetic stirring. the first PEG solution Da was added, after stirring for one minute, the second PEG solution (40%, average molecular weight 20,000 Da, which was left to stabilize at room temperature to the next 10 ml) was added to the emulsion day. The microspheres formed were processed in two different ways to show their ability to maintain an adequate protein charge. The first washing methodology consisted of centrifugal washes with buffer solutions and resulted in virtually all protein leaking out of the starch microspheres. In the second reprocessing method, isopropanol was also used to increase the charge of lactoglobulin in the microspheres. In this was washed first three times with 70% isopropanol, then once with 5 mM phosphate buffered saline solution containing 1 mm calcium chloride and 0.02% sodium azide, then once more with the same buffer leaving them to stand in the buffer under a hour with rocking, then 5 times with buffer and finally 3 times with 100% isopropanol. The microspheres were then dried in vacuo. The protein loading using the isopropanol wash was 3.6%, which corresponds to a theoretical yield of 18%.

The example shows i.a. on significant practical difficulties in making microspheres containing protein from goods; 10 15 20 25 30 35 51 7 61 0 40 amylose when the starch solution must be exposed to very high temperatures to dissolve completely, as the starch changes easily chemically at these high temperatures and gels very quickly after cooling to temperatures which are reasonable low so that the starch solution can be mixed with a sensitive protein. The manufacture is further complicated by the need to use two different solutions of PEG to enable the formation of microspheres and the capture of the protein in them. An additional serious disadvantage is the need to use isopropanol in the reprocessing of the microspheres in order to obtain an acceptable protein charge as most sensitive proteins cannot be exposed to this or similar organic solvents. Starch microspheres made from this amylose are not completely biodegradable by æ amylase in vitro nor in vivo.

Example 4 Preparation of amylose from pea.

Amylose was prepared by leaching from starch granules. NUTRIO-P-star 33 (300 g, Nordfalk) was suspended in water (7200 g) and the suspension was heated to 75 ° C for one hour. The swollen granules were removed by centrifugation and the resulting solution was filtered through first a carbon filter, then a pre-filter (Filtron, 1.5 μm) and finally a sterile filter (Millipore, 0.2 μm).

The filtered solution was placed in a refrigerator overnight, whereupon the amylose precipitated and was recovered by centrifugation. The resulting precipitate was washed twice with ethanol (95%, 700 ml) and then dried under vacuum. About 30 g of amylose were obtained.

Example 5 Starch microspheres were made from amylose prepared according to Example 4 in order to investigate their biodegradability in vitro and in vivo. When amylose gels too fast for manual manufacture to be possible, a machine was used which enables continuous manufacture.

The central unit of the machine is a microwave unit that allows the starch to heat up rapidly to about 150 ° C, followed by cooling to around 45 ° C before mixing with protein or buffer solution. The starch solution was prepared in distilled water as it would otherwise turn brown on heating in the microwave unit. The starch consisted of pregelatinized amylose from pea (4%) and despite repeated homogenization, several lumps remained. The flows were adjusted according to: starch (5 ml / min), Tris buffer, pH 7.8 (1.2 ml / min) and the PEG solution (2.4% by weight PEG with an average molecular weight of 300,000 Da) which also contained 6 , 9% sodium chloride and 14.3% mannitol. The microparticles were allowed to stabilize at room temperature for a few hours and then in the refrigerator overnight. They were worked up by the following centrifuge washes: three times with 70% ethanol, three times with 5 mm phosphate buffered saline containing 1 mm calcium chloride and 0.02% sodium azide, pH 7.4, and finally 99.5% ethanol three times. The microparticles were dried in vacuo. As there were some very small particles in the preparation, these were attempted to be removed by sedimentation in 99.5% ethanol three times, which did not lead to their complete removal. The yield was 8.5 g of particles after sedimentation, during which 1 g of microspheres were purified.

The particle size distribution was determined with a Malvern Mastersizer and was found to be wide, with the smallest microspheres around 5 μm and the largest over 160 μm. The mean diameter calculated from the volume was 25 μm.

