- Email: [email protected]
Optimized dose delivery of the peptide cyclosporine using hydrofluoroalkane‐based metered dose inhalers
Optimized Dose Delivery of the Peptide Cyclosporine Using Hydrofluoroalkane-Based Metered Dose Inhalers PAUL B. MYRDAL,1 KELLY L. KARLAGE,1 STEVE W. STEIN,2 BETH A. BROWN,2 ALFRED HAYNES3 1
College of Pharmacy, University of Arizona, 1703 East Mabel Street, Tucson, Arizona 85721
2
Early Pharmaceutics and Technology, 3M Center, St. Paul, Minnesota 55144
3
College of Pharmacy, Florida A&M University, Tallahassee, Florida 32307
Received 23 June 2003; revised 13 November 2003; accepted 5 December 2003
ABSTRACT: The goal of this study was to illustrate the potential to deliver relatively high doses of a therapeutic peptide using hydrofluoroalkane (HFA) metered dose inhaler (MDI) drug delivery systems. For the purposes of this study, cyclosporine was used as the model compound. Cyclosporine formulations, varying in peptide concentration, ethanol cosolvent concentration, and propellant type, were evaluated and optimized for product performance. As ethanol concentration was decreased from 10 to 3% by weight, fine particle fraction (the mass of cyclosporine which passes through a 4.7-micron cut point divided by the total mass of cyclosporine delivered ex-valve) increased from 34 to 68% for 227 and 33 to 52% for 134a formulations. Because of the excellent solubility properties of cyclosporine in HFA-based systems, minimal or no ethanol was needed as a cosolvent to achieve cyclosporine concentrations of 1.5% w/w. With these formulations, it was possible to obtain a fine particle mass (mass of particles <4.7 microns) greater than 500 mg per actuation. In addition, one formulation was chosen for stability analysis: 0.09% w/w cyclosporine, 10% w/w ethanol, 134a. Three different types of container closure systems (stainless steel, aluminum, and epoxy-coated canisters) and two storage configurations (upright and inverted) were evaluated. Cyclosporine was determined to be stable in HFA 134a-based MDI systems, regardless of container closure system and configuration, over a 2-year period. Cyclosporine represents a compelling example of how significant peptide doses are attainable through the use of solution-based MDIs. It has been shown that through formulation optimization, 2–3 mg of the peptide, cyclosporine, may be delivered in five actuations to the lung for local or systemic therapy. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1054–1061, 2004
Keywords: pulmonary drug delivery; fine particle fraction; peptide delivery; protein delivery; aerosol; metered dose inhalers; hydrofluoroalkanes; cyclosporine
INTRODUCTION Pulmonary drug delivery is an important delivery route for the treatment of lung diseases, such as asthma and chronic obstructive pulmonary disease. This administration route is also being Correspondence to: Paul B. Myrdal (Telephone: 520-6261296; Fax: 520-626-4063; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 93, 1054–1061 (2004) ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association
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investigated as a potentially advantageous delivery route for proteins and peptides, examples of which are insulin,1–3 leuprolide,4,5 and cyclosporine.6 Degradation due to the acidic stomach environment and enzymatic degradation throughout the gastrointestinal tract have traditionally made oral delivery of therapeutic proteins and peptides problematic. In addition, the low permeability, inadequate stability, and relatively short half-lives of these compounds often necessitate administration via injection. With inhalation
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drug delivery, these compounds can either be directly administered to the lungs for localized treatment of respiratory diseases, or can be administered to the lungs as a route for systemic delivery.7–12 The most widely used inhalation drug delivery system is the metered dose inhaler (MDI). Because of the phasing out of chlorofluorocarbon systems, as called for by the Montreal Protocol (1987), current research has focused on developing new MDI systems for the delivery of traditional drugs, as well as for new therapies, such as delivery of proteins and peptides. The new MDI systems utilize hydrofluoroalkanes (HFAs) as propellants. Through formulation optimization, these systems can provide advantages over the chlorofluorocarbon-based MDIs,13 such as enhanced deep lung deposition, the ability to deliver drug doses above 1 mg, and compatibility with proteins and peptides.14–16 The goal of this study was to illustrate that it is possible to deliver relatively high peptide concentrations directly to the lung using HFA-based MDI systems. Cyclosporine, a cyclic undecapeptide, was chosen as a model drug for this study. This peptide is also of particular interest because of its uses as an immunosuppressant agent for tissue transplants, particularly for lung tissue, and for treatment of asthma.17–19 Using aerosolized cyclosporine can provide local immunosuppression directly to the lung, thereby eliminating the effects of nephrotoxicity, which are associated with oral administration of cyclosporine.20,21 Cyclosporine formulations, varying in peptide concentration, ethanol cosolvent concentration, and propellant type were evaluated for product performance. In addition, a representative formulation was evaluated for stability over a 2-year period.
