- Email: [email protected]
FDA Perspective on Peptide Formulation and Stability Issues
FDA Perspective on Peptide Formulation and Stability Issues CHIEN-HUA NIU*
AND
YUAN-YUAN CHIU
Contribution from Office of New Drug Chemistry, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, Food and Drug Administration, Parklawn Building, Room 14B-45 5600 Fishers Lane, Rockville, Maryland 20857. Received March 16, 1998. Accepted for publication July 20, 1998. Abstract 0 Traditionally, peptide drugs are prepared as sterile solutions and administered to patients by daily injection. However, this form of drug delivery causes pain and inconvenience to patients and thus has been poorly accepted. In addition to improving patient compliance, many novel delivery systems have been developed to address the need for prolonged, localized (targeted), or pulsatile drug action. Examples include, but are not limited to oral, nasal, or longacting controlled release injectable dosage forms; a number of them have been approved by FDA recently. The unique characteristics and the relevant regulatory issues with respect to each type of delivery system are presented.
Introduction Over the past two decades developments in the field of peptide synthesis and chromatographic methodology have led to commercial availability of many clinically useful peptides. However, the advances in the large-scale manufacture of peptides has outpaced our ability to deliver these molecules systemically in convenient and effective delivery systems. The main obstacles to delivery of peptides are related to their chemical and physical properties. For example, transdermal delivery of peptides is not feasible due to their relatively large molecular size. The oral route has not been suitable because of their susceptibility to hydrolysis and proteolytic breakdown. Also, progress is further hindered by a lack of complete understanding of the unique requirements and restrictions posed by the physicochemical and biological properties of peptide drugs, during the manufacturing and the storage of the formulations for different delivery systems. In contrast to conventional synthetic pharmaceuticals, peptides demonstrate more complex chemical modifications and are often more susceptible to denaturation and degradation.1 Recently, demands for effective delivery systems for peptide drugs has led to research and development in the area of new drug delivery technology. In addition to parenteral administration, other peptide delivery systems have been developed to administer peptides through the gut, sinus, mouth, and lungs.2-7 To complement different delivery systems, various peptide formulations are required. Each formulation has its own unique features. The following discussion focuses on three delivery systems, parenteral, oral, and nasal, and their FDA-approved formulations and their stability issues.
Formulation A. Parenteral Delivery Systems. Parenteral delivery of peptides has been the most common route for systemic delivery, because of ease of administration and the avoid* Corresponding author; Telephone: (301)827-6390; FAX: (301)4430072.
Published 1998, American Chemical Society and American Pharmaceutical Association
ance of biological barriers. Examples of peptides the FDA has approved for parenteral administration to patients are DDAVP (desmopressin), Sandostatin (octreotide), and Lupron (leuprolide). Currently, three different formulations are used in parenteral delivery systems; These are, (1) solution formulation, (2) powder formulation, and (3) depot formulation. There are common features among these formulations, and discussion will touch on these similarities; however, the focus will be to elaborate on the unique and important features of each of these formulations. 1. Solution Formulation. Dosage strength/potency, integrity, and stability of peptide drugs are of the utmost concern in the design of parenteral formulations. The most common sources that cause changes in peptide dosage and integrity are adsorption, sterilization, and chemical properties of the peptide. For peptides in solution, it is known that many peptides are absorbed or attached to manufacturing equipment or delivery devices.8-11 The absorption losses are most significant when the peptide concentration is relatively low. The result of loss due to absorption is a reduction in the dose to the patient if the loss is not corrected by some manner such as overage and/or overfill. A second common source of peptide loss can occur during sterilization. Many peptides cannot undergo terminal sterilization because of problems of degradation. To overcome this problem, sterile filtration and aspectic filling must be used to sterilize peptide drugs. In this case, it is possible that the peptide may be retained by membrane filters. Thus, the effect of membrane filtration on retention of peptide needs to be validated by modern analytical methods. Finally, factors that affect the dosage strength of a peptide drug include the physical properties of the peptide itself. For example, when a peptide (e.g., LHRH antagonists12,13) contains a large number of hydrophobic amino acid residues, there is an intrinsic propensity of the peptide to form aggregates, resulting in a lower effective concentration and, therefore, serious safety concerns. Several factors of the peptide in solution that one must consider and often affect adsorption loss and/or gel formation are ionic strength, excipients, pH, temperature, and osmotic pressure.14 To ensure that any accidentally introduced microorganism cannot grow in the multiple-dose injectable solution, a preservative is added to the formulation. The most commonly acceptable preservatives includes phenolic compounds (phenol, m-cresol, and benzyl alcohol). Since these preservatives are susceptible to oxidation and light, carefully monitoring the phenolic compound in the drug product is highly recommended during storage. 2. Lyophilized Powder Formulation. The stability problem of many peptides in solution is serious enough that peptides are commonly formulated as a solid by lyophilization and reconstituted with a sterile diluent prior to administration. Due to its high potency, often a small amount of peptide provides a sufficient therapeutic dose. Therefore, in this formulation, a bulking agent as a carrier is usually required. For example, lactose and mannitol are
10.1021/js9800782 Not subject to U.S. copyright.
