Pharmacokinetic Interactions Augment Toxicities of
`Sirolimus/Cyclosporine Combinations
`
`J Am Soc Nephrol 12: 1059 –1071, 2001
`
`HEMANGSHU PODDER,* STANISLAW M. STEPKOWSKI,* KIMBERLY L. NAPOLI,*
`JAMES CLARK,† REGINA R. VERANI,† TING-CHAO CHOU,‡ and
`BARRY D. KAHAN*
`*Department of Surgery, Division of Immunology and Organ Transplantation, and †Department of Pathology
`and Laboratory Medicine, The University of Texas Medical School at Houston, Houston, Texas; and
`‡Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York,
`New York.
`
`Abstract. This study correlated the dynamic effects of siroli-
`mus (rapamycin; RAPA) and cyclosporine (CsA) alone versus
`in combination to produce renal dysfunction, myelosuppres-
`sion, or hyperlipidemia, with their corresponding blood and
`tissue concentrations. After salt-depleted rats were treated with
`RAPA (0.4 to 6.4 mg/kg per d) and/or CsA (2.5 to 20.0 mg/kg
`per d) for 14 d, the GFR, lipid levels, bone marrow cellularity,
`and CsA/RAPA concentrations in whole blood versus liver or
`renal tissues were measured, and the median effect model was
`used to discern the type of drug interactions. Compared with
`vehicle controls (1.98 6 0.34 ml/min), GFR values were
`reduced only by large doses of drug monotherapy, namely
`RAPA (3.2 mg/kg per d 5 1.2 6 0.02 ml/min or 6.4 mg/kg per
`d 5 1.3 6 0.2 ml/min; both P , 0.01) or CsA (10.0 mg/kg per
`d 5 1.2 6 0.1 ml/min or 20.0 mg/kg per d 5 0.8 6 0.4 ml/min;
`both P , 0.01). In contrast, hosts that were treated with smaller
`
`doses of CsA/RAPA combinations showed more pronounced
`effects in reduction of GFR values: 2.5/0.4 mg/kg per d,
`modestly (1.5 6 0.5 ml/min; P , 0.01); 5.0/0.8 mg/kg per d,
`moderately (0.23 6 0.01 ml/min; P , 0.001); and higher-dose
`groups, markedly. The exacerbation of renal dysfunction
`seemed to be due to a pharmacokinetic interaction of RAPA to
`greatly increase CsA concentrations in whole blood and, par-
`ticularly, in kidney tissue. In contrast, the pharmacodynamic
`effects of CsA to potentiate two RAPA-mediated toxicities—
`myelosuppression and increased serum cholesterol/low-density
`lipoprotein cholesterol— occurred independently of pharmaco-
`kinetic interactions. RAPA aggravates CsA-induced renal dys-
`function owing to a pharmacokinetic interaction, whereas CsA
`produces a pharmacodynamic effect that augments RAPA-
`induced myelosuppression and hyperlipidemia.
`
`The development of agents that produce synergistic immuno-
`suppression with the calcineurin antagonists (CNA)— cyclo-
`sporine (CsA) or tacrolimus—is based on the hypothesis that
`drug-dose sparing mitigates renal dysfunction, as well as the
`pleiotropic neural and hepatic toxicities associated with CNA
`administration (1,2). Furthermore, the addition of a synergistic
`drug may reduce the adverse impact of the large variations in
`the pharmacokinetic (3,4) and pharmacodynamic (5) behavior
`of CsA among renal transplant recipients. Although the addi-
`tion of sirolimus (rapamycin; RAPA) to a CsA-based regimen
`has fulfilled the expectations of synergistic immunosuppres-
`sion (6,7), randomized, blinded trials document inferior renal
`function, compared with CsA/azathioprine (Aza)/prednisone
`(Pred)- or CsA/placebo/Pred-treated patients (8,9). However,
`CsA and RAPA show a pharmacokinetic interaction that is
`
`Received August 8, 2000. Accepted September 30, 2000.
`Correspondence to Dr. Barry D. Kahan, Division of Immunology and Organ
`Transplantation, The University of Texas Medical School at Houston, 6431
`Fannin, Suite 6.240, Houston, TX 77030. Phone: 713-500-7400; Fax: 713-
`500-0785; E-mail: bkahan@orgtx71.med.uth.tmc.edu
`1046-6673/1205-1059
`Journal of the American Society of Nephrology
`Copyright © 2001 by the American Society of Nephrology
`
`due, at least in part, to common metabolism by cytochrome
`P450 3A4. Therefore, the present study used an animal model
`to dissect the pharmacokinetic from the pharmacodynamic
`components of
`the
`toxicity produced by CsA/RAPA
`combinations.