EXPERIMENTAL Product Performance Materials and Formulations Twenty-two formulations were evaluated (Table 1). These formulations varied in cyclosporine (Research Products International, Mt. Prospect, IL) concentration, ethanol (AAPER, Shelbyville, KY) cosolvent concentration, and propellant type, either HFA 227 (ICI Klea, Cheshire, UK) or HFA 134a (DuPont, Wilmington, DE). Canisters were prepared by directly weighing the desired amount of cyclosporine and ethanol into pressure-resistant glass canisters. The desired propellant was then
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Table 1. HFA-Based Solution Formulations Used in the Study Formulation No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Cyclosporine (% w/w)
Ethanol (% w/w)
Propellant Type
0.10 0.10 0.10 0.10 1.00 1.00 1.00 1.00 1.50 1.50 1.50 1.50 0.10 0.10 0.10 0.10 1.00 1.00 1.00 1.50 1.50 1.50
0 3 6 10 0 3 6 10 0 3 6 10 0 3 6 10 3 6 10 3 6 10
227 227 227 227 227 227 227 227 227 227 227 227 134a 134a 134a 134a 134a 134a 134a 134a 134a 134a
added to the canisters. Afterward, the canisters were immediately crimped with 50-mL SpraymiserTM valves (3M Drug Delivery Systems, St. Paul, MN), using a small-scale bottle crimper, model 3000B (Aero-Tech Laboratory Equipment Company, Maryland, NY). A QVAR1 actuator (3M Drug Delivery Systems) was used with all of the formulations. High-Performance Liquid Chromatography (HPLC) Assay The HPLC system consisted of a Waters 2690 Separations Module (Waters, Milford, MA) coupled with a Waters 996 Photodiode array detector. Sample analysis was done by a reversed phase HPLC assay using a 150 3.2, 5-m, Alltech platinum C18 column, which was maintained at 608C. Acetonitrile/water (70:30) was used as the mobile phase, at a flow rate of 1.0 mL/min, with an injection volume of 100 mL. Ultraviolet detection was at 210 nm. The retention time for the cyclosporine peak was 2.7 min. Quantitation was done by peak area, using a standard curve that was prepared daily. System suitability tests, which JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
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were also run daily, verified that the reproducibility of the system was adequate. Product Performance Testing Procedure Pharmaceutical performance of the formulations was determined by using a TSI model 3306 Respirable Impactor Inlet (‘‘RI’’; TSI Inc., Shoreview, MN) coupled with a TSI model 3321 Aerodynamic Particle Sizer (‘‘APS 3321’’; TSI Inc.).22 The model 3306 RI isokinetically samples <1% of the aerosol for particle size measurement with the APS 3321. The remainder of the aerosol passes through a single-stage impactor with a cut point of 4.7 microns for characterization of the fine particle mass of the MDI system, which was then used to calculate the fine particle fraction. For this study, the ‘‘fine particle fraction’’ is defined as the fine particle mass divided by the total drug mass delivered, ex-valve, from the MDI system. The results for particle size are reported as mass median aerodynamic diameter (MMAD) in microns. The flow rate through the 3306 was 28.3 Lpm. Along with the USP throat,23 a 20-cm vertical extension was also used to ensure that the particles were fully evaporated before passing through the single stage impactor in the 3306 RI.24 The formulation was actuated five times, over a 40-s interval. After the 40-s interval, the deposited cyclosporine was collected from each component by rinsing with a known volume of diluent (70:30 acetonitrile/water). All formulations were tested in triplicate. The samples were then analyzed using the reversed phase HPLC method previously described. Stability HPLC Assay The HPLC system consisted of a Waters 2690 Separations Module (Waters) coupled with a Waters 996 Photodiode array detector. Sample analysis was performed with a modified USP stability-indicating reversed phase HPLC assay,25 using a 150 3.2, 5-m, Alltech platinum C18 column, which was maintained at 608C. The mobile phase consisted of acetonitrile/water/ methyl tert-butyl ether (340:590:70), pH 6.0, at a flow rate of 1.0 mL/min, with an injection volume of 100 mL. Ultraviolet detection was at 210 nm. The retention time for the cyclosporine peak was 35 min. Quantitation was done by peak area, using a standard curve that was prepared daily. System suitability tests, which were also run daily, verified the reproducibility of the system. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
Testing Procedures The stability of a single HFA-based solution MDI was evaluated over a 2-year period. The formulation consisted of 0.09% w/w cyclosporine and 10% w/w ethanol, using HFA 134a as a propellant. Different container closure systems and storage configurations were examined. Container closure systems consisted of aluminum, stainless steel, and an epoxy-coated canister, all crimped with 50mL valves after filling. Canisters were stored either in an upright or inverted position for the duration of the study. All canisters were filled and provided by 3M Drug Delivery Systems. Samples were taken at an initial time point, 2 weeks, 19 months, and 24 months. Before opening the canisters, they were placed in a 608C freezer for a minimum of 12 h. The valves were removed and the contents poured into a chilled 100-mL volumetric flask. Canisters were rinsed with 70:30 acetonitrile/water. The samples were then analyzed using the reversed phase HPLC method previously described.
RESULTS Product Performance Pharmaceutical performance testing was completed for all of the formulations, as described in Table 1. Particle size, actuator fraction, coarse particle fraction, fine particle fraction, and fine particle mass were evaluated. The MMAD particle sizes for the formulations are shown in Figures 1 and 2. The particle size distribution delivered from solution MDIs has been shown to be dependent on formulation parameters, with MMAD increasing with increasing drug concentration, increasing size of the initial atomized droplets, increasing formulation
Figure 1. Particle size for cyclosporine formulations in HFA 227. Error bars represent standard deviations.
