European Journal of Pharmaceutical Sciences 15 (2002) 497–502www.elsevier.nl / locate /ejps

P harmacokinetic and pharmacodynamic evaluation of cyclosporinA O/W-emulsion and microsphere formulations in rabbits

a b c a ,*Soo-Jin Kim , Hoo-Kyun Choi , Soon-Pal Suh , Yong-Bok LeeaCollege of Pharmacy, Chonnam National University, 300 Yongbong-dong, Buk-gu, Kwangju 500-757, South Korea

bCollege of Pharmacy, Chosun University, 375 Seosuk-dong, Dong-gu, Kwangju 501-759, South KoreacDepartment 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 newformulations. 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 orintravenous 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 ) was increased after intravenous administration of CSA O/W-emulsiontcompared 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 of CSAmax

microspheres and O/W-emulsion were significantly increased (P,0.05). There were no significant differences in the area between thebaseline and effect curves (ABEC) among these formulations (P.0.05), but the pharmacodynamic availability (F ) of CSAPD

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 suchformulations 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 and nephrotoxicity due to Cremophor EL , a solubilizingagent used in the commercial intravenous formulation

Cyclosporin A (CSA) is a poorly water-soluble cyclic (Cavanak and Sucker, 1986; Besarab et al., 1987). Owingpeptide comprising 11 amino acids. It inhibits T-lympho- to the above mentioned disadvantages of commercialcyte function that plays an important role in the induction products, there is a great interest in the development ofof immune response. The potent immunosuppressive ac- alternative dosage forms.tivity of CSA has been used for the prevention of rejection Several oral and intravenous formulations of CSAfollowing transplantation of liver, kidney and bone mar- including liposomes (Vadiei et al., 1989; Al-Meshal et al.,row, etc. (Tibell and Norrlind, 1994; Noble and Markham, 1998; Lee et al., 1999), fat carrier (Tibell and Norrlind,1995; Kahan, 1999). The use of CSA has been often 1994; Tibell et al., 1995), microspheres (Urata et al.,limited by several disadvantages including low bioavail- 1999), and microemulsion (Sarciaux et al., 1995) haveability, narrow therapeutic window, nephrotoxicity, hepa- been investigated to improve the therapeutic efficacy andtotoxicity and neurotoxicity (Yee and Salomon, 1992; to reduce the toxicity. It has been reported that CSA-Gijtenbeek et al., 1999). Moreover, CSA injection is containing microspheres and liposomes showed sustainedlimited to patients who are unable to take the oral depot characteristics (Urata et al., 1999; Lee et al., 1999),preparations, because it has a risk of anaphylactic shock but liposomes have several problems during storage such

as phospholipid hydrolysis, decomposition of encapsulateddrug, separation of drug from liposome, sedimentation,*Corresponding author. Tel.: 182-62-530-2931; fax: 182-62-530-aggregation and fusion of liposomes (Lee et al., 1999). In2911.

E-mail address: leeyb@chonnam.chonnam.ac.kr (Y.-B. Lee). case of intravenous formulation, nephrotoxicity caused by

0928-0987/02/$ – see front matter 2002 Published by Elsevier Science B.V.PI I : S0928-0987( 02 )00048-9

498 S.-J. Kim et al. / European Journal of Pharmaceutical Sciences 15 (2002) 497 –502

CSA or Cremophor EL could be avoided by using a method (Lee et al., 2001). Organic solvent mixture wassoybean oil-based fat emulsion carrier (Tibell et al., 1993). prepared by mixing ethanol, isopropyl alcohol, dichloro-

In this study, we prepared CSA microspheres and O/W- methane, and Labrafill 1944 (12:8:10:3, w/w). CSA (200emulsion using soybean oil. We also evaluated phar- mg) and 1 g of Eudragit S100 were dissolved in 10 ml of

macokinetic and pharmacodynamic characteristics of these the above solvent mixture. The polymer solution con-two formulations in rabbits and the results were compared taining CSA was slowly introduced into 1000 ml of 0.4%

to those of two commercial products, Sandimmun Neoral (w/v) poly(vinyl alcohol) aqueous solution while beingfor oral administration and CIPOL Inj. for intravenous stirred at 250 rev. /min using a mechanical stirrer (RZR

administration. 2000, Cafrimo, Canada) equipped with a three-bladedpropeller at room temperature. The solution was stirred for10 min and the microspheres were collected by filtration

2 . Materials and methods (Whatman No. 2., Whatman, UK). The collected micro-spheres were dried for 12 h at 50 8C. The final content of

2 .1. Materials CSA was measured by HPLC.

