Fatty liver disease is associated with underlying cardiovascular disease in HIV-infected persons(*).

BACKGROUND: Cardiovascular disease is an increasing concern among HIV-infected persons and their providers. We determined if fatty liver disease is a marker for underlying coronary atherosclerosis among HIV-infected persons. METHODS: We performed a cross-sectional study in HIV-infected adults to evaluate the prevalence of and factors, including fatty liver disease, associated with subclinical coronary atherosclerosis. All participants underwent computed tomography for determination of coronary artery calcium (CAC; positive defined as a score >0) and fatty liver disease (defined as a liver-to-spleen ratio <1.0). Factors associated with CAC were determined using multivariate logistic regression models. RESULTS: We included in the study 223 HIV-infected adults with a median age of 43 years [interquartile range (IQR) 36-50 years]; 96% were male and 49% were Caucasian. The median CD4 count was 586 cells/µL and 83% were receiving antiretroviral medications. Seventy-five (34%) had a positive CAC score and 29 (13%) subjects had fatty liver disease. Among those with CAC scores of 0, 1-100 and >100, the percentage with concurrent fatty liver disease was 8, 18 and 41%, respectively (P=0.001). In the multivariate model, CAC was associated with increasing age [odds ratio (OR) 4.3 per 10 years; P<0.01], hypertension (OR 2.6; P<0.01) and fatty liver disease (OR 3.8; P<0.01). CONCLUSIONS: Coronary atherosclerosis as detected using CAC is prevalent among young HIV-infected persons. The detection of fatty liver disease among HIV-infected adults should prompt consideration of assessment for underlying cardiovascular disease and risk factor reduction.

Population pharmacokinetics and bioavailability of motexafin gadolinium (Xcytrin) in CD1 mice following intravenous and intraperitoneal injection.

Motexafin gadolinium (Xcytrin) is an expanded porphyrin macrocyclic compound under development for the treatment of several types of cancer. Currently clinical trials and non-clinical pharmacology and toxicology studies are ongoing. The goals of this open label, four arm, non-crossover bioavailability study were to explore motexafin gadolinium pharmacokinetics, determine the i.p. bioavailability, and define a pharmacokinetic model suitable for descriptive and predictive use. Mice received one or seven daily i.v. or i.p. injections (40 mg/kg) then blood samples were collected and analyzed. Plasma concentration data were modelled using population pharmacokinetic methods and a two compartment model was the most appropriate model. The stability and predictive performance of the model were evaluated using bootstrap procedures. The accuracy of the predicted concentrations was 8.3%. Motexafin gadolinium was rapidly cleared from the plasma and although T(1/2beta) was 12.9 h there was no accumulation following seven doses. The i.p. bioavailability was 87.4% and higher plasma concentrations were sustainable for a longer period with i.p. dosing. V(c) was larger than the blood volume and the tissue compartment volume was 38% of V(c), suggesting motexafin gadolinium was not widely distributed into less well perfused tissues. The pharmacokinetic profile in this study was similar to that in oncology patients administered multiple doses of motexafin gadolinium. The unbiased model yields reliable parameter estimates and insight into the pharmacokinetics of motexafin gadolinium in mice, is suitable for both descriptive and predictive purposes, and is a valuable tool in the planning, analysis, and interpretation of pharmacology and toxicology studies in mice.

Novel 3-phenylprop-2-ynylamines as inhibitors of mammalian squalene epoxidase.

The synthesis of a novel series of 3-phenylprop-2-ynylamines as selective mammalian squalene epoxidase inhibitors is described. Structure activity relationship studies led to the discovery of compound 19, 1-[3-(3,5-dichlorophenyl)-prop-2-ynyl]-3- methylpiperidine hydrochloride with an IC50 of 2.8 +/- 0.6 microM against rat liver squalene epoxidase. Against 23 strains of fungal squalene epoxidase compound 19 was found to be inactive.

Neuromuscular blocking activity and therapeutic potential of mixed-tetrahydroisoquinolinium halofumarates and halosuccinates in rhesus monkeys.