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Figure 2. Particle size for cyclosporine formulations in HFA 134a. Error bars represent standard deviations.
density, and decreasing drug density.26 The measured MMAD increased approximately proportional to cyclosporine concentration to the onethird power as previously discussed.26 The MMAD for the formulations using HFA 227 were on average about 8–17% higher than for formulations using HFA 134a. Some of this difference can be explained by the fact that the density of HFA 227 is higher than the density of HFA 134a (1.39 g/cm3 versus 1.206 g/cm3 at 258C). Because particle size is dependent on nonvolatile mass, the 227 formulations, at a given concentration, will have more drug mass and a correspondingly larger particle size. This explains a difference of approximately 5% in the final MMAD. The remaining difference is likely attributable to differences in the diameter of the droplets immediately after atomization. The actuator fraction is the mass of cyclosporine deposited on the actuator, divided by the total mass of cyclosporine delivered ex-valve. As can be seen from Figures 3 and 4, there is a modest increase in actuator deposition as the amount of
Figure 3. The actuator fraction [the mass of cyclosporine (CSP) deposited on the actuator divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 227. Error bars represent standard deviations.
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Figure 4. The actuator fraction [the mass of cyclosporine (CSP) deposited on the actuator divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 134a. Error bars represent standard deviations.
ethanol in the formulation increases. No practical differences were observed for the different drug concentrations. The average actuator fraction increased by 7.5% for HFA 227 and by 4.8% for HFA 134a as the ethanol concentration was increased from 0 to 10%. The coarse particle fraction is the summation of the mass of cyclosporine deposited on the throat and impaction plate (particle mass >4.7 microns) divided by the total amount of cyclosporine delivered ex-valve. Figures 5 and 6 depict the coarse particle fraction for the HFA 227 and HFA 134a formulations, respectively. In both cases, the coarse particle fraction increases significantly as the concentration of the semivolatile ethanol increases. In addition, the coarse particle fraction also increases with increasing cyclosporine concentration. This is likely because of the increase in particle size observed with increasing cyclosporine
Figure 5. Coarse particle fraction [the combined mass of cyclosporine (CSP) deposited on the throat and impaction plate divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 227. Error bars represent standard deviations. There is a significant difference (p < 0.05) between ethanol levels for a given drug concentration. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
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Figure 6. Coarse particle fraction [the combined mass of cyclosporine (CSP) deposited on the throat and impaction plate divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 134a. Error bars represent standard deviations. With the exception of the data points between 3 and 6% ethanol, there is a significant difference (p < 0.05) between ethanol levels for a given drug concentration.
Figure 8. Fine particle fraction [the mass of cyclosporine (CSP) <4.7 microns divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 134a. The error bars represent standard deviations. With the exception of the data for 1% CSP between 3 and 6% ethanol, there is a significant difference (p < 0.05) between ethanol levels for a given drug concentration.
concentration. As the MMAD increases, a larger fraction of the aerosol exceeds the 4.7-micron aerodynamic diameter being used to define the fine particle and coarse particle fractions. The fine particle fraction is the ratio of the fine particle mass (the drug collecting on the RI filter) divided by the total amount of drug delivered exvalve. The fine particle fractions for formulations in HFA 227 and HFA 134a are presented in Figures 7 and 8, respectively. In both cases, the fine particle fraction decreases as ethanol concentration increases. As ethanol concentration was decreased from 10 to 3% by weight, fine particle fraction increased from 34 to 68% for 227 and from 33 to 52% for 134a. In the range of ethanol
concentrations examined, the fine particle fraction decreased in a nearly linear manner with increasing ethanol concentration. Statistical analysis of the data between ethanol groups was performed using single factor ANOVA. The statistically significant (p < 0.05) change in fine particle fraction as a function of ethanol concentration in the formulation is consistent with previously reported results.27 Because of the high solubility of cyclosporine in HFA propellants and ethanol, a minimal amount of ethanol was needed as a cosolvent. In fact, for propellant HFA 227, concentrations of 1.5% w/w cyclosporine were achievable with no ethanol. For these formulations, the fine particle fraction ranged from 80% (for the 0.1% cyclosporine formulation) to 65% (for the 1.5% cyclosporine formulation). Once again, the increase in particle size for the 1.5% formulation is the probable cause for the decrease in fine particle fraction. The total fine particle mass is defined as the mass of drug with aerodynamic particle diameters <4.7 microns. The fine particle mass for each formulation is given in Tables 2 and 3. In the case of the ethanol-free HFA 227 formulation (at 1.5% w/w cyclosporine), it was possible to obtain a fine particle mass of 3.75 mg from five actuations (correspondingly, 750 mg per actuation). For 134a, the formulation having the highest drug concentration (1.5% w/w) and lowest ethanol concentration (3% w/w) gave a fine particle mass of 2.52 mg from five actuations (correspondingly, 504 mg per actuation).
Figure 7. Fine particle fraction [the mass of cyclosporine (CSP) <4.7 microns divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 227. The error bars represent standard deviations. There is a significant difference (p < 0.05) between ethanol levels for a given drug concentration. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
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Table 2. Fine Particle Mass Obtained for Cyclosporine HFA 227 Formulations Using a 50-mL Valve Cyclosporine (% w/w) 0.10 0.10 0.10 0.10 1.00 1.00 1.00 1.00 1.50 1.50 1.50 1.50
Ethanol (% w/w)
Fine Particle Mass in Five Actuations, Average SD (mg)
Average Fine Particle Mass per Actuation (mg)
0 3 6 10 0 3 6 10 0 3 6 10
0.349 0.013 0.245 0.014 0.195 0.025 0.173 0.020 2.635 0.104 2.357 0.013 1.737 0.022 1.314 0.047 3.751 0.025 3.362 0.022 2.374 0.150 1.744 0.036
69.8 49.0 39.0 34.6 527.0 471.4 347.4 262.8 750.2 672.4 474.8 348.8
Stability
CONCLUSIONS
The chemical stability of cyclosporine is shown in Figure 9. Three different types of canisters were used: aluminum, epoxy-coated, and stainless steel. Canisters were stored in either an inverted or upright position over the 2-year duration. Chemical analysis of each formulation revealed that the peptide, cyclosporine, is very stable in HFA systems. There was no detectable degradation in any of the three different canister closures or with storage orientation. Photodiode array analysis confirmed the purity and identity of the cyclosporine peak. A slight increase in cyclosporine concentration was observed over the 2-year period. This increase may be attributable to a nonoptimal canister-to-vial crimp for these experimental formulations, which led to an elevated leak rate.
