558
`
`TRANSPLANTATION
`
`Vol. 45, No.3
`
`11. Simmons RL, Thompson EJ, Yunis EJ, et al. 115 patients with
`first cadaver kidney transplants followed two to seven and a half
`years. Am J Med 1977; 62: 234.
`12. Simmons RL, Van Hook EJ, Yunis EJ, et al. 100 sibling kidney
`transplants followed 2 to 71f2 years. Ann Surg 1977; 185: 196.
`13. Kirkman RL, Strom TB, Weir MR, Tilney NL. Late mortality and
`morbidity in recipients of long-term renal allografts. Transplan(cid:173)
`tation 1982; 34: 347.
`14. Penn 1. Tumor incidence in human allograft recipients. Transplant
`Proc 1979; 11: 1047.
`15. Penn I. Malignant lymphomas in organ transplant recipients.
`Transplant Proc 1981; 13: 736.
`16. Adler M, Delhaye M, Hardy N, et al. Severe portal hypertension
`
`due to vascular disease of the liver in two renal transplant
`patients receiving azathioprine. Nephrol Dial Transplant 1987;
`2: 183.
`17. Vereerstraeten P, Kinnaert P, Dupont E, et al. Vital prognosis in
`hemodialysis and transplant patients. Dial Transplant 1981; 10:
`117.
`18. Rubin RH, Wolfson JS, Cosimi AB, Tolkoff-Rubin NE. Infection
`in the renal transplant patient. Am J Med 1981; 70; 405.
`19. Medical Research Council Working Party. MRC trial of treatment
`of mild hypertension: principal results. Br Med J 1985; 291: 97.
`
`Received 6 March 1987.
`Accepted 23 June 1987.
`
`0041-1337/88/4503-0558$02.00/0
`TRANSPLANTATION
`Copyright © 1988 by The Williams & Wilkins Co.
`
`Vol. 45, 558-561, No.3, March 1988
`Printed in U.S.A.
`
`PHARMACOKINETICS OF CYCLOSPORINE G IN PATIENTS
`WITH RENAL FAILURE
`
`M. WENK,!,2 M. BINDSCHEDLER,! E. COSTA,! M. ZUBER,! S. VOZEH,! G. THIEL,3 E. ABISCH,4
`H. P. KELLER,4 T. BEVERIDGE,4 AND F. FOLLATH1
`
`Divisions of Clinical Pharmacology and Nephrology, Department of Medicine, University Hospital (Kantonsspital), CH-4031 Basel;
`and Sandoz Ltd., Pharmaceutical Division, CH-4002 Basel, Switzerland
`
`The pharmacokinetics of the cyclosporine A (CsA,
`Sandimmune) analogue Nva2-cyclosporine, or cyclo(cid:173)
`sporine G (CsG) was investigated in 6 patients with
`terminal renal failure after a 4-hr intravenous infusion
`(3.5 mg/kg) and after oral administration (600 mg) of
`the drug. Blood samples were collected up to 38 hr and
`CsG concentrations were measured by radioimmuno(cid:173)
`assay and high-performance liquid chromatography.
`The resulting pharmacokinetic parameters of CsG were
`similar to those described for CsA in the same patient
`population. Based on HPLC determinations, a mean ter(cid:173)
`minal elimination half-life of 18.9 hr was calculated.
`The total body clearance was 0.55 L/hr/kg, the volume
`of the central compartment was 0.32 Ljkg, and the
`steady-state volume of distribution was 5.97 Ljkg. After
`oral administration maximum CsG concentrations in
`blood were reached between 2.5 and 3 hr, and the bio(cid:173)
`availability was in the range of 24-55% (mean 36%).
`The ratios between the polivalent RIA and HPLC deter(cid:173)
`minations were considerably larger after oral dosing
`than after i.v. infusion. The blood-to-plasma ratio was
`1.23, which is smaller than that observed for CsA. These
`results suggest that in patients undergoing renal trans(cid:173)
`plantation the same dosing strategies can be applied for
`CsG as have been established for CsA.