CSA and CIPOL Inj. were kindly supplied by Chong 2 .4. Animal studyKun Dang Pharm. (Seoul, Korea) and Sandimmun Neoral

(Novartis Pharma Schweiz, Basle, Switzerland) was pur- New Zealand White male rabbits weighing 1.5–2.5 kg ´chased from the local market. Labrafill 1944 (Gatteposse were used in this study. The rabbits were fasted overnight

¨Korea, Seoul, Korea), Eudragit S100 (Rohom Pharma, but were allowed free access to water. The animals wereDarmstadt, Germany), soybean oil (Junsei Chemical, divided into four groups (three to five animals each), andTokyo, Japan), egg lecithin (Asahi Chemical Industry, each animal received 10 mg/kg of CSA dose in one of theCharlotte, NC, USA), ethanol (99.8%, Oriental Chemical following dosage forms: (1) CSA commercial intravenous

Industries, Seoul, Korea) and glycerin (Sigma, St. Louis, CIPOL Inj. ; (2) CSA commercial oral SandimmunMO, USA) were purchased and used as received. All other Neoral ; (3) CSA microspheres for oral administration;

reagents used in this study were of analytical grade. (4) CSA O/W-emulsion administered orally or intraven-ously. The oral doses were administered using a catheter

2 .2. Preparation of CSA-containing O /W-emulsion (CAMEL 10FR, DAE-BO Industries, Kyoungki-do,Korea), and the marginal ear vein was used for the

O/W-emulsion was prepared in accordance with the intravenous dosing. The CSA formulations were adminis-method reported in the literature with some modifications tered at the same time (09:30 h) to avoid chronophar-using a microfluidizer (EmulsiFlex-B3, Avestin, Ottawa, macokinetic effects (Malmary et al., 1995). The bloodCanada) (Tibell et al., 1995; Maa and Hsu, 1999). The samples (about 1 ml) were withdrawn via ear artery into alipid phase was prepared by dissolving CSA in soybean oil 7.5% EDTA (sodium salt) polypropylene tube (13375at 70 8C. Purified egg lecithin dissolved in ethanol was mm, Becton Dickinson, Meylan, UK) with the aid of anslowly added into the lipid phase. The water phase was implanted Angiocatheter (22G, JELCOE, Johnson &prepared by mixing water and glycerol at 70 8C. Glycerol Johnson Medical, Pomezia, Italia). The samples were(25 mg/ml) was used to make the emulsion isotonic. After collected at predetermined time intervals up to 24 h afterremoving ethanol from the lipid phase using a centrifugal the drug administration. The whole blood samples wereevaporator (CVE-200D, Tokyo Rikakikai, Tokyo, Japan) at lightly shaken and stored at 4 8C until assay.room temperature, the water phase and the lipid phasewere mixed at 10,000 rev. /min for 10 min using a 2 .5. Determination of cyclosporin A in whole blood

homogenizer (Polytron PT 3100, Kinematica, Littau,Switzerland). A fine emulsion was prepared at 4 8C using a CSA concentration in whole blood was assayed by usingmicrofluidizer. The final concentration of CSA in the a monoclonal antibody fluorescence polarization immuno-

prepared emulsion was 3 mg/ml and the concentrations of assay (monoclonal TDxFLx , Abbott Laboratories, Abbottegg lecithin and soybean oil were 2% and 10%, respective- Park, IL, USA) (Agarwal, 1985). To an aliquot of 150 mlly. The particle size of the prepared CSA O/W-emulsion of the sample, 50 ml of solubilizing agent were added towas 221.5612.5 nm as determined by a dynamic light dissolve the cells, and 300 ml of precipitation agent werescattering analyzer (Autosizer Lo-C, Malvern Instruments, added to precipitate protein. The solution was centrifugedWorcestershire, UK). to obtain a clear supernatant. The supernatant was used for

CSA monoclonal whole blood assay using TDxFLx .2 .3. Preparation of CSA-containing microspheres Calibration curves (0–1500 ng/ml) were obtained each

time a set of samples was analyzed, and their qualityFloating CSA microspheres containing Labrafill 1944 control was assessed by analyzing the standard samples

as a core material were prepared by a solvent evaporation provided by the instrument manufacturer. Under these