This study illustrates the feasibility of delivering relatively large peptide doses to the lung using HFA-based MDI systems. Cyclosporine formulations, varying in peptide concentration, ethanol cosolvent concentration, and propellant type were evaluated for product performance and stability. Because of the excellent solubility properties of cyclosporine in HFA-based systems, minimal cosolvent was needed, and cyclosporine concentrations of 1.5% w/w were achievable. With these formulations, it was possible to obtain a fine particle mass (mass of particles <4.7 microns) greater than 500 mg per actuation. In addition, cyclosporine was found to be remarkably stable over a 2-year period, regardless of container closure system or configuration.
Table 3. Fine Particle Mass Obtained for Cyclosporine HFA 134a Formulations Using a 50-mL Valve Cyclosporine (% w/w) 0.10 0.10 0.10 0.10 1.00 1.00 1.00 1.50 1.50 1.50
Ethanol (% w/w)
Fine Particle Mass in Five Actuations, Average SD (mg)
Average Fine Particle Mass per Actuation (mg)
0 3 6 10 3 6 10 3 6 10
0.274 0.003 0.146 0.016 0.188 0.018 0.165 0.008 1.669 0.129 1.507 0.027 1.254 0.033 2.523 0.150 2.306 0.006 1.610 0.143
54.8 29.2 37.6 33.0 333.8 301.4 250.8 504.6 461.2 322.0
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Figure 9. Percent cyclosporine remaining in aluminum, epoxy-coated, and stainless steel canisters over a 2-year period at room temperature. Formulations were stored either inverted (inv) or upright (up) for the duration of the study.
Cyclosporine represents a compelling example of how significant peptide doses are attainable through the HFA-based MDIs. Through formulation optimization, peptide doses of the magnitude of 2–3 mg can be delivered in five actuations to the lung for local or systemic therapy.
ACKNOWLEDGMENTS We thank Professor Mandip Sachdeva, with the College of Pharmacy at Florida A&M University, Tallahassee, FL, for initial discussions on this project and for the initial supply of cyclosporine.
REFERENCES 1. Belmin J, Valensi P. 2003. Novel drug delivery systems for insulin: Clinical potential for use in the elderly. Drugs Aging 20(4):303–312. 2. Harsch IA, Hahn EG, Konturek PC. 2001. Syringe, pen, inhaler: The evolution of insulin therapy. Med Sci Monit 7(4):833–836. 3. Heinemann L, Klappoth W, Rave K, Hompesch B, Linkeschowa R, Heise T. 2000. Intra-individual variability of the metabolic effect of inhaled insulin together with an absorption enhancer. Diabetes Care 23(9):1343–1347. 4. Zheng JY, Fulu M, Lee DY, Barber TE, Adjei AL. 2001. Pulmonary peptide delivery: Effect of taste-masking excipients on leuprolide suspension metered-dose inhalers. Pharm Dev Technol 6(4): 521–530. 5. Adjei AL, Gupta PK. 1997. Developmental pharmaceutics of inhaled peptides and proteins. Inhalation delivery of therapeutic peptides and proteins. Lung Biol Health Dis 107:735–769. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
6. Burckart GJ, Keenan R, Griffith BP, Iacono AT. 1997. Cyclosporine. Inhalation delivery of therapeutic peptides and proteins. Lung Biol Health Dis 107:281–299. 7. Agu RU, Ugwoke MI, Armand M, Kinget R, Verbeke N. 2001. The lung as a route for systemic delivery of therapeutic proteins and peptides. Respir Res 2:198–209. 8. Groneberg DA, Witt C, Chung KF, Fisher A. 2003. Fundamentals of pulmonary drug delivery. Respir Med 97:382–387. 9. Smith PL. 1997. Peptide delivery via the pulmonary route: A valid approach for local and systemic delivery. J Control Release 46:99–106. 10. Johnson KA. 1997. Preparation of peptide and protein powders for inhalation. Adv Drug Delivery Rev 26:3–15. 11. Timsina MP, Martin GP, Marriott C, Ganderton D, Yianneskis M. 1994. Drug delivery to the respiratory tract using dry powder inhalers. Int J Pharm 101:1–13. 12. Edwards DA, Ben-Jebria A, Langer R. 1998. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Phys 85:379– 385. 13. Brown BAS. 2002. 5 Myths about MDIs. Drug Deliv 2(7):52–59. 14. Brown AR, George DW. 1997. Tetrafluoroethane (HFC 134a) propellant-driven aerosols of proteins. Pharm Res 14(11):1542–1547. 15. Williams RO III, Liu J. 1998. Formulation of a protein with propellant HFA 134a for aerosol delivery. Eur J Pharm Sci 7:137–144. 16. Williams RO III, Barron MK, Alonso MJ, Remunan-Lopez C. 1998. Investigation of a pMDI system containing chitosan microspheres and P134a. Int J Pharm 174:209–222. 17. Shirbacheh MV, Jones JW, Breidenbach WC, McCabe S, Barker JH, Gruber SA. 1998. The case for local immunosuppression in composite tissue allotransplantation. Transplant Proc 30: 2739–2742. 18. Mitruka SN, Won A, McCurry KR, Zeevi A, McKaveney T, Venkataramanan R, Iacono A, Griffith BP, Burckart GJ. 2000. In the lung aerosol cyclosporine provides a regional concentration advantage over intramuscular cyclosporine. J Heart Lung Transplant 19(10):969–975. 19. Underwood SL, McMillan S, Reeves R, Hunt J, Brealey CJ, Webber S, Foster M, Sargent CA. 2001. Effects of cyclosporin A administered into the airways against antigen-induced airway inflammation and hyperreactivity in the rat. Eur J Pharm 420:165–173. 20. Rohatagi S, Calic F, Harding N, Ozoux ML, Bouriot JP, Kirkesseli S, DeLeij L, Jensen B. 2000. Pharmacokinetics, pharmacodynamics, and safety of inhaled cyclosporin A (ADI628) after single and