Published on Web 09/16/1998
Journal of Pharmaceutical Sciences / 1331 Vol. 87, No. 11, November 1998
added during the formulation of Acthrel and Geref, respectively. The same precautions as described in the liquid formulation should be taken during the manufacture of the lyophilized powder because the manufacturing processes are generally identical between liquid formulation and lyophilized powder formulation, except the final process of lyophilization. After lyophilization, the peptide must also be analyzed for dosage strength as well as physicochemical and biological integrity. 3. Depot Formulation. To alleviate the pain caused by daily injections and to improve patient compliance, there has been interest in the use of biodegradable polymers for controlled release of peptides to be administrated via a parenteral route. In this delivery system, peptide drugs are embedded in polymer matrixes (microspheres) that undergo hydrolysis or enzymatic digestion, resulting in controlled release of the peptide into the human body.13 Commonly used in this delivery system is poly(lactic-coglycolic) (PLGA) copolymer. On exposure to water, the copolymer undergoes random chain scission by simple hydrolysis of the ester bond linkage and the drug is released.15 To date, a number of peptide drug products formulated for depot delivery, including Lupron Depot and Zoladex, have been approved by the FDA for marketing. These depot formulations are designed for steady release of the peptide over a period of 1 to 3 months after administration. During manufacture of the PLGA depot formulation, the following seven common assurances should be considered: (1) Copolymer: The chemical composition and ratio of monomers used in the polycondensation reaction strongly influences the degradation characteristics of the PLGA copolymer and thus drug release kinetics. Because the copolymer is a noncompendial excipient, it is important to determine the purity, chemical composition, and average molecular weight of the polymer (for details, see Guideline for Submitting Documentation for the Manufacture of and Controls for Drug Products, published by FDA, February, 1987). (2) Organic Solvents: Because many copolymers can only be dissolved in organic solvents, a residual amount of organic solvent (e.g., methylene chloride) will typically remain in the finished product. Because there is no therapeutic benefit from residual solvents and they may in fact cause inflammation or other adverse biological effects, all residual solvents should be removed to described safe levels (For details, see ICH Draft Guideline on Impurities: Residual Solvents; Availability Notice, published in Federal Register, Dec 4, 1997). (3) Copolymer-Peptide Complexes: Entrapped water in microspheres may cause degradation of the PLGA copolymer and shorten the shelf life of the finished drug product during storage. To avoid this problem, the residual water in the drug product needs to be removed under heat and vacuum. However, prolonged heating at or near the glass transition temperature of the polymer may increase the chance of reaction between the copolymer and the peptide to form conjugated complexes. The resultant products may be antigenic to the patient and may lower the effective dose of the peptide. One should also consider the effect of heat on the stability of peptide itself. To monitor the existence of these complexes or peptide degradation products, a sensitive analytical method, such as HPLC, should be used to test the final product. (4) Sterilization: The sterility of a depot formulation is another important quality issue that should be addressed. It is known that the γ-irradiation is unsuitable to accomplish the terminal sterilization of a depot formulation because it causes degradation of both the copolymer and peptide.16 Therefore, sterilization of the drug is carried out 1332 / Journal of Pharmaceutical Sciences Vol. 87, No. 11, November 1998
by membrane filtration and followed by an aseptic process. It is important to demonstrate sterility of both the external surfaces and internal core of the microspheres. To validate external sterility of the drug, a general compendial method can be used. For validation of internal sterility, the microspheres are dissolved in an appropriate organic solvent which will not kill spores, and the solution is then cultivated in a growth medium to detect microorganisms. (5) In Vitro and In Vivo Correlation of Peptide Release: For quality control of the microspheres, the in vitro release pattern should be determined. One can also examine the in vivo release rate in test animals. From these studies, a correlation between the in vitro and in vivo release pattern can be established. Ideally, both the in vitro and in vivo release patterns should follow approximately zero-order kinetics after an initial “burst” of the peptide adsorbed to the surface of the microspheres. (6) Particle Size: Problems related to microsphere particle size may occur at the extremes. For large particles, occasional clogging of the syringe tip and needle may occur. Very small particle sizes will not only generate greater initial “burst”, but also provide a high surface area that may be difficult to wet with the suspending vehicle.15 Therefore, careful selection of target particle size range is essential. Cautious control of the particle size distribution is an important feature during manufacture of the finished drug product. (7) Diluent (Suspending Vehicle): The vehicle needs to have the properties capable of keeping the microspheres well suspended in it. Optimization of the suspending vehicle plays an essential role in the successful delivery of the microspheres of the depot formulations to the human body through intramuscular or subcutaneous injection. The most critical factors for the selection of a vehicle include its wetting property, isotonicity, specific gravity, and viscosity.15 Each of these factors should be considered to minimize sedimentation rate. B. Oral Delivery Systems: Tablets. The most convenient route for the systemic delivery of pharmaceuticals is oral; however, attempts to deliver peptides orally have not been widely successful. Bioavailability via this route is poor because peptides are susceptible to hydrolysis and modification at gastric pH levels, and they can be degraded by proteolytic enzymes in the gastrointestinal (GI) tract. One way to overcome the enzymatic barriers is to modify the peptide structure in a way to increase its resistance to enzymatic degradation but not to reduce its biological potency. So far, there have been a few examples of modified peptide drugs with improved oral activity. One example is DDAVP (desmopression) tablets approved in 1995 for the treatment of patients with cranial diabetes insipidus (CDI). DDAVP differs structurally from the naturally occurring peptide, vasopressin, in two positions. DDAVP has β-mercaptopropoinic acid instead of hemicystine in position 1 and D-arginine in place of L-arginine in position 8. These modifications lead to enhanced stability against proteolytic degradation and increased membrane penetration.17 However, the peptide dose required for the tablet still is much higher than those needed by parenteral and nasal routes. There are two issues involved with respect to the manufacture of these peptide tablets: (1) Heat Granulation: Tablets can be manufactured using either a dry or a wet granulation process. If a wet granulation process is utilized, heat is usually applied to remove the water solvent from granules prior to compression into tablets. Similar to the problems encountered in depot formulation, precautions need to be taken to monitor the possible degradation of the peptide during the drying process.