`Although the exact mechanisms of CsA-induced nephrotox-
`icity are not understood fully, important components include
`increased vascular resistance, which produces decreased renal
`blood flow (10,11); generation of reactive free radicals, which
`causes both oxidative stress (12) and cytochrome P450 activa-
`tion (13); upregulated expression of the profibrogenic principle
`transforming growth factor-b (14); increased generation and
`responses of smooth muscle cell calcium to vasoconstrictive
`stimuli (15); upregulated synthesis and expression of angioten-
`sin II receptors (15); and depressed nitric oxide production by
`both endothelial and inducible nitric oxide synthases (16).
`Furthermore, CsA has been reported to promote Fas-mediated
`(17) apoptosis of LLC-PK1-cultured renal tubular cells in
`vitro, an effect that is blocked by peptide inhibitors of caspases
`3, 8, and 9 (18). Thus, increased vasoconstriction and apoptosis
`characterize CsA nephrotoxicity.
`Because the synergistic immunosuppressive interactions be-
`tween CsA and RAPA were first documented in rats (19,20),
`this species represented a logical model to investigate the
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`impact of the drug combination on the kidney. The salt-de-
`pleted rat model has been reported to produce functional and
`histopathologic effects that resemble the dose-dependent im-
`pairment of renal function observed in CsA-treated kidney
`transplant patients (1,10,21–23). In contrast, administration of
`therapeutic doses of RAPA (0.04 to 0.8 mg/kg per d intrave-
`nously) caused no significant renal changes in rats (24). At 0.8
`mg/kg per d RAPA, normal rats displayed only a marginal
`elevation in serum creatinine (SCr) values, and spontaneously
`hypertensive rats showed neither accelerated necrotizing vas-
`culopathy nor tubular atrophy (25). Only supratherapeutic (1.6
`to 6.4 mg/kg per d) RAPA doses produced transmural fibrinoid
`necrosis of vessels in the gastrointestinal submucosa and in the
`kidney, as well as juxtaglomerular hypertrophy, tubular dila-
`tion, basement membrane thickening, vacuolization, and atro-
`phy (25).
`Owing to its use in combination with CNA, there is consid-
`erable interest in understanding the impact of RAPA on CsA-
`induced nephrotoxic injuries. Andoh et al. (22) reported that
`subcutaneous coadministration of RAPA potentiated CsA-in-
`duced nephrotoxicity in salt-depleted rats and postulated that
`the effect was due to enhanced hyperglycemia. In contrast, we
`suggested that increased drug concentrations contribute to the
`adverse renal effects displayed by the CsA/RAPA combina-
`tion, because we had documented elsewhere that a portion of
`the synergistic effect was due to pharmacokinetic interactions
`(26), particularly after oral coadministration (27). The present
`study revealed a predominant role of pharmacokinetic interac-
`tions to produce toxic renal exposures of CsA and of dynamic
`effects to potentiate the lipid as well as the myelosuppressive
`toxicities.
`
`Materials and Methods
`Animals
`Male Wistar Furth (RT1u) rats (160 to 200 g), obtained from Harlan
`Sprague Dawley (Indianapolis, IN), were housed in cages in a tem-
`perature- and light-controlled environment. The animals, which were
`maintained ad libitum on either regular or low-salt chow (0.05%
`sodium; Teklad Premier, Madison, WI) with access to tap water, were
`weighed and examined daily.
`
`Drugs
`Commercial oral formulations of CsA (Sandimmune; Novartis
`Research, East Hanover, NJ) and RAPA (Rapamune; Wyeth-Ayerst,
`Princeton, NJ) were administered by oral gavage in a constant volume
`of 0.2 ml daily for 14 d.
`
`Experimental Groups
`After a 7-d conditioning period on low-salt chow, groups of six rats
`were assigned randomly to treatment for 14 d with CsA alone (2.5,
`5.0, 7.5, 10.0, 15.0, or 20.0 mg/kg per d), RAPA alone (0.4, 0.8, 1.2,
`1.6, 3.2, or 6.4 mg/kg per d), or CsA/RAPA combinations at a fixed
`6.25:1 ratio (2.5/0.4, 5.0/0.8, 7.5/1.2, 10.0/1.6, 15.0/3.2, or 20.0/6.4
`mg/kg per d), which had been shown to be the optimal ratio to
`document synergistic immunosuppression, or at varying ratios of
`fixed 5.0 or 10.0 mg/kg per d CsA doses with ascending amounts of
`RAPA (0.4, 0.8, 1.2, 1.6, 3.2, or 6.4 mg/kg per d). In addition, there
`
`were two untreated control groups, each composed of six rats: one fed
`a low-salt diet and the other fed a normal diet.