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repeated administration in healthy male and female subjects and asthmatic patients. J Clin Pharmacol 40:1211–1226. 21. Burckart GJ, Smaldone GC, Eldon MA, Venkataramanan JD, Zeevi A, McCurry K, McKaveney TP, Corcoran TE, Griffith BP, Iacono AT. 2003. Lung deposition and pharmacokinetics of cyclosporine after aerosolization in lung transplant patients. Pharm Res 20(2):252–256. 22. Myrdal PB, Gupta A, Stein SW, Gabrio BJ, Beck TJ. 2002. Evaluation of a new aerodynamic particle sizer for MDI distribution measurements. In: Byron PR, Dalby RN, Farr SJ, Peart J, editors. Respiratory drug delivery VIII. Raleigh, NC: Serentec Press, pp 663–666. 23. U.S. Pharmacopeia. 1996. Physical tests and determinations h601i aerosols, metered dose inhalers, and dry powders. Pharmac Forum 22:3065– 3095.
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24. Gupta A, Myrdal PB, Stein SW, Gabrio BJ, Beck TJ. 2002. Comparison of the TSI model 3306 impactor inlet with the Andersen Cascade Impactor; solution metered dose inhalers. In: Byron PR, Dalby RN, Farr SJ, Peart J, editors. Respiratory drug delivery VIII. Raleigh, NC: Serentec Press, pp 659–662. 25. Husek A. 1997. High-performance liquid chromatographic analysis of cyclosporin A and its oral solutions. J Chromatogr A 759:217–224. 26. Stein SW, Myrdal PB, Gabrio BJ, Obereit D, Beck TJ. 2003. Evaluation of a new aerodynamic particle sizer spectrometer for size distribution measurement of solution metered dose inhalers. J Aerosol Med 16(2):107–119. 27. Gupta A, Stein SW, Myrdal PB. 2003. Balancing ethanol cosolvent concentration with product performance in 134a-based pressurized metered dose inhalers. J Aerosol Med 16(2):167–174.
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College of Pharmacy, University of Arizona, 1703 East Mabel Street, Tucson, Arizona 85721
2
Early Pharmaceutics and Technology, 3M Center, St. Paul, Minnesota 55144
3
College of Pharmacy, Florida A&M University, Tallahassee, Florida 32307
Received 23 June 2003; revised 13 November 2003; accepted 5 December 2003
ABSTRACT: The goal of this study was to illustrate the potential to deliver relatively high doses of a therapeutic peptide using hydrofluoroalkane (HFA) metered dose inhaler (MDI) drug delivery systems. For the purposes of this study, cyclosporine was used as the model compound. Cyclosporine formulations, varying in peptide concentration, ethanol cosolvent concentration, and propellant type, were evaluated and optimized for product performance. As ethanol concentration was decreased from 10 to 3% by weight, fine particle fraction (the mass of cyclosporine which passes through a 4.7-micron cut point divided by the total mass of cyclosporine delivered ex-valve) increased from 34 to 68% for 227 and 33 to 52% for 134a formulations. Because of the excellent solubility properties of cyclosporine in HFA-based systems, minimal or no ethanol was needed as a cosolvent to achieve cyclosporine concentrations of 1.5% w/w. With these formulations, it was possible to obtain a fine particle mass (mass of particles <4.7 microns) greater than 500 mg per actuation. In addition, one formulation was chosen for stability analysis: 0.09% w/w cyclosporine, 10% w/w ethanol, 134a. Three different types of container closure systems (stainless steel, aluminum, and epoxy-coated canisters) and two storage configurations (upright and inverted) were evaluated. Cyclosporine was determined to be stable in HFA 134a-based MDI systems, regardless of container closure system and configuration, over a 2-year period. Cyclosporine represents a compelling example of how significant peptide doses are attainable through the use of solution-based MDIs. It has been shown that through formulation optimization, 2–3 mg of the peptide, cyclosporine, may be delivered in five actuations to the lung for local or systemic therapy. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1054–1061, 2004
Keywords: pulmonary drug delivery; fine particle fraction; peptide delivery; protein delivery; aerosol; metered dose inhalers; hydrofluoroalkanes; cyclosporine
INTRODUCTION Pulmonary drug delivery is an important delivery route for the treatment of lung diseases, such as asthma and chronic obstructive pulmonary disease. This administration route is also being Correspondence to: Paul B. Myrdal (Telephone: 520-6261296; Fax: 520-626-4063; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 93, 1054–1061 (2004) ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association
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investigated as a potentially advantageous delivery route for proteins and peptides, examples of which are insulin,1–3 leuprolide,4,5 and cyclosporine.6 Degradation due to the acidic stomach environment and enzymatic degradation throughout the gastrointestinal tract have traditionally made oral delivery of therapeutic proteins and peptides problematic. In addition, the low permeability, inadequate stability, and relatively short half-lives of these compounds often necessitate administration via injection. With inhalation
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