(2) Dissolution Test: The bioavailability of tablets in vitro is usually determined by the USP dissolution test. However, presence of a low quantity of the drug substance, such as in the case of DDAVP tablets (0.1 and 0.2 mg), may require a modified version of the USP dissolution test to accommodate the low dose strength present in the tablets. C. Nasal Delivery System. A promising alternative for delivering peptide drugs is the nasal route. The advantages of intranasal delivery are (1) the large surface area of the nasal mucosa for absorption and (2) the rapid onset of action.18,19 The FDA has approved a number of peptide drugs formulated in intranasal dosage form, such as Diapid (lypressin), DDAVP (desmopressin), and nafarelin (Synarel). During formulation of the intranasal dosage for these peptide drugs, a number of important issues need to be considered: (1) Particle Size: Particle size distribution is an important parameter to be determined. Particle size determines whether the dispensed particles containing the peptide drug can penetrate into the nasal cavities. For optimal deposition in the nasal cavity, the mass median diameter should be >10 µm when an aerosol formulation is used.4,18 (2) Preservatives: Preservatives are needed to avoid microbial contamination during multiple application. A number of preservatives, including chlorobutanol and benzalkonium chloride, have been used. However, the stability of these preservatives in an aqueous solution varies. Therefore, it is essential to monitor the stability of preservatives in the drug product during storage. (3) Adsorption: As already discussed for solution formulations, it is a common phenomenon that peptides will adsorb onto plastic surfaces of containers.8,20,21 The adsorption of peptides onto containers results in underdosage to patients. Therefore, it is important to monitor the dosage strength during formulation and storage.
Stability To fully establish the stability of peptide drug products formulated for special delivery systems, one must consider all major degradation routes. The most common degradation pathways for peptides are aggregation, deamidation, isomerization, cleavage, oxidation, disulfide exchange, and β-elimination.22 The extent of chemical and physical degradation of peptides depends on temperature, the amino acid sequence, composition, pH, and conformation, along with the dosage forms. For liquid formulations, the major degradation pathways are hydrolysis, deamidation, and isomerization.23 In the case of lyophilized powder and tablet formulations, the general degradation patterns are similar to those observed in solution, although the removal of water by lyophilization or drying processes results in a reduction of the degradation rate constant and in alternation of certain degradation pathways (e.g., hydrolysis) that occur predominantly in the solution formulation.24 With respect to the microspheres depot formulation, aggregation and oxidation are the main causes of degradation of the peptide. Therefore, in stability studies of peptide formulations during storage, the physicochemical integrity and, in some instances, potency of the peptide needs to be demonstrated. The drug product must meet its specifications throughout its shelf life of the drug product.
Summary Many peptide delivery systems have been approved for marketing. Formulations for each route of administration are unique and have special characteristics, but in careful examination of these delivery systems, common features
appear. For example, in both liquid and lyophilized powder formulations, the sterilization process and loss of peptide concentration due to adsorption are critical issues during formulation. During manufacture of PLGA depot formulations, issues of purity and average molecular weight of the PLGA copolymer, residual organic solvents, duration and temperature used to remove solvents and water, particle size, and sterilization process need to be considered. In all cases, for a successful formulation, one should consider the clinical indication, pharmacokinetics, toxicity, and physicochemical stability of the drug product in the specific delivery system. Moreover, research on new peptides, peptide drug formulation, and delivery technology have progressed rapidly during the past few years. As new therapeutic peptides are discovered and formulated for commercialization, each new formulation for each route of delivery must be evaluated for its physicochemical properties, stability, safety, and efficacy.