S.-J. Kim et al. / European Journal of Pharmaceutical Sciences 15 (2002) 497 –502 499

conditions, percentage recovery of CSA in the samples dR C] ]]]ranged from 95.22 to 102.35% and within-day and be- 5 k ? 1 2 2 k ? R (1)H Jin outdt C 1 IC50tween-day coefficient of variation did not exceed 5% forwhere R is the observed response, i.e. lymphocyte fractionthe same batch of reagents. Samples exceeding the upper(LY/WBC, %), k is the zero-order rate constant for thelimit of calibration concentration range were diluted with in

production of the response and k is the first-order ratephosphate buffered saline and re-assayed. out

constant for the loss of the response, IC is the whole50

blood CSA concentration that leads to 50% of maximum2 .6. Determination of lymphocyte population in whole inhibition and C is the whole blood CSA concentration atblood the time of the observed response. When no CSA is

present, this model yields the following relation for theThe lymphocyte fraction (LY/WBC), the ratio of response production rate constant (k ) at steady-statein

lymphocyte population to white blood cell population ink 5 k ? R (2)in out 0whole blood, served as pharmacodynamic marker for CSAwhere R is a no-dose baseline of the lymphocyte fractionsince the immunosuppressive activity of CSA is primarily 0

at steady-state. Therefore, the rate of change of CSAdue to its effect on T cells and B cells (Awni, 1992). Thepharmacological response can be described by the follow-total cell counts in whole blood were measured by an

ing equationautomated hemocytometer (Coulter STKS, Coulter Elec-tronics, Northwell, UK). The whole blood samples were RdR 0

] ]]]5 k ? 2 R . (3)assayed within 24 h after collection to avoid cell destruc- outdt C]]51 1 6tion. IC50

The area between the baseline and effect curves (ABEC)2 .7. Analysis of pharmacokinetic parameters was calculated using the trapezoidal rule. Two different

metrics, pharmacodynamic efficacy (EFF) and pharmaco-The pharmacokinetic parameters associated with in- dynamic availability (F ), were used to evaluate thePD

travenous and oral formulations were estimated by both pharmacological response of each formulation (Gobburucompartmental and noncompartmental methods using Win- and Jusko, 1998). EFF (response /concentration units) wasNonlin (Version 1.1, Scientific Consulting, Cary, NC, calculated as ABEC/AUC, and F was determined as:PDUSA) (Gabrielsson and Weiner, 1997). In case of intraven-

ABEC AUC EFFtest ref testous formulations, the lowest Akaike’s number which ]]] ]]] ]]F 5 ? 5 . (4)PD ABEC AUC EFFref test refindicates the goodness of fit was obtained when the CSAconcentration–time profiles were fitted to a two-compart- The EFF provides an overall measure of effect per unitment open model with bolus intravenous input and first- dose and the F provides a dimensionless quantitation ofPD

order output from central compartment. The corresponding relative efficiency of test formulation when compared topharmacokinetic parameters of intravenous formulations that of a reference formulation. In a linear pharmacokineticwere calculated using standard equations (Gibaldi and system, an F value less than 1.0 indicates lower pharma-PD

Perrier, 1982). Area under the whole blood concentration– codynamic availability and a value greater than 1.0 indi-time curve (AUC) and area under the first moment curve cates that the test formulation is more efficient than the(AUMC) were calculated using trapezoidal rule. The mean reference formulation.residence time (MRT) was calculated as AUMC/AUC. The pharmacodynamic model parameters were estimatedMaximum whole blood concentration (C ) and time for by fitting lymphocyte fraction and whole blood CSAmax

maximum whole blood concentration (T ) were obtained concentration to the above model equation using WinNon-max

directly from the experimental data. The absolute bioavail- lin (Version 1.1, Scientific Consulting).ability (F, %) for each formulation was calculated as the

2 .9. Statistical evaluationratio of AUC of each formulation to AUC of CIPOL Inj. .