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drug delivery, these compounds can either be directly administered to the lungs for localized treatment of respiratory diseases, or can be administered to the lungs as a route for systemic delivery.7–12 The most widely used inhalation drug delivery system is the metered dose inhaler (MDI). Because of the phasing out of chlorofluorocarbon systems, as called for by the Montreal Protocol (1987), current research has focused on developing new MDI systems for the delivery of traditional drugs, as well as for new therapies, such as delivery of proteins and peptides. The new MDI systems utilize hydrofluoroalkanes (HFAs) as propellants. Through formulation optimization, these systems can provide advantages over the chlorofluorocarbon-based MDIs,13 such as enhanced deep lung deposition, the ability to deliver drug doses above 1 mg, and compatibility with proteins and peptides.14–16 The goal of this study was to illustrate that it is possible to deliver relatively high peptide concentrations directly to the lung using HFA-based MDI systems. Cyclosporine, a cyclic undecapeptide, was chosen as a model drug for this study. This peptide is also of particular interest because of its uses as an immunosuppressant agent for tissue transplants, particularly for lung tissue, and for treatment of asthma.17–19 Using aerosolized cyclosporine can provide local immunosuppression directly to the lung, thereby eliminating the effects of nephrotoxicity, which are associated with oral administration of cyclosporine.20,21 Cyclosporine formulations, varying in peptide concentration, ethanol cosolvent concentration, and propellant type were evaluated for product performance. In addition, a representative formulation was evaluated for stability over a 2-year period.
EXPERIMENTAL Product Performance Materials and Formulations Twenty-two formulations were evaluated (Table 1). These formulations varied in cyclosporine (Research Products International, Mt. Prospect, IL) concentration, ethanol (AAPER, Shelbyville, KY) cosolvent concentration, and propellant type, either HFA 227 (ICI Klea, Cheshire, UK) or HFA 134a (DuPont, Wilmington, DE). Canisters were prepared by directly weighing the desired amount of cyclosporine and ethanol into pressure-resistant glass canisters. The desired propellant was then
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Table 1. HFA-Based Solution Formulations Used in the Study Formulation No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Cyclosporine (% w/w)
Ethanol (% w/w)
Propellant Type
0.10 0.10 0.10 0.10 1.00 1.00 1.00 1.00 1.50 1.50 1.50 1.50 0.10 0.10 0.10 0.10 1.00 1.00 1.00 1.50 1.50 1.50
0 3 6 10 0 3 6 10 0 3 6 10 0 3 6 10 3 6 10 3 6 10
227 227 227 227 227 227 227 227 227 227 227 227 134a 134a 134a 134a 134a 134a 134a 134a 134a 134a
added to the canisters. Afterward, the canisters were immediately crimped with 50-mL SpraymiserTM valves (3M Drug Delivery Systems, St. Paul, MN), using a small-scale bottle crimper, model 3000B (Aero-Tech Laboratory Equipment Company, Maryland, NY). A QVAR1 actuator (3M Drug Delivery Systems) was used with all of the formulations. High-Performance Liquid Chromatography (HPLC) Assay The HPLC system consisted of a Waters 2690 Separations Module (Waters, Milford, MA) coupled with a Waters 996 Photodiode array detector. Sample analysis was done by a reversed phase HPLC assay using a 150 3.2, 5-m, Alltech platinum C18 column, which was maintained at 608C. Acetonitrile/water (70:30) was used as the mobile phase, at a flow rate of 1.0 mL/min, with an injection volume of 100 mL. Ultraviolet detection was at 210 nm. The retention time for the cyclosporine peak was 2.7 min. Quantitation was done by peak area, using a standard curve that was prepared daily. System suitability tests, which JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
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were also run daily, verified that the reproducibility of the system was adequate. Product Performance Testing Procedure Pharmaceutical performance of the formulations was determined by using a TSI model 3306 Respirable Impactor Inlet (‘‘RI’’; TSI Inc., Shoreview, MN) coupled with a TSI model 3321 Aerodynamic Particle Sizer (‘‘APS 3321’’; TSI Inc.).22 The model 3306 RI isokinetically samples <1% of the aerosol for particle size measurement with the APS 3321. The remainder of the aerosol passes through a single-stage impactor with a cut point of 4.7 microns for characterization of the fine particle mass of the MDI system, which was then used to calculate the fine particle fraction. For this study, the ‘‘fine particle fraction’’ is defined as the fine particle mass divided by the total drug mass delivered, ex-valve, from the MDI system. The results for particle size are reported as mass median aerodynamic diameter (MMAD) in microns. The flow rate through the 3306 was 28.3 Lpm. Along with the USP throat,23 a 20-cm vertical extension was also used to ensure that the particles were fully evaporated before passing through the single stage impactor in the 3306 RI.24 The formulation was actuated five times, over a 40-s interval. After the 40-s interval, the deposited cyclosporine was collected from each component by rinsing with a known volume of diluent (70:30 acetonitrile/water). All formulations were tested in triplicate. The samples were then analyzed using the reversed phase HPLC method previously described. Stability HPLC Assay The HPLC system consisted of a Waters 2690 Separations Module (Waters) coupled with a Waters 996 Photodiode array detector. Sample analysis was performed with a modified USP stability-indicating reversed phase HPLC assay,25 using a 150 3.2, 5-m, Alltech platinum C18 column, which was maintained at 608C. The mobile phase consisted of acetonitrile/water/ methyl tert-butyl ether (340:590:70), pH 6.0, at a flow rate of 1.0 mL/min, with an injection volume of 100 mL. Ultraviolet detection was at 210 nm. The retention time for the cyclosporine peak was 35 min. Quantitation was done by peak area, using a standard curve that was prepared daily. System suitability tests, which were also run daily, verified the reproducibility of the system. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