Structure-activity relationships in rhesus monkeys for a novel mixed-onium class of ultra-short-acting nondepolarizing tetrahydroisoquinolinium neuromuscular blockers (NMBs) are described. Bis-onium chlorofumarate 20a with (1R,2S)-benzyltetrahydroisoquinolinium groups was a potent lead compound (ED(95) = 0.079 mg/kg) with an ultra-short duration of NMB effect (7.1 min) and a selectivity index (SI: defined as a ratio of the cardiovascular threshold dose to the ED(95)) similar to that of mivacurium (3). The mean threshold dose for cardiovascular effects with 20a was ca. 20 times its ED(95) value (SI = 20). A novel mixed-onium analogue of 20a was prepared by replacing the benzyltetrahydroisoquinolinium group distal to the fumarate chlorine atom with a (1S,2R)-phenyltetrahydroisoquinolinium moiety. The resulting mixed-onium chlorofumarate 24a displayed good NMB potency (ED(95) = 0.063 mg/kg), ultra-short duration of action (5.6 min) and an improved selectivity index (SI = 57). Several other mixed-onium derivatives containing octanedioate (25a; ED(95) = 0.103 mg/kg), difluorosuccinate (27c; ED(95) = 0.056 mg/kg), and fluorofumarate (28a; ED(95) = 0.137 mg/kg) linkers were also potent, ultra-short-acting NMBs with good to excellent selectivity index values (SI = 37-96). Octanedioate 25a was longer acting at higher doses compared to difluorosuccinate 27c and chlorofumarate 24a. Durations of NMB effect following a 0.4 mg/kg bolus dose (100% block) of 25a, 27c, and 24a were 16.9, 13.0, and 10.0 min, respectively. Recovery time for mixed-onium chlorofumarate 24a following a 1 h continuous infusion at 10-20 microg/kg/min (95-100% block) was ca. 5 min which is similar to that observed following a 0.2 mg/kg bolus dose of this compound and indicates a lack of cummulative effects. Preliminary studies with chlorofumarate 24a in whole human blood revealed that mixed-onium thiazolidine 29 was the major metabolite and that plasma cholinesterases do not play the primary role in duration of NMB effect. The NMB properties of 24a in rhesus monkeys led to its clinical evaluation as a possible alternative to succinylcholine.

3-(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-alkyl-N-arylbenzamides: potent, non-peptidic agonists of both the micro and delta opioid receptors.

Opioid analgesics with both micro and delta opioid receptor activation represent a new approach to the treatment of severe pain with an improved safety profile. Compounds with this profile may exhibit strong analgesic properties due to micro agonism, with a reduced side effect profile resulting from delta agonism. Replacing the p-diethylamide of the known potent delta opioid receptor selective agonist BW373U86 with a m-diethylamide resulted in a compound with agonist activity at both the micro and delta opioid receptors. Modifying the amide to an N-methyl-N-phenylamide increased agonist potency at both receptors. A series of 3-(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-alkyl-N-arylbenzamides have been made to explore the structure-activity relationship (SAR) around the N-methyl-N-phenylamide. Several potent agonists of both the micro and delta opioid receptors have been identified, including (+)-3-((alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N-(4-fluorophenyl)-N-methylbenzamide (23), which has EC50 values of 0.67 and 1.1 nM at the micro (guinea pig ileum assay) and delta (mouse vas deferens assay) opioid receptors, respectively.

Arms races and the evolution of big fierce societies.

The causes of biological gigantism have received much attention, but only for individual organisms. What selection pressures might favour the evolution of gigantic societies? Here we consider the largest single-queen insect societies, those of the Old World army ant Dorylus, single colonies of which can have 20 million workers. We propose that colony gigantism in Dorylus arises as a result of an arms race and test this prediction by developing a size-structured mathematical model. We use this model for exploring and potentially explaining differences in colony size, colony aggression and colony propagation strategies in populations of New World army ants Eciton and Old World army ants Dorylus. The model shows that, by determining evolutionarily stable strategies (ESSs), differences in the trophic levels at which these army ants live feed forwards into differences in their densities and collision rates and, hence, into different strategies of growth, aggression and propagation. The model predicts large colony size and the occurrence of battles and a colony-propagation strategy involving highly asymmetrical divisions in Dorylus and that Eciton colonies should be smaller, non-combative and exhibit equitable binary fission. These ESSs are in excellent agreement with field observations and demonstrate that gargantuan societies can arise through arms races.

