MXPA99000499A - Temperature-sensitive gel for sustained supply of medicines with protei - Google Patents

Abstract

The present invention provides pharmaceutical compositions for the administration of pharmacologically active proteins. The compositions of the present invention comprise a polymeric matrix having thermal gelatinization properties in which a discrete of at least one biologically active macromolecular polypeptide is incorporated which retains more than 90% of its biological activity. In addition, the concentration of the macromolecular polypeptide is greater than 0.5% of the weight of the composition

Description

GEL SENSITIVE TO TEMPERATURE. FOR SUSTAINED SUPPLY OF PROTEIN DRUGS DESCRIPTION OF THE INVENTION The present invention relates to temperature sensitive polymers for the sustained delivery of pharmacological agents and, more particularly, to poloxamers comprising suspensions of native macromolecular polypeptide agents. Recent advances in recombinant DNA techniques and, consequently, the proliferation of many pharmaceuticals with proteins available for a variety of therapeutic needs, have been observed in recent years. In effect, proteins or polypeptides are the largest class of drugs that are currently considered for FDA approval. However, the therapeutic and commercial potential of drugs with polypeptides will only be understood if these advances are accompanied by formulations designs that lead to effective administration and stability. Proteins are large organic molecules or macromolecules made up of amino acid residues covalently linked together by peptide bonds in a linear, unbranched polypeptide chain with the relative molecular mass in the range of a few thousand to millions. The useful properties of proteins as drugs depend on the polypeptide chain adopting a unique three-dimensional bent conformation, that is, the tertiary structure of the protein. It will be recognized this unique fold that is responsible for the protein to be highly selective in the molecules. However, the ability to maintain a unique three-dimensional structure is precisely one of the obstacles that makes the use of polypeptides in human and animal diseases full of problems. Traditionally, the most widely used method for administration of therapeutic agents is by the oral route. However, such a supply is not feasible, in the case of macromolecular drugs, since these are rapidly degraded and deactivated by hydrolytic enzymes in the alimentary tract. Even if it is stable to enzymatic digestion, its molecular weights are very high for absorption through the intestinal wall. Consequently, they are usually administered parentally; but, since such drugs often have short half-lives in vivo, frequent injections are required to produce effective therapy. Unfortunately, while the parental route is the most efficient means for drug introduction, this route has several disadvantages such as that the injections are painful; they can lead to an infection; and can lead to severe vascular problems as a result of repeated intravenous injections. For these reasons, biodegradable polymer matrices have been considered as sustained release delivery systems for a variety of active agents or drugs. Once implanted, the matrix dissolves slowly or erodes, releasing the drug. An alternative procedure is to use small implantable pumps, which slowly extrude the drug and matrix components, which dissolve after contact with body fluids. With both systems it is crucial that the drug remains uniformly distributed throughout the matrix since the heterogeneous distribution of the drug (for example, the formation of large lumps and voids) can lead to erratic dosing. Additionally, both systems require polymers that remain somewhat fluid in such a way that they can? be easily manipulated before implantation or loading into a device. The use of polymers as solid implants and for use in small implantable pumps for the delivery of various therapeutic agents in scientific publications and patent literature has been described. See, for example, Kent, et al., "In vivo controlled release of an LHRH analog from injected polymeric microcapsules", Contracept, Deliv. Syst. 3:58 (1982); Sanders, et al., "Controlled reléase of a luteinizing hormone releasing hormone analogue from poly (d, 1-lactide-co-glycolide) -microspheres", J. Pharmacut. Sci., 73: 1294-1297 (1984); Johnston, T.P., et al., "Sustained delivery of Interleukin-2 from a poloxamer 407 gel matrix following intraperitoneal injection in mice", Pharmaceut. Res., 9 (3): 425-434 (1992); Yolks, et al., "Timed relay depot for anti-cancer agents II", Acta Pharm. Svec. , 15: 382-388 (1978); Krezanoski, "Clear, water-miscible, liquid pharmaceutical vehicles and compositions which gel at body temperature for drug delivery to mucous membranes", Patent of the United States NO. 4,474,752. However, polymers that have the greatest potential for use in drug delivery with proteins may exhibit reverse thermal gel formation and have good drug release characteristics. There is a class of block copolymers which can be generally classified