Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/5089—Processes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/5005—Wall or coating material
  • A61K9/5021—Organic macromolecular compounds
  • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)

Definitions

• the present invention lies within the field of galenic formulations for the administration of biologically active substances, more specifically microparticles for controlled release primarily intended for parenteral administration of biologically active substances, especially drugs. More specifically, it relates to a novel production process for such particles containing a biologically active substance and to novel particles for controlled release which are thereby obtainable .
• PLGA linear polyesters based on lactic acid, glycolic acid and mixtures thereof. These polymers will also hereinafter be referred to as PLGA.
• PLGA is degraded by ester hydrolysis into lactic acid and glycolic acid and has been shown to possess excellent biocompatibility.
• the innocuous nature of PLGA can be exemplified, moreover, by the approval by the regulating authorities, including the US Food and Drug Administration, of several parenteral delayed release preparations based on these polymers .
• Parenterally administrable delayed release products currently on the market and based on PLGA include Decapeptyl TM (Ibsen Biotech) , Prostap SRTM
• the drugs in these preparations are all peptides. In other words, they consist of amino acids condensed into a polymer having a relatively low degree of polymerization and they do not have any well-defined three-dimensional structure. This, in turn, usually allows the use of relatively stringent conditions during the production of these products. For example, extrusion and subsequent size-reduction can be utilized, which techniques would probably 1 not be allowed in connection with proteins, since these do not, generally speaking, withstand such stringent conditions. Consequently, there is also a need for controlled release preparations for proteins .
• Proteins are similar to peptides in that they also consist of amino acids, but the molecules are larger and the majority of proteins are dependent on a well-defined three-dimensional structure as regards many of their properties, including biological activity and immunogenicity. Their three-dimensional structure can be destroyed relatively easily, for example by high temperatures, surface-induced denaturation and, in many cases, exposure to organic solvents.
• a very serious drawback connected 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 stability of the protein will be compromised and that conformation changes in the protein will lead to an immunological reaction in the patient, which can produce both a loss of therapeutic effect, through the formation of inhibitory antibodies, and toxic side effects. Since it is extremely difficult to determine with certainty whether a complex protein has retained its three- dimensional structure in every respect, it is very important to avoid exposing the protein to conditions which might induce conformation changes .
• PLGA microspheres containing proteins are described in WO-A1-9013780, in which the main feature is the use of very low temperatures during the production of the microspheres for the purpose of preserving high biological activity in the proteins.
• the activity for encapsulated superoxide dismutation is measured, but only on the part which has been released from the particles.
• This method has been used to produce PLGA microspheres containing human growth hormone in WO-Al-9412158, wherein human growth hormone is dispersed in methylene chloride containing PLGA, the obtained dispersion is sprayed into a container of frozen ethanol beneath a layer of liquid nitrogen in order to freeze the fine droplets and said droplets are allowed to settle in the nitrogen on the ethanol .
• Starch granules naturally contain impurities, such as starch proteins, which makes them unsuitable for injection parenterally. In the event of unintentional depositing of insufficiently purified starch, such as can occur in operations where many types of operating gloves are powdered with stabilized starch granules, very serious secondary effects can arise. Neither are starch granules intrinsically suitable for repeated parenteral administrations, for the reason that they are not fully biodegradable within acceptable time spans .
• WO 99/00425 describes the use of heat-resistant proteolytic enzymes with a broad pH-optimum to purge starch granules of surface-associated proteins.
• the obtained granules are not suitable for parenteral administration, since they still contain the starch proteins which are present within the granules and there is a r.isk that residues of the added proteolytic enzymes will be left in the granules.
• biologically active substances Even though it is generally possible to incorporate biologically active substances in microparticles in a highly effective manner, since the biologically active substance is present in soluble form during the entrapment stage, it is in certain cases preferable for the biologically active substance to be converted into solid form.
• substances which are well known within the field and are acceptable for parenteral use can also be used, for example to adjust ionic strength and osmolarity.
• the obtained solution can be sterilized by means of, for example, sterile filtration.
• step b) is performed such that the solidification of the biologically active substance results in a highly viscous solution, which has the ability of forming droplets which can be handled at room temperature .