References and Notes 1. Lee, V. H. L. Peptide and Protein Drug Delivery: Opportunities and Challenges. Pharm. Int. 1986, 7, 208-212. 2. Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Marcel Dekker: New York, 1990. 3. Peptide and Protein Drug Delivery; Lee, V. H. L., Ed.; Marcel Dekker: New York, 1990. 4. Novel Drug Delivery Systems; Chien, Y. W., Ed.; Mercel Dekker: New York, 1992. 5. Formulation and Delivery of Proteins and Peptides; Cleland, J. L.; Langer, R., Eds.; American Chemical Society: Washington, DC, 1994. 6. Ranade, V. V.; Hollinger, M. A. Drug Delivery Systems; CRC: Florida, 1996. 7. Inhalation Delivery of Therapeutic Peptides and Proteins; Adjei, A. L.; Gupta, P. K., Eds.; Marcel Dekker: New York, 1997. 8. Anik, S. T.; Hwang, J.-Y. Adsorption of D-Nal(2)6LHRH, a Decapeptide, onto Glass and Other Surface. Int. J. Pharm. 1983, 16, 181-190. 9. McElnay, J. C.; Elliott, D. S.; D’Arcy, P. F. Binding of Human Insulin to Buret Administration Sets. Int. J. Pharm. 1987, 36, 199-203. 10. Adams, P. S.; Haines-Nutt, R. F.; Town, R. Stability of Insulin Mixtures in Disposable Plastic Insulin Syringes. J. Pharm. Pharmacol. 1987, 39, 158-163. 11. Chantelau, E.; Lange, G.; Gasthaus, M.; Boxberger, M.; Berger, M. Interaction between Plastic Catheter Tubings and Regular Insulin Preparations Used for Continuous Subcutaneous Insulin-Infusion Therapy. Diabetes Care 1987, 10, 348-351. 12. Nestor, J. J., Jr.; Tahilramani, R.; Ho, T. L.; McRae, G. I.; Vickery, B. H. New Luteinizing Hormone-Releasing Factor Antagonists. In Peptides, Structure and Function, Proceeding of the 8th American Peptide Symposium; Hruby, V. J.; Rich, D. H., Eds.; Pierce Chemical Company: Rockford, IL, 1983; pp 861-864. 13. Haviv, F., et al. The Effect of NMeTyr5 Substitution in Luteinizing Hormone-Releasing Hormone Antgonists. J. Med. Chem. 1993, 36, 928-933. 14. Banerjee, P. S.; Hosny, E. A.; Robinson, J. R. Parenteral Delivery of Peptide and Protein Drugs. In Peptide and Protein Drug Delivery, Lee, V. H. L., Ed.; Marcel Dekker: New York, 1991; pp 487-540. 15. Sanders, L. M. Controlled Delivery Systems of Peptides. In Peptide and Protein Drug Delivery, Lee, V. H. L., Ed.; Marcel Dekker: New York, 1991; pp 785-803. 16. Lewis, D. H. Controlled Release of Bioactive Agents from Lactide/Glycolide Polymers. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M., Langer, R., Eds.; Marcel Dekker: New York, 1990; pp 1-41. 17. Vilhard, H.; Lundin, H. In Vitro Intestinal Transport of Vasopressin and its Analogues. Acta Physiol. Scand. 1986, 126, 601-607. 18. Su, K. S. E. Nasal Route of Peptide and Protein Drug Delivery. In Peptide and Protein Drug Delivery; Lee. H. L., Ed.; Marcel Dekker: New York, 1991; pp 595-631. 19. Chien, Y. W. Novel Drug Delivery Systems, 2nd ed.; Marcel Dekker: New York, 1992; Chapter 5. 20. Mizutani, T. Estimation of Protein and Drug Adsorption onto Silicone-Coated Glass Surface. J. Pharm. Sci. 1981, 70, 493496.
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21. Ogino, J.; Noguchi, K.; Terato, K. Adsorption of Secretin on Glass Surfaces. Chem. Pharm. Bull. 1979, 27, 3160-3163. 22. Cleland, J. L.; Langer, R. Formulation and Delivery of Proteins and Peptides. In Formulation and Delivery of Proteins and Peptides; Cleland, J. L.; Langer, R., Eds.; American Chemical Society: Washington, DC, 1994; pp 1-19. 23. Powell, M. F. Peptide Stability in Aqueous Parenteral Formulation: Prediction of Chemical Stability Based on Primary Sequence. In Formulation and Delivery of Proteins and Peptides; Cleland, J. L.; Langer, R., Eds.; American Chemical Society: Washington, DC, 1994; pp 100-117.
1334 / Journal of Pharmaceutical Sciences Vol. 87, No. 11, November 1998
24. Oliyai, C.; Patel, J. P.; Carr, L.; Borchardt, R. T. Chemical Pathways of Peptide Degradation. VII. Solid State Chemical Instability of an Aspartyl Residue in a Model Hexapeptide. Pharm. Res. 1994, 11, 901-908.