as polyoxyethylene polyoxypropylene condensates, ie Pluronic polyols or poloxamers. These are formed by the condensation of propylene oxide in a propylene glycol core followed by the condensation of ethylene oxide at both ends of the polyoxypropylene base. The polyoxyethylene hydrophilic groups at the ends of the molecule are controlled in length to constitute anywhere from 10 percent to 80 percent by weight of the final molecule. Poloxamers, which have the ability to form a gel as a function of polymer temperature and concentration, can be represented empirically by the formula: HO (C2H40) b (C2HsO) a (C2H40) bH (I) Where a and b are integers such that the hydrophobic base represented by (C3HsO) a has a molecular weight in the range of about 900 to 4,000, as determined by the hydroxyl number; and the polyoxyethylene chain consisting of at least 60 weight percent to 70 weight percent of the copolymer, and the copolymer having a total average molecular weight of about 4,000 to 15,000. Table 1, below, refers to the minimum concentrations of several poloxamers necessary to form a gel in water at room temperature. Table 1 Poloxamer * Molecular weight Minimum concentration Pluronic® F-68 8,400 50% -60% Pluronic® F-88 11,400 40% Pluronic®F-108 14,600 30% Pluronic® F-127 12,600 20% * Poloxamers are commercially available under the reference marks through BASF Corporation, Parsippany, NJ Low molecular weight poloxamers, ie, below 10,000 PM, do not form gels at any concentration in water. While poloxamers, and more specifically Pluronic® F-127 or Poloxamer 407, have been used to deliver non-peptidic drugs as well as biologically active proteins, see U.S. Patent Nos. 4,100,271 and 5,457,093, respectively, Sustained supply of biologically active macromolecules for six months and months has not been possible for reasons that double. First, the previous references which describe the incorporation of proteins in a Pluronic® matrix only describe solutions of a protein, with concentrations lower than approximately 2 mg / ml and second, the formulation methods used to incorporate proteins in polymeric systems result in frequency in irreversible inactivation of the protein due to the presence of organic solvents, changes in pH, and thermal effects. Consequently, the above references which teach the use of poloxamers as pharmaceutical carriers for protein delivery have suffered two serious limitations; (i) low initial protein concentrations are used, and (ii) an unacceptable percentage of the protein loses its biological activity during use or storage. These two limitations have a direct impact on the ability to produce a polypeptide drug delivery system which can be stowed for long periods of time before the use and administration of controlled doses of protein for a period of weeks or more preferably months. Additionally, degraded proteins may have reduced efficacy as a drug, and may also produce adverse reactions, such as sensitization and adverse immune response. There is still a need, therefore, for a polypeptide drug delivery device or composition having high concentrations of fully native macromolecular polypeptides which can be released regularly over a long period of time. Accordingly, it is an object of this invention to provide a polypeptide drug delivery system. It is a further object of this invention to provide a polypeptide drug delivery device or composition having protein or peptide concentrations greater than 5 mg / ml. It is a further object of the present invention to incorporate protein stabilizers into the drug delivery device. Additional objects, advantages and novel features of this invention may be indicated in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following specification or may be learned by the practice of the invention. The objects and advantages of the invention may be understood and obtained by means of instruments, combinations, compositions, and methods particularly indicated in the appended claims. In order to achieve the foregoing and other objects and in accordance with the purposes of the present invention, as exemplified and broadly described herein, the composition of the present invention comprises a polymeric matrix having thermal gelatinization properties in which incorporates a suspension of at least one biologically active macromolecular polypeptide having a concentration of 0.5 percent or more by weight of the composition. DESCRIPTION OF THE INVENTION The accompanying drawing, which is incorporated in and forms part of the specification, illustrates the prepared embodiments of the present invention, and together with the description serves to explain the principles of the invention. In the drawing: Figure 1 is a graphic representation of the effects that various additives have on the transition temperature of the gel solution (sol gel) of the present invention, wherein each additive is represented by the following symbols: - 0.1% of PEG 800, -x- Sucrose 0.5 M, - - 1.0% of PEG 800, - * - Sucrose l.OM, - - Pluronic® F-127, - - Sucrose 1.5 M. The pharmaceutical device or composition of the