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W-emulsion and microsphere formulations in rabbits
European Journal of Pharmaceutical Sciences 15 (2002) 497–502 www.elsevier.nl / locate / ejps
Pharmacokinetic and pharmacodynamic evaluation of cyclosporin A O / W-emulsion and microsphere formulations in rabbits a b c a, Soo-Jin Kim , Hoo-Kyun Choi , Soon-Pal Suh , Yong-Bok Lee * a
College of Pharmacy, Chonnam National University, 300 Yongbong-dong, Buk-gu, Kwangju 500 -757, South Korea b College of Pharmacy, Chosun University, 375 Seosuk-dong, Dong-gu, Kwangju 501 -759, South Korea c Department of Clinical Pathology, Chonnam National University, Medical School, 8 Hak-dong, Dong-gu, Kwangju 501 -757, South Korea Received 4 September 2001; received in revised form 2 April 2002; accepted 10 April 2002
Abstract O / W-emulsion and microspheres containing cyclosporin A (CSA) were prepared to investigate the feasibility of developing new formulations. The pharmacokinetic and pharmacodynamic characteristics of these preparations were evaluated in rabbits and compared to two commercial products, Sandimmun Neoral for oral administration and CIPOL Inj. for intravenous administration. After oral or intravenous administration (10 mg / kg) to male rabbits, CSA concentration and lymphocyte population in whole blood were measured by TDxFLx and Coulter STKS , respectively. Total clearance (CL t ) was increased after intravenous administration of CSA O / W-emulsion compared with intravenous administration of CIPOL Inj . In case of oral administration, AUC and bioavailability of CSA microspheres and O / W-emulsion were not significantly different (P.0.05) from those of Sandimmun Neoral , however, MRT and T max of CSA microspheres and O / W-emulsion were significantly increased (P,0.05). There were no significant differences in the area between the baseline and effect curves (ABEC) among these formulations (P.0.05), but the pharmacodynamic availability (FPD ) of CSA O / W-emulsion was 5.51-fold higher than that of CIPOL Inj. and was significantly greater than that of Sandimmun Neoral (P,0.05). These results suggest that CSA microspheres and O / W-emulsion have sustained release characteristics and may be used as such formulations for oral or intravenous administration of CSA. 2002 Published by Elsevier Science B.V. Keywords: Cyclosporin A; Pharmacokinetics; Pharmacodynamics; O / W-emulsion; Microsphere
1. Introduction Cyclosporin A (CSA) is a poorly water-soluble cyclic peptide comprising 11 amino acids. It inhibits T-lymphocyte function that plays an important role in the induction of immune response. The potent immunosuppressive activity of CSA has been used for the prevention of rejection following transplantation of liver, kidney and bone marrow, etc. (Tibell and Norrlind, 1994; Noble and Markham, 1995; Kahan, 1999). The use of CSA has been often limited by several disadvantages including low bioavailability, narrow therapeutic window, nephrotoxicity, hepatotoxicity and neurotoxicity (Yee and Salomon, 1992; Gijtenbeek et al., 1999). Moreover, CSA injection is limited to patients who are unable to take the oral preparations, because it has a risk of anaphylactic shock *Corresponding author. Tel.: 182-62-530-2931; fax: 182-62-5302911. E-mail address: [email protected] (Y.-B. Lee).
and nephrotoxicity due to Cremophor EL , a solubilizing agent used in the commercial intravenous formulation (Cavanak and Sucker, 1986; Besarab et al., 1987). Owing to the above mentioned disadvantages of commercial products, there is a great interest in the development of alternative dosage forms. Several oral and intravenous formulations of CSA including liposomes (Vadiei et al., 1989; Al-Meshal et al., 1998; Lee et al., 1999), fat carrier (Tibell and Norrlind, 1994; Tibell et al., 1995), microspheres (Urata et al., 1999), and microemulsion (Sarciaux et al., 1995) have been investigated to improve the therapeutic efficacy and to reduce the toxicity. It has been reported that CSAcontaining microspheres and liposomes showed sustained depot characteristics (Urata et al., 1999; Lee et al., 1999), but liposomes have several problems during storage such as phospholipid hydrolysis, decomposition of encapsulated drug, separation of drug from liposome, sedimentation, aggregation and fusion of liposomes (Lee et al., 1999). In case of intravenous formulation, nephrotoxicity caused by
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CSA or Cremophor EL could be avoided by using a soybean oil-based fat emulsion carrier (Tibell et al., 1993). In this study, we prepared CSA microspheres and O / Wemulsion using soybean oil. We also evaluated pharmacokinetic and pharmacodynamic characteristics of these two formulations in rabbits and the results were compared to those of two commercial products, Sandimmun Neoral for oral administration and CIPOL Inj. for intravenous administration.
2. Materials and methods
2.1. Materials CSA and CIPOL Inj. were kindly supplied by Chong Kun Dang Pharm. (Seoul, Korea) and Sandimmun Neoral (Novartis Pharma Schweiz, Basle, Switzerland) was purchased from the local market. Labrafill 1944 (Gatteposse´ ¨ Korea, Seoul, Korea), Eudragit S100 (Rohom Pharma, Darmstadt, Germany), soybean oil (Junsei Chemical, Tokyo, Japan), egg lecithin (Asahi Chemical Industry, Charlotte, NC, USA), ethanol (99.8%, Oriental Chemical Industries, Seoul, Korea) and glycerin (Sigma, St. Louis, MO, USA) were purchased and used as received. All other reagents used in this study were of analytical grade.