`
`1 Division of Clinical Pharmacology.
`2 Address reprint requests to Markus Wenk, Clinical Pharmacology,
`Department of Medicine, University Hospital (Kantonsspital), CH-
`4031 Basel, Switzerland.
`3 Division of Nephrology.
`• Sandoz Ltd., Pharmaceutical Division, Basel.
`
`Cyclosporine G (CsG),* or Nva2-cyclosporine is, like cyclo(cid:173)
`sporine A (CsA, Sandimmun) produced by the fungus Tolypo(cid:173)
`cladium in/latum GAMS (1). It differs from CsA by having a
`norvaline residue instead of alphaaminobutyric acid in position
`2 of the molecule. In Wistar rats (2) and dogs (3) the immu(cid:173)
`nosuppressive activity of CsG was found to be similar to that
`of CsA, but no nephrotoxicity was observed. On the other hand,
`in Sprague-Dawley rats the nephrotoxic and hepatotoxic side(cid:173)
`effects were comparable to those observed with CsA (4), indi(cid:173)
`cating that there are important differences between species and
`between animal strains. Therefore, carefully monitored clinical
`trials in man are indicated to confirm the lack of toxic side(cid:173)
`effects that have been demonstrated at least in some animals.
`However, as a prerequisite for clinical investigations some
`knowledge of the relationship between dosage and CsG blood
`concentrations has first to be gathered. Thus, the aim of the
`present study was to investigate the pharmacokinetics of CsG
`after intravenous and oral administration in patients with
`terminal renal failure who were awaiting a renal transplanta(cid:173)
`tion and to compare the results with CsA.
`
`MATERIALS AND METHODS
`This investigation was designed as a randomized crossover study
`with a one-week washout period between the two CsG administrations.
`
`* Abbreviations used: AUC, area under the blood concentration
`curve; Cl, clearance; CsA, cyclosporine A; CsG, cyclosporine G, Nva2
`cyclosporine; f, bioavailability; HPLC, high-performance liquid chro(cid:173)
`matography; RIA, radioimmunoassay; V" volume of the central com(cid:173)
`partment; Vdss, volume of distribution at steady-state.
`
`-
`
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`

`
`March,1988
`
`WENK ET AL.
`
`559
`
`To obtain some dosage guidelines for patients, a pilot study was carried
`out using the same protocol in healthy volunteers. For ethical reasons
`this group was restricted to two subjects. The protocol was approved
`by the Ethical Committee for Human Research of the Medical Depart(cid:173)
`ment of the University Hospital and informed consent was obtained
`from all participants in the study.
`The two healthy male volunteers were 19 and 20 years of age, and
`weighed 63 and 67 kg, respectively. The 6 patients were chronically
`hemodialyzed candidates for kidney transplantation with a mean age
`of 46 (34-59) years and a mean weight of 68 (51-89) kg. CsG was
`provided by Sandoz Ltd, Basel, Switzerland. The drug was infused via
`central venous line at a constant rate over 4 hr using a calibrated
`pump. All subjects received 3.5 mg/kg CsG, which was diluted in 500
`ml glucose (5%). For the oral administration 600 mg CsG in 50 ml milk
`containing drinking chocolate (Caotina, Wander) was given after a 10
`hr fasting period. A light breakfast was served after 2 hr and lunch 4
`hr after drug administration.