present invention provides a delivery system for the controlled and sustained administration of totally native macromolecular polypeptides or therapeutic agents to a human or animal. The biodegradable, biocompatible matrix for drug delivery is formed by suspending insoluble and soluble particles of totally native macromolecular polypeptides having a concentration of 5 mg / ml or more, and other protein stabilizing components uniformly and discretely throughout the pharmaceutical carrier or matrix polymer that exhibits characteristics of reverse thermal gelatinization. As with previously known systems, suspended particles and other components are released through the combination of diffusion and dissolution mechanisms as soon as the device is hydrated and subsequently erodes or dissolves. However, unlike the known polymer matrix systems which supply macromolecules, the composition of this invention comprises a suspension opposite to a solution of native polypeptides. Consequently, high concentrations of polypeptide are obtained as a result of the suspension thereby achieving the ability for sustained administration of the therapeutic agent for a period of days, weeks or months opposed to hours. Additionally, stabilizing agents can be incorporated into the composition of the present invention whereby the degradation of these drugs is further minimized, which directly impacts the efficacy of the drug and the ability to store or transport the device to world markets. The pharmaceutical composition of the present invention is a serepndipiti discovery that is made during a research project, the goal of which is simply to identify a drug delivery system that is suitable for studying the effect of polymer matrices on the structure of the protein . In this study, infrared spectroscopy with Fourier transform is used, since it can be used to analyze the secondary protein structure in solutions, suspensions and solids. Of the various drug-protein delivery matrices described in the literature, those that use the polymeric detergent, Pluronic® F-127, which forms a temperature sensitive gel, are the most attractive for infrared spectroscopy studies, since it does not appear (based on the structures of the poloxamer) that the poloxamers may have an infrared absorbance that may interfere with the protein absorbance signal, which is necessary for structural evaluation. Additionally, it has been previously established that poloxamers, in sufficient concentrations, have the characteristics of being liquid at temperatures below room temperature but will gel as soon as they are heated. In this way, an additional objective of the study is to determine what effect this transition from the liquid to the poloxamer gel can have on the structure of the protein. In order to study the structure of the protein with infrared spectroscopy it is necessary to have protein concentrations of at least 15-20 mg / ml, in this way a protein concentration of 20 mg / ml is prepared in the presence of sufficient poloxamer to allow gelation during heating. The resulting protein solution forms a fine, milky suspension, which is initially very unpleasant, since the formation of such suspensions often indicates that a component of the solution (e.g., polymeric detergent) causes the protein to denature and form non-native or inactive protein aggregates, thus indicating a failure of the test. Nevertheless, infrared spectroscopy can be used, fortunately, to analyze protein structures in suspension, therefore the suspension is analyzed in any way. Surprisingly, when analyzing the protein suspension with infrared spectroscopy with Fourier transform, instead of finding non-native or inactive aggregates of expected proteins, it is unexpectedly found that they are totally native. In accordance with the present invention, the pharmaceutical composition of the present invention comprises a polymer such as a polyoxyalkylene block copolymer of the formula: HO (C2H40) b (C2HsO) a (C2H40) bH (I) Which has the unique feature, in the preferred embodiment, of being liquid at room temperature or lower temperatures and existing as a semi-solid gel at mammalian body temperatures where a and b are integers in the range of 20 to 80 and 15 to 60, respectively . A preferred polyoxyalkylene block copolymer for use as the pharmaceutical carrier of this invention is a polyoxyethylene polyoxypropylene block copolymer having the following formula: HO- (CH2CH20) b- (CH2 (CH3) CHO) a- (CH2CH20) bH (II) Where a and b are integers such that the hydrophobic base represented by (CH 2 (CH 3) CHO) a has a molecular weight of at least about 4,000, as determined by the hydroxyl number; the polyoxyethylene chain that constitutes approximately 70 percent of the total number of monomer units in the molecule and where the copolymer has an average molecular weight of about 12,600. Pluronic® F-127, also known as Poloxamer 407, is such a material. The procedures used to prepare aqueous solutions which form polyoxyalkylene block copolymer gels are well conformed. For example, it can be used either a hot or cold process to