• step b) is performed to form a reversibly solidified active substance .
• the solidified biologically active substance forms a pellet or a highly viscous or solid bottom phase . in centrifugation or ultracentrifugation.
• the starch In order that fully biodegradable microparticles with high active substance yield shall be formed in a two-phase aqueous system and in order that the obtained starch microparticles shall have the properties to be described below, the starch must generally predominantly consist of highly branched starch, which, in the natural state in the starch granule, is referred to as amylopectin. It should also have a molecular weight distribution which ' makes it possible to achieve desired concentrations and gelation rates . In the two cases referred to in the previous paragraph said molecular weight distribution can be accomplished by means of shearing or acid hydrolysis, respectively.
• biodegradable means that the microparticles, after parenteral administration, are dissolved in the body to form endogenic substances, ultimately, for example, glucose.
• the biodegradability can be determined or examined through incubation with a suitable enzyme, for example alpha-amylase, in vitro. It is in this case appropriate to add the enzyme a number of times during the incubation period, so as thereby to ensure that there is active enzyme permanently present in the incubation mixture.
• the biodegradability can also be examined through parenteral injection of the microparticles, for example subcutaneously or intramuscularly, and histological examination of the tissue as a function of time.
• biodegradable starch microparticles disappear from the tissue within a few weeks and often within one week.
• the starch microparticles are coated with a release-controlling shell, for example by the application of a thin layer, it is generally this shell which determines the biodegradability rate, which then, in turn, determines when alpha-amylase becomes available to the starch matrix.
• the biocompatibility can also be examined through parenteral administration of the microparticles, for example subcutaneously or intramuscularly, and histological evaluation of the tissue, it being important to bear in mind that the biologically active substance, which often is a protein, has in itself the capacity to induce, for example, an immune response if administered to another species.
• the starch must further have a purity which is acceptable for the manufacture of a parenterally administrable preparation. It must also be able to form sufficiently stable solutions in sufficiently high concentration to enable the biologically active substance to be mixed in under conditions allowing the retention of the bioactivity of the substance, at the same time as it must spontaneously be able to be solidified in a controlled manner in order to achieve stable, yet at the same time biodegradable, microparticles. High concentration of the starch is also important to prevent the biologically active substance from being distributed out to an unacceptable extent to the outer phase or to the interface between the inner and the outer phases .
• the starch preferably has an amylopectin content exceeding 85% by weight, the molecular weight of said amylopectin being reduced so that at least 80% by weight of the material lies within the range of 10-10 000 kDa.
• Another preferred embodiment involves the starch having an endotoxin content of less than 25 EU/g.
• step d) heating according to a technique which is known per se is in general used to dissolve the starch.
• An especially preferred embodiment simultaneously involves the starch being dissolved under autoclaving, it also preferably being sterilized. This autoclaving is realized in aqueous solutions, for example water for injection or suitable buffer.
• the active substance (s) is/are therefore combined with the starch solution at a temperature of at most 60 °C, more preferably at most 55°C, and preferably within the range of 20-45°C, especially 30-37°C.-'
• a weight ratio of starch:biologically active substance within the range of 3:1 to 10 000:1 is expediently used.
• the active substance is concentrated/solidified with the use of a PEG solution before being mixed with the starch solution. It is possible to add the starch solution to the biologically active substance or vice versa. After this, a homogeneous distribution of the concentrated/solidified active substance in the starch solution is created by means of a suitable technique. Such a technique is well known within the field, examples which might be quoted being magnetic agitation, propeller agitation or the use of one or more static mixers.
• the concentration of starch in the solution which is to be converted to solid form and in which the biologically active substance is to be incorporated should be at least 20% by weight to enable the formation of starch microparticles having good properties .
• Exactly what starch concentration works best in each individual case can be titrated out in a simple manner for each individual biologically active substance, where the load in the microparticles is that which is required in the individual case.
• the biologically active substance to be incorporated in the microparticles can affect the two- phase system and the gelation properties of the starch, which also means that customary preparatory trials are conducted for the purpose of determining the optimal conditions in the individual case.