`After receiving the final drug doses on day 14, the animals were
`placed in metabolic cages for 24-h urine collections and GFR mea-
`surements, by use of iohexol in the Renalyzer PRX 90 (Provalid AB,
`Lund, Sweden). Inman et al. (28) documented in rats that this method
`provides an accurate and reliable measure of GFR, compared with
`inulin determinations. In addition, urinary sodium, potassium, mag-
`nesium, calcium, phosphate, and creatinine levels were quantified by
`use of established methods in our clinical chemistry laboratory. Upon
`completion of the urine collection, the animals were anesthetized with
`intraperitoneal pentobarbital (Abbott, Chicago, IL) to obtain whole-
`blood samples for complete blood counts, CsA and RAPA concen-
`tration measurements, and serum aliquots for sodium, potassium,
`magnesium, calcium, phosphate, uric acid, cholesterol, creatinine, and
`lipoproteins. The laboratory results, presented as mean values 6
`deviants, were compared for statistical significance by use of
`ANOVA; P , 0.05 was accepted as significant.
`The left kidney was removed and split in half. One half of the left
`kidney and a 2-g sample of the right lateral lobe of the liver were used
`for drug concentration measurements. The other half of the left kidney
`was fixed in buffered 10% formalin and processed overnight; 3-mm
`histologic sections were stained with progressive hematoxylin-eosin,
`periodic acid-Schiff, or Masson’s Trichrome reagents. Two indepen-
`dent pathologists (J.C. and R.V.), who were blinded to treatment
`assignments, used semiquantitative scales of light microscopic criteria
`to assess the degree of vasculopathy, glomerular changes, and tubu-
`lointerstitial damage in multiple kidney sections. Tubular and glomer-
`ular changes were graded separately as follows: 0, no changes; 11,
`,5%; 21, 5 to 25%; 31, 26 to 50%; and 41, .50% involvement. A
`similar vascular scale included the following: 0, none; 11, minimal;
`21, mild; 31, moderate; and 41, severe. Although the scores gen-
`erally were concordant, when they were disparate, a mean value was
`chosen as the histopathologic grade.
`
`Renal Function
`GFR was measured by the iohexol method (29). The femoral vein
`and artery, as well as the transplant ureter, were cannulated individ-
`ually by use of 10-0 silicone tubing (Baxter, Deerfield, IL). BP, heart
`rate, and urine output were monitored with the use of a Micro-Med
`apparatus (Louisville, KY) and analyzed with the use of a DMSI 2004
`computer program (Micro-Med). BP was recorded automatically ev-
`ery 30 s and urine output every 5 min. A loading dose of 1000 mg/kg
`iohexol (Omnipaque, 300 mg/ml; Nycomed, Inc., Princeton, NJ) was
`administered intravenously over 5 min, followed by infusion of 600
`mg/kg over 90 min, as recommended by Inman et al. (28). Urine
`samples, collected at 20-min intervals after completion of the loading
`dose, were analyzed for iohexol concentrations. Whole-blood samples
`were obtained at the midpoints of the urine collections. GFR values
`(ml/min) were calculated by the formula (U 3 V)/P, where U is
`urinary iohexol concentration (mg/ml), V is urine output (ml/24 h),
`and P is plasma iohexol concentration (mg/ml). The results were
`presented as mean 6 deviants, and statistical significance was as-
`sessed by t test.
`
`Bone Marrow Cellularity
`The right femur was harvested, fixed in 10% buffered formalin,
`decalcified in formic acid for approximately 1 wk, sectioned (3 to 5
`mm), and stained with hematoxylin and eosin by use of standard
`techniques. Hematopoiesis was estimated as the percentage of the
`marrow space occupied by cellular as opposed to adipose tissue
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`Sirolimus/Cyclosporine Interactions
`
`1061
`
`elements. The average number of megakaryocytes in four high-power
`fields (403) was used to estimate the effects of RAPA with or without
`concomitant administration of CsA on platelet formation.
`
`Drug Concentration Measurements
`For CsA measurements, whole-blood samples (0.5 ml) were col-
`lected into ethylenediaminetetraacetate-containing tubes (Becton
`Dickinson, Mountain View, CA); 1- to 2-g aliquots of hepatic and
`renal tissues were disrupted by use of an Ultrasound Homogenizer
`(Fisher Scientific, Pittsburgh, PA). CsA determinations were per-
`formed by use of an automated fluorescence polarization immunoas-
`say (TDx; Abbott, Chicago, IL). In contrast to human specimens and
`on the basis of supporting data of others (30 –35), we documented
`elsewhere that the TDx technology provides results similar to HPLC
`because rats do not produce CsA metabolites that cross-react signif-
`icantly in the TDx assay (36). CsA concentrations were expressed as
`ng/ml for whole blood or ng/g for wet-tissue weight. The intra-/
`interassay coefficients of variation for blood and tissue CsA measure-
`
`ments were 3.2% at 150 ng/ml and 1.7% at 800 ng/ml, and 2.3% at
`150 ng/ml and 1.6% at 800 ng/ml, respectively (Napoli KL, Kahan
`BD, unpublished observations).