The biodegradability of the starch microspheres was examined by incubation with u-amylase in vitro. Initially, the degradation was rapid and after one day about 35-45% had been transferred in soluble form. Thereafter, the rate of degradation decreased and after 6 days approximately 50% had dissolved. Thereafter, biodegradability was negligible and after 25 days, approximately 50% of the starch microspheres remained in undissolved form. 42 This comparative example shows the difficulties in making microspheres from amylose due to the fact that it is difficult to prepare homogeneous solutions of, requires very high temperatures to dissolve and is sensitive to degradation during exposure to these high temperatures, and gels a lot. fast. The example also shows that the resulting microspheres have too wide a size distribution, both immediately after manufacture and after attempts to narrow the size distribution, and an excessively high content of microspheres with a size of less than 10 μm to be well suited for elongation. release after injection subcutaneously or intramuscularly. The example also shows that the biodegradability, examined in vitro, is not complete. Example 6 Investigation of the biodegradability of starch microspheres made from amylose in vivo Starch microspheres (3.0 μmg) made from the amylose-2O in Example 4 were injected subcutaneously into the neck in a volume of 10 10 ul on eight rat phraophysectomies. Small lumps could be felt under the skin of all rats at the injection sites 8-9 days after injection. At dissection 9 days after injection, a 5.3 macroscopic inspection was performed and small white cysts were found at the injection site in all rats. The macroscopic changes were fixed in 4% phosphate-buffered formaldehyde, embedded in paraffin, sectioned with a nominal thick HH. 5 μm, stained with hematoxylin and eosin, and examined under a light microscope. In the paraffin sections where the starch microspheres could be found, they were seen as eosinophilic small spheres surrounded by a zone of granulating tissue containing giant cells and the tissue reaction was characterized as a chronic "granulomatous" inflammation in the subcutaneous tissue. Starch microspheres could also be observed inside macrophages. The experiment shows that starch microspheres made of amylose are found in the subcutaneous tissue 9 days after injection and thus have not been biodegradable enough to have dissolved during that time, and that at least a proportion of the microspheres were small enough for to be able to be phagocytosed by macrophages.

Example 7 Investigation of the biodegradability of starch microspheres made from amylose in vivo.

Starch microspheres made from amylose according to Example 4 and suspended in 10 ml of phosphate buffered (5 mM) physiological saline (0, 15 M), pH 7.2. was injected intramuscularly into the pig thigh. Two pigs were dosed with 120 mg microspheres and two pigs with 600 mg microspheres. The animals were sacrificed on day 14 and the tissue was examined for macroscopic changes and, following the usual embedding and staining procedure, for microscopic changes. On day 14, microspheres 20 were found in the tissue in a dose-dependent manner. Some microparticles had been phagocytosed by macrophages and giant cells, and could be found intracellularly, indicating very slow degradation of the microspheres.

The experiment shows that starch microspheres made from: amylose are found in the intramuscular tissue two weeks after injection, which indicates that their biodegradability is very slow. Example 7b 30 Starch microspheres were made from acid hydrolysis Potato amylose (Reppal PSM60U), which was ultrafiltered to remove low molecular weight components, in order to test their biodegradability and ability to incorporate protein without exposing it. this for an organic solvent. B-lactoglobulin was used as the model protein. The starch concentration was 24% and 4 ml of this was mixed with 1.6 ml of the protein solution as being; 517 610: f- 44 had a concentration of 50 mg / ml at 37 ° C. The microspheres were solidified while stirring at room temperature overnight.

All protein leaked out of the microspheres during the buffer washes in buffer (5 mM sodium phosphate, pH 7.8). If only isopropanol or a series consisting of first 15% PEG with a molecular weight of 100,000, followed by 40% PEG with a molecular weight of 10,000 Da and finally isopropanol were used, protein corresponding to between 1.5 and 3% could be recovered. in the microspheres. The biodegradability of the microspheres was investigated in vitro. This was initially fast and after 24 hours about 60% of the matrix had been converted to soluble form. Thereafter the degradation stopped and after 7 days about 70% of the matrix had been biodegradable under these conditions.

To obtain any charge of the protein in these microspheres, it was necessary to precipitate the protein with an organic solvent, which is not an acceptable procedure for sensitive proteins. The resulting microspheres were also not completely biodegradable in vitro.