Comparative tacrolimus pharmacokinetics: normal versus mildly hepatically impaired subjects.

Tacrolimus (FK506, Prograf), marketed for the prophylaxis of organ rejection following allogenic liver or kidney transplantation, is virtually completely metabolized. The major metabolic pathways are P450 3A4-mediated hydroxylation and demethylation. Since P450 hepatic drug-metabolizing enzymes may be impaired in hepatic dysfunction, a study was conducted to characterize oral and intravenous tacrolimus pharmacokinetics in 6 patients with mild hepatic dysfunction and compared with parameters to those from normal subjects obtained in a separate study. Patients received two treatments: a single 0.020 mg/kg ideal body weight (IBW) i.v. dose infused over 4 hours and approximately 0.12 mg/kg IBW orally; normal subjects were dosed at 0.02 mg/kg 4-hour i.v. and 5 mg (0.065 mg/kg) p.o. Mean blood pharmacokinetic parameters with mild hepatic dysfunction were as follows: clearance = 0.035 L/h/kg, terminal exponential volume of distribution = 2.59 L/kg, terminal exponential half-life = 60.6 hours (i.v.), p.o. maximum blood concentration = 48.2 ng/mL, time of p.o. maximum blood concentration = 1.5 hours, and absolute bioavailability = 22.3%. The respective parameters in normal subjects were as follows: 0.040 L/h/kg, 1.91 L/kg, 34.2 hours (i.v.), 29.7 ng/mL, 1.6 hours, and 17.8%. Inasmuch as clearance and bioavailability were not substantially different from that in normal subjects, patients with mild hepatic impairment may initially be treated with conventional tacrolimus doses, with subsequent dosage adjustments based on response, toxicity, and therapeutic drug monitoring.

A contract research organization's response to the new FDA guidances for bioequivalence/bioavailability studies for orally administered drug products.

The new FDA Guidance for Industry BA and BE Studies for Orally Administered Drug Products--General Considerations and Average, Population, and Individual Approaches to Establishing Bioequivalence imply significant changes in the areas of enrollment, cost, ethics, time, entry, validation applications (EVAs), and statistical and pharmacokinetic methods. The changes from three-period to two-period design for food effect studies, the elimination of most steady state studies, and the analyses of only the active moiety or ingredient are welcome. However, if the current guidances are adopted, additional time will be needed for participants, and more participants will be needed, resulting in higher costs to drug developers. The PK parameters needed to assess BE and the need for replicate designs for drugs with long t1/2 are still unclear. Finally, the advantages of the aggregate property of the FDA metric versus the disaggregate criteria are challenged, and four bioequivalence criteria are proposed.

Coadministration of tacrolimus and mycophenolate mofetil in stable kidney transplant patients: pharmacokinetics and tolerability.

The tolerance and pharmacokinetics (PK) of tacrolimus (T) by the addition of mycophenolate mofetil (MMF) in stable kidney transplant patients (6/group) on long-term tacrolimus-based therapy were investigated. Patients received combination T and MMF therapy at three MMF doses: 1, 1.5, and 2 g/day administered twice daily. A 12-hour blood PK profile for T was obtained prior to MMF dosing; concomitant 12-hour profiles for T, mycophenolic acid (MPA), and mycophenolic acid glucuronide (MPAG) were obtained after 2 weeks of administration. Tolerance was monitored through 3 months. The intra- and intergroup PK of T were variable. The mean AUC0-12 of T for each group was increased after 2 weeks of concomitant MMF administration, but the increase was not statistically significant. Both drugs were well tolerated. Gastrointestinal events were of interest as such have been attributed to both T and MMF. Events reported were diarrhea, nausea, dyspepsia, and vomiting. Other common adverse events were headache, hypomagnesemia, and tremors. Most were mild, although a few were considered to be moderate. There was no apparent relationship between the incidence of any adverse event and MMF treatment group. In the present study, the coadministration of T and MMF did not significantly alter T pharmacokinetics.

Safety, toxicokinetics and tissue distribution of long-term intravenous liposomal amphotericin B (AmBisome): a 91-day study in rats.