`RAPA concentrations were estimated by use of our published
`method of HPLC with ultraviolet detection (37). Owing to the pho-
`tosensitivity of RAPA, left kidney and liver samples (1 to 2 g) had to
`be protected from light during ultrasonic disruption. Briefly, 1 ml of
`0.1 M sodium carbonate and 20 ml of methanolic-estradiol-3-methyl
`ether, an internal standard, were added to 1 ml of whole blood. After
`double extraction with 10 ml of t-butyl methyl ether, the pooled
`supernates were evaporated, reconstituted twice with 150 ml of abso-
`lute ethanol, and finally suspended in 100 ml of mobile phase buffer
`composed of an 85:15 ratio of methanol/water. After centrifugation,
`85-ml aliquots of supernates were injected onto tandem Supelosil C18
`columns (Supelco, Bellefonte, PA) heated to 40°C. During elution at
`a flow rate of 0.5 ml/min, ultraviolet absorbance was monitored at 276
`nm. RAPA concentrations were estimated on the basis of a calibration
`curve consisting of 8 drug-free whole-blood (or tissue) samples that
`
`Figure 1. Effect of cyclosporine (CsA) and rapamycin (RAPA) alone or in combination on animal weight and renal function. Animals that were
`fed a low-salt diet either were untreated (s) or were treated for 14 d with CsA alone at doses of 2.5, 5.0, 7.5, 10.0, 15.0, or 20.0 mg/kg per
`d (m); RAPA alone at doses of 0.4, 0.8, 1.2, 1.6, 3.2, or 6.4 mg/kg per d (h); or CsA/RAPA combination at doses of 2.5/0.4, 5.0/0.8, 7.5/1.2,
`10.0/1.6, 15.0/3.2, or 20.0/6.4 mg/kg per d (u). In addition, some rats were treated with a constant dose of 5 (^) or 10 (z) mg/kg per d CsA
`with ascending RAPA doses, namely 0.4, 0.8, 1.2, 1.6, 3.2, and 6.4 mg/kg per d. After 14 d, we measured percentage of weight change in
`comparison with the weight at the beginning of therapy (A), 24-h urine output (B), serum creatinine levels (SCr; C), and GFR (D). For details,
`see Materials and Methods section.
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`had been spiked with 0, 2, 5, 10, 20, 30, 40, or 50 ng of RAPA. The
`assays for tissue concentrations added 0.5 ml of drug-containing (or,
`for calibrators, exogenously spiked) homogenates to 0.5 ml of sodium
`carbonate. The intra-/interassay coefficients of variation for blood and
`tissue RAPA measurements were 6.4% at 4.0 ng/ml and 4.2% at 32
`ng/ml, and 7.8% at 4.0 ng/ml and 5.6% at 32 ng/ml (38).
`
`relates dose (or concentration) to biologic effect, this model was
`chosen to assess the nephrotoxic interactions between CsA and
`RAPA. The relationship is described by the following equation:
`
`~ fa/fu! 5 ~D/Dm!m
`
`(1)
`
`Statistical Analyses
`Because all immunosuppressive agents studied to date (39) have
`obeyed the median-effect equation of Chou and Talalay (40), which
`
`where fa is the fraction affected, the percentage of inhibition (reduc-
`tion from the normal value), fu is the uninhibited fraction (1 2 fa), D
`is the administered drug dose (concentration), Dm is the dose (con-
`centration) required for 50% inhibition (the median effect), and m is
`
`Figure 2. Pharmacokinetic interactions between CsA and RAPA. Whole-blood levels of CsA (A) and RAPA (B), kidney tissue levels of CsA
`(C) and RAPA (D), and liver tissue levels of CsA (E) and RAPA (F) were measured after a 14-d course of immunosuppressive therapy with
`CsA and RAPA alone or in combination. Experimental groups are described in Figure 1 legend.
`
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`Sirolimus/Cyclosporine Interactions
`
`1063
`
`index (CI) analysis, which assessed the dose of each drug necessary to
`achieve x% inhibition:
`
`CIx 5
`
`D1C
`D1A
`
`1
`
`D2C
`D2A
`
`1
`
`D1C 3 D2C
`D1A 3 D2A
`
`(3)
`
`where D1C and D2C are the doses (or concentrations) of drugs when
`used in combination and D1A and D2A are the corresponding doses (or
`concentrations) of drugs used alone. CI values ,1 reflect synergistic
`interactions, CI values 51 reflect additive interactions, and CI values
`.1 reflect antagonistic interactions.