2.2. Preparation of CSA-containing O /W-emulsion O / W-emulsion was prepared in accordance with the method reported in the literature with some modifications using a microfluidizer (EmulsiFlex-B3, Avestin, Ottawa, Canada) (Tibell et al., 1995; Maa and Hsu, 1999). The lipid phase was prepared by dissolving CSA in soybean oil at 70 8C. Purified egg lecithin dissolved in ethanol was slowly added into the lipid phase. The water phase was prepared by mixing water and glycerol at 70 8C. Glycerol (25 mg / ml) was used to make the emulsion isotonic. After removing ethanol from the lipid phase using a centrifugal evaporator (CVE-200D, Tokyo Rikakikai, Tokyo, Japan) at room temperature, the water phase and the lipid phase were mixed at 10,000 rev. / min for 10 min using a homogenizer (Polytron PT 3100, Kinematica, Littau, Switzerland). A fine emulsion was prepared at 4 8C using a microfluidizer. The final concentration of CSA in the prepared emulsion was 3 mg / ml and the concentrations of egg lecithin and soybean oil were 2% and 10%, respectively. The particle size of the prepared CSA O / W-emulsion was 221.5612.5 nm as determined by a dynamic light scattering analyzer (Autosizer Lo-C, Malvern Instruments, Worcestershire, UK).
2.3. Preparation of CSA-containing microspheres Floating CSA microspheres containing Labrafill 1944 as a core material were prepared by a solvent evaporation
method (Lee et al., 2001). Organic solvent mixture was prepared by mixing ethanol, isopropyl alcohol, dichloromethane, and Labrafill 1944 (12:8:10:3, w / w). CSA (200 mg) and 1 g of Eudragit S100 were dissolved in 10 ml of the above solvent mixture. The polymer solution containing CSA was slowly introduced into 1000 ml of 0.4% (w / v) poly(vinyl alcohol) aqueous solution while being stirred at 250 rev. / min using a mechanical stirrer (RZR 2000, Cafrimo, Canada) equipped with a three-bladed propeller at room temperature. The solution was stirred for 10 min and the microspheres were collected by filtration (Whatman No. 2., Whatman, UK). The collected microspheres were dried for 12 h at 50 8C. The final content of CSA was measured by HPLC.
2.4. Animal study New Zealand White male rabbits weighing 1.5–2.5 kg were used in this study. The rabbits were fasted overnight but were allowed free access to water. The animals were divided into four groups (three to five animals each), and each animal received 10 mg / kg of CSA dose in one of the following dosage forms: (1) CSA commercial intravenous CIPOL Inj. ; (2) CSA commercial oral Sandimmun Neoral ; (3) CSA microspheres for oral administration; (4) CSA O / W-emulsion administered orally or intravenously. The oral doses were administered using a catheter (CAMEL 10FR, DAE-BO Industries, Kyoungki-do, Korea), and the marginal ear vein was used for the intravenous dosing. The CSA formulations were administered at the same time (09:30 h) to avoid chronopharmacokinetic effects (Malmary et al., 1995). The blood samples (about 1 ml) were withdrawn via ear artery into a 7.5% EDTA (sodium salt) polypropylene tube (13375 mm, Becton Dickinson, Meylan, UK) with the aid of an implanted Angiocatheter (22G, JELCOE, Johnson & Johnson Medical, Pomezia, Italia). The samples were collected at predetermined time intervals up to 24 h after the drug administration. The whole blood samples were lightly shaken and stored at 4 8C until assay.
2.5. Determination of cyclosporin A in whole blood CSA concentration in whole blood was assayed by using a monoclonal antibody fluorescence polarization immunoassay (monoclonal TDxFLx , Abbott Laboratories, Abbott Park, IL, USA) (Agarwal, 1985). To an aliquot of 150 ml of the sample, 50 ml of solubilizing agent were added to dissolve the cells, and 300 ml of precipitation agent were added to precipitate protein. The solution was centrifuged to obtain a clear supernatant. The supernatant was used for CSA monoclonal whole blood assay using TDxFLx . Calibration curves (0–1500 ng / ml) were obtained each time a set of samples was analyzed, and their quality control was assessed by analyzing the standard samples provided by the instrument manufacturer. Under these
S.-J. Kim et al. / European Journal of Pharmaceutical Sciences 15 (2002) 497 – 502
conditions, percentage recovery of CSA in the samples ranged from 95.22 to 102.35% and within-day and between-day coefficient of variation did not exceed 5% for the same batch of reagents. Samples exceeding the upper limit of calibration concentration range were diluted with phosphate buffered saline and re-assayed.
2.6. Determination of lymphocyte population in whole blood The lymphocyte fraction (LY/ WBC), the ratio of lymphocyte population to white blood cell population in whole blood, served as pharmacodynamic marker for CSA since the immunosuppressive activity of CSA is primarily due to its effect on T cells and B cells (Awni, 1992). The total cell counts in whole blood were measured by an automated hemocytometer (Coulter STKS, Coulter Electronics, Northwell, UK). The whole blood samples were assayed within 24 h after collection to avoid cell destruction.
2.7. Analysis of pharmacokinetic parameters The pharmacokinetic parameters associated with intravenous and oral formulations were estimated by both compartmental and noncompartmental methods using WinNonlin (Version 1.1, Scientific Consulting, Cary, NC, USA) (Gabrielsson and Weiner, 1997). In case of intravenous formulations, the lowest Akaike’s number which indicates the goodness of fit was obtained when the CSA concentration–time profiles were fitted to a two-compartment open model with bolus intravenous input and firstorder output from central compartment. The corresponding pharmacokinetic parameters of intravenous formulations were calculated using standard equations (Gibaldi and Perrier, 1982). Area under the whole blood concentration– time curve (AUC) and area under the first moment curve (AUMC) were calculated using trapezoidal rule. The mean residence time (MRT) was calculated as AUMC /AUC. Maximum whole blood concentration (Cmax ) and time for maximum whole blood concentration (T max ) were obtained directly from the experimental data. The absolute bioavailability (F, %) for each formulation was calculated as the ratio of AUC of each formulation to AUC of CIPOL Inj. .