Acknowledgments The authors thank Dr. Joseph T. Woitach of National Cancer Institute for his comments and suggestions to improve the manuscript.
JS9800782
AND
YUAN-YUAN CHIU
Contribution from Office of New Drug Chemistry, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, Food and Drug Administration, Parklawn Building, Room 14B-45 5600 Fishers Lane, Rockville, Maryland 20857. Received March 16, 1998. Accepted for publication July 20, 1998. Abstract 0 Traditionally, peptide drugs are prepared as sterile solutions and administered to patients by daily injection. However, this form of drug delivery causes pain and inconvenience to patients and thus has been poorly accepted. In addition to improving patient compliance, many novel delivery systems have been developed to address the need for prolonged, localized (targeted), or pulsatile drug action. Examples include, but are not limited to oral, nasal, or longacting controlled release injectable dosage forms; a number of them have been approved by FDA recently. The unique characteristics and the relevant regulatory issues with respect to each type of delivery system are presented.
Introduction Over the past two decades developments in the field of peptide synthesis and chromatographic methodology have led to commercial availability of many clinically useful peptides. However, the advances in the large-scale manufacture of peptides has outpaced our ability to deliver these molecules systemically in convenient and effective delivery systems. The main obstacles to delivery of peptides are related to their chemical and physical properties. For example, transdermal delivery of peptides is not feasible due to their relatively large molecular size. The oral route has not been suitable because of their susceptibility to hydrolysis and proteolytic breakdown. Also, progress is further hindered by a lack of complete understanding of the unique requirements and restrictions posed by the physicochemical and biological properties of peptide drugs, during the manufacturing and the storage of the formulations for different delivery systems. In contrast to conventional synthetic pharmaceuticals, peptides demonstrate more complex chemical modifications and are often more susceptible to denaturation and degradation.1 Recently, demands for effective delivery systems for peptide drugs has led to research and development in the area of new drug delivery technology. In addition to parenteral administration, other peptide delivery systems have been developed to administer peptides through the gut, sinus, mouth, and lungs.2-7 To complement different delivery systems, various peptide formulations are required. Each formulation has its own unique features. The following discussion focuses on three delivery systems, parenteral, oral, and nasal, and their FDA-approved formulations and their stability issues.
Formulation A. Parenteral Delivery Systems. Parenteral delivery of peptides has been the most common route for systemic delivery, because of ease of administration and the avoid* Corresponding author; Telephone: (301)827-6390; FAX: (301)4430072.
Published 1998, American Chemical Society and American Pharmaceutical Association
ance of biological barriers. Examples of peptides the FDA has approved for parenteral administration to patients are DDAVP (desmopressin), Sandostatin (octreotide), and Lupron (leuprolide). Currently, three different formulations are used in parenteral delivery systems; These are, (1) solution formulation, (2) powder formulation, and (3) depot formulation. There are common features among these formulations, and discussion will touch on these similarities; however, the focus will be to elaborate on the unique and important features of each of these formulations. 1. Solution Formulation. Dosage strength/potency, integrity, and stability of peptide drugs are of the utmost concern in the design of parenteral formulations. The most common sources that cause changes in peptide dosage and integrity are adsorption, sterilization, and chemical properties of the peptide. For peptides in solution, it is known that many peptides are absorbed or attached to manufacturing equipment or delivery devices.8-11 The absorption losses are most significant when the peptide concentration is relatively low. The result of loss due to absorption is a reduction in the dose to the patient if the loss is not corrected by some manner such as overage and/or overfill. A second common source of peptide loss can occur during sterilization. Many peptides cannot undergo terminal sterilization because of problems of degradation. To overcome this problem, sterile filtration and aspectic filling must be used to sterilize peptide drugs. In this case, it is possible that the peptide may be retained by membrane filters. Thus, the effect of membrane filtration on retention of peptide needs to be validated by modern analytical methods. Finally, factors that affect the dosage strength of a peptide drug include the physical properties of the peptide itself. For example, when a peptide (e.g., LHRH antagonists12,13) contains a large number of hydrophobic amino acid residues, there is an intrinsic propensity of the peptide to form aggregates, resulting in a lower effective concentration and, therefore, serious safety concerns. Several factors of the peptide in solution that one must consider and often affect adsorption loss and/or gel formation are ionic strength, excipients, pH, temperature, and osmotic pressure.14 To ensure that any accidentally introduced microorganism cannot grow in the multiple-dose injectable solution, a preservative is added to the formulation. The most commonly acceptable preservatives includes phenolic compounds (phenol, m-cresol, and benzyl alcohol). Since these preservatives are susceptible to oxidation and light, carefully monitoring the phenolic compound in the drug product is highly recommended during storage. 2. Lyophilized Powder Formulation. The stability problem of many peptides in solution is serious enough that peptides are commonly formulated as a solid by lyophilization and reconstituted with a sterile diluent prior to administration. Due to its high potency, often a small amount of peptide provides a sufficient therapeutic dose. Therefore, in this formulation, a bulking agent as a carrier is usually required. For example, lactose and mannitol are
10.1021/js9800782 Not subject to U.S. copyright.