`
`Results
`Body Weight Changes
`Control (untreated) animals that were maintained on a low-
`salt diet for 21 d showed a mean weight gain of 41.5 6 22.8%
`(Figure 1A). In contrast, hosts that were treated with 2.5 or 5.0
`mg/kg per d CsA showed less weight gain, and those that were
`given 10.0, 15.0, or 20.0 mg/kg per d showed a maximal
`weight loss of 10% (versus untreated hosts; all groups, P ,
`0.001). Although animals that were treated with 0.4 mg/kg per
`d RAPA showed a slight weight gain, those that received 0.8,
`1.2, 1.6, 3.2, or 6.4 mg/kg per d RAPA showed a maximal
`weight loss of 20% (all groups, P , 0.001). Rats that were
`treated with CsA/RAPA drug combinations displayed a max-
`imal weight loss of 27% (all groups, P , 0.005). The weight
`loss seemed more likely to reflect metabolic causes than an-
`orexia, because all experimental animals had food in their
`stomachs and stool in their colons. Furthermore, there was no
`evidence of drug-induced diarrhea or dehydration, as evident
`on examination and by blood chemistries (vide infra).
`
`Renal Function Changes
`Animals that were treated with either CsA or RAPA mono-
`therapy showed greater urine output than hosts in the control
`groups (Figure 1B; P , 0.002), suggesting the presence of
`renal injury. Because blood glucose levels were similar among
`rats in each group, there was no evidence that hyperglycemia
`was producing a diuretic effect. In contrast to the control
`group, which showed a mean SCr value of 0.25 6 0.05 mg/dl,
`treatment with ascending 2.5 to 20.0 mg/kg per d doses of CsA
`increases in SCr values (all groups, P ,
`produced serial
`0.0007; Figure 1C). In contrast, rats that were treated with the
`smaller RAPA doses (0.4, 0.8, or 1.6 mg/kg per d) showed
`insignificant changes; only animals that received 3.2 or 6.4
`mg/kg per d showed significantly increased SCr values (P 5
`0.001). The CsA/RAPA groups showed higher SCr concentra-
`tions than either monotherapy group, increasing from 0.35 6
`0.05 mg/dl for the 2.5/0.4 mg/kg per d group to 2.35 6 0.37
`mg/dl for the 20.0/6.4 mg/kg per d group. Interestingly, as-
`cending RAPA doses added to fixed amounts of CsA (5 or 10
`mg/kg per d) produced less adverse effects (P 5 0.005) than
`those observed in groups with simultaneously increasing doses
`of both drugs (P 5 0.001).
`The SCr results were confirmed by the GFR values. In
`comparison to the normal GFR values (1.98 6 0.34 ml/min;
`Figure 1D), animals that were treated with CsA doses of 5.0
`mg/kg per d (1.1 6 0.2 ml/min), 7.5 mg/kg per d (0.92 6 0.37
`
`Figure 3. Correlation between CsA and RAPA tissue concentrations
`and SCr levels. Kidney tissue levels were measured after a 14-d
`course of immunosuppression with CsA alone at doses of 2.5, 5.0, 7.5,
`10.0, 15.0, or 20.0 mg/kg per d as shown sequentially on the z-axis;
`RAPA alone at doses of 0.4, 0.8, 1.2, 1.6, 3.2, or 6.4 mg/kg per d as
`shown sequentially on the x-axis; or CsA/RAPA combination at doses
`of 2.5/0.4, 5.0/0.8, 7.5/1.2, 10.0/1.6, 15.0/3.2, or 20.0/6.4 mg/kg per d
`(p). In addition, some rats were treated with a constant dose of 5 or
`10 mg/kg per d CsA with ascending RAPA doses, namely 0.4, 0.8,
`1.2, 1.6, and 3.2 mg/kg per d. Kidney drug concentrations (ng/g wet
`tissue) either for treatment with each drug alone or in combination are
`plotted versus creatinine levels.
`
`Figure 4. Impact of RAPA on CsA-induced nephrotoxicity. The arrow
`indicates the impact of addition of RAPA to shift the SCr values. (A)
`Increase in SCr as a function of CsA dose in the absence (h) or
`presence (F) of 0.8 mg/kg RAPA. (B) Increase in SCr as a function
`of CsA kidney concentrations. With use of regression lines to assess
`statistical differences between RAPA Yes/No, the P values are 0.0001
`for A and 0.002 for B.