2.8. Analysis of pharmacodynamic parameters The indirect pharmacodynamic response model (Dayneka et al., 1993; Gobburu and Jusko, 1998) was used to describe the whole blood CSA concentration–pharmacodynamic activity relationship. The mathematical expression that describes the relationship between the lymphocyte fraction as the pharmacological response and the CSA concentration is:
H
J
dR C ] 5 k in ? 1 2 ]]] 2 k out ? R dt C 1 IC 50
499
(1)
where R is the observed response, i.e. lymphocyte fraction (LY/ WBC, %), k in is the zero-order rate constant for the production of the response and k out is the first-order rate constant for the loss of the response, IC 50 is the whole blood CSA concentration that leads to 50% of maximum inhibition and C is the whole blood CSA concentration at the time of the observed response. When no CSA is present, this model yields the following relation for the response production rate constant (k in ) at steady-state k in 5 k out ? R 0
(2)
where R 0 is a no-dose baseline of the lymphocyte fraction at steady-state. Therefore, the rate of change of CSA pharmacological response can be described by the following equation R0 dR ] 5 k out ? ]]] 2R . dt C 1 1 ]] IC 50
5
6
(3)
The area between the baseline and effect curves (ABEC) was calculated using the trapezoidal rule. Two different metrics, pharmacodynamic efficacy (EFF) and pharmacodynamic availability (FPD ), were used to evaluate the pharmacological response of each formulation (Gobburu and Jusko, 1998). EFF (response / concentration units) was calculated as ABEC /AUC, and FPD was determined as: ABEC test AUC ref EFF test FPD 5 ]]] ? ]]] 5 ]]. ABEC ref AUC test EFF ref
(4)
The EFF provides an overall measure of effect per unit dose and the FPD provides a dimensionless quantitation of relative efficiency of test formulation when compared to that of a reference formulation. In a linear pharmacokinetic system, an FPD value less than 1.0 indicates lower pharmacodynamic availability and a value greater than 1.0 indicates that the test formulation is more efficient than the reference formulation. The pharmacodynamic model parameters were estimated by fitting lymphocyte fraction and whole blood CSA concentration to the above model equation using WinNonlin (Version 1.1, Scientific Consulting).
2.9. Statistical evaluation All data were analyzed for statistical significance by the Student’s t-test (P,0.05). All calculated values were expressed as their mean6S.E. 3. Results and discussion
3.1. Pharmacokinetics Fig. 1 shows the mean whole blood concentration–time
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Fig. 1. (A) The whole blood concentration–time profiles of cyclosporin A after intravenous administration (10 mg / kg) of CIPOL Inj. (d) and CSA O / W-emulsion (s) to rabbits. The points are the experimental data (6S.E.), the lines are the pharmacokinetic model fitted curves; (B) the whole blood concentration–time profiles of cyclosporin A after oral administration (10 mg / kg) of Sandimmun Neoral (d), CSA microsphere (.) and O / W-emulsion (s) to rabbits. Each point represents the mean6S.E. (n53–5).
profiles of CSA after intravenous administration (A) of O / W-emulsion and CIPOL Inj. , and oral administration (B) of CSA microsphere, O / W-emulsion and Sandimmun Neoral to rabbits at 10 mg / kg (n53–5). Table 1 lists the pharmacokinetic parameters of CSA that were evaluated by the two-compartment model and noncompartmental method using intravenous and oral administration data, respectively. After intravenous administration, the AUC of CSA O / W-emulsion was significantly smaller than that of CIPOL Inj. (P,0.05). However, the CL t , MRT and half-life of CSA O / W-emulsion were significantly increased (9.03-fold, 2.24-fold and 1.97-fold, respectively) when compared with those of CIPOL Inj. (P,0.05). The CL t and half-life of CIPOL Inj. were 777.766131.28 ml / h / kg and 5.8962.65 h, respectively, and these results were similar to those of other investigator’s (El-Sayed et al., 1995). In the case of oral administration, the half-lives,
MRT and T max obtained from CSA microspheres and O / W-emulsion were significantly prolonged (P,0.05) when compared with those of Sandimmun Neoral . The bioavailabilities (F ), related to the AUC of CIPOL Inj. , were 11.58% in CSA O / W-emulsion for intravenous administration, 1.33%, 2.17% and 0.83% in Sandimmun Neoral , CSA microspheres and O / W-emulsion for oral administration, respectively. Similar or higher bioavailability, delayed MRT, and extended T max of CSA microspheres and O / W-emulsion for oral administration when compared with those of Sandimmun Neoral suggested that CSA microspheres and O / W-emulsion could provide sustained release characteristics, and they might be used as oral sustained release formulations of CSA.
3.2. Pharmacodynamics The lymphocyte fraction–time profiles for various CSA formulations are shown in Fig. 2. The pharmacodynamic parameters of intravenous formulations were calculated by Eq. (3) using predicted CSA concentration from the twocompartment model as the whole blood concentration of CSA. The experimental data and the respective fitted data of the lymphocyte fraction in whole blood showed good correlation with the correlation coefficient of 0.979 and 0.912 in case of the intravenous administration of CIPOL Inj. and CSA O / W-emulsion, respectively. The IC 50 of CSA O / W-emulsion, which represents the intrinsic sensitivity of CSA effects on the proliferation of lymphocyte, was smaller than that of CIPOL Inj. , however, there was no significant difference between the two formulations (P.0.05) due to large variation from CIPOL Inj. (Table 2). Regardless of routes of administration, there were no significant differences in ABEC among all formulations tested in this study (P.0.05). However, the pharmacodynamic efficacy (EFF), the effect per unit dose, of CSA O / W-emulsion for intravenous administration was significantly increased when compared with that of CIPOL Inj. (P,0.05). In the case of oral administration, EFF of
Table 1 Pharmacokinetic parameters of cyclosporin A (mean6S.E., n53–5) Parameters a
AUC t (ng / ml / h) MRT (h) Cmax (ng / ml) T max (h) T 1 / 2 (h) CL t (ml / h / kg) F (%)
Intravenous administration
Oral administration
O / W-emulsion
CIPOL Inj.