Example 7c Starch microspheres were made from extensively acid hydrolysed amylose from potatoes (Reppal PSM25, Reppe Glykos, Växjö) in order to investigate their biodegradability and ability to incorporate protein without exposing it to an organic solvent. Β-lactoglobulin was used as the model protein. The starch concentration was 20% and 4 ml of this was mixed with 1.6 ml of the protein solution having a concentration of 50 mg / ml at 37 ° C.

The microspheres were solidified while stirring at room temperature overnight.

All protein leaked out of the microspheres during the buffer washes in buffer (5 mM sodium phosphate, pH 7.8). If only isopropanol or a series consisting of first 15% PEG with a molecular weight of 100,000, followed by 40% PEG now »- 10 l5 20 25 30 35 5 17 6 10 = ¿:: = 45 with a molecular weight of 10,000 Da and finally isopropanol was used, protein corresponding to approximately between 1.5 and 2.5% could be found in the microspheres. The biodegradability of the microspheres was examined in vitro. This was initially fast and after 24 hours, about 55% of the matrix had been converted to a soluble form. Thereafter the degradation stopped and after 7 days about 70% of the starch matrix had been biodegradable under these conditions.

To obtain any charge of the protein in these microspheres, it was necessary to precipitate the protein with an organic solvent, which is not an acceptable procedure for sensitive proteins. The resulting microspheres were also not completely biodegradable in vitro.

Example 8 Immobilization of BSA with high charge in starch microspheres made from highly branched sheared starch.

A starch solution (40%) of sheared highly branched starch with an average molecular weight of 1600 kDa, a solution (38%) of PEG 20,000 Da in average molecular weight and a solution of BSA (14%) were diluted in 50 mM sodium phosphate, pH 8.3. The starch solution was tempered to 50-55 ° C, the PEG and BSA solution to about 33 ° C. The starch solution (2g) was mixed with the BSA solution (0.7 ml). The resulting solution was aspirated into a syringe mounted in a syringe pump. A solution of PEG (29g) was mounted in another syringe in another syringe pump. The starch microspheres were manufactured by pumping the starch / BSA and PEG mixtures by means of the syringe pump through static mixers into a carrier where the emulsion is stirred with a propeller (100 rpm).

This part of the process took 2 minutes from start until everything was mixed. Stirring in the beaker was allowed to continue for 10 minutes and then the sample was moved to 4 ° C, where it was allowed to stir for about 4 hours. Then the pH of the solution was adjusted down to about 5.5 and the preparation was set at 37 ° C overnight without stirring. The starch microspheres were washed by filtration in an Amicon (Amicon ultrafiltration cell) 5 mM sodium phosphate, pG 4.5 and lyophilized.

The dried microspheres were enzymatically dissolved with d-amylase and amyloglucosidase to determine protein and starch yield, as well as protein loading. The yield of protein was 94%, of the starch 89% and the obtained charge 10%. The average particle size determined with a Malvern Mastersizer was 90 μm and with less than 10% of the distribution below 35 μm. Upon incubation with α-amylase or α-amylase and amyloglucosidase, the microspheres completely dissolved within two days.

The example shows that a protein, BSA, can be immobilized in high yield and that the resulting microspheres have a high charge of the protein. The microspheres are biodegradable as they are completely dissolved by α-amylase in vitro and this can be done under mild conditions which allows accurate chemical analysis of the immobilized protein without the introduction of artifacts due to the extraction process itself. The example also shows that all captured protein can be found after dissolution of the microspheres.

Example 9 Immobilization of high charge BSA in starch microspheres made from highly branched sheared starch.

Starch microspheres containing BSA were made using a starch solution (40%) of highly branched, sheared starch with an average molecular weight of 1600 kDa, one (38%, average molecular weight 20,000 Da) (16%) was made of 50 mM phosphate, pH 8.3.

The starch solution was tempered to 50-55 ° C and the PEG and PEG solution and a solution of the BSA BSA solution to about 30%. Starch microspheres forward (IKA lab reactor LR250). The starch solution (20g) was pumped into the reactor vessel while stirred in an IKA reactor (100 rpm) for about 6 minutes and stirring was continued for about 15 minutes. The preparation was transferred to 4 ° C and allowed to stand under stirring overnight. The pH of the solution was adjusted down to about 5.5 and this was transferred to 37 ° C where it was allowed to stand for about 7 hours without stirring. The starch microspheres containing BSA were washed with 5 mM sodium phosphate, pH 4.5 and lyophilized.