`
`the slope coefficient. Logarithmic conversion of the median effect
`equation linearizes the following relationship:
`
`log~ fa/fu! 5 m log~D! 2 m log~Dm!
`
`(2)
`
`When the data display a Pearson’s correlation coefficient (r) .0.75,
`the equation is believed to predict the drug dose D (or concentration,
`C) necessary to achieve any arbitrary effect level. The nature of the
`interaction between RAPA and CsA was assessed by a combination
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`Table 1. Median effect analysis of CsA/RAPA-induced nephrotoxicitya
`
`Drug Measurement
`
`Dose
`CsA
`RAPA
`CsA/RAPA
`Blood concentration
`CsA
`RAPA
`CsA/RAPA
`Kidney concentration
`CsA
`RAPA
`CsA/RAPA
`
`Serum Creatinine Levels
`
`Dm/Cm
`
`r
`
`CI Rangeb
`
`Dm/Cm
`
`GFR
`
`r
`
`CI Rangeb
`
`19.85 mg/kg
`109.18 mg/kg
`—
`
`5921.0 ng/ml
`45.16 ng/ml
`—
`
`3757.0 ng/g
`85.50 ng/g
`—
`
`0.92
`0.76
`—
`
`0.91
`0.65
`—
`
`0.90
`0.77
`
`—
`—
`0.5–0.14 (S)
`
`9.58 mg/kg
`7.43 mg/kg
`—
`
`—
`—
`0.95–0.3 (AD/S)
`
`3913.3 ng/ml
`53.9 ng/ml
`—
`
`—
`—
`9.4–4.4 (AN)
`
`2469.7 ng/ml
`1001.8 ng/ml
`—
`
`0.90
`0.97
`—
`
`0.91
`0.92
`—
`
`0.85
`0.98
`—
`
`—
`—
`0.05–0.09 (SS)
`
`—
`—
`0.98–0.62 (AD)
`
`—
`—
`8.3–3.6 (AN)
`
`a The median effect equation was used to assess drug interactions, as described in Materials and Methods section. Dm, the dose (mg/kg
`per d) that produces 50% of the maximal effect; Cm, (ng/ml) the concentration necessary to produce the effect; CsA, cyclosporine; RAPA,
`rapamycin.
`b The interaction between the drugs was assessed by the combination index (CI) analysis of the doses necessary to achieve x%
`inhibition: CIx 5 D1 combined/Dx1 alone 1 D1 combined/Dx2 alone 1 (D1 combined) (D2 combined)/[Dx1 alone][Dx2 alone]; CI ,1
`reflect synergistic interaction; CI 5 1 demonstrates additive interaction; and CI .1 shows antagonistic interaction. Results are presented as
`mean 6 SD with minimal and maximal range, as calculated using Microsoft Excel. Interactions were qualified as strongly synergistic (SS)
`with CI ,0.1; synergistic (S) with CI 0.11 to 0.7; antagonistic (AN) with CI .1.2; or additive (AD) with CI 5 0.71 to 1.19.
`
`ml/min), or 20.0 mg/kg per d (0.75 6 0.24 ml/min) displayed
`significantly reduced GFR values. Although RAPA alone
`caused only modest effects, the GFR values were significantly
`lower among CsA/RAPA combination groups. Thus, RAPA
`potentiates the dose-dependent nephrotoxicity of CsA (CsA
`versus CsA/RAPA, P 5 0.025 and RAPA versus CsA/RAPA,
`P 5 0.0045).
`
`Blood and Tissue CsA and RAPA Concentrations
`Twenty-four h after the last dose of a 14-d course of therapy,
`CsA and RAPA concentrations were measured in whole blood
`
`as well as in liver and kidney tissues. Whole-blood trough
`concentrations increased proportionate to the CsA dose. At
`each dose level, concomitant administration of RAPA pro-
`duced a significant pharmacokinetic interaction to increase
`further the CsA concentrations by approximately twofold
`above those found in hosts that were treated with CsA alone
`(Figure 2A; all, P , 0.006).
`Similarly, the whole-blood concentrations of RAPA, which
`displayed dose-dependent increases, were exaggerated when
`the drug was administered concomitantly with CsA (Figure
`2B). However, kidney and liver tissue drug concentrations
`
`Table 2. Median effect analysis of CsA/RAPA effect on blood parameters
`
`Drug
`Measurement
`
`Serum Sodium
`
`Serum Phosphate
`
`LDLa Cholesterol
`
`Dm/Cm
`
`r
`
`CI Range
`
`Dm/Cm
`
`r
`
`CI Range
`
`Dm/Cm
`
`r
`
`CI Range
`
`49.4 mg/kg 0.93
`11.8 mg/kg 0.92
`
`Dose
`CsA
`RAPA
`CsA/RAPA
`Blood concentration
`CsA
`8560.0 ng/ml 0.88
`RAPA
`69.7 ng/ml 0.98
`CsA/RAPA
`Kidney concentration
`CsA
`56.98 ng/g 0.86
`RAPA
`1332.0 ng/g
`0.88
`CsA/RAPA
`
`a LDL, low-density lipoprotein.