Microsphere
O / W-emulsion
Sandimmun Neoral
1518.956435.63 b 10.84614.28 –e – 11.58615.67 7030.9362350.11 b 11.5863.32
13122.0962359.04 4.8362.01 – – 5.8962.65 777.766131.28 100
284.29646.20 c 12.4161.02 d 15.5062.81 d 8.0063.46 d 111.63693.61 – 2.1760.35
130.59651.09 12.1460.72 d 9.3863.16 d 6.0062.00 d 79.51688.96 – 0.8360.67
174.77659.20 7.7060.85 25.0960.74 1.3060.08 9.9465.35 – 1.3360.45
a AUC t , area under the whole blood concentration–time curve until time is t; MRT, mean residence time; Cmax , maximum whole blood concentration; T max , time for Cmax ; T 1 / 2 , terminal half-life; CL t , total clearance; F, absolute bioavailability. b P,0.05 from intravenous administration of CIPOL Inj. . c P,0.05 from oral administration of CSA O / W-emulsion. d P,0.05 from oral administration of Sandimmun Neoral . e Not determined.
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from the blood concentration may not be used to evaluate the pharmacodynamic effects of immunomodulatory drugs like CSA. Since the lymphocytes, the pharmacological action site of CSA, are mainly distributed in the lymphatic system (Adair and Guyton, 1985), we need to evaluate the correlation between the pharmacokinetics and pharmacodynamics in the lymphatic system in order to understand the immunosuppressive activity of CSA.
Acknowledgements This work was partly supported by the Brain-Korea 21 Project in 2001, and by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic Korea (01-PJ1-PG3-21400-0020).
Fig. 2. (A) The pharmacodynamic profiles of cyclosporin A after intravenous administration (10 mg / kg) of CIPOL Inj. (d) and CSA O / W-emulsion (s) to rabbits. The points are the experimental data (6S.E.), the lines are the model fitted curves; (B) the whole blood concentration–time profiles of cyclosporin A after oral administration (10 mg / kg) of Sandimmun Neoral (d), CSA microsphere (.) and O / Wemulsion (s) to rabbits. Each point represents the mean6S.E. (n53–5).
References
CSA microspheres was significantly decreased and that of CSA O / W-emulsion was significantly increased when compared with Sandimmun Neoral (P,0.05). Pharmacokinetic bioavailability (F ) was compared with pharmacodynamic availability (FPD ) using CIPOL Inj. as a reference. The F of CSA O / W-emulsion administered intravenously was 11.5863.32%, on the contrary, the FPD was 5.51-fold higher than that of CIPOL Inj. . The mean F of Sandimmun Neoral , CSA microspheres and O / Wemulsion was only 1.33%, 2.17% and 0.83%, respectively, but the FPD of these formulations were 75.89-, 35.91- and 100.16-fold higher than that of CIPOL Inj. (P,0.05). These results would imply two things. One is that the sustained release from CSA O / W-emulsion and microspheres offers higher efficiencies than Sandimmun Neoral since maintaining an effective concentration above IC 50 would result in higher efficiencies. The other is that the CSA concentration in whole blood may not reflect the CSA concentration in the pharmacological action site. Accordingly, the pharmacokinetic parameters obtained
Adair, T.H., Guyton, A.C., 1985. Lymph Formation and its Modification in the Lymphatic System: Experimental Biology of the Lymphatic Circulation. Elsevier Science, New York. Agarwal, R.P., 1985. Assessment of cyclosporin A in whole blood and plasma in five patients with different hematocrits. Ther. Drug Monit. 7, 61. Al-Meshal, M., Khidr, S.H., Bayomi, M.A., Al-Angary, A.A., 1998. Oral administration of liposomes containing cyclosporine: a pharmacokinetic study. Int. J. Pharm. 168, 163–168. Awni, W.M., 1992. Pharmacodynamic monitoring of cyclosporin. Clin. Pharmacokinet. 23, 428–448. Besarab, A., Jarrell, B.E., Hirsch, S., Carabasi, R.A., Cressman, M.D., Green, P., 1987. Use of the isolated perfused kidney model to assess the acute pharmacologic effects of cyclosporine and its vehicle, Cremophor EL. Transplantation 44, 195–201. Cavanak, T., Sucker, H., 1986. Formulation of dosage forms. Prog. Allerg. 38, 65–72. Dayneka, N.L., Garg, V., Jusko, W.J., 1993. Comparison of four basic models of indirect pharmacodynamic responses. J. Pharmacokinet. Biopharm. 21, 457–478. El-Sayed, Y.M., Tabbara, K.F., Gouda, M.W., 1995. Effect of acetazolamide on the pharmacokinetics of cyclosporin in rabbits. Int. J. Pharm. 121, 181–186.
Table 2 Pharmacodynamic parameters of cyclosporin A (mean6S.E., n53–5) Parameters a
ABEC IC 50 (ng / ml) k out (h 21 ) EFF FPD
Intravenous administration
Oral administration
O / W-emulsion
CIPOL Inj.
Microsphere
O / W-emulsion
Sandimmun Neoral
436.556168.40 25.9768.78 2.1660.19 c 0.3060.04 c 5.5160.40
687.78685.81 93.81640.57 0.8860.30 0.0560.00 1
599.266135.18 –b 1.0160.74 d 1.9060.26 d 35.9162.34 d
641.286112.10 – 4.0760.78 d 5.3561.63 d 100.16621.90 d
666.636100.23 – 0.2460.07 4.0461.06 75.89613.83
a ABEC (%lymphocyte fraction?h), area between the baseline and effect curves; IC 50 , whole blood concentration that led to 50% of maximum inhibition; k out , first-order rate constant for the loss of the response; EFF (% lymphocyte fraction?ml / ng), pharmacodynamic efficacy; FPD , pharmacodynamic availability. b Not determined. c P,0.05 from intravenous administration of CIPOL Inj. . d P,0.05 from oral administration of Sandimmun Neoral .