PURPOSE: Amphotericin B in small, unilamellar liposomes (AmBisome) is safer and produces higher plasma concentrations than other formulations. Because liposomes may increase and prolong tissue exposures, the potential for drug accumulation or delayed toxicity after chronic AmBisome was investigated. METHODS: Rats (174/sex) received intravenous AmBisome (1, 4, or 12 mg/kg), dextrose, or empty liposomes for 91 days with a 30-day recovery. Safety (including clinical and microscopic pathology) and toxicokinetics in plasma and tissues were evaluated. RESULTS: Chemical and histopathologic changes demonstrated that the kidneys and liver were the target organs for chronic AmBisome toxicity. Nephrotoxicity was moderate (urean nitrogen [BUN] < or = 51 mg/dl; creatinine unchanged). Liposome-related changes (vacuolated macrophages and hypercholesterolemia) were also observed. Although plasma and tissue accumulation was nonlinear and progressive (clearance and volume decreased, half-life increased with dose and time), most toxic changes occurred early, stabilized by the end of dosing, and reversed during recovery. There were no delayed toxicities. Concentrations in liver and spleen greatly exceeded those in plasma: kidney and lung concentrations were similar to those in plasma. Elimination half-lives were 1-4 weeks in all tissues. CONCLUSIONS: Despite nonlinear accumulation, AmBisome revealed predictable hepatic and renal toxicities after 91 days, with no new or delayed effects after prolonged treatment at high doses that resulted in plasma levels >200 microg/ml and tissue levels >3000 microg/g.

Safety and toxicokinetics of intravenous liposomal amphotericin B (AmBisome) in beagle dogs.

PURPOSE: Amphotericin B (AmB) in small, unilamellar liposomes (AmBisome) has an improved therapeutic index, and altered pharmacokinetics. The repeat-dose safety and toxicokinetic profiles of AmBisome were studied at clinically relevant doses. METHODS: Beagle dogs (5/sex/group) received intravenous AmBisome (0.25, 1, 4, 8, and 16 mg/kg/day), empty liposomes or vehicle for 30 days. AmB was determined in plasma on days 1, 14, and 30, and in tissues on day 31. Safety parameters included body weight, clinical chemistry, hematology and microscopic pathology. RESULTS: Seventeen of twenty animals receiving 8 and 16 mg/kg were sacrificed early due to weight loss caused by reduced food intake. Dose-dependent renal tubular nephrosis, and other effects characteristic of conventional AmB occurred at 1 mg/kg/day or higher. Although empty liposomes and AmBisome increased plasma cholesterol, no toxicities unique to AmBisome were revealed. Plasma ultrafiltrates contained no AmB. AmBisome achieved plasma levels 100-fold higher than other AmB formulations. AmBisome kinetics were non-linear, with clearance and distribution volumes decreasing with increasing dose. This, and nonlinear tissue uptake, suggest AmBisome disposition was saturable. CONCLUSIONS: AmBisome has the same toxic effects as conventional AmB, but they appear at much higher plasma exposures. AmBisome's non-linear pharmacokinetics are not associated with increased risk, as toxicity increases linearly with dosage. Dogs tolerated AmBisome with minimal to moderate changes in renal function at doses (4 mg/kg/day) producing peak plasma concentrations of 18-94 microg/mL.

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 2. Structure-activity relationship and optimization of the phenyl alkyl ether moiety.

We previously reported the identification of (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (2) (PPARgamma pKi = 8.94, PPARgamma pEC50 = 9.47) as a potent and selective PPARgamma agonist. We now report the expanded structure-activity relationship around the phenyl alkyl ether moiety by pursuing both a classical medicinal chemistry approach and a solid-phase chemistry approach for analogue synthesis. The solution-phase strategy focused on evaluating the effects of oxazole and phenyl ring replacements of the 2-(5-methyl-2-phenyloxazol-4-yl)ethyl side chain of 2 with several replacements providing potent and selective PPARgamma agonists with improved aqueous solubility. Specifically, replacement of the phenyl ring of the phenyloxazole moiety with a 4-pyridyl group to give 2(S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-pyridin-4-yloxazol+ ++- 4-yl)ethoxy]phenyl¿propionic acid (16) (PPARgamma pKi = 8.85, PPARgamma pEC50 = 8.74) or a 4-methylpiperazine to give 2(S)-((2-benzoylphenyl)amino)-3-(4-¿2-[5-methyl-2-(4-methylpiperazin+ ++- 1-yl)thiazol-4-yl]ethoxy¿phenyl)propionic acid (24) (PPARgamma pKi = 8.66, PPARgamma pEC50 = 8.89) provided two potent and selective PPARgamma agonists with increased solubility in pH 7.4 phosphate buffer and simulated gastric fluid as compared to 2. The second strategy took advantage of the speed and ease of parallel solid-phase analogue synthesis to generate a more diverse set of phenyl alkyl ethers which led to the identification of a number of novel, high-affinity PPARgamma ligands (PPARgamma pKi's 6.98-8.03). The combined structure-activity data derived from the two strategies provide valuable insight on the requirements for PPARgamma binding, functional activity, selectivity, and aqueous solubility.