`Blood samples were drawn into ethylenediaminotetraacetate tubes
`at 0, 0.5, 1, 2, 3, and 4 hr during infusion, then 4.33, 4.66, 5, 5.5, 6, 7,
`8, 10, 12, 14, 16, 22, 28, and 34 hr after the start of infusion. After oral
`dosing the sampling times were 0, 0.25, 0.5, 0.75, 1, 1.5, 2. 2.5, 3, 4, 6,
`8, 10, 12, 15, 18, 24, and 30 hr after drug administration. All samples
`were split into two portions for RIA and HPLC analysis and stored at
`-20'C until assayed. In addition, 0.5 and 12 hr after the start of the
`infusion, and 3 and 12 hr after oral dosing, one aliquot of the blood
`samples was stored at room temperature (20-22' C) for 2 hr and
`centrifuged at 4000 rpm for 10 min to separate plasma. All samples
`(whole-blood and plasma) were analyzed by RIA and HPLC. The RIA
`method was performed with the commercially available RIA kit for
`CsA (5) using CsG as standards. The RIA detection limit was 22 ng/
`m!. In the concentration range from 100 to 2000 ng/ml the intraassay
`and interassay coefficients of variation (CV) were between 3.3 and 11 %
`and between 4.2 and 11.3%, respectively. The HPLC method was
`similar to the technique described by Smith and Robinson (6), with
`the exception that an internal standard (CsA) was used and that the
`automated sample wash was replaced by a manual treatment with n"
`hexane. The interassay CV of the HPLC method for concentrations
`above 150 ng/ml was <10%, and the accuracy of spiked samples was
`found to be within 100±25% · of the expected concentrations. The
`
`detection limit was 20 ng/m!. Regression analysis of 20 control samples
`measured by HPLC(x) and RIA(y) yielded in the equation y = 0.944 x
`+26.9 (r = 0.976).
`Data analysis. Blood concentration-time curves after intravenous
`infusion measured by HPLC and RIA were fitted according to a three(cid:173)
`compartment open-body model by the following equation:
`
`C _ ~ Ci (1 - eX,,") . &,
`i:-'
`-Xi
`b -
`
`- A-t
`. e '
`
`where Ci is the coefficient corresponding to a single intravenous bolus
`injection, Ro is the infusion rate, Xi the exponential rate constant, and
`t ' is the time from beginning of infusion (during infusion t' = t;
`thereafter t' becomes a constant equal to 4 hr). Data analysis was
`performed by the extended least-square fit program (ELSFIT, version
`3.0) on an HP 9816 computer (7) . All subsequently derived pharma(cid:173)
`cokinetic parameters were calculated using standard formulas (8).
`After oral administration only model-independent pharmacokinetic
`parameters were calculated from blood concentration data of CsG.
`Bioavailability was calculated using the model independent area under
`the blood concentration curves (AUCs) from 0 to 30 hr. For calculation
`of the AUCi.v. the concentration at 30 hr was obtained by interpolation
`between 26 and 32 hr.
`
`RESULTS
`The pharmacokinetic results from the pilot study with two
`healthy volunteers were in the expected range and comparable
`with CsA. Peak CsG concentrations were 1264 ng/ml and 1558
`ng/ml after i.v. infusion measured by HPLC and 712 ng/ml
`and 976 ng/ml after oral administration (Table 1). Reliable
`measurements were obtained up to 30 hr after oral administra(cid:173)
`tion and up to 32 hr and 38 hr after start of infusion using
`HPLC and RIA, respectively. The bioavailability was estimated
`at 21 %. The elimination half-lives after i.v. infusion were rather
`short, being 6.7 and 3.7 hr, respectively. After these acceptable
`results, the study was continued with 6 patients using the
`identical protocol. Both routes of CsG administration were well
`tolerated by all patients except for a slight sensation of warmth
`
`TABLE 1. Pharmacokinetic p'arameters of CsG after a single dose (3.5 mg/kg) administered as a 4-hr infusion in two healthy volunteers and in
`6 patients with terminal renal failure
`
`Patient
`
`CsG determined by
`HPLC:
`Bpa
`TDa
`
`HE
`WS
`GJ
`Bjo
`SV
`BJ
`Mean ± SD'
`
`CsG determined by
`RIA:
`Mean ± SDe
`
`Dose
`(mg)
`
`emu •
`(ng/ mi)
`
`tl/'I
`(hr)
`
`t lf2.