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Gabrielsson, J., Weiner, D., 1997. In: 2nd Edition. Pharmacokinetic and Pharmacodynamic Data Analysis: Concepts and Applications. Swedish Pharmaceutical Press, Stockholm, Sweden. Gibaldi, M., Perrier, D., 1982. In: 2nd Edition. Pharmacokinetics. Dekker, New York. Gijtenbeek, J.M.M., van den Bent, M.J., Vecht, C.J., 1999. Cyclosporine neurotoxicity: a review. J. Neurol. 246, 339–346. Gobburu, J.V.S., Jusko, W.J., 1998. Role of dosage regimen in controlling indirect pharmacodynamic response. Adv. Drug Deliv. Rev. 33, 221– 233. Kahan, B.D., 1999. Cyclosporine: a revolution in transplantation. Transplant. Proc. 31 (Suppl.), 14S–15S. Lee, M.K., Choi, L., Kim, M., Kim, C., 1999. Pharmacokinetics and organ distribution of cyclosporin A incorporated in liposomes and mixed micelles. Int. J. Pharm. 191, 87–93. Lee, J.H., Park, T.G., Lee, Y.B., Shin, S.C., Choi, H.K., 2001. Effect of adding non-volatile oil as a core material for the floating microspheres prepared by emulsion solvent diffusion method. J. Microencaps. 18 (1), 65–75. Maa, Y., Hsu, C.C., 1999. Performance of sonication and microfluidization for liquid–liquid emulsification. Pharm. Dev. Technol. 4, 233– 240. Malmary, M.F., Houti, I., Labat, C., Batalla, A., Moussamih, S., Bouguettaya, D., Oustrin, J., Houin, G., 1995. Chronopharmacokinetics of cyclosporine A following a single i.v. dose in the Wistar rat. Eur. J. Pharm. Sci. 3, 49–56.
Noble, S., Markham, A., 1995. Cyclosporin: a review of the pharmacokinetic properties, clinical efficacy and tolerability of a microemulsion-based formulation (Neoral ). Drugs 5, 924–941. Sarciaux, J.M., Acar, L., Sado, P.A., 1995. Using microemulsion formulations for oral drug delivery of therapeutic peptides. Int. J. Pharm. 120, 127–136. Tibell, A., Larsson, M., Alvestrand, A., 1993. Dissolving intravenous cyclosporin A in a fat emulsion carrier prevents acute renal side effects in the rat. Transplant. Int. 6, 69–72. ¨ Tibell, A., Lindholm, A., Sawe, J., Chen, G., Norrlind, B., 1995. Cyclosporin A in fat emulsion carriers: experimental studies on pharmacokinetics and tissue distribution. Pharmacol. Toxicol. 76, 115–121. Tibell, A., Norrlind, B., 1994. Cyclosporin A in fat emulsion carrier: studies on the immunosuppressive potential, using the heterotopic heart transplant model in rats. Transplant. Int. 7, 438–441. Urata, T., Arimori, K., Nakano, M., 1999. Modification of release rates of cyclosporin A from poly ( L-lactic acid) microspheres by fatty acid esters and in-vivo evaluation of the microspheres. J. Control. Release 58 (2), 133–141. Vadiei, K., Perez-Soler, R., Lopez-Berestein, G., Luke, D.R., 1989. Pharmacokinetic and pharmacodynamic evaluation of liposomal cyclosporine. Int. J. Pharm. 57, 125–131. Yee, G.C., Salomon, D.R., 1992. In: 3rd Edition. Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring. Applied Therapeutics, Vancouver.
Pharmacokinetic and pharmacodynamic evaluation of cyclosporin A O / W-emulsion and microsphere formulations in rabbits a b c a, Soo-Jin Kim , Hoo-Kyun Choi , Soon-Pal Suh , Yong-Bok Lee * a
College of Pharmacy, Chonnam National University, 300 Yongbong-dong, Buk-gu, Kwangju 500 -757, South Korea b College of Pharmacy, Chosun University, 375 Seosuk-dong, Dong-gu, Kwangju 501 -759, South Korea c Department of Clinical Pathology, Chonnam National University, Medical School, 8 Hak-dong, Dong-gu, Kwangju 501 -757, South Korea Received 4 September 2001; received in revised form 2 April 2002; accepted 10 April 2002
Abstract O / W-emulsion and microspheres containing cyclosporin A (CSA) were prepared to investigate the feasibility of developing new formulations. The pharmacokinetic and pharmacodynamic characteristics of these preparations were evaluated in rabbits and compared to two commercial products, Sandimmun Neoral for oral administration and CIPOL Inj. for intravenous administration. After oral or intravenous administration (10 mg / kg) to male rabbits, CSA concentration and lymphocyte population in whole blood were measured by TDxFLx and Coulter STKS , respectively. Total clearance (CL t ) was increased after intravenous administration of CSA O / W-emulsion compared with intravenous administration of CIPOL Inj . In case of oral administration, AUC and bioavailability of CSA microspheres and O / W-emulsion were not significantly different (P.0.05) from those of Sandimmun Neoral , however, MRT and T max of CSA microspheres and O / W-emulsion were significantly increased (P,0.05). There were no significant differences in the area between the baseline and effect curves (ABEC) among these formulations (P.0.05), but the pharmacodynamic availability (FPD ) of CSA O / W-emulsion was 5.51-fold higher than that of CIPOL Inj. and was significantly greater than that of Sandimmun Neoral (P,0.05). These results suggest that CSA microspheres and O / W-emulsion have sustained release characteristics and may be used as such formulations for oral or intravenous administration of CSA. 2002 Published by Elsevier Science B.V. Keywords: Cyclosporin A; Pharmacokinetics; Pharmacodynamics; O / W-emulsion; Microsphere
1. Introduction Cyclosporin A (CSA) is a poorly water-soluble cyclic peptide comprising 11 amino acids. It inhibits T-lymphocyte function that plays an important role in the induction of immune response. The potent immunosuppressive activity of CSA has been used for the prevention of rejection following transplantation of liver, kidney and bone marrow, etc. (Tibell and Norrlind, 1994; Noble and Markham, 1995; Kahan, 1999). The use of CSA has been often limited by several disadvantages including low bioavailability, narrow therapeutic window, nephrotoxicity, hepatotoxicity and neurotoxicity (Yee and Salomon, 1992; Gijtenbeek et al., 1999). Moreover, CSA injection is limited to patients who are unable to take the oral preparations, because it has a risk of anaphylactic shock *Corresponding author. Tel.: 182-62-530-2931; fax: 182-62-5302911. E-mail address: [email protected] (Y.-B. Lee).