form the solutions. The cold technique involves the steps of dissolving the polyoxyalkylene block copolymer at a temperature of about 5 ° C to 10 ° C in water or in a buffer, such as a phosphate buffer. If water is used, in the formation of the aqueous solution, it is preferably purified, such as by distillation, filtration, ion exchange or the like. When the solution is complete it is brought to room temperature where it then forms a gel. If the hot process is used to form the gel, the polymer is added to the water or a buffer and heated to a temperature of about 75 ° C to 85 ° C with slow stirring until the clear homogenous solution is obtained, after cooling the gel is formed. Any macromolecular polypeptide can be mixed with the pharmaceutical carrier to form the pharmaceutical composition of this invention wherein the concentration of the macromolecular polypeptide is in the range of 0.5 to 50 weight percent of the composition. The choice of polypeptides which can be delivered in accordance with the practice of this invention is limited only by requirements that are at least very slightly soluble in an aqueous physiological medium such as plasma, intestinal fluid, and intra and extracellular fluids. of the subcutaneous space and mucosal tissues. Specific examples of polypeptides suitable for incorporation into the delivery system of the present invention include the following biologically active macromolecules: interferons, interleukins, insulin, enzyme inhibitors, colony stimulating factors, plasminogen activators, growth factors and polypeptide hormones The list of the macromolecular polypeptides indicated above is provided only to illustrate the types of active agents which are suitable for use in the practice of the present invention, and are not intended to limit the scope of the present invention. The pharmaceutical composition of the present invention can be easily prepared using any solution formation technique which achieves the concentration of the polyoxyalkylene block copolymer necessary to gel. Preferably the pharmaceutical carrier and polypeptide mixture are prepared separately and the polypeptide mixture having a concentration of 5 mg / ml or more is added thereto at a temperature of about 0 ° C to 10 ° C. When the protein is combined, it forms a homogeneous suspension of fine particles in the polymer solution, which then has a "milky" appearance. By light microscopy the particles are approximately 5-10 microns in diameter. Increasing the temperature of the sample above the poloxamer gel point results in a uniform distribution of protein particles throughout the polymer gel. Due to the high viscosity of the gel matrix, the particles remain homogeneously distributed and do not "settle". The transition from liquid to gel is completely reversible after cooling. Additionally, when the gel is exposed to an aqueous solution, the gel matrix and protein particles will dissolve, releasing the fully native protein which retains more than 90% of its biological activity. The pharmaceutical composition of the present invention can be implanted directly into the body by injecting it as a liquid, wherein the pharmaceutical composition will gel once it is inside the body. In the alternative form, the pharmaceutical composition can be introduced into a small implantable pump which is then introduced into the body. In another embodiment, the protein stabilizer solutes can be incorporated into the pharmaceutical device of the present invention described above. Initially, stabilizers are added to the pharmaceutical device of the present invention to increase the stability of the macromolecular polypeptides since such stabilization may be crucial for use of the present invention for sustained delivery of the protein in the body. However, by doing this it is discovered that protein stabilizing solutes, such as sucrose, not only aid in the protection and stabilization of the protein, but also allow the poloxamer to form suitable gels at concentrations lower than those required in water or buffer. alone. In this way, the working range of the polymer concentration can be extended. As described previously, the concentration of the polyoxyalkylene block copolymer is an important parameter. It is known that a gel will not form when the concentration of the polyoxyethylene polyoxypropylene block copolymer in water or dilute buffer is outside the range of about 20 to 30 weight percent, as shown in Figure 1 and exemplified by the line that It has open triangles. However, the sol gel transition temperature can be manipulated by introducing protein stabilization solutes to the pharmaceutical device of the present invention, while decreasing the concentration of the polyoxyethylene polyoxypropylene block copolymer which is necessary to form a gel. In a third embodiment, the concentrations of the polypeptide at the extreme end of the range of 0.5 to 50 weight percent can be obtained by centrifuging the pharmaceutical composition of the present invention at low temperatures in the range of -10 ° C to 10 °. C, and preferably 0-4 ° C for a