All data were analyzed for statistical significance by the2 .8. Analysis of pharmacodynamic parameters Student’s t-test (P,0.05). All calculated values were

expressed as their mean6S.E.The indirect pharmacodynamic response model (Day-

neka et al., 1993; Gobburu and Jusko, 1998) was used todescribe the whole blood CSA concentration–pharmaco- 3 . Results and discussiondynamic activity relationship. The mathematical expres-sion that describes the relationship between the lympho- 3 .1. Pharmacokineticscyte fraction as the pharmacological response and the CSAconcentration is: Fig. 1 shows the mean whole blood concentration–time

500 S.-J. Kim et al. / European Journal of Pharmaceutical Sciences 15 (2002) 497 –502

MRT and T obtained from CSA microspheres andmax

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 intravenousadministration, 1.33%, 2.17% and 0.83% in Sandimmun

Neoral , CSA microspheres and O/W-emulsion for oraladministration, respectively. Similar or higher bioavail-ability, delayed MRT, and extended T of CSA micro-max

spheres and O/W-emulsion for oral administration whencompared with those of Sandimmun Neoral suggested

that CSA microspheres and O/W-emulsion could providesustained release characteristics, and they might be used asoral sustained release formulations of CSA.Fig. 1. (A) The whole blood concentration–time profiles of cyclosporin A

after intravenous administration (10 mg/kg) of CIPOL Inj. (d) andCSA O/W-emulsion (s) to rabbits. The points are the experimental data 3 .2. Pharmacodynamics(6S.E.), the lines are the pharmacokinetic model fitted curves; (B) thewhole blood concentration–time profiles of cyclosporin A after oral

The lymphocyte fraction–time profiles for various CSAadministration (10 mg/kg) of Sandimmun Neoral (d), CSA micro-formulations are shown in Fig. 2. The pharmacodynamicsphere (.) and O/W-emulsion (s) to rabbits. Each point represents theparameters of intravenous formulations were calculated bymean6S.E. (n53–5).

Eq. (3) using predicted CSA concentration from the two-compartment model as the whole blood concentration of

profiles of CSA after intravenous administration (A) of CSA. The experimental data and the respective fitted dataO/W-emulsion and CIPOL Inj. , and oral administration of the lymphocyte fraction in whole blood showed good

(B) of CSA microsphere, O/W-emulsion and Sandimmun correlation with the correlation coefficient of 0.979 andNeoral to rabbits at 10 mg/kg (n53–5). Table 1 lists the 0.912 in case of the intravenous administration of CIPOL

pharmacokinetic parameters of CSA that were evaluated Inj. and CSA O/W-emulsion, respectively. The IC of50

by the two-compartment model and noncompartmental CSA O/W-emulsion, which represents the intrinsic sen-method using intravenous and oral administration data, sitivity of CSA effects on the proliferation of lymphocyte,

respectively. After intravenous administration, the AUC of was smaller than that of CIPOL Inj. , however, there wasCSA O/W-emulsion was significantly smaller than that of no significant difference between the two formulations

CIPOL Inj. (P,0.05). However, the CL , MRT and (P.0.05) due to large variation from CIPOL Inj. (Tablet

half-life of CSA O/W-emulsion were significantly in- 2). Regardless of routes of administration, there were nocreased (9.03-fold, 2.24-fold and 1.97-fold, respectively) significant differences in ABEC among all formulations

when compared with those of CIPOL Inj. (P,0.05). The tested in this study (P.0.05). However, the pharmaco-CL and half-life of CIPOL Inj. were 777.766131.28 dynamic efficacy (EFF), the effect per unit dose, of CSAt

ml /h /kg and 5.8962.65 h, respectively, and these results O/W-emulsion for intravenous administration was sig-were similar to those of other investigator’s (El-Sayed et nificantly increased when compared with that of CIPOL

al., 1995). In the case of oral administration, the half-lives, Inj. (P,0.05). In the case of oral administration, EFF of

Table 1Pharmacokinetic parameters of cyclosporin A (mean6S.E., n53–5)

aParameters Intravenous administration Oral administration O/W-emulsion CIPOL Inj. Microsphere O/W-emulsion Sandimmun Neoral

b cAUC (ng/ml /h) 1518.956435.63 13122.0962359.04 284.29646.20 130.59651.09 174.77659.20td dMRT (h) 10.84614.28 4.8362.01 12.4161.02 12.1460.72 7.7060.85

e d dC (ng/ml) – – 15.5062.81 9.3863.16 25.0960.74maxd dT (h) – – 8.0063.46 6.0062.00 1.3060.08max

T (h) 11.58615.67 5.8962.65 111.63693.61 79.51688.96 9.9465.351 / 2bCL (ml /h /kg) 7030.9362350.11 777.766131.28 – – –t

F (%) 11.5863.32 100 2.1760.35 0.8360.67 1.3360.45a AUC , area under the whole blood concentration–time curve until time is t; MRT, mean residence time; C , maximum whole blood concentration;t max

T , time for C ; T , terminal half-life; CL , total clearance; F, absolute bioavailability.max max 1 / 2 tb 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 evaluatethe pharmacodynamic effects of immunomodulatory drugslike CSA. Since the lymphocytes, the pharmacologicalaction site of CSA, are mainly distributed in the lymphaticsystem (Adair and Guyton, 1985), we need to evaluate thecorrelation between the pharmacokinetics and pharmaco-dynamics in the lymphatic system in order to understandthe immunosuppressive activity of CSA.