and nephrotoxicity due to Cremophor EL , a solubilizing agent used in the commercial intravenous formulation (Cavanak and Sucker, 1986; Besarab et al., 1987). Owing to the above mentioned disadvantages of commercial products, there is a great interest in the development of alternative dosage forms. Several oral and intravenous formulations of CSA including liposomes (Vadiei et al., 1989; Al-Meshal et al., 1998; Lee et al., 1999), fat carrier (Tibell and Norrlind, 1994; Tibell et al., 1995), microspheres (Urata et al., 1999), and microemulsion (Sarciaux et al., 1995) have been investigated to improve the therapeutic efficacy and to reduce the toxicity. It has been reported that CSAcontaining microspheres and liposomes showed sustained depot characteristics (Urata et al., 1999; Lee et al., 1999), but liposomes have several problems during storage such as phospholipid hydrolysis, decomposition of encapsulated drug, separation of drug from liposome, sedimentation, aggregation and fusion of liposomes (Lee et al., 1999). In case of intravenous formulation, nephrotoxicity caused by
0928-0987 / 02 / $ – see front matter 2002 Published by Elsevier Science B.V. PII: S0928-0987( 02 )00048-9
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CSA or Cremophor EL could be avoided by using a soybean oil-based fat emulsion carrier (Tibell et al., 1993). In this study, we prepared CSA microspheres and O / Wemulsion using soybean oil. We also evaluated pharmacokinetic and pharmacodynamic characteristics of these two formulations in rabbits and the results were compared to those of two commercial products, Sandimmun Neoral for oral administration and CIPOL Inj. for intravenous administration.
2. Materials and methods
2.1. Materials CSA and CIPOL Inj. were kindly supplied by Chong Kun Dang Pharm. (Seoul, Korea) and Sandimmun Neoral (Novartis Pharma Schweiz, Basle, Switzerland) was purchased from the local market. Labrafill 1944 (Gatteposse´ ¨ Korea, Seoul, Korea), Eudragit S100 (Rohom Pharma, Darmstadt, Germany), soybean oil (Junsei Chemical, Tokyo, Japan), egg lecithin (Asahi Chemical Industry, Charlotte, NC, USA), ethanol (99.8%, Oriental Chemical Industries, Seoul, Korea) and glycerin (Sigma, St. Louis, MO, USA) were purchased and used as received. All other reagents used in this study were of analytical grade.
2.2. Preparation of CSA-containing O /W-emulsion O / W-emulsion was prepared in accordance with the method reported in the literature with some modifications using a microfluidizer (EmulsiFlex-B3, Avestin, Ottawa, Canada) (Tibell et al., 1995; Maa and Hsu, 1999). The lipid phase was prepared by dissolving CSA in soybean oil at 70 8C. Purified egg lecithin dissolved in ethanol was slowly added into the lipid phase. The water phase was prepared by mixing water and glycerol at 70 8C. Glycerol (25 mg / ml) was used to make the emulsion isotonic. After removing ethanol from the lipid phase using a centrifugal evaporator (CVE-200D, Tokyo Rikakikai, Tokyo, Japan) at room temperature, the water phase and the lipid phase were mixed at 10,000 rev. / min for 10 min using a homogenizer (Polytron PT 3100, Kinematica, Littau, Switzerland). A fine emulsion was prepared at 4 8C using a microfluidizer. The final concentration of CSA in the prepared emulsion was 3 mg / ml and the concentrations of egg lecithin and soybean oil were 2% and 10%, respectively. The particle size of the prepared CSA O / W-emulsion was 221.5612.5 nm as determined by a dynamic light scattering analyzer (Autosizer Lo-C, Malvern Instruments, Worcestershire, UK).
2.3. Preparation of CSA-containing microspheres Floating CSA microspheres containing Labrafill 1944 as a core material were prepared by a solvent evaporation
method (Lee et al., 2001). Organic solvent mixture was prepared by mixing ethanol, isopropyl alcohol, dichloromethane, and Labrafill 1944 (12:8:10:3, w / w). CSA (200 mg) and 1 g of Eudragit S100 were dissolved in 10 ml of the above solvent mixture. The polymer solution containing CSA was slowly introduced into 1000 ml of 0.4% (w / v) poly(vinyl alcohol) aqueous solution while being stirred at 250 rev. / min using a mechanical stirrer (RZR 2000, Cafrimo, Canada) equipped with a three-bladed propeller at room temperature. The solution was stirred for 10 min and the microspheres were collected by filtration (Whatman No. 2., Whatman, UK). The collected microspheres were dried for 12 h at 50 8C. The final content of CSA was measured by HPLC.
2.4. Animal study New Zealand White male rabbits weighing 1.5–2.5 kg were used in this study. The rabbits were fasted overnight but were allowed free access to water. The animals were divided into four groups (three to five animals each), and each animal received 10 mg / kg of CSA dose in one of the following dosage forms: (1) CSA commercial intravenous CIPOL Inj. ; (2) CSA commercial oral Sandimmun Neoral ; (3) CSA microspheres for oral administration; (4) CSA O / W-emulsion administered orally or intravenously. The oral doses were administered using a catheter (CAMEL 10FR, DAE-BO Industries, Kyoungki-do, Korea), and the marginal ear vein was used for the intravenous dosing. The CSA formulations were administered at the same time (09:30 h) to avoid chronopharmacokinetic effects (Malmary et al., 1995). The blood samples (about 1 ml) were withdrawn via ear artery into a 7.5% EDTA (sodium salt) polypropylene tube (13375 mm, Becton Dickinson, Meylan, UK) with the aid of an implanted Angiocatheter (22G, JELCOE, Johnson & Johnson Medical, Pomezia, Italia). The samples were collected at predetermined time intervals up to 24 h after the drug administration. The whole blood samples were lightly shaken and stored at 4 8C until assay.