sufficient period of time to pellet the protein particles. For example, a sample of the previously described pharmaceutical composition comprising 20 mg / ml of protein at 4 ° C can be brought to centrifugation in such a way that the insoluble protein particles settle. The supernatably equivalent to half the volume can then be removed and the sediment resuspended in the remaining liquid. This will result in a suspension containing almost 40 mg / ml. In the following examples, the pharmaceutical composition of the present invention is prepared according to the following preparation procedure. Since the polyoxyalkylenes dissolve more completely at reduced temperatures, preferred methods of solubilization are to add the required amount of the copolymer to the amount of water or buffer to be used. Generally after moistening the copolymer by stirring, the mixture is capped and placed in a cold chamber or thermostatic vessel at about 0 ° C to 10 ° C in order to dissolve the copolymer. The mixture can be stirred or mixed to arrive at approximately a faster solution of the polymer. Subsequently, the polypeptides and various additives such as stabilizers and dissolved ones can be added to form a suspension. The following non-limiting examples provide methods for preparing temperature sensitive polymers for the sustained delivery of pharmaceutical agents comprising high concentrations of totally native macromolecular polypeptide agents. All scientific and technical terms have the meanings as understood by a person with ordinary skill in the art. The specific examples that follow illustrate the representative polypeptides and concentrations capable of being reached by the present invention and are not constructed as limiting the invention in sphere or scope. The methods can be adapted to variation in order to produce compositions or devices encompassed by this invention but not specifically described. Variations of the methods for producing the same compositions in some different way by one skilled in the art will be apparent. It is understood that all temperatures are in centigrade (° C) when not specified. The infrared spectral description (IF) is measured on a Nicolet Magna-IR 550 Spectrometer. Commercially available chemicals are used without purification. In the examples that follow, the solution is prepared at 27.5% (w / w) of Pluronic® F-127 in the following manner: 7.59 g of dry Pluronic® F-127 is added to a sterile tube containing 20 grams of a phosphate buffer cooled in ice (pH 7.4, 30 mM). The tube is capped and the mixture is stirred well before being stored overnight at 4 ° C.

EXAMPLE 1 Preparation and characterization of a suspension of chyme ripsin in Pluronic F-127 a) Preparation of the suspension: When 50 μl of a 100 mg / ml suspension of chymotrypsin in 30 mM phosphate buffer at pH 7.4 is mixed with 200 μl of a solution at 27.5 percent (w / w) of Pluronic ® F-127 is formed or immediately a white, uniform, milky suspension. The suspension contains 20 mg / ml of protein and 22 percent (w / w) of Pluronic® F-127 and is a viscous liquid below about 18 ° C and a soft, solid gel above about 20 ° C. The crystalline portion of the liquid suspension settles the solution when it is allowed to stand for a day or two at 4 ° C or when it is brought to cold centrifugation but can be easily resuspended by mixing while it is cold. The fact that the suspended material can settle and be resuspended in a volume smaller than the original suspension allows the formation of suspensions of significantly higher concentrations, as discussed previously. In order to determine the solubility of chymotrypsin in the liquid phase of the suspension, the aliquots of the total suspension and of the supernatant are tested after centifugation at 1000-5000 rpm for 5 minutes at 4 ° C spectrophotometrically for enzymatic activity. The concentration of chymotrypsin remaining soluble in Pluronic® F-127 is shown below in Table 2. Table 2 Enzyme Solubility (mg / ml) 1 Dev. Est. Chymotrypsin 0.132 0.062 1 Determined by dividing the activity measured in the supernatant by the activity calculated per mg of protein in the total suspension. 2 n = 3. b) Suspended enzyme exhibits secondary structure similar to native co or is measured by FTIR: Infrared spectra obtained using a Nicolet Magna-IR 550 spectrophotometer are used at a regulated temperature, 6 μM long cells are used to examine and compare the structure of chymotrypsin in the phosphate buffer (30 mM, pH 7.4) and in 22 percent (w / w) of Pluronic ® F-127 in the same buffer. The concentration of the protein in these studies is 20 mg / ml. Careful examination of the spectra shows that the secondary structure of chymotrypsin is not altered due to the suspension of the protein in Pluronic® F-127. Further spectroscopic examinations of the FTIR structure of chymotrypsin in Pluronic® F-127 at temperatures below and above the gel transition (approximately 15 ° C to 18 ° C) shows that gel formation does not alter the structure. c) Suspended enzyme maintains its biological activity For this experiment, a chymotrypsin suspension