Safety, tolerance, and pharmacokinetics of a small unilamellar liposomal formulation of amphotericin B (AmBisome) in neutropenic patients.

The safety, tolerance, and pharmacokinetics of a small unilamellar liposomal formulation of amphotericin B (AmBisome) administered for empirical antifungal therapy were evaluated for 36 persistently febrile neutropenic adults receiving cancer chemotherapy and bone marrow transplantation. The protocol was an open-label, sequential-dose-escalation, multidose pharmacokinetic study which enrolled a total of 8 to 12 patients in each of the four dosage cohorts. Each cohort received daily doses of either 1.0, 2.5, 5.0, or 7.5 mg of amphotericin B in the form of AmBisome/kg of body weight. The study population consisted of patients between the ages of 13 and 80 years with neutropenia (absolute neutrophil count, <500/mm3) who were eligible to receive empirical antifungal therapy. Patients were monitored for safety and tolerance by frequent laboratory examinations and the monitoring of infusion-related reactions. Efficacy was assessed by monitoring for the development of invasive fungal infection. The pharmacokinetic parameters of AmBisome were measured as those of amphotericin B by high-performance liquid chromatography. Noncompartmental methods were used to calculate pharmacokinetic parameters. AmBisome administered as a 1-h infusion in this population was well tolerated and was seldom associated with infusion-related toxicity. Infusion-related side effects occurred in 15 (5%) of all 331 infusions, and only two patients (5%) required premedication. Serum creatinine, potassium, and magnesium levels were not significantly changed from baseline in any of the dosage cohorts, and there was no net increase in serum transaminase levels. AmBisome followed a nonlinear dosage relationship that was consistent with reticuloendothelial uptake and redistribution. There were no breakthrough fungal infections during empirical therapy with AmBisome. AmBisome administered to febrile neutropenic patients in this study was well tolerated, was seldom associated with infusion-related toxicity, was characterized by nonlinear saturation kinetics, and was effective in preventing breakthrough fungal infections.

AmBisome (liposomal amphotericin B): a comparative review.

AmBisome (NeXstarPharmaceuticals, San Dimas, CA) is a unilamellar liposomal formulation of amphotericin B that was recently approved for use as empirical treatment for presumed fungal infections in febrile neutropenic patients and for aspergillosis, candidiasis, and cryptococcosis infections refractory to amphotericin B. It is a small closed microscopic sphere (<100 nm in diameter) with an inner aqueous core (i.e., a true liposome). AmBisome remains as an intact sphere in vitro and for prolonged periods of time in vivo during the processes of systemic transport and pharmacologic action. As a consequence of its size and in vivo stability, AmBisome has physiochemical properties and a pharmacokinetic profile that are considerably different from those of currently available lipid-complexed amphotericin B formulations, with greatly increased area under the plasma concentration-time curve and much lower clearance at equivalent doses. AmBisome liposomes can be seen to accumulate at sites of fungal infection. Disruption of AmBisome liposomes occurs after attachment to the fungal cell wall and results in amphotericin B binding to fungal cell membrane ergosterol with subsequent cell lysis. AmBisome has been shown to penetrate the cell wall of both extracellular and intracellular forms of susceptible fungi.

Toxicological profile and pharmacokinetics of a unilamellar liposomal vesicle formulation of amphotericin B in rats.