`(hr)
`
`til.,
`(hr)
`
`k 1{
`(hr-I)
`
`k"
`(hr-I)
`
`k13
`c
`(hr-I)
`
`k .. <
`(hr-I)
`
`Vd..
`(L/ kg)
`
`VI
`(L/kg)
`
`Cl
`(L/ hr/ kg)
`
`AUCO-3Oh!
`(ng/ml · hr)
`
`234
`220
`273
`174
`198
`217
`239
`283
`
`1264
`1558
`3172
`1024
`1168
`1204
`1704
`1610
`1647
`793
`
`0.044
`0.072
`
`0.201
`0.067
`0.148
`0.175
`0.119
`0.112
`0.137
`0.047
`
`0.70
`0.87
`1.56
`0.55
`1.62
`1.62
`1.20
`1.43
`1.33
`0.41
`
`6.7
`3.7
`
`19.7
`27.5
`22.9
`20.9
`14.0
`8.4
`18.9
`6.7
`
`8.85
`4.47
`1.34
`2.66
`1.81
`1.30
`2.25
`3.15
`2.08
`0.74
`
`2.81
`1.65
`0.95
`1.80
`0.78
`0.71
`1.08
`1.25
`1.09
`0.39
`
`2.51
`1.31
`0.33
`3.04
`0.97
`0.52
`0.67
`0.69
`1.04
`1.00
`
`0.230
`0.303
`
`0.449
`0.045
`0.051
`0.043
`0.064
`0.126
`0.130
`0.159
`
`2.26
`1.34
`3.30
`11.60
`9.17
`5.07
`2.41
`4.25
`5.97
`3.62
`
`0.150
`0.167
`
`0.338
`0.166
`0.411
`0.339
`0.179
`0.474
`0.318
`0.123
`
`0.414
`0.497
`
`0.428
`0.651
`0.445
`0.627
`0.427
`0.739
`0.553
`0.136
`
`9061
`7414
`12282
`4927
`7810
`6638
`8874
`9782
`8385
`2558
`
`1891
`624
`
`0.112
`0.048
`
`1.93
`0.55
`
`26.2
`13.1
`
`4.74
`4.94
`
`0.998
`0.223
`
`0.715
`0.316
`
`0.098
`0.135
`
`6.14
`4.28
`
`0.247
`0.113
`
`0.434
`0.096
`
`10514
`3051
`
`a Healthy volunteers.
`b C .... at the end of infusion.
`c k12, k2" k13, and k31 are transfer rate constants between compartments.
`d Interpolated between 26 hr and 32 hr.
`, Patients only.
`
`NOVARTIS EXHIBIT 2002
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`
`

`
`560
`
`TRANSPLANTATION
`
`Vol. 45,No. 3
`
`during the first hours of the trial. Representative blood concen(cid:173)
`tration curves of one patient after intravenous infusion and
`oral administration are shown in Figures 1 and 2. Cyclosporine
`G concentrations measured by the polyvalent RIA were higher
`than those measured by HPLC in all patients. The mean ratios
`(±SD) between the two analytical methods are displayed in
`Figure 3. The considerably higher ratios after oral dosing in(cid:173)
`dicate that there is a greater quantity of crossreacting metab(cid:173)
`olites after this route of cyclosporine G administration com(cid:173)
`pared with i.v. infusion. The pharmacokinetic parameters after
`i.v. infusion are shown in Table 1. There is a large interindivid(cid:173)
`ual variability in most of the parameters. The terminal elimi(cid:173)
`nation half-life (tlI23) measured by HPLC was 18.9 hr (range
`8.4-27.5 hr). The volume of distribution at steady-state (Vd..)