`
`—
`—
`2.2–0.6 (AD)
`
`—
`—
`2.4–0.6 (AD)
`
`39.4 mg/kg 0.97
`1655.0 mg/kg 0.98
`
`8710.0 ng/ml 0.98
`12750.0 ng/ml 0.90
`
`—
`—
`1–0.003 (S)
`
`—
`—
`1–0.03 (S)
`
`102.0 mg/kg 0.96
`11.0 mg/kg 0.89
`
`14236.0 ng/ml 0.97
`59.9 ng/ml 0.91
`
`—
`—
`2–1 (AD)
`
`5142.0 ng/g
`10983.0 ng/g
`
`0.99
`0.99
`
`—
`—
`1.5–0.04 (AD/S)
`
`9630.0 ng/g
`1430.0 ng/g
`
`0.94
`0.94
`
`—
`—
`0.95–0.007 (S)
`
`—
`—
`1.8–0.05 (AD/S)
`
`—
`—
`1.9–0.07 (AD/S)
`
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`J Am Soc Nephrol 12: 1059 –1071, 2001
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`Sirolimus/Cyclosporine Interactions
`
`1065
`
`effect model for both SCr and GFR values (CsA r 5 0.92/
`RAPA r 5 0.76 and CsA r 5 0.90/RAPA r 5 0.97, respec-
`tively). The median effect analysis also showed good correla-
`tions between kidney tissue concentrations and SCr levels or
`GFR (CsA r 5 0.90/RAPA r 5 0.77 or CsA r 5 0.85/RAPA
`r 5 0.98, respectively). Although the CI values that were
`calculated on the basis of drug doses suggested a synergistic
`interaction between CsA and RAPA, those that were based on
`whole-blood concentrations, which were markedly increased
`by the drug combination, showed less synergism and those that
`were based on kidney tissue concentrations demonstrated phar-
`macodynamic antagonism. These findings suggest
`that
`the
`alteration in renal function associated with CsA/RAPA dual-
`drug
`therapy
`primarily
`represents
`a
`pharmacokinetic
`interaction.
`
`Renal Analytes
`Serum magnesium, uric acid, and phosphate concentrations
`were measured as potential indicators of altered tubular func-
`tion. In a dose-dependent manner, CsA but not RAPA reduced
`serum magnesium concentrations (P , 0.00001; data not
`shown). Interestingly, virtually all animals in the CsA/RAPA
`combination groups displayed magnesium concentrations sim-
`ilar to those of hosts that were treated with RAPA alone. Uric
`acid concentrations were increased by CsA monotherapy, es-
`pecially at 15 and 20 mg/kg per d doses, although not signif-
`icantly by RAPA alone. However, all animals in the CsA/
`RAPA combination groups displayed a dose-dependent
`increase in serum uric acid concentrations even greater than
`that for animals that were given CsA alone (P 5 0.02 to 0.002;
`data not shown). A similar picture was observed for phosphate
`levels: only hosts that were treated with high doses of CsA
`alone displayed increased phosphate levels, whereas animals
`that received combination therapy displayed a dose-dependent
`increase in this analyte. When corrected for renal tissue con-
`centrations, the effect of the drug combinations was found to
`be predominantly additive; only serum phosphate concentra-
`tions showed synergistic interactions (Table 2). Combination
`therapy had no significant impact on serum sodium (Table 2)
`or serum potassium concentrations (Figure 5). Furthermore, the
`dose-dependent increase in blood glucose levels produced by
`CsA but not by RAPA monotherapy was only slightly greater
`among the combination treatment groups. Overall, pharmaco-
`kinetic interactions of RAPA to increase renal tissue CsA
`concentrations readily accounted for the observed changes in
`renal analytes.
`
`Renal Histology
`Kidneys from rats that were given a low-salt diet alone or
`treated with the smallest drug doses showed scant evidence of
`pathologic abnormalities (Figure 6). Ascending doses from 0.8
`to 6.4 mg/kg per d RAPA alone or 5.0 to 20.0 mg/kg per d CsA
`alone were associated with progressive tubular and glomerular
`abnormalities, as well as arterial wall thickening. Renal sec-
`tions from hosts that were treated with the 10 mg/kg per d CsA
`dose showed increased glomerular cellularity accompanied by
`modest (,25%) thickening of vessels, focal tubular dilation,
`
`Figure 5. Lack of effect of RAPA on CsA-induced hyperkalemia.