is prepared in 22 percent Pluronic ® F-127 as described above but in two different concentrations, 20 mg / ml and 2 mg / ml. Solutions with the same protein concentrations are prepared using phosphate buffer (30 mM, pH 7.4) without Pluronic® F-127. Aliquots of each of these are diluted as necessary and assayed for enzymatic activity as described above. As can be seen from Table 3, below, the enzyme incorporated in the gels can be recovered quantitatively and without loss of activity after the gels are dissolved in a buffer. Table 3 1 Phosphate, 30 mM, pH 7.4 2 22% (w / w) in 30 mM phosphate buffer, pH 7.4 3 expressed as mAU / min at 410 nm 4 n = 9 Biological activity is not damaged by inclusion in Pluronic® F-127. A slight increase in activity is observed when low levels of the detergent are present in the enzyme assay mixtures. It is possible that the difference in the percent recovery observed between the 2 mg / ml and 20 mg / ml samples shown in Table 3 is due to the higher dilution of the gel in the 20 mg / ml sample and thus levels of Pluronic ® F-127 in the 2 mg / ml samples. d) Suspended enzyme exhibits increased storage stability: In order to demonstrate that proteins suspended in 22 percent Pluronic ® F-127 have a stabilizing action, 20 mg / ml chymotrypsin is prepared in either 30 mM phosphate buffer, pH 7.4 or 22 percent Pluronic ® F-127 in this buffer as described above. These dilutions are diluted as necessary and the enzymatic activity is assayed. The results shown in Table 4 below demonstrate that the enzyme incubated in the presence of Pluronic® F-127 retains more activity than the enzyme incubated in the presence of the phosphate buffer alone. Table 4 Time (h) Amorti- Pluronic1 Amortigua Pluronic Amortigua Pluronic 1 phosphate 2 22% (w / w) in phosphate buffer, 30 mM, pH 7.4 3 expressed as a percentage of value of 1 hour 4 value not available In this way , Pluronic® F-127 provides a stabilizing environment, which is most evident at prolonged times and high temperatures, in which peptide and protein containing drugs can be suspended before and during delivery. Additionally, other protein stabilizing agents can be added to the formulation as described further in Example 6 below. EXAMPLE 2 Preparation and Characterization of a Suspension of Subtilisin in Pluronic® F-127 a) Preparation of the suspension: A suspension of subtilisin is made by the same procedure described in detail above for chymotrypsin. The physical properties are the same in terms of formation and behavior of the milky white suspension and sun gel transition temperature. The solubility of subtilisin in the liquid phase of the suspension is examined using the method described above and found to be different as might be expected for a different protein. The results are shown in Table 5 below. Table 5 Enzyme Solubility (mg / ml) 1 Dev. Est. Subtilisin 6.482 l.ll2 1 Determined by dividing the measured activity in the supernatant by the activity calculated per mg of protein in the entire suspension. 2 n = 2. b) Suspended enzyme exhibits secondary structure similar to native as measured by FTIR; The subtilisin suspensions, such as those of the chymotrypsin described above, are examined by FTIR spectroscopy to determine if the inclusion in the gel has left any effect on the structure and if the gel-gel transition influences the structure. As in the case of chymotrypsin, there is no effect on the structure. s) Suspended enzyme maintains biological activity; In experiments similar to those described above for chymotrypsin, the suspensions of subtilisin in both phosphate buffer and 22 percent of Pluronic® F-127 in phosphate buffer are prepared and then dissolved by dilution with buffer. In these cases as in the case of chymotrypsin, 100 percent of enzyme activity is recovered showing that the biological activity is stable while it is incorporated into the gel suspension d) Suspended enzyme exhibits increased stability in storage In order to demonstrate that subtilisin suspended in 22 percent Pluronic F-127 has a stabilizing action similar to that demonstrated by chymotrypsin, 20 mg / ml subtilisin is prepared in either 30 mM phosphate buffer, pH 7.4 or 22 percent Pluronic (®F-127 in phosphate buffer as described above.) These suspensions are incubated at 8, 25, and 37 ° C for times up to 118 hours, the gels or suspensions are diluted as necessary and the enzymatic activity is assayed. The results, shown below in Table 6, demonstrate that although subtilisin autocatalyzes and loses activity more rapidly than chymotrypsin, it still retains more activity when incubated in the presence of Pluronic® F-127 than when incubated in the presence of the phosphate buffer alone. Table 6 1 phosphate, 30 mM, pH 7.4 2 22% (w / w) in phosphate buffer, 30 mM, pH 7.4 3 expressed as a percentage of value of 0.5 hour EXAMPLE 3 Preparation and characterization of a suspension of lactate dehydrogenase in Pluronic® F-127 a) Preparation of the suspension: A suspension of lactate dehydrogenase is made by the same procedure described in detail above for Chymotrypsin The visual