2.5. Determination of cyclosporin A in whole blood CSA concentration in whole blood was assayed by using a monoclonal antibody fluorescence polarization immunoassay (monoclonal TDxFLx , Abbott Laboratories, Abbott Park, IL, USA) (Agarwal, 1985). To an aliquot of 150 ml of the sample, 50 ml of solubilizing agent were added to dissolve the cells, and 300 ml of precipitation agent were added to precipitate protein. The solution was centrifuged to obtain a clear supernatant. The supernatant was used for CSA monoclonal whole blood assay using TDxFLx . Calibration curves (0–1500 ng / ml) were obtained each time a set of samples was analyzed, and their quality control was assessed by analyzing the standard samples provided by the instrument manufacturer. Under these
S.-J. Kim et al. / European Journal of Pharmaceutical Sciences 15 (2002) 497 – 502
conditions, percentage recovery of CSA in the samples ranged from 95.22 to 102.35% and within-day and between-day coefficient of variation did not exceed 5% for the same batch of reagents. Samples exceeding the upper limit of calibration concentration range were diluted with phosphate buffered saline and re-assayed.
2.6. Determination of lymphocyte population in whole blood The lymphocyte fraction (LY/ WBC), the ratio of lymphocyte population to white blood cell population in whole blood, served as pharmacodynamic marker for CSA since the immunosuppressive activity of CSA is primarily due to its effect on T cells and B cells (Awni, 1992). The total cell counts in whole blood were measured by an automated hemocytometer (Coulter STKS, Coulter Electronics, Northwell, UK). The whole blood samples were assayed within 24 h after collection to avoid cell destruction.
2.7. Analysis of pharmacokinetic parameters The pharmacokinetic parameters associated with intravenous and oral formulations were estimated by both compartmental and noncompartmental methods using WinNonlin (Version 1.1, Scientific Consulting, Cary, NC, USA) (Gabrielsson and Weiner, 1997). In case of intravenous formulations, the lowest Akaike’s number which indicates the goodness of fit was obtained when the CSA concentration–time profiles were fitted to a two-compartment open model with bolus intravenous input and firstorder output from central compartment. The corresponding pharmacokinetic parameters of intravenous formulations were calculated using standard equations (Gibaldi and Perrier, 1982). Area under the whole blood concentration– time curve (AUC) and area under the first moment curve (AUMC) were calculated using trapezoidal rule. The mean residence time (MRT) was calculated as AUMC /AUC. Maximum whole blood concentration (Cmax ) and time for maximum whole blood concentration (T max ) were obtained directly from the experimental data. The absolute bioavailability (F, %) for each formulation was calculated as the ratio of AUC of each formulation to AUC of CIPOL Inj. .
2.8. Analysis of pharmacodynamic parameters The indirect pharmacodynamic response model (Dayneka et al., 1993; Gobburu and Jusko, 1998) was used to describe the whole blood CSA concentration–pharmacodynamic activity relationship. The mathematical expression that describes the relationship between the lymphocyte fraction as the pharmacological response and the CSA concentration is:
H
J
dR C ] 5 k in ? 1 2 ]]] 2 k out ? R dt C 1 IC 50
499
(1)
where R is the observed response, i.e. lymphocyte fraction (LY/ WBC, %), k in is the zero-order rate constant for the production of the response and k out is the first-order rate constant for the loss of the response, IC 50 is the whole blood CSA concentration that leads to 50% of maximum inhibition and C is the whole blood CSA concentration at the time of the observed response. When no CSA is present, this model yields the following relation for the response production rate constant (k in ) at steady-state k in 5 k out ? R 0
(2)
where R 0 is a no-dose baseline of the lymphocyte fraction at steady-state. Therefore, the rate of change of CSA pharmacological response can be described by the following equation R0 dR ] 5 k out ? ]]] 2R . dt C 1 1 ]] IC 50
5
6
(3)
The area between the baseline and effect curves (ABEC) was calculated using the trapezoidal rule. Two different metrics, pharmacodynamic efficacy (EFF) and pharmacodynamic availability (FPD ), were used to evaluate the pharmacological response of each formulation (Gobburu and Jusko, 1998). EFF (response / concentration units) was calculated as ABEC /AUC, and FPD was determined as: ABEC test AUC ref EFF test FPD 5 ]]] ? ]]] 5 ]]. ABEC ref AUC test EFF ref
(4)
The EFF provides an overall measure of effect per unit dose and the FPD provides a dimensionless quantitation of relative efficiency of test formulation when compared to that of a reference formulation. In a linear pharmacokinetic system, an FPD value less than 1.0 indicates lower pharmacodynamic availability and a value greater than 1.0 indicates that the test formulation is more efficient than the reference formulation. The pharmacodynamic model parameters were estimated by fitting lymphocyte fraction and whole blood CSA concentration to the above model equation using WinNonlin (Version 1.1, Scientific Consulting).
2.9. Statistical evaluation All data were analyzed for statistical significance by the Student’s t-test (P,0.05). All calculated values were expressed as their mean6S.E. 3. Results and discussion
3.1. Pharmacokinetics Fig. 1 shows the mean whole blood concentration–time
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Fig. 1. (A) The whole blood concentration–time profiles of cyclosporin A after intravenous administration (10 mg / kg) of CIPOL Inj. (d) and CSA O / W-emulsion (s) to rabbits. The points are the experimental data (6S.E.), the lines are the pharmacokinetic model fitted curves; (B) the whole blood concentration–time profiles of cyclosporin A after oral administration (10 mg / kg) of Sandimmun Neoral (d), CSA microsphere (.) and O / W-emulsion (s) to rabbits. Each point represents the mean6S.E. (n53–5).