The dried microspheres were enzymatically dissolved with α-amylase and amyloglucosidase to determine protein and starch yield, as well as protein loading. The yield of protein was 99%, of the starch 91% and the obtained charge l1.5%. The average particle size determined with a Malvern Mastersizer was 48 μm and with less than 10% of the distribution below 17 μm. Upon incubation with d-amylase or α-amylase and amyloglucosidase, the microspheres completely dissolved within 2 days.

Example 10 Immobilization of BSA with high charge in starch microspheres made of highly branched sheared starch.

A starch solution (20%) of highly branched sheared starch with an average molecular weight of 1930 kDa, a PEG solution (38%, average molecular weight 20,000 Da) (20%) was prepared in 50 mM sodium carbonate, pH 9.8. The starch and a BSA solution of the cell solution were tempered to 50-55 ° C and the others to about 37 ° C. The starch solution (3 g) was mixed with the BSA solution (0.7 ml). The mixture was sucked into a syringe and added to the PEG solution (28 g) in a beaker with stirring. Preparation was transferred to 4 ° C where they were allowed to stand for 4 hours and then to 37 ° C where they were allowed to stand overnight. The starch microspheres containing BSA were washed with 5 mM sodium phosphate, pH 4.5, and lyophilized.

The dried microspheres were enzymatically dissolved with d-amylase and amyloglucosidase to determine protein and starch yield, of 90% starch and the resulting 10.6% charge. The average particle size determined with a grinding and protein charge. The yield of protein was 91%, the protection Mastersizer was 44 μm and with less than 10% of the distribution during 21 μm. Upon incubation with α-amylase or d-amylase and amyloglucosidase, the microspheres completely dissolved within 2 days.

Example 11 Immobilization of crystalline hGH in starch microspheres made of highly branched sheared starch with high starch and PEG concentration and with temperature cycling.

Crystals of zinc-hGH were prepared according to EP 0 540 582 B1. A suspension of these crystals was prepared in 10 mM Na acetate, pH 6.4, containing 2 mM zinc acetate.

A starch solution (40%) sheared starch with an average molecular weight of 378 kDa in 10 mM sodium phosphate, pH 6.4, was made from highly branched and a solution of PEG with an average molecular weight of 20,000 at a concentration of 30%. This was adjusted to pH 6.4 with 1 M HCl. The solutions were tempered as follows: the starch solution to 50-55 ° C, the PEG solution to 37 ° C and the suspension of Zn-hGH crystals to 37 ° C. To 7 ml of suspension of Zn-hGH crystals was added 4.9 g of the starch solution, with stirring.

After about 20 seconds, 28 g of the PEG solution was added by means of a syringe pump for about 6 minutes and still with stirring at 400 rpm (Eurostar digital). The microspheres began to form immediately and were moved after about 15 minutes from 37 ° C to 4 ° C, and were kept there for about 4 hours with stirring. After stabilization for 4 hours, the microspheres were so stable that they could be transferred to 37 ° C and kept there without stirring overnight. The resulting microspheres were washed three times with 10 mM sodium acetate, pH 6.4, containing 2 mM zinc acetate, after stabilizing at 37 ° C for about 17-20 hours by filtration in an Amicon, and lyophilized.

The dried microspheres were enzymatically dissolved with α-amylase and amyloglucosidase to determine protein and starch yield, as well as protein loading and protein quality. The yield of protein was 95.2%, the yield of starch was 68% and the charge of protein in the microspheres was 27.8%.

The average particle size determined with a Malvern Mastersizer: true was 15 microns and with less than 10% of the distribution under 29 microns. Upon incubation with α-amylase or α-amylase and amyoglucosidase, the microspheres completely dissolved within 2 days. The protein content of dimers was 0.75% and of polymer The experiment shows that even proteins that have been transferred in solid form can be immobilized in high yield and resulting in starch microspheres with a high charge of the protein according to the present invention. The experiment also shows that the starch microspheres obtained can be dissolved under mild conditions, which enables rigorous quality control of the properties of the immobilized protein without the introduction of sample preparation-induced artifacts, and that the protein is not degraded during the process. The resulting protein quality is acceptable for parenteral administration in humans.