observations indicate that the physical properties are the same in terms of the formation and behavior of the milky white suspension and sol gel transition temperature. b) Suspended enzyme maintains biological activity; In experiments similar to those described above for chymotrypsin, suspensions of lactate dehydrogenase are prepared in both phosphate buffer and 22 percent Pluronic® F-127 in phosphate buffer and then dissolved by dilution with buffer. In these cases as in the case of chymotrypsin, 100 percent of the enzyme activity is recovered showing that the biological activity is stable while it is incorporated into the gel suspension. c) Suspended enzyme exhibits increased storage stability Experiments similar to those performed with chymotrypsin are performed to illustrate the effect of Pluronic ® F-127 on the stabilization of lactate dehydrogenase enzymatic activity during storage at elevated temperatures. In this case, 0.5 M sucrose is included in some samples and in this way it is necessary to reduce the concentration of Pluronic ® F-127 to 18 percent in order to maintain the transition temperature of gel sol. Table 7 below shows the results obtained when lactate dehydrogenase is stored for 48 hours at 37 ° C. Table 7 Composition1 of the sample Activity Dev. Enzymatic enzyme2 (n = 3) 18% Pluronic 59.3 1.7 18% pluronic + sucrose 0.5 M 46.1 2.7 sucrose 0.5 M 39.1 3.3 buffer only 40.0 1.7 1 buffer used in all is tris: 30 mM HCl, pH 7.35 with 0.1% NaN3. 2 expressed in arbitrary units EXAMPLE 4 Preparation and characterization of a suspension of bovine serum albumin in Pluronic® F-127 a) Preparation of the suspension: Various mixtures of Pluronic® F-127 and bovine serum albumin are made in attempts to find a combination that could be a clear gel with a protein concentration of 15 mg / ml or more at room temperature. Concentrations of Pluronic ® F-127 below 20 percent (w / w) may not gel at 25 ° C or below and concentrations of 20 percent and above result in the milky, white suspension when serum albumin is added of bovine at concentrations of 14 mg / ml and above. In this way, no combination is found that can provide the desired properties. Subsequent studies of structure and capacity in several proteins demonstrate that it is not necessary to have a clear gel to have a satisfactory drug-protein delivery formulation with Pluronic® F-127. b) Suspended protein exhibits secondary structure similar to native as measured by FTIR Preliminary FTIR spectroscopic examination of the structure of a 20 mg / ml suspension of bovine serum albumin in 22 percent of Pluronic® F-127 shows no difference of a similar suspension of the protein in shock absorber alone. This result is similar to the results of more prolonged examinations of the chymotrypsin and subtilisin structures suspended in the gel. EXAMPLE 5 Preparation and Characterization of an Insulin Suspension in Pluronic® F-127 a) Preparation of the suspension: An insulin suspension is made by the same procedure described in detail above for chymotrypsin. Visual observation indicates that the physical properties are the same in terms of the formation and behavior of the milky white suspension and gel transition temperature. b) Suspended protein exhibits secondary structure similar to native as measured by FTIR Insulin suspensions are examined, like those of chymotrypsin described above, by FTIR spectroscopy to determine if the inclusion in the gel has any effect on the structure and if the transition of sun gel influences the structure. As in the case of chymotrypsin, there is no effect on the structure. EXAMPLE 6 Protein stabilizing agents used to manipulate the properties of the gels Several sucrose concentrates are incorporated into solutions of Pluronic® 127 as an example of the use of known protein stabilizing agents to manipulate the properties of the gels. Typical results for sol gel transition temperature are shown in Table 8 below: Table 8 1 p / p, in 30 mM phosphate, pH 7.4 It is clear from Table 8 that the transition temperature of the sol gel can be manipulated in the range below 0 ° C to about 25 ° C as required by the present invention. The description mentioned above is considered as illustrative only of the principles of the invention. Additionally, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and processes shown as described above. Accordingly, all suitable modifications and equivalents may fall within the scope of the invention as defined by the claims that follow.

Claims (32)

1. CLAIMS 1. A pharmaceutical composition for the controlled and sustained administration of biologically active macromolecular polypeptides characterized in that it comprises a polymeric matrix having thermal gelatinization properties in which the soluble aqueous particles of at least one biologically macromolecular polypeptide are incorporated into the polymer mixture. active at a sufficient concentration to form a suspension when incorporated into the polymer matrix.
2. 2. The composition according to claim 1, characterized in that the polymer matrix is a polyoxyalkylene block copolymer.