Published on Web 09/16/1998
Journal of Pharmaceutical Sciences / 1331 Vol. 87, No. 11, November 1998
added during the formulation of Acthrel and Geref, respectively. The same precautions as described in the liquid formulation should be taken during the manufacture of the lyophilized powder because the manufacturing processes are generally identical between liquid formulation and lyophilized powder formulation, except the final process of lyophilization. After lyophilization, the peptide must also be analyzed for dosage strength as well as physicochemical and biological integrity. 3. Depot Formulation. To alleviate the pain caused by daily injections and to improve patient compliance, there has been interest in the use of biodegradable polymers for controlled release of peptides to be administrated via a parenteral route. In this delivery system, peptide drugs are embedded in polymer matrixes (microspheres) that undergo hydrolysis or enzymatic digestion, resulting in controlled release of the peptide into the human body.13 Commonly used in this delivery system is poly(lactic-coglycolic) (PLGA) copolymer. On exposure to water, the copolymer undergoes random chain scission by simple hydrolysis of the ester bond linkage and the drug is released.15 To date, a number of peptide drug products formulated for depot delivery, including Lupron Depot and Zoladex, have been approved by the FDA for marketing. These depot formulations are designed for steady release of the peptide over a period of 1 to 3 months after administration. During manufacture of the PLGA depot formulation, the following seven common assurances should be considered: (1) Copolymer: The chemical composition and ratio of monomers used in the polycondensation reaction strongly influences the degradation characteristics of the PLGA copolymer and thus drug release kinetics. Because the copolymer is a noncompendial excipient, it is important to determine the purity, chemical composition, and average molecular weight of the polymer (for details, see Guideline for Submitting Documentation for the Manufacture of and Controls for Drug Products, published by FDA, February, 1987). (2) Organic Solvents: Because many copolymers can only be dissolved in organic solvents, a residual amount of organic solvent (e.g., methylene chloride) will typically remain in the finished product. Because there is no therapeutic benefit from residual solvents and they may in fact cause inflammation or other adverse biological effects, all residual solvents should be removed to described safe levels (For details, see ICH Draft Guideline on Impurities: Residual Solvents; Availability Notice, published in Federal Register, Dec 4, 1997). (3) Copolymer-Peptide Complexes: Entrapped water in microspheres may cause degradation of the PLGA copolymer and shorten the shelf life of the finished drug product during storage. To avoid this problem, the residual water in the drug product needs to be removed under heat and vacuum. However, prolonged heating at or near the glass transition temperature of the polymer may increase the chance of reaction between the copolymer and the peptide to form conjugated complexes. The resultant products may be antigenic to the patient and may lower the effective dose of the peptide. One should also consider the effect of heat on the stability of peptide itself. To monitor the existence of these complexes or peptide degradation products, a sensitive analytical method, such as HPLC, should be used to test the final product. (4) Sterilization: The sterility of a depot formulation is another important quality issue that should be addressed. It is known that the γ-irradiation is unsuitable to accomplish the terminal sterilization of a depot formulation because it causes degradation of both the copolymer and peptide.16 Therefore, sterilization of the drug is carried out 1332 / Journal of Pharmaceutical Sciences Vol. 87, No. 11, November 1998
by membrane filtration and followed by an aseptic process. It is important to demonstrate sterility of both the external surfaces and internal core of the microspheres. To validate external sterility of the drug, a general compendial method can be used. For validation of internal sterility, the microspheres are dissolved in an appropriate organic solvent which will not kill spores, and the solution is then cultivated in a growth medium to detect microorganisms. (5) In Vitro and In Vivo Correlation of Peptide Release: For quality control of the microspheres, the in vitro release pattern should be determined. One can also examine the in vivo release rate in test animals. From these studies, a correlation between the in vitro and in vivo release pattern can be established. Ideally, both the in vitro and in vivo release patterns should follow approximately zero-order kinetics after an initial “burst” of the peptide adsorbed to the surface of the microspheres. (6) Particle Size: Problems related to microsphere particle size may occur at the extremes. For large particles, occasional clogging of the syringe tip and needle may occur. Very small particle sizes will not only generate greater initial “burst”, but also provide a high surface area that may be difficult to wet with the suspending vehicle.15 Therefore, careful selection of target particle size range is essential. Cautious control of the particle size distribution is an important feature during manufacture of the finished drug product. (7) Diluent (Suspending Vehicle): The vehicle needs to have the properties capable of keeping the microspheres well suspended in it. Optimization of the suspending vehicle plays an essential role in the successful delivery of the microspheres of the depot formulations to the human body through intramuscular or subcutaneous injection. The most critical factors for the selection of a vehicle include its wetting property, isotonicity, specific gravity, and viscosity.15 Each of these factors should be considered to minimize sedimentation rate. B. Oral Delivery Systems: Tablets. The most convenient route for the systemic delivery of pharmaceuticals is oral; however, attempts to deliver peptides orally have not been widely successful. Bioavailability via this route is poor because peptides are susceptible to hydrolysis and modification at gastric pH levels, and they can be degraded by proteolytic enzymes in the gastrointestinal (GI) tract. One way to overcome the enzymatic barriers is to modify the peptide structure in a way to increase its resistance to enzymatic degradation but not to reduce its biological potency. So far, there have been a few examples of modified peptide drugs with improved oral activity. One example is DDAVP (desmopression) tablets approved in 1995 for the treatment of patients with cranial diabetes insipidus (CDI). DDAVP differs structurally from the naturally occurring peptide, vasopressin, in two positions. DDAVP has β-mercaptopropoinic acid instead of hemicystine in position 1 and D-arginine in place of L-arginine in position 8. These modifications lead to enhanced stability against proteolytic degradation and increased membrane penetration.17 However, the peptide dose required for the tablet still is much higher than those needed by parenteral and nasal routes. There are two issues involved with respect to the manufacture of these peptide tablets: (1) Heat Granulation: Tablets can be manufactured using either a dry or a wet granulation process. If a wet granulation process is utilized, heat is usually applied to remove the water solvent from granules prior to compression into tablets. Similar to the problems encountered in depot formulation, precautions need to be taken to monitor the possible degradation of the peptide during the drying process.