Testing Procedures The stability of a single HFA-based solution MDI was evaluated over a 2-year period. The formulation consisted of 0.09% w/w cyclosporine and 10% w/w ethanol, using HFA 134a as a propellant. Different container closure systems and storage configurations were examined. Container closure systems consisted of aluminum, stainless steel, and an epoxy-coated canister, all crimped with 50mL valves after filling. Canisters were stored either in an upright or inverted position for the duration of the study. All canisters were filled and provided by 3M Drug Delivery Systems. Samples were taken at an initial time point, 2 weeks, 19 months, and 24 months. Before opening the canisters, they were placed in a 608C freezer for a minimum of 12 h. The valves were removed and the contents poured into a chilled 100-mL volumetric flask. Canisters were rinsed with 70:30 acetonitrile/water. The samples were then analyzed using the reversed phase HPLC method previously described.
RESULTS Product Performance Pharmaceutical performance testing was completed for all of the formulations, as described in Table 1. Particle size, actuator fraction, coarse particle fraction, fine particle fraction, and fine particle mass were evaluated. The MMAD particle sizes for the formulations are shown in Figures 1 and 2. The particle size distribution delivered from solution MDIs has been shown to be dependent on formulation parameters, with MMAD increasing with increasing drug concentration, increasing size of the initial atomized droplets, increasing formulation
Figure 1. Particle size for cyclosporine formulations in HFA 227. Error bars represent standard deviations.
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Figure 2. Particle size for cyclosporine formulations in HFA 134a. Error bars represent standard deviations.
density, and decreasing drug density.26 The measured MMAD increased approximately proportional to cyclosporine concentration to the onethird power as previously discussed.26 The MMAD for the formulations using HFA 227 were on average about 8–17% higher than for formulations using HFA 134a. Some of this difference can be explained by the fact that the density of HFA 227 is higher than the density of HFA 134a (1.39 g/cm3 versus 1.206 g/cm3 at 258C). Because particle size is dependent on nonvolatile mass, the 227 formulations, at a given concentration, will have more drug mass and a correspondingly larger particle size. This explains a difference of approximately 5% in the final MMAD. The remaining difference is likely attributable to differences in the diameter of the droplets immediately after atomization. The actuator fraction is the mass of cyclosporine deposited on the actuator, divided by the total mass of cyclosporine delivered ex-valve. As can be seen from Figures 3 and 4, there is a modest increase in actuator deposition as the amount of
Figure 3. The actuator fraction [the mass of cyclosporine (CSP) deposited on the actuator divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 227. Error bars represent standard deviations.
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Figure 4. The actuator fraction [the mass of cyclosporine (CSP) deposited on the actuator divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 134a. Error bars represent standard deviations.
ethanol in the formulation increases. No practical differences were observed for the different drug concentrations. The average actuator fraction increased by 7.5% for HFA 227 and by 4.8% for HFA 134a as the ethanol concentration was increased from 0 to 10%. The coarse particle fraction is the summation of the mass of cyclosporine deposited on the throat and impaction plate (particle mass >4.7 microns) divided by the total amount of cyclosporine delivered ex-valve. Figures 5 and 6 depict the coarse particle fraction for the HFA 227 and HFA 134a formulations, respectively. In both cases, the coarse particle fraction increases significantly as the concentration of the semivolatile ethanol increases. In addition, the coarse particle fraction also increases with increasing cyclosporine concentration. This is likely because of the increase in particle size observed with increasing cyclosporine
Figure 5. Coarse particle fraction [the combined mass of cyclosporine (CSP) deposited on the throat and impaction plate divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 227. Error bars represent standard deviations. There is a significant difference (p < 0.05) between ethanol levels for a given drug concentration. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
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Figure 6. Coarse particle fraction [the combined mass of cyclosporine (CSP) deposited on the throat and impaction plate divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 134a. Error bars represent standard deviations. With the exception of the data points between 3 and 6% ethanol, there is a significant difference (p < 0.05) between ethanol levels for a given drug concentration.