Example 12 Immobilization of BSA in starch microspheres of highly branched sheared starch and study of biodegradability in vivo.

Starch microspheres containing BSA were made from highly branched sheared starch having an average molecular weight of 1930 kDa under the following conditions: the starch (30%, 100 ml) was mixed with PEG (38%, 1466 ml, average molecular weight 20 kDa) and stirred for 6 hours at 20 ° C and then overnight at 37 ° C. These were administered subcutaneously and intramuscularly to rats at a dose of 30 mg in an injection vehicle consisting of 0.6% sodium hyaluronic acid (MW 2000 kDa, Kraeber GMBH, Hamburg) and the injection site was prepared for histological evaluation after 3 and 7 days. On day 3, cellular infiltration was observed at the injection site and already by day 7, these changes had disappeared.

This experiment shows that starch microspheres made from highly branched sheared starch are rapidly biodegradable, within a week, in vivo and that the tissue rapidly normalizes.

Example 13 Determination of the biodegradability of starch microspheres in 1 pig.

Starch microspheres were made from sheared starch with an average molecular weight of 529 kDa. The starch was weighed into 10 mM sodium phosphate, pH 6.4, so that the concentration after dissolution was 30% and PEG 20 with an average molecular weight in the same buffer so that the final concentration after dissolution became 27%. The solutions were then prepared by autoclaving. The production was carried out in an IKA reactor with Eurostar digital agitation control (Labasco). During production, 14.35 g of the starch solution, which was kept warm at 50 ° C after dissolution, were charged, after which 200 g of the PEG solution were fed to the reactor.

The emulsion was formed by stirring at 160 rpm with propeller and after 8 minutes the stirring speed was adjusted to 140 rpm and after 5 hours to 110 rpm, and the temperature was set at 20 ° C for 7 hours and then 38 hours. ° C for 17 hours. Then, the obtained starch microspheres (Amicon ultrafiltration unit 8400) were washed four times with 300 ml of water and lyophilized. The dry starch microspheres were sieved (sieve machine Retsch) with 38 and 100 μm sieves. The total amount of starch microspheres obtained before sieving was 3.61 g, which corresponds to a yield of approximately 86% and after sieving to 2.54 g, which corresponds to a yield of approximately 59%.

The starch microspheres were resuspended in 1 ml of 0.11% sodium hyaluronic acid, 4% mannitol, in water for injection and 100 mg of microspheres were injected subcutaneously into pigs. The injection site was prepared for histological evaluation.

No starch microspheres could be observed 7 days after injection. anna: 5 10 15 20 25 30 35 51 7 e 1 o 51 This experiment shows that the starch microspheres biodegradable rapidly and have disappeared from the injection site within a week.

Example 14 Preparation of coated starch microspheres containing hGH in starch microspheres made from highly branched sheared starch with temperature cycling, coating them and testing their ability to control the captured protein after parenteral administration in pigs.

Starch microspheres containing crystalline hGH were prepared according to Example 11.

The resulting starch microspheres were coated with a PLGA release control casing by air suspension technique according to WO97 / 14408 using different casing compositions to obtain different release rates. The following mixtures of PLGA are used; Batch 1 (+) 75% RG502H and 25% RG756, Batch 2 ('u_) 60% RG502H and 40% RG756, Batch 3 (_ ° _) 75% RG504H and 25% Rcvss and Batch 4 ("°') 1oo % RG5o4H.