(2) Dissolution Test: The bioavailability of tablets in vitro is usually determined by the USP dissolution test. However, presence of a low quantity of the drug substance, such as in the case of DDAVP tablets (0.1 and 0.2 mg), may require a modified version of the USP dissolution test to accommodate the low dose strength present in the tablets. C. Nasal Delivery System. A promising alternative for delivering peptide drugs is the nasal route. The advantages of intranasal delivery are (1) the large surface area of the nasal mucosa for absorption and (2) the rapid onset of action.18,19 The FDA has approved a number of peptide drugs formulated in intranasal dosage form, such as Diapid (lypressin), DDAVP (desmopressin), and nafarelin (Synarel). During formulation of the intranasal dosage for these peptide drugs, a number of important issues need to be considered: (1) Particle Size: Particle size distribution is an important parameter to be determined. Particle size determines whether the dispensed particles containing the peptide drug can penetrate into the nasal cavities. For optimal deposition in the nasal cavity, the mass median diameter should be >10 µm when an aerosol formulation is used.4,18 (2) Preservatives: Preservatives are needed to avoid microbial contamination during multiple application. A number of preservatives, including chlorobutanol and benzalkonium chloride, have been used. However, the stability of these preservatives in an aqueous solution varies. Therefore, it is essential to monitor the stability of preservatives in the drug product during storage. (3) Adsorption: As already discussed for solution formulations, it is a common phenomenon that peptides will adsorb onto plastic surfaces of containers.8,20,21 The adsorption of peptides onto containers results in underdosage to patients. Therefore, it is important to monitor the dosage strength during formulation and storage.
Stability To fully establish the stability of peptide drug products formulated for special delivery systems, one must consider all major degradation routes. The most common degradation pathways for peptides are aggregation, deamidation, isomerization, cleavage, oxidation, disulfide exchange, and β-elimination.22 The extent of chemical and physical degradation of peptides depends on temperature, the amino acid sequence, composition, pH, and conformation, along with the dosage forms. For liquid formulations, the major degradation pathways are hydrolysis, deamidation, and isomerization.23 In the case of lyophilized powder and tablet formulations, the general degradation patterns are similar to those observed in solution, although the removal of water by lyophilization or drying processes results in a reduction of the degradation rate constant and in alternation of certain degradation pathways (e.g., hydrolysis) that occur predominantly in the solution formulation.24 With respect to the microspheres depot formulation, aggregation and oxidation are the main causes of degradation of the peptide. Therefore, in stability studies of peptide formulations during storage, the physicochemical integrity and, in some instances, potency of the peptide needs to be demonstrated. The drug product must meet its specifications throughout its shelf life of the drug product.
Summary Many peptide delivery systems have been approved for marketing. Formulations for each route of administration are unique and have special characteristics, but in careful examination of these delivery systems, common features
appear. For example, in both liquid and lyophilized powder formulations, the sterilization process and loss of peptide concentration due to adsorption are critical issues during formulation. During manufacture of PLGA depot formulations, issues of purity and average molecular weight of the PLGA copolymer, residual organic solvents, duration and temperature used to remove solvents and water, particle size, and sterilization process need to be considered. In all cases, for a successful formulation, one should consider the clinical indication, pharmacokinetics, toxicity, and physicochemical stability of the drug product in the specific delivery system. Moreover, research on new peptides, peptide drug formulation, and delivery technology have progressed rapidly during the past few years. As new therapeutic peptides are discovered and formulated for commercialization, each new formulation for each route of delivery must be evaluated for its physicochemical properties, stability, safety, and efficacy.