profiles of CSA after intravenous administration (A) of O / W-emulsion and CIPOL Inj. , and oral administration (B) of CSA microsphere, O / W-emulsion and Sandimmun Neoral to rabbits at 10 mg / kg (n53–5). Table 1 lists the pharmacokinetic parameters of CSA that were evaluated by the two-compartment model and noncompartmental method using intravenous and oral administration data, respectively. After intravenous administration, the AUC of CSA O / W-emulsion was significantly smaller than that of CIPOL Inj. (P,0.05). However, the CL t , MRT and half-life of CSA O / W-emulsion were significantly increased (9.03-fold, 2.24-fold and 1.97-fold, respectively) when compared with those of CIPOL Inj. (P,0.05). The CL t and half-life of CIPOL Inj. were 777.766131.28 ml / h / kg and 5.8962.65 h, respectively, and these results were similar to those of other investigator’s (El-Sayed et al., 1995). In the case of oral administration, the half-lives,
MRT and T max obtained from CSA microspheres and O / W-emulsion were significantly prolonged (P,0.05) when compared with those of Sandimmun Neoral . The bioavailabilities (F ), related to the AUC of CIPOL Inj. , were 11.58% in CSA O / W-emulsion for intravenous administration, 1.33%, 2.17% and 0.83% in Sandimmun Neoral , CSA microspheres and O / W-emulsion for oral administration, respectively. Similar or higher bioavailability, delayed MRT, and extended T max of CSA microspheres and O / W-emulsion for oral administration when compared with those of Sandimmun Neoral suggested that CSA microspheres and O / W-emulsion could provide sustained release characteristics, and they might be used as oral sustained release formulations of CSA.
3.2. Pharmacodynamics The lymphocyte fraction–time profiles for various CSA formulations are shown in Fig. 2. The pharmacodynamic parameters of intravenous formulations were calculated by Eq. (3) using predicted CSA concentration from the twocompartment model as the whole blood concentration of CSA. The experimental data and the respective fitted data of the lymphocyte fraction in whole blood showed good correlation with the correlation coefficient of 0.979 and 0.912 in case of the intravenous administration of CIPOL Inj. and CSA O / W-emulsion, respectively. The IC 50 of CSA O / W-emulsion, which represents the intrinsic sensitivity of CSA effects on the proliferation of lymphocyte, was smaller than that of CIPOL Inj. , however, there was no significant difference between the two formulations (P.0.05) due to large variation from CIPOL Inj. (Table 2). Regardless of routes of administration, there were no significant differences in ABEC among all formulations tested in this study (P.0.05). However, the pharmacodynamic efficacy (EFF), the effect per unit dose, of CSA O / W-emulsion for intravenous administration was significantly increased when compared with that of CIPOL Inj. (P,0.05). In the case of oral administration, EFF of
Table 1 Pharmacokinetic parameters of cyclosporin A (mean6S.E., n53–5) Parameters a
AUC t (ng / ml / h) MRT (h) Cmax (ng / ml) T max (h) T 1 / 2 (h) CL t (ml / h / kg) F (%)
Intravenous administration
Oral administration
O / W-emulsion
CIPOL Inj.
Microsphere
O / W-emulsion
Sandimmun Neoral
1518.956435.63 b 10.84614.28 –e – 11.58615.67 7030.9362350.11 b 11.5863.32
13122.0962359.04 4.8362.01 – – 5.8962.65 777.766131.28 100
284.29646.20 c 12.4161.02 d 15.5062.81 d 8.0063.46 d 111.63693.61 – 2.1760.35
130.59651.09 12.1460.72 d 9.3863.16 d 6.0062.00 d 79.51688.96 – 0.8360.67
174.77659.20 7.7060.85 25.0960.74 1.3060.08 9.9465.35 – 1.3360.45
a AUC t , area under the whole blood concentration–time curve until time is t; MRT, mean residence time; Cmax , maximum whole blood concentration; T max , time for Cmax ; T 1 / 2 , terminal half-life; CL t , total clearance; F, absolute bioavailability. b P,0.05 from intravenous administration of CIPOL Inj. . c P,0.05 from oral administration of CSA O / W-emulsion. d P,0.05 from oral administration of Sandimmun Neoral . e Not determined.
S.-J. Kim et al. / European Journal of Pharmaceutical Sciences 15 (2002) 497 – 502
501
from the blood concentration may not be used to evaluate the pharmacodynamic effects of immunomodulatory drugs like CSA. Since the lymphocytes, the pharmacological action site of CSA, are mainly distributed in the lymphatic system (Adair and Guyton, 1985), we need to evaluate the correlation between the pharmacokinetics and pharmacodynamics in the lymphatic system in order to understand the immunosuppressive activity of CSA.
Acknowledgements This work was partly supported by the Brain-Korea 21 Project in 2001, and by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic Korea (01-PJ1-PG3-21400-0020).
Fig. 2. (A) The pharmacodynamic profiles of cyclosporin A after intravenous administration (10 mg / kg) of CIPOL Inj. (d) and CSA O / W-emulsion (s) to rabbits. The points are the experimental data (6S.E.), the lines are the model fitted curves; (B) the whole blood concentration–time profiles of cyclosporin A after oral administration (10 mg / kg) of Sandimmun Neoral (d), CSA microsphere (.) and O / Wemulsion (s) to rabbits. Each point represents the mean6S.E. (n53–5).
References
CSA microspheres was significantly decreased and that of CSA O / W-emulsion was significantly increased when compared with Sandimmun Neoral (P,0.05). Pharmacokinetic bioavailability (F ) was compared with pharmacodynamic availability (FPD ) using CIPOL Inj. as a reference. The F of CSA O / W-emulsion administered intravenously was 11.5863.32%, on the contrary, the FPD was 5.51-fold higher than that of CIPOL Inj. . The mean F of Sandimmun Neoral , CSA microspheres and O / Wemulsion was only 1.33%, 2.17% and 0.83%, respectively, but the FPD of these formulations were 75.89-, 35.91- and 100.16-fold higher than that of CIPOL Inj. (P,0.05). These results would imply two things. One is that the sustained release from CSA O / W-emulsion and microspheres offers higher efficiencies than Sandimmun Neoral since maintaining an effective concentration above IC 50 would result in higher efficiencies. The other is that the CSA concentration in whole blood may not reflect the CSA concentration in the pharmacological action site. Accordingly, the pharmacokinetic parameters obtained
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Intravenous administration
Oral administration
O / W-emulsion
CIPOL Inj.
Microsphere
O / W-emulsion
Sandimmun Neoral
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687.78685.81 93.81640.57 0.8860.30 0.0560.00 1
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666.636100.23 – 0.2460.07 4.0461.06 75.89613.83
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