The pharmacokinetics of hGH were studied in pigs whose immune system reactivity was attenuated by oral administration of cyclosporin A (20 mg / kg), azathioprine (2 mg / kg) and prednisolone (2 mg / kg) to avoid the formation of antibodies to the human growth hormone which could affect pharmacokinetics. The coated microspheres were administered subcutaneously in the neck with a 19G cannula at a dose of 1.25 mg hGH / kg. For each formulation, five pigs were studied. Blood samples were taken before the injection and after 0.02, 0.04, 0.08, 0.17, 0.25, 0.33 days and then daily until the study was completed 29 days after dosing. The serum concentration of hGH was determined. Figure 1 below shows the mean value of the serum concentration of hGH obtained for the different formulations. »Vasa 10 15 20 51 7 e 1 o 52 FIG. 25 Ni OI 15- Plasma concentration of hGH (ng / ml) 'fi d (day fi The experiment shows that starch microspheres coated with PLGA after parenteral administration in pigs give rise to a controlled prolonged release of hGH. level, 0.2 ng / ml, which induces elevated levels of insulin-like growth factor (IGF-1), which is an indicator of biological effect, in humans (Putney, Current Opinion in Chemical Biology 1998, 2: 548-552 The example also shows that the pharmacokinetics of hGH can be varied considerably by varying the composition of the coating envelope. The example also shows that, compared to previously available parenteral preparations for prolonged release, extremely low initial release, so called The experiment also shows that high bioavailability can be obtained in general, in the range 19- 52%, and with an exceptionally high proportion, 72-96%, in the phase of extended release that occurs after you the initial release, the so-called "burst". The analysis of plasma hGH was performed according to Agersö et al. (J. Pharmacol Toxicol 41 (1999) 1-8).

Example 15 The following example shows how to optimize a controlled release formulation by mixing two different formulations with different properties.

Starch microspheres containing human growth hormone were first prepared according to Example 14, except that the obtained load for Batch 1 and Batch 2 was 7.7 and 7.6%, respectively, and Batch 1 was coated with a single polymer, RG502H (Boehringer Ingelheim). and solid particles of zinc carbonate were mixed into the coating emulsion used to create the innermost third of the coating, while Batch 2 was coated with a polymer blend consisting of 75% RG502H and 25% RG756 (Boehinger Ingelheim).

The release of the protein incorporated into the microspheres was studied in hypophysectomized rats (MOL: Wist). containing 0.15% hyaluronic acid, 30 mM sodium phosphate buffer The microspheres were suspended in a solution in pH 7.4 containing 80 mM NaCl. The animals were injected with a volume of 200 μl subcutaneously in the neck with an 18G needle. Five animals per batch were injected. The dose was 750 pg hGH / animal. Blood samples (250 ul) were taken from the orbital plexus under carbon dioxide anesthesia. Serum was prepared and stored below -18 ° C. The serum samples were analyzed for growth hormone using a specific growth hormone-antibody-based RIA method (Tandhem-R hGH, Hybritech).

After Batch 1 and 2 had been tested and analyzed in the animal model and a plasma concentration curve was developed, it was calculated what a plasma concentration curve would look like if equal parts of Batch 1 and 2 were mixed.

The plasma concentration value of the two batches at each time point was easily calculated by taking the mean of point. The experiment described above was then repeated with a mixture of the two. The resulting plasma concentration curve was plotted, which has been plotted in Figures 2A and 2B. This shows that by mixing two batches with different release characteristics in a controlled manner, a desired release profile and concentration of the biologically active substance in serum or plasma can be obtained.

Claims (24)