3. 3. The composition according to claim 2, characterized in that the polyoxyalkylene block copolymer has the formula: HO (C2H40) b (C2H60) a (C2H40) bH Where the polyoxyethylene portion constitutes at least about 70 percent by weight of the polyoxyalkylene block copolymer, wherein a and b are integers such that the hydrophobic base represented by (C3HsO) a has a molecular weight of at least about 4,000, and wherein the copolymer has an average molecular weight of about 12,600.
4. 4. The composition according to claim 2, characterized in that the polyoxyalkylene block copolymer has the formula: CH3 H (0CH2CH2) b (0CHCH2) s (0CH2CH2) b0H wherein a is 20 to 80 and b is 15 to 60.
5. The composition according to claim 4, characterized in that a is 67 and b is 49.
6. 6. The composition according to claim 1, characterized in that the macromolecular polypeptide particles constitute more than 0.5 weight percent of the composition.
7. The composition according to claim 1, characterized in that the incorporated macromolecular polypeptide retains more than 90 percent of the biological activity which the polypeptide possesses before being incorporated.
8. 8. The composition according to claim 1, characterized in that the transition temperature exists, therefore if the composition is maintained at a first temperature below the transition temperature the composition will exist in a liquid phase and if the composition is maintained at a second temperature above the transition temperature the composition will exist in a gelatinization phase.
9. 9. The composition according to claim 8, characterized in that it also comprises a solute that varies the transition temperature at which the composition will exist in either the liquid or gelatinous phase
10. 10. The composition according to claim 9, characterized in that the transition temperature decreases when the solute is sucrose.
11. 11. The composition according to claim 10, characterized in that the sucrose has a molar concentration of 0.5 to 1.5.
12. 12. The composition according to claim 9, characterized in that the transition temperature increases when the solute is polyethylene glycol.
13. The composition according to claim 9, characterized in that the minimum concentration of the polymer matrix necessary to gelify decreases after the addition of the solute.
14. 14. A pharmaceutical composition for controlled and sustained administration of a biologically active macromolecular polypeptide comprising a polymer matrix in which a suspension of soluble aqueous macromolecular polypeptide particles is incorporated.
15. 15. The composition according to claim 14, characterized in that the polypeptide retains more than 90 percent of the biological activity which the polypeptide possesses before being incorporated into the polymer matrix.
16. 16. The composition according to claim 14, characterized in that the matrix possesses thermal gelatinization properties.
17. 17. The composition according to claim 16, characterized in that the polymer matrix is a polyoxyethylene polyoxypropylene block copolymer.
18. 18. The composition according to claim 16, characterized in that the concentration of the polymer matrix necessary to form a gel can be decreased with the addition of a solute.
19. 19. The composition according to claim 18, characterized in that the solute is sucrose.
20. 20. The composition according to claim 9, characterized in that the solute is a protein stabilizer.
21. The composition according to claim 16, characterized in that there is a transition temperature where the polymer matrix will form a gel if it is in a liquid phase or alternatively the polymer matrix will form a liquid if it is in a gelatinous phase.
22. 22. The composition according to claim 21, characterized in that the transition temperature can be varied with the addition of a solute.
23. 23. The composition according to claim 22, characterized in that the transition temperature decreases when the solute is sucrose.
24. 24. The composition according to claim 22, characterized in that the transition temperature is increased when the solute is propylene glycol.
25. 25. The composition according to claim 22, characterized in that the solute is a protein stabilizer.
26. 26. The composition according to claim 22, characterized in that the solute is sucrose.
27. 27. The composition according to claim 22, characterized in that the solute is propylene glycol.
28. 28. A method for preparing the pharmaceutical composition according to claim 1, characterized in that it comprises: Preparing a mixture of a polymer matrix having thermal gelatinization properties; and Adding the mixture to at least one soluble biologically active macromolecular polypeptide.
29. 29. The method according to claim 28, characterized in that the polymer matrix is a polyoxyalkylene block copolymer.
30. 30. The method according to claim 29, characterized in that the polyoxyalkylene block copolymer has the formula: HO (C2H40) b (C2H60) a (C2H40) bH Where a is 20 to 80 and b is 15 to 60.
31. 31 The method according to claim 28, characterized in that it additionally comprises adding sucrose.
32. 32. The method according to claim 28, characterized in that it comprises additionally adding polyethylene glycol.