Florfenicol residues in three species of fish after 10-day oral dosing in feed.

Nile tilapia Oreochromis niloticus, walleye Sander vitreus, and hybrid striped bass (female white bass Morone chrysops x male striped bass M. saxatilis) were medicated with florfenicol (AQUAFLOR type A medicated article; Schering-Plough Animal Health, Summit, New Jersey) via a medicated ration of 10 mg florfenicol x kg fish body weight(-1) d(-1) for 10 d to compare the elimination kinetics of the test article. This study was part of a larger effort in support of a species grouping concept that could contribute to the regulatory approval process for therapeutic compounds for cultured fishes. The trials in this study were conducted at the ideal water temperature for each species and at the temperature 5 degrees C lower than the ideal. The test temperatures were 30 degrees C and 25 degrees C for Nile tilapia, 25 degrees C and 20 degrees C for both walleyes and hybrid striped bass. In all cases, the elimination kinetics of florfenicol were more rapid at higher temperatures. The time to reach the tolerance of 1 microg/g in muscle-skin, as set by the U.S. Food and Drug Administration for channel catfish Ictalurus punctatus and salmonids, ranged from 6.1 to 4.1 d for Nile tilapia, from 12.6 to 9.7 d for walleyes, and from 2.6 to 0.7 d for hybrid striped bass at temperatures between 20 degrees C and 30 degrees C.

Florfenicol residues in Nile tilapia after 10-d oral dosing in feed: effect of fish size.

Nile tilapia Oreochromis niloticus were medicated with florfenicol (AQUAFLOR type A medicated article; Schering-Plough Animal Health, Summit, New Jersey) via a medicated ration at 15 mg florfenicol x kg fish body weight(-1) d(-1) for 10 d to compare the elimination kinetics of the test article in different size fish held at 25 degrees C. The groups of fish used in the study had mean weights of approximately 100, 250, and 500 g. In each trial, the fish were provided the medicated ration and 15 fish were processed at each of seven time points postfeeding for determination of the florfenicol concentration in serum and the florfenicol residue in the edible portion (composite muscle and skin). There was a trend toward shorter half-lives of elimination in the smaller fish. The elimination times in muscle-skin (times to reach the established tolerance concentration for channel catfish Ictalurus punctatus and salmonids of 1.0 microg florfenicol residue/g) and half-lives were 9.2 and 1.2 d (100 g), 8.6 and 1.7 d (250 g), and 12.7 and 2.2 d (500 g), respectively.

A PBPK model for midazolam in four avian species.

A physiologically based pharmacokinetic (PBPK) model was developed for midazolam in the chicken and extended to three other species. Physiological parameters included organ weights obtained from 10 birds of each species and blood flows obtained from the literature. Partition coefficients for midazolam in tissues vs. plasma were estimated from drug residue data obtained at slaughter. The avian models include separate compartments for venous plasma, liver, kidney, muscle, fat and all other tissues. An estimate of total body clearance from an earlier in vitro study was used as a starting value in the model, assuming almost complete removal of the parent compound by liver metabolism. The model was optimized for the chicken with plasma and tissue data from a pharmacokinetic study after intravenous midazolam (5 mg/kg) dose. To determine which parameters had the most influence on the goodness of fit, a sensitivity analysis was performed. The optimized chicken model was then modified for the turkey, pheasant and quail. The models were validated with midazolam plasma and tissue residue data in the turkey, pheasant and quail. The PBPK models in the turkey, pheasant and quail provided good predictions of the observed tissue residues in each species, in particular for liver and kidney.

Enantioselective pharmacokinetics of racemic carprofen in New Zealand white rabbits.