10 l5 20 25 30 35 n nan-o 517 610 ss' PATENTKRAV 1. l. Parenterally administrable, biodegradable microparticle preparation containing a biologically active substance which is sensitive to or unstable in organic solvents, the preparation showing during the first 24 hours after injection a release of the active substance which is less than 20% of the total release, determined from a concentration-time curve in the form of the ratio between area under the curve during said first 24 hours and total area during the curve in question. A formulation according to claim 1, wherein the release during the first 24 hours after the injection is less than 15%, preferably less than 10%, and most preferably less than 5% of the total release. Parenterally administrable, biodegradable microparticle preparation containing a biologically active substance, which is sensitive to or unstable in organic solvents, the preparation during the first 48 hours after injection showing a release of the active substance where the maximum the concentration in plasma or serum is less than 300% of the maximum concentration of the biologically active substance at any time exceeding 48 hours after injection. A formulation according to claim 3, wherein said maximum concentration is less than 200%, preferably less than 100%, of said maximum concentration. A formulation according to claim 3 or 4, which has the release defined in any one of claims 1 and 2. A parenterally administrable, biodegradable microparticle preparation containing a biologically active substance which is sensitive to or unstable in organic solvents, wherein the microparticles consist essentially of starch having a content exceeding 85% by weight of amylopectin, for which at least 80% by weight have an average molecular weight in the range of 10-10 000 kDa, which have an amino acid nitrogen content of less than 50 pg / g dry weight starch and which lack covalent chemical crosslinking between the starch molecules, the preparation having a release of the biologically active substance wherein the bioavailability of said substance is at least 35% of the bioavailability obtained when the substance in question is injected intravenously in soluble form. A formulation according to claim 6, wherein said bioavailability is at least 45%, preferably at least 50%, of the bioavailability obtained when the biologically active substance is injected intravenously. A parenterally administrable, biodegradable microparticle preparation containing a biologically active substance, which is sensitive to or unstable in organic solvents, wherein the microparticles consist essentially of starch, having a content exceeding 85% by weight of amylopectin, for which at least 80% by weight have an average molecular weight in the range of 10-10 000 kDa, which have an amino acid nitrogen content of less than 50 pg / g dry weight and which lack covalent chemical crosslinking between the starch molecules, the preparation showing a release of the active substance in the release that occurs during any continuous seven-day period, the ratio between the highest concentration of the biologically active substance in serum or plasma divided by the mean concentration during said seven-day period is less than 5, provided that the selected seven-day period does not include the first 24 hours after injection. A formulation according to claim 8, wherein said release is less than four times, preferably less than three times, most preferably less than twice. A parenterally administrable, biodegradable microparticle preparation containing a biologically active substance which is sensitive to or unstable in organic solvents, wherein the microparticles consist essentially of starch having a content exceeding 85% by weight of amylopectin, for which at least 80% by weight has an average molecular weight in the range 10-10 000 kDa, which have a 10 15 20 25 30 35 517 610 5? now have amino acid nitrogen contents of less than 50 pg per g dry weight starch and which lack covalent chemical crosslinking between the starch molecules, the preparation showing a release of the biologically active substance where the average residence time of said biologically active substance is at least four days. A preparation according to claim 10, wherein said average residence time is at least seven days, preferably at least nine days, more preferably eleven days, most preferably at least thirteen days. A formulation according to any one of the preceding claims, wherein the biologically active substance is a protein, a peptide or a polynucleotide, preferably a protein. The formulation of claim 12, wherein the protein is a recombinantly produced protein. A formulation according to any one of claims 12 and 13, wherein said substance is a hormone, preferably human growth hormone. A formulation according to any one of claims 12 and 13, wherein said substance is selected from growth factors, insulin, erythropoietin, interferon d, interferon ß, interferon 7, Factor VII, Factor VIII, glucagon-like peptide 1 or 2 and C-peptide. A preparation according to any one of claims 1-5, wherein the microparticles consist essentially of starch, which has a content exceeding 85% by weight of amylopectin, for which at least 80% by weight has an average molecular weight in the range 10-10,000 kDa, which have an amino acid nitrogen content of less than 50 pg per g of dry weight starch and which lack covalent chemical crosslinking between the starch semolecules. A formulation according to any one of claims 6-16, wherein said average molecular weight is in the range of 100-4000 kDa, preferably 100-1000 kDa. A formulation according to claim 17, wherein the average molecular weight is in the range of 200-1000 kDa, preferably 300-600 kDa. 10 15 20 517 610 oc. no 58 A formulation according to any one of the preceding claims, wherein the bioactivity of the biological substance is at least 80%, preferably at least 90% and most preferably substantially retained, compared to the bioactivity which the substance exhibited before it was incorporated into the microparticles. A preparation according to any one of the preceding claims, which is biodegradable in vitro in the presence of alpha-amylase and / or amyloglucosidase. A preparation according to any one of the preceding claims, which is biodegradable and eliminated from tissue after subcutaneous or intramuscular administration. A formulation according to any one of the preceding claims, which has a release-regulating coating of at least one film-forming biocompatible and biodegradable polymer. A formulation according to claim 22, wherein the polymer is a homo- or copolymer containing alpha-hydroxy acid units. A formulation according to claim 23, wherein the alpha hydroxy acid is lactic acid and / or glycolic acid.