`Serum potassium levels were measured after a 14-d course of immu-
`nosuppression with no treatment (s); CsA alone at doses of 2.5, 5.0,
`7.5, 10.0, 15.0, or 20.0 mg/kg per d as shown sequentially on the
`z-axis (m); RAPA alone at doses of 0.4, 0.8, 1.2, 1.6, 3.2, or 6.4
`mg/kg per d as shown sequentially on the x-axis (h); or CsA/RAPA
`combination at doses of 5.0/0.8, 10.0/1.6, or 15.0/3.2 mg/kg per d (p).
`
`showed even greater degrees of pharmacokinetic interactions
`among all combination groups (Figure 2, C through F; all, P ,
`0.001). These findings document that individual drug doses are
`a poor index of CsA and RAPA exposure in whole blood as
`well as in tissues when the drugs are used in combination
`therapy.
`
`Median Effect Analysis of Renal Function
`Figure 3 shows a concentration-dependent increase in SCr
`values among hosts that were treated with CsA/RAPA combi-
`nations. Because the effect showed the best correlation with
`CsA concentrations in kidney tissue samples, it seems likely
`that the reduction in renal function was due to the impact of
`RAPA to increase CsA levels. Indeed, the pharmacokinetic
`interaction produced a 12-fold increase in renal CsA concen-
`trations, compared with the only 4-fold increase in CsA doses.
`Figure 4A shows an increase in SCr upon addition of a fixed
`0.8 mg/kg per d dose of RAPA to ascending doses of CsA (P
`5 0.0001). However, when the data were expressed as kidney
`tissue CsA concentrations, the increase actually was less than
`would have been predicted. Thus, RAPA seemed to exert a
`protective effect (Figure 4B; P 5 0.002).
`A median effect analysis was performed to examine rigor-
`ously dose- and concentration-dependent effects of CsA and/or
`RAPA on renal function, expressed as SCr levels or GFR
`(Table 1). The Pearson’s correlation coefficient values docu-
`mented a good relation between drug doses and the median
`
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`Page 7 of 13
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`1066
`
`Journal of the American Society of Nephrology
`
`J Am Soc Nephrol 12: 1059 –1071, 2001
`
`Figure 6. Histopathologic effects of administration of CsA or RAPA alone or in combination. Rats were treated with 2.5, 5.0, 7.5, 10.0, 15.0,
`or 20.0 CsA alone; 0.4, 1.6, or 6.4 RAPA alone; or 2.5/0.4, 5.0/0.8, 7.5/1.2, 10.0/1.6, 15.0/3.2 or 20.0/6.4 CsA/RAPA combination. The
`photomicrographs (2003) show representative sections from rats that were treated with 2.5, 10.0, or 20.0 mg/kg per d CsA; 0.4, 1.6, or 6.4
`mg/kg per d RAPA alone; and 2.5/0.4, 10.0/1.6, or 20.0/6.4 mg/kg per d CsA/RAPA combinations. The table shows the overall scores for
`kidneys in different therapeutic groups, as graded on the basis of tubular and glomerular (number on the left side) and vascular changes (number
`on the right side). Tubular and glomerular changes were graded as follows: 0, no changes; 11, ,5%; 21, 5 to 25%; 31, 26 to 50%; and 41,
`.50% involvement. A vascular scale included the following: 0, none; 11, minimal; 21, mild; 31, moderate; and 41, severe. Although the
`scores generally were concordant, when they were disparate, a mean value was chosen as the histopathologic grade. See Materials and Methods
`section.
`
`and significantly increased interstitial fibrosis. The next higher
`CsA dose (15 mg/kg per d) caused significant arteriolar thick-
`ening, as well as focal necrotic changes, tubular dilation, and
`mild inflammatory cell infiltrates. The highest CsA dose (20
`mg/kg per d) was associated with arteriolar vacuolization and
`hyalinosis, glomerular hypercellularity, tubular dilation, and
`diffuse interstitial inflammation and fibrosis. In contrast, only
`
`moderate changes in renal histopathology were present even at
`the highest RAPA dose (6.4 mg/kg per d).
`Although kidneys from rats that were treated with the com-
`bination of 2.5 mg/kg per d CsA and 0.4 mg/kg per d RAPA
`did not show tubular or glomerular changes, they did display
`focal vasculitis, including cellular infiltration into the walls of
`medium- to large-sized vessels. Increasing doses of the CsA/
`
`NOVARTIS EXHIBIT 2056
`Par v Nov