`was 5.97 L/kg (range 2.41-11.6 L/kg), and total clearance (Cl)
`was 0.553 L/hr/kg (range 0.427-0.739 L/hr/kg). As expected,
`the pharmacokinetic parameters based on RIA determinations
`differed considerably from the HPLC derived data, with the
`most dominant difference for tlI23 (mean value 26.2 hr; range
`13.4-50.6 hr). Model-independent pharmacokinetic parameters
`after oral administration of CsG are shown in Table 2. Maxi(cid:173)
`mum CsG blood concentrations were reached between 2.5 and
`3 hr, and the bioavailability (f) was 0.36 (range 0.24-0.55). A
`total of 24 plasma samples separated from whole blood at room
`
`5000
`
`'"
`~ 1000
`0>
`C
`'-' 500
`U
`Z
`0
`U
`C
`0
`0
`...J
`m
`Cl
`'" U
`
`100
`
`50
`
`10
`
`0
`
`4
`
`8
`
`12
`TIME
`
`20
`16
`(HOURS)
`
`24
`
`28
`
`32
`
`36
`
`FIGURE 1. Cyciosporine G blood concentration in a patient (B.J.)
`after a 4-hr infusion (3.5 mg/kg) measured by RIA (0) and HPLC (*).
`The solid lines represent the computer fitted curves.
`
`5000
`
`,....
`E
`til 1000
`c
`"" 500
`<.,)
`Z
`0
`U
`0
`0
`0
`......
`III
`
`100
`
`50
`
`Cl
`<II
`U
`
`10
`
`0
`
`4
`
`8
`
`20
`16
`12
`TIME (HOURS)
`
`24
`
`28
`
`32
`
`FIGURE 2. Cyciosporine G blood concentrations in a patient (B.J.)
`after oral administration (600 mg) measured by RIA (0) and HPLC
`(*).
`
`3 .0
`
`2.6
`
`2
`I-
`eI: 2.2
`a::
`
`(J
`...... 1.8
`Q.
`:l:
`~1.4
`a::
`
`1.0
`
`0.6 a
`
`4
`
`8
`
`20
`16
`12
`TIME (HOURS)
`
`24
`
`28
`
`FIGURE 3. Ratios between CsG concentrations measured by RIA
`and HPLC after oral (*) and intravenous (0) dosing.
`
`TABLE 2. Model independent pharmacokinetic parameters of CsG
`after a single oral dose (600 mg) in two healthy volunteers and in 6
`patients with terminal renal failure
`
`Patient
`
`t=x
`(hr)
`
`Cm=
`(ng/ ml)
`
`AUC0- 30lu
`(ng/ ml.hr)
`
`CsG determined by
`HPLC:
`Bpa
`TDa
`HE
`WS
`GJ
`BJo
`SV
`BJ
`Mean ± SD b
`
`GsG determined by
`RIA:
`Mean ± SD b
`
`a Healthy volunteers.
`b Patients only.
`
`3.0
`3.0
`2.5
`3.0
`3.0
`2.5
`2.5
`2.5
`
`2.67
`0.41
`
`712
`976
`1539
`1134
`844
`1466
`1362
`1392
`
`1289
`258
`
`4994
`4123
`8067
`6837
`5570
`10120
`7399
`7046
`
`7506
`1521
`
`0.215
`0.204
`0.299
`0.402
`0.235
`0.551
`0.332
`0.340
`
`0.360
`0.108
`
`2.83
`0.26
`
`1791
`196
`
`12142
`2725
`
`0.463
`0.138
`
`temperature were also measured by both methods. Mean blood:
`plasma ratios (±SD) were 1.23±0.18 (HPLC) and 1.23±0.23
`(RIA).
`
`DISCUSSION
`In this study CsG was applied for the first time in man.
`Single doses of CsG were well tolerated by the patients and the
`volunteers of the pilot study. The pharmacokinetic parameters
`of CsG derived from patients with end-stage renal disease were
`similar to those found for CsA in the same patient population
`(9). After Lv. infusion a terminal average half-life (tlI2a) of 18.9
`hr was calculated that is in good agreement with the half-life
`of 15.8 hr found for esA. The values for the volume of distri(cid:173)
`bution VI (0.318 L/kg) and for the total clearance CI (0.553 L/
`hr/kg) are both larger than estimated for esA (0.18 L/kg and
`0.369 L/hr/kg). The pharmacokinetics ofCsG after oral admin(cid:173)
`istration were calculated using a model-independent analysis.