A cknowledgements

This work was partly supported by the Brain-Korea 21Project in 2001, and by a grant of the Korea Health 21Fig. 2. (A) The pharmacodynamic profiles of cyclosporin A after

intravenous administration (10 mg/kg) of CIPOL Inj. (d) and CSA R&D Project, Ministry of Health & Welfare, RepublicO/W-emulsion (s) to rabbits. The points are the experimental data Korea (01-PJ1-PG3-21400-0020).(6S.E.), the lines are the model fitted curves; (B) the whole bloodconcentration–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). R eferences

CSA microspheres was significantly decreased and that ofAdair, T.H., Guyton, A.C., 1985. Lymph Formation and its ModificationCSA O/W-emulsion was significantly increased when

in the Lymphatic System: Experimental Biology of the Lymphaticcompared with Sandimmun Neoral (P,0.05). Circulation. Elsevier Science, New York.Pharmacokinetic bioavailability (F ) was compared with Agarwal, R.P., 1985. Assessment of cyclosporin A in whole blood and

pharmacodynamic availability (F ) using CIPOL Inj. as plasma in five patients with different hematocrits. Ther. Drug Monit. 7,PD61.a reference. The F of CSA O/W-emulsion administered

Al-Meshal, M., Khidr, S.H., Bayomi, M.A., Al-Angary, A.A., 1998. Oralintravenously was 11.5863.32%, on the contrary, the FPD administration of liposomes containing cyclosporine: a phar-was 5.51-fold higher than that of CIPOL Inj. . The mean F macokinetic study. Int. J. Pharm. 168, 163–168.of Sandimmun Neoral , CSA microspheres and O/W- Awni, W.M., 1992. Pharmacodynamic monitoring of cyclosporin. Clin.emulsion was only 1.33%, 2.17% and 0.83%, respectively, Pharmacokinet. 23, 428–448.

Besarab, A., Jarrell, B.E., Hirsch, S., Carabasi, R.A., Cressman, M.D.,but the F of these formulations were 75.89-, 35.91- andPD Green, P., 1987. Use of the isolated perfused kidney model to assess100.16-fold higher than that of CIPOL Inj. (P,0.05).

the acute pharmacologic effects of cyclosporine and its vehicle,These results would imply two things. One is that the Cremophor EL. Transplantation 44, 195–201.sustained release from CSA O/W-emulsion and micro- Cavanak, T., Sucker, H., 1986. Formulation of dosage forms. Prog.

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CSA concentration in whole blood may not reflect the El-Sayed, Y.M., Tabbara, K.F., Gouda, M.W., 1995. Effect of acetazola-CSA concentration in the pharmacological action site. mide on the pharmacokinetics of cyclosporin in rabbits. Int. J. Pharm.Accordingly, the pharmacokinetic parameters obtained 121, 181–186.

Table 2Pharmacodynamic parameters of cyclosporin A (mean6S.E., n53–5)

aParameters Intravenous administration Oral administration O/W-emulsion CIPOL Inj. Microsphere O/W-emulsion Sandimmun Neoral

ABEC 436.556168.40 687.78685.81 599.266135.18 641.286112.10 666.636100.23bIC (ng/ml) 25.9768.78 93.81640.57 – – –50

21 c d dk (h ) 2.1660.19 0.8860.30 1.0160.74 4.0760.78 0.2460.07outc d dEFF 0.3060.04 0.0560.00 1.9060.26 5.3561.63 4.0461.06

d dF 5.5160.40 1 35.9162.34 100.16621.90 75.89613.83PD

a ABEC (%lymphocyte fraction?h), area between the baseline and effect curves; IC , whole blood concentration that led to 50% of maximum inhibition;50

k , first-order rate constant for the loss of the response; EFF (% lymphocyte fraction?ml/ng), pharmacodynamic efficacy; F , pharmacodynamicout PD

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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