• the polymer is dissolved in suitable concentration in water or aqueous solution, which expression also includes buffer solution, and is temperature-adjusted to a suitable temperature.
• This temperature lies preferably within the range of 4-50°C, more preferably 10-40°C and often 10-37°C.
• concentration of the polymer in the water-based solution is expediently at least 20% by weight and preferably at least 30% by weight, and expediently at most 45% by weight. An especially preferred range is 30-40% by weight.
• Suitable enzymes for use in connection with starch are alpha-amylase and amyloglucosidase, singly or in combination, it being important to establish, where appropriate, that they are free from possible proteases, which can degrade proteins.
• the presence of proteases can be detected with methods known within the field and, for example, by mixing the biologically active substance in control trials and determining its integrity in the usual manner after incubation with the intended enzyme mixture under the conditions which will afterwards be used to dissolve the microparticles. Where the preparation is found to contain protease contamination, it can be replaced by a preparation which offers higher purity or is purged of proteases.
• the produced microparticles When the produced microparticles are used, either they are coated with a release-controlling outer shell or not, and the dry microparticles are suspended in a suitable medium, specifically to permit injection. Such media and processes in these regards are well known within the field and will not need here to be described in further detail.
• the actual injection can be given through a suitable needle or with a needle-free injector. It is also possible to inject the microparticles using a dry powder injector, without prior resuspension in an injection medium.
• biologically active substances of the above-specified type are growth hormone, erythropoietin, interferon ( ⁇ , ⁇ , ⁇ -type) , vaccine, epidermal growth hormone, Factors IV, V, VI, VII, VIII and IX, LHRH- analogue, insulin, macrophage-colony-stimulating factor, granulocyte-colony-stimulating factor and interleukin.
• Another embodiment is represented by microparticles in which the starch has an amino acid nitrogen content of less than 50 ⁇ g per g dry weight of starch and which microparticles have no covalent chemical cross-linking between the starch molecules.
• the starch can otherwise have the features which have been discussed in connection with the process.
• the protein is a growth hormone, especially a human growth hormone (hGH) .
• hGH human growth hormone
• the microparticles according to the invention have the advantage that they are essentially or wholly lacking in such zinc.
• a human growth hormone constitutes the biologically active substance
• this is preferably of the type whose dimers content is less than 2% by weight, and more preferably less than 1% by weight, and whose polymers content is less than 0.2% by weight, preferably less than 0.1% by weight.
• the said mean residence time is at least 7 days, more preferably at least 9 days, for example at least 11 days, or especially at least 13 days.
• the features which have been specified for the above-presented microparticle preparations can be combined in any suitable combinations whatsoever.
• Absolute bioavailability is defined according to the following formula:
• the obtained suspension are added 12 g of a PEG solution of 42% concentration, in which the average molecular weight of PEG is 20 kDa.
• the solidification is initiated at 4°C for 17 hours and concluded at 37°C for 6 hours.
• the obtained starch microspheres containing hGH are washed three times with 38 ml 10 mM sodium acetate buffer containing 2 mM zinc acetate, pH 6.4, and freeze-dried.
• the obtained microspheres are dissolved with ⁇ -amylase and the quantity of incorporated hGH is determined, for example by means of analysis with high-pressure-liquid chromatography. "The fraction of dimer and polymer is also determined, for example using high-pressure-liquid chromatography.
• the yield of starch microspheres containing hGH is generally at least . 80% and the hGH content, expressed as dry weight, is around 15 percent by weight.
• the dimer content of the protein is generally ⁇ 1% and the polymer content ⁇ 0.1%, which shows that the protein is acceptable for parenteral administration to humans .

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• 2001
  • 2001-10-05 EP EP01972893A patent/EP1333814A1/en not_active Withdrawn
  • 2001-10-05 CA CA002429100A patent/CA2429100A1/en not_active Abandoned
  • 2001-10-05 AU AU2001292527A patent/AU2001292527A1/en not_active Abandoned
  • 2001-10-05 JP JP2002542360A patent/JP2004513914A/ja not_active Ceased
  • 2001-10-05 WO PCT/SE2001/002166 patent/WO2002039985A1/en active Application Filing

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