The enantioselective pharmacokinetics of single dose (2 mg/kg) racemic carprofen (CPF) were evaluated in adult New Zealand white rabbits after intravenous (i.v.) and subcutaneous (s.c.) dose. Six rabbits were utilized in a two-way randomized crossover study and serial blood samples were collected. Plasma CPF concentrations were determined by high-performance liquid chromatography. After i.v. and s.c. racemic CPF administration, plasma concentration-time curves were best described by a two-compartment open model and a one-compartment model, respectively. The S(+) CPF enantiomer predominated in plasma following both routes of administration. Mean observed clearance of R(-)-CPF (82.17 +/- 13.70 mL/h.kg) was more rapid than for S(+)-CPF (27.92 +/- 7.07 mL/h.kg; P < 0.001). T(1/2)lambda z was shorter for R(-)-CPF than S(+)-CPF after both i.v. (1.03 and 2.99 h, respectively) and s.c. (1.94 and 4.14 h, respectively) dosing. Mean AUC(0-->infinity) ratios for R(-):S(+)-CPF were approximately 1:3 for both routes of administration. Mean residence time of R(-)-CPF was shorter than of S(+)-CPF (1.06 +/- 0.29 h, 3.45 +/- 0.50 h; P < 0.001) and R(-)- and S(+)-CPF volumes of distribution at steady state were 85.00 +/- 14.42 and 94.39 +/- 18.66 mL/kg, respectively after i.v. administration. The mean s.c. bioavailability [F (%)] for both R(-)- and S(+)-CPF was high, 94.4 +/- 22.8 and 91.0 +/- 35.7%, respectively.

Plasma pharmacokinetics of midazolam in chickens, turkeys, pheasants and bobwhite quail.

In vivo plasma pharmacokinetics of midazolam hydrochloride (5 mg/kg i.v.) were determined in commercially raised broiler chickens, turkeys, ring-necked pheasants and bobwhite quail. Pharmacokinetic profiles of midazolam were similar for all four species, especially with regard to the area under the plasma drug concentration-time curve. Estimates of the half-life of elimination of midazolam were 0.42, 1.45, 1.90, and 9.71 h for turkeys, chickens, bobwhite quail, and pheasant, respectively. This was similar to the major metabolite (1-hydroxymidazolam). Elimination half-lives for 1-hydroxymidazolam were 1.35, 1.86, 1.97, and 13.97 h for turkey, chicken, bobwhite quail and pheasant, respectively. Elimination half-lives for 4-hydroxymidazolam were 0.76, 1.23, 2.85, and 13.82 h for chicken, turkey, pheasant, and bobwhite quail, respectively. In addition to traditional pharmacokinetic approaches to parameter estimation, a bootstrapping technique was employed to attempt to achieve more realistic approximations of the concentrations at later time-points.

Pharmacokinetics of ruminally dosed sodium [36Cl]chlorate in beef cattle.

The recently recognized potential of sodium chlorate as a possible preharvest food safety tool for pathogen reduction in meat animals has spurred interest in the pharmacokinetics of intraruminally dosed chlorate. Six Loala cattle were assigned (one heifer and one steer per treatment) to one of three intraruminal doses of radiolabeled sodium [36Cl]chlorate (21, 42, or 63 mg/kg body weight) administered in four equal aliquots over a 24-h period. Blood and serum were collected (29 samples in 48 h). Total radioactive residues were measured and the radioactive moieties were speciated. Chlorate appeared rapidly in blood and serum after dosing. For animals administered a dose of 42 or 63 mg/kg, the half-life of absorption was estimated at 0.6-0.9 h. Serum chlorate concentrations progressively increased with aliquot administration until peaking at 6-21 parts per million at 26 h. Between aliquot administrations, serum chlorate levels typically peaked in 3.5 h or less. The half-life of chlorate elimination ranged between 6.9 and 11 h, depending on the dose. Ultimately, absorption of chlorate removes it from its desired site of action, the lower gastrointestinal tract, thereby reducing its efficacy. Further research is needed to develop a chlorate formulation that will allow passage to the lower gastrointestinal tract.

Sulfadimethoxine and ormetoprim residues in three species of fish after oral dosing in feed.