`A bioavailability (f) of 36% (range 24-55%) was calculated that
`is in the same range as that reported for CsA (a review of the
`pharmacokinetic data of esA) has been published elsewhere
`
`NOVARTIS EXHIBIT 2002
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`
`

`
`March,1988
`
`WENK ET AL.
`
`561
`
`[10]). For CsA a temperature-dependent uptake into blood cells
`has been observed (11), which is the main reason why it is
`recommended to measure this drug in whole blood rather than
`in serum or plasma. In the present study the concentration
`ratio between blood and plasma separated at 22°C was only
`1.23, which is considerably lower than the ratio described for
`CsA. Similar to CsA all concentrations measured by RIA were
`higher than those measured by the specific HPLC. This signi(cid:173)
`fies that the CsG metabolites crossreact also with the antiserum
`of the CsA-RIA used in this study. However, if the RIA:HPLC
`ratios between the two routes of administration are compared
`(Fig. 3) it is evident that these ratios are significantly (P<O.Ol)
`higher after p.o. administration than after i.v. infusion, which
`can be attributed to first-pass metabolism. A similar result was
`recently described for CsA in the dog (12), suggesting an
`additional metabolism in the gastrointestinal tract. Therefore,
`CsA or CsG blood concentrations measured by the polyvalent
`RIA should be interpreted with caution if the routes of drug
`administration are changed. It also means that determinations
`of blood cyclosporine concentrations by RIA to estimate the
`absolute bioavailability are not reliable.
`In conclusion, the results of this study indicate that, in
`patients with renal failure, the pharmacokinetic behavior of
`CsG is similar to CsA-and, as a consequence, the same dosing
`strategies can be chosen until more clinical relevant data for
`CsG are available. In addition, clinical studies over longer
`periods are needed to confirm the optimistic results concerning
`reduced toxicity found in some animals.
`
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`G, hr and 1. Helv Chir Acta 1982; 65: 1655.
`
`2. Hiestand PC, Gunn H, Gale J, et al. The immunosuppressive
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`3. Todo S, Porter KA, Kam I, et al. Canine liver transplantation
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`5. Donatsch P, Abisch E, Hornberger M, Traber R, Trapp M, Voges
`R. A radioimmunoassay to measure cyclosporin A in plasma and
`serum samples. J Immunoassay 1981; 2: 19.
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`ine in plasma and blood using column switching. J Chromatogr
`1984; 305: 353.
`7. Sheiner LB. ELSFIT. A program for the extended least squares fit
`to individual pharmacokinetic data: user's manual. A technical
`report of the Division of Clinical Pharmacology. San Francisco:
`University of California, 1983.
`8. Gibaldi M, Perrier D. Pharmacokinetics. 2nd ed. New York: Marcel
`Dekker, 1982: 84.
`9. Follath F, Wenk M, Vozeh S, et al. Intravenous cyclosporine
`kinetics in renal failure. Clin Pharmacol Ther 1983; 34: 638.
`10. Ptachcinski RJ, Venkataramanan R, Burckart GJ. Clinical phar(cid:173)
`macokinetics of cyclosporin. Clin Pharmacokinet 1986; 11: 107.
`11. Wenk M, Follath F, Abisch E. Temperature dependency of appar(cid:173)
`ent cyclosporin A concentrations in plasma. Clin Chern 1983; 29:
`1865.
`12. Gridelli B, Scanlon L, Pellicci R, et al. Cyclosporine metabolism
`and pharmacokinetics following intravenous and oral adminis(cid:173)
`tration in the dog. Transplantation 1986; 41: 388.
`
`Received 29 May 1987.
`Accepted 6 July 1987.
`
`NOVARTIS EXHIBIT 2002
`Par v Novartis, IPR 2016-00084
`Page 4 of 4