References and Notes 1. Lee, V. H. L. Peptide and Protein Drug Delivery: Opportunities and Challenges. Pharm. Int. 1986, 7, 208-212. 2. Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Marcel Dekker: New York, 1990. 3. Peptide and Protein Drug Delivery; Lee, V. H. L., Ed.; Marcel Dekker: New York, 1990. 4. Novel Drug Delivery Systems; Chien, Y. W., Ed.; Mercel Dekker: New York, 1992. 5. Formulation and Delivery of Proteins and Peptides; Cleland, J. L.; Langer, R., Eds.; American Chemical Society: Washington, DC, 1994. 6. Ranade, V. V.; Hollinger, M. A. Drug Delivery Systems; CRC: Florida, 1996. 7. Inhalation Delivery of Therapeutic Peptides and Proteins; Adjei, A. L.; Gupta, P. K., Eds.; Marcel Dekker: New York, 1997. 8. Anik, S. T.; Hwang, J.-Y. Adsorption of D-Nal(2)6LHRH, a Decapeptide, onto Glass and Other Surface. Int. J. Pharm. 1983, 16, 181-190. 9. McElnay, J. C.; Elliott, D. S.; D’Arcy, P. F. Binding of Human Insulin to Buret Administration Sets. Int. J. Pharm. 1987, 36, 199-203. 10. Adams, P. S.; Haines-Nutt, R. F.; Town, R. Stability of Insulin Mixtures in Disposable Plastic Insulin Syringes. J. Pharm. Pharmacol. 1987, 39, 158-163. 11. Chantelau, E.; Lange, G.; Gasthaus, M.; Boxberger, M.; Berger, M. Interaction between Plastic Catheter Tubings and Regular Insulin Preparations Used for Continuous Subcutaneous Insulin-Infusion Therapy. Diabetes Care 1987, 10, 348-351. 12. Nestor, J. J., Jr.; Tahilramani, R.; Ho, T. L.; McRae, G. I.; Vickery, B. H. New Luteinizing Hormone-Releasing Factor Antagonists. In Peptides, Structure and Function, Proceeding of the 8th American Peptide Symposium; Hruby, V. J.; Rich, D. H., Eds.; Pierce Chemical Company: Rockford, IL, 1983; pp 861-864. 13. Haviv, F., et al. The Effect of NMeTyr5 Substitution in Luteinizing Hormone-Releasing Hormone Antgonists. J. Med. Chem. 1993, 36, 928-933. 14. Banerjee, P. S.; Hosny, E. A.; Robinson, J. R. Parenteral Delivery of Peptide and Protein Drugs. In Peptide and Protein Drug Delivery, Lee, V. H. L., Ed.; Marcel Dekker: New York, 1991; pp 487-540. 15. Sanders, L. M. Controlled Delivery Systems of Peptides. In Peptide and Protein Drug Delivery, Lee, V. H. L., Ed.; Marcel Dekker: New York, 1991; pp 785-803. 16. Lewis, D. H. Controlled Release of Bioactive Agents from Lactide/Glycolide Polymers. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M., Langer, R., Eds.; Marcel Dekker: New York, 1990; pp 1-41. 17. Vilhard, H.; Lundin, H. In Vitro Intestinal Transport of Vasopressin and its Analogues. Acta Physiol. Scand. 1986, 126, 601-607. 18. Su, K. S. E. Nasal Route of Peptide and Protein Drug Delivery. In Peptide and Protein Drug Delivery; Lee. H. L., Ed.; Marcel Dekker: New York, 1991; pp 595-631. 19. Chien, Y. W. Novel Drug Delivery Systems, 2nd ed.; Marcel Dekker: New York, 1992; Chapter 5. 20. Mizutani, T. Estimation of Protein and Drug Adsorption onto Silicone-Coated Glass Surface. J. Pharm. Sci. 1981, 70, 493496.
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21. Ogino, J.; Noguchi, K.; Terato, K. Adsorption of Secretin on Glass Surfaces. Chem. Pharm. Bull. 1979, 27, 3160-3163. 22. Cleland, J. L.; Langer, R. Formulation and Delivery of Proteins and Peptides. In Formulation and Delivery of Proteins and Peptides; Cleland, J. L.; Langer, R., Eds.; American Chemical Society: Washington, DC, 1994; pp 1-19. 23. Powell, M. F. Peptide Stability in Aqueous Parenteral Formulation: Prediction of Chemical Stability Based on Primary Sequence. In Formulation and Delivery of Proteins and Peptides; Cleland, J. L.; Langer, R., Eds.; American Chemical Society: Washington, DC, 1994; pp 100-117.
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24. Oliyai, C.; Patel, J. P.; Carr, L.; Borchardt, R. T. Chemical Pathways of Peptide Degradation. VII. Solid State Chemical Instability of an Aspartyl Residue in a Model Hexapeptide. Pharm. Res. 1994, 11, 901-908.
Acknowledgments The authors thank Dr. Joseph T. Woitach of National Cancer Institute for his comments and suggestions to improve the manuscript.
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