Figure 8. Fine particle fraction [the mass of cyclosporine (CSP) <4.7 microns divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 134a. The error bars represent standard deviations. With the exception of the data for 1% CSP between 3 and 6% ethanol, there is a significant difference (p < 0.05) between ethanol levels for a given drug concentration.
concentration. As the MMAD increases, a larger fraction of the aerosol exceeds the 4.7-micron aerodynamic diameter being used to define the fine particle and coarse particle fractions. The fine particle fraction is the ratio of the fine particle mass (the drug collecting on the RI filter) divided by the total amount of drug delivered exvalve. The fine particle fractions for formulations in HFA 227 and HFA 134a are presented in Figures 7 and 8, respectively. In both cases, the fine particle fraction decreases as ethanol concentration increases. As ethanol concentration was decreased from 10 to 3% by weight, fine particle fraction increased from 34 to 68% for 227 and from 33 to 52% for 134a. In the range of ethanol
concentrations examined, the fine particle fraction decreased in a nearly linear manner with increasing ethanol concentration. Statistical analysis of the data between ethanol groups was performed using single factor ANOVA. The statistically significant (p < 0.05) change in fine particle fraction as a function of ethanol concentration in the formulation is consistent with previously reported results.27 Because of the high solubility of cyclosporine in HFA propellants and ethanol, a minimal amount of ethanol was needed as a cosolvent. In fact, for propellant HFA 227, concentrations of 1.5% w/w cyclosporine were achievable with no ethanol. For these formulations, the fine particle fraction ranged from 80% (for the 0.1% cyclosporine formulation) to 65% (for the 1.5% cyclosporine formulation). Once again, the increase in particle size for the 1.5% formulation is the probable cause for the decrease in fine particle fraction. The total fine particle mass is defined as the mass of drug with aerodynamic particle diameters <4.7 microns. The fine particle mass for each formulation is given in Tables 2 and 3. In the case of the ethanol-free HFA 227 formulation (at 1.5% w/w cyclosporine), it was possible to obtain a fine particle mass of 3.75 mg from five actuations (correspondingly, 750 mg per actuation). For 134a, the formulation having the highest drug concentration (1.5% w/w) and lowest ethanol concentration (3% w/w) gave a fine particle mass of 2.52 mg from five actuations (correspondingly, 504 mg per actuation).
Figure 7. Fine particle fraction [the mass of cyclosporine (CSP) <4.7 microns divided by the total mass of cyclosporine delivered ex-valve] for cyclosporine formulations in HFA 227. The error bars represent standard deviations. There is a significant difference (p < 0.05) between ethanol levels for a given drug concentration. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, APRIL 2004
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Table 2. Fine Particle Mass Obtained for Cyclosporine HFA 227 Formulations Using a 50-mL Valve Cyclosporine (% w/w) 0.10 0.10 0.10 0.10 1.00 1.00 1.00 1.00 1.50 1.50 1.50 1.50
Ethanol (% w/w)
Fine Particle Mass in Five Actuations, Average SD (mg)
Average Fine Particle Mass per Actuation (mg)
0 3 6 10 0 3 6 10 0 3 6 10
0.349 0.013 0.245 0.014 0.195 0.025 0.173 0.020 2.635 0.104 2.357 0.013 1.737 0.022 1.314 0.047 3.751 0.025 3.362 0.022 2.374 0.150 1.744 0.036
69.8 49.0 39.0 34.6 527.0 471.4 347.4 262.8 750.2 672.4 474.8 348.8
Stability
CONCLUSIONS
The chemical stability of cyclosporine is shown in Figure 9. Three different types of canisters were used: aluminum, epoxy-coated, and stainless steel. Canisters were stored in either an inverted or upright position over the 2-year duration. Chemical analysis of each formulation revealed that the peptide, cyclosporine, is very stable in HFA systems. There was no detectable degradation in any of the three different canister closures or with storage orientation. Photodiode array analysis confirmed the purity and identity of the cyclosporine peak. A slight increase in cyclosporine concentration was observed over the 2-year period. This increase may be attributable to a nonoptimal canister-to-vial crimp for these experimental formulations, which led to an elevated leak rate.
This study illustrates the feasibility of delivering relatively large peptide doses to the lung using HFA-based MDI systems. Cyclosporine formulations, varying in peptide concentration, ethanol cosolvent concentration, and propellant type were evaluated for product performance and stability. Because of the excellent solubility properties of cyclosporine in HFA-based systems, minimal cosolvent was needed, and cyclosporine concentrations of 1.5% w/w were achievable. With these formulations, it was possible to obtain a fine particle mass (mass of particles <4.7 microns) greater than 500 mg per actuation. In addition, cyclosporine was found to be remarkably stable over a 2-year period, regardless of container closure system or configuration.
Table 3. Fine Particle Mass Obtained for Cyclosporine HFA 134a Formulations Using a 50-mL Valve Cyclosporine (% w/w) 0.10 0.10 0.10 0.10 1.00 1.00 1.00 1.50 1.50 1.50
Ethanol (% w/w)
Fine Particle Mass in Five Actuations, Average SD (mg)
Average Fine Particle Mass per Actuation (mg)
0 3 6 10 3 6 10 3 6 10
0.274 0.003 0.146 0.016 0.188 0.018 0.165 0.008 1.669 0.129 1.507 0.027 1.254 0.033 2.523 0.150 2.306 0.006 1.610 0.143
54.8 29.2 37.6 33.0 333.8 301.4 250.8 504.6 461.2 322.0
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Figure 9. Percent cyclosporine remaining in aluminum, epoxy-coated, and stainless steel canisters over a 2-year period at room temperature. Formulations were stored either inverted (inv) or upright (up) for the duration of the study.
Cyclosporine represents a compelling example of how significant peptide doses are attainable through the HFA-based MDIs. Through formulation optimization, peptide doses of the magnitude of 2–3 mg can be delivered in five actuations to the lung for local or systemic therapy.
ACKNOWLEDGMENTS We thank Professor Mandip Sachdeva, with the College of Pharmacy at Florida A&M University, Tallahassee, FL, for initial discussions on this project and for the initial supply of cyclosporine.
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