AmBisome (ABLP) is a unilamellar liposomal preparation of amphotericin B that has demonstrated an improved safety profile compared to conventional amphotericin B. Single- and multiple-dose pharmacokinetics were determined by using noncompartmental methods for rats administered ABLP at 1, 3, 9, and 20 mg/kg/day. The toxicological profile was evaluated following 30 consecutive days of intravenous ABLP administration. Mean plasma amphotericin B concentrations reached 500 and 380 microg/ml (males and females, respectively) following 30 days of ABLP administration at 20 mg/kg. The overall apparent half-life was 11.2+/-4.5 h (males) or 8.7+/-2.2 h (females), and the overall clearance (CL) was 9.4+/-5.5 ml/h/kg (males) or 10.2+/-4.1 ml/h/kg (females). ABLP appears to undergo saturable disposition, resulting in a non-dose-proportional amphotericin B area under the curve and a lower CL at higher doses. Histopathological examination revealed dose-related transitional-cell hyperplasia in the transitional epithelium of the urinary tract (kidney, ureters, and urinary bladder) and moderate hepatocellular necrosis at the 20 mg/kg/day dose. Administration of ABLP in doses of up to 20 mg/kg/day resulted in 100-fold higher plasma amphotericin B concentrations, with significantly lower toxicity than that reported with conventional amphotericin B therapy.

Tacrolimus pharmacokinetics in BMT patients.

The pharmacokinetics of tacrolimus following its administration as monotherapy or in combination with corticosteroids or methotrexate to 31 BMT patients are presented. All patients received i.v. tacrolimus initially and were subsequently switched to p.o. dosing. Patients received methotrexate by i.v. bolus on post-transplantation days 1, 3, 6 and 11. Patients were started on i.v. corticosteroids beginning on post-transplantation day 7. The noncompartmental pharmacokinetics of tacrolimus based on whole blood concentrations were determined following the i.v. and p.o. doses and were not different at steady-state compared to a single dose. The mean terminal elimination half-life of tacrolimus was 18.2 h following i.v. administration; the total body clearance was 71 ml/h/kg, the volume of distribution was 1.67 1/kg. Co-administration of methylprednisolone or methotrexate did not significantly alter tacrolimus pharmacokinetics. The p.o. bioavailability was 31-49%. Trough blood concentrations (Cmin) at 0 h (pre-dose) and 12 h (post-dose) correlated well to AUC(0-12)indicating that, as in solid organ transplantation, Cmin was a good index of drug exposure. Correlation at 0 h (r = 0.92) and at 12 h (r = 0.93) indicate that either time point can be used for therapeutic drug monitoring in patient management.

The characterization of potent novel warfarin analogs.

Racemic sodium warfarin, Coumadin, is widely used in the prevention of thromboembolic disease. The present study was undertaken to characterize three novel classes of warfarin analogs, and to compare them with the warfarin enantiomers. All three classes of compounds inhibit vitamin K epoxide reductase, the enzyme inhibited by racemic warfarin. The alcohol and the ester analogs have reduced protein binding compared with R-(+)-warfarin. The ester and the fluoro-derivatives have similar in vivo anticoagulant activity in the rat to that of S-(-)-warfarin. Thus, it is possible to synthesize novel warfarin analogs that differ from racemic warfarin or its enantiomers in certain selected properties.

Lack of interaction between tacrolimus (FK506) and methotrexate in bone marrow transplant recipients.

Tacrolimus (FK506) is a macrolide lactone effective in the control of graft-versus-host disease (GVHD). An interaction between high-dose methotrexate and a macrolide antibiotic (pristinamycin) leading to prolonged methotrexate exposure has been described. Because a randomized prospective trial comparing tacrolimus with cyclosporine (both in combination with methotrexate) following allogeneic BMT showed the tacrolimus plus methotrexate regimen to be more effective in prevention of GVHD, we assessed methotrexate pharmacokinetics in a subgroup of the participants of this trial to evaluate the possibility that an interaction of FK506 and methotrexate was the explanation for the clinical findings. Mean and median methotrexate levels at various time-points after the day 1 and 6 methotrexate doses were comparable in the tacrolimus and cyclosporine cohorts and were elevated in only three of 70 study patients. Area under the curve (AUC) concentrations were also similar after the day 1 and 6 methotrexate doses. Thus, no significant interaction between tacrolimus and methotrexate is apparent and the differences in efficacy between tacrolimus and cyclosporine are unlikely to be attributable to pharmacologic interactions with methotrexate.