Nile tilapia Oreochromis niloticus, summer flounder Paralichthys dentatus, and walleyes Sander vitreus were treated with Romet-30 (PHARMAQ AS, Oslo, Norway) via a medicated ration at 50 mg Romet-30 kg fish body weight(- 1) d(-1) for 10 d to compare the elimination kinetics of the test substance. This study was part of a larger effort to develop a species grouping concept for the labeling of therapeutic compounds for cultured fishes. The fish tests were conducted at the ideal water temperature for each species and at 5 degrees C lower than the ideal temperature except for summer flounder, which would not feed at the lower temperature of 15 degrees C. Test temperatures were 30 degrees C and 25 degrees C for Nile tilapia, 20 degrees C and 17 degrees C for summer flounder, and 25 degrees C and 20 degrees C for walleyes. Neither component of Romet-30 (sulfadimethoxine and ormetoprim) could be detected in samples of the edible portion of walleyes (muscle plus skin) collected at day 10 posttreatment or thereafter. In studies with summer flounder, only one fish had a detectable concentration of either component on day 21 or thereafter. Elimination of Romet-30 by Nile tilapia was extremely rapid. The limited number of Nile tilapia with detectable sulfadimethoxine or ormetoprim during the posttreatment period prevented the determination of elimination half-life or elimination in this species.

Cytochrome P450-dependent metabolism of midazolam in hepatic microsomes from chickens, turkeys, pheasant and bobwhite quail.

In vitro putative cytochrome P450 3A mediated activity, and inhibition thereof, were measured in four avian species using midazolam (MDZ) as a substrate and ketoconazole as an inhibitor. All species produced 1-hydroxymidazolam (1-OH MDZ) to a much greater extent than 4-hydroxymidazolam (4-OH MDZ). Calculated Vmaxapparent values for formation of 1-OH MDZ were 117+/-17, 239+/-108, 437+/-168, and 201+/-55 pmol/mg protein*min and Kmapparent values were 2.1+/-0.8, 2.4+/-1.6, 6.7+/-5.1 and 3.2+/-2.1 microm for chicken, turkey, pheasant and bobwhite quail, respectively. For the formation of 4-OH MDZ the Vmaxapparent values were 21+/-10, 94+/-46, 144+/-112, and 68+/-30 pmol/mg protein*min and Kmapparent values for 4-OH MDZ formation were 12.4+/-10.1, 18.0+/-10.8, 38.6+/-34.7 and 29.1+/-10.1 microm for chicken, turkey, pheasant and bobwhite quail, respectively. In all four species, ketoconazole inhibited the production of both major metabolites of MDZ, with 4-OH MDZ formation more sensitive to inhibition than 1-OH MDZ. Pheasant and bobwhite quail appeared most sensitive to ketoconazole inhibition.

Investigation of Japanese quail (Coturnix japonica) as a pharmacokinetic model for cockatiels (Nymphicus hollandicus) and Poicephalus parrots via comparison of the pharmacokinetics of a single intravenous injection of oxytetracycline hydrochloride.

The purpose of this study was to determine whether Japanese quail (Coturnix japonica) would serve as a pharmacokinetic animal model for two small companion parrots: cockatiels (Nymphicus hollandicus) and Poicephalus parrots. Oxytetracycline (OTC) was the pharmacologic agent chosen for this study as it is eliminated primarily by renal glomerular filtration and undergoes minimal metabolism. A single intravenous injection of 20 mg/kg oxytetracycline hydrochloride was administered to the three study groups and blood samples were obtained at 5, 10, 15, and 30 min post-OTC injection as well as 1, 2, 4, 8, 12 and 24 h post-OTC injection. Quantification of plasma OTC was accomplished using a standardized microbial inhibition assay. Naïve-pooled data (NPD) analysis of the plasma concentration-time profile of OTC best fit a two-compartment open model for all three avian species. Noncompartmental analysis of the mean data yielded the following parameters for quail, cockatiels and Poicephalus parrots respectively: lambda(z) = 3.14, 4.57, 3.71 h; AUC = 38.9, 42.7, 49.6 microg x h/mL; and Cl = 514, 468, 403 mL/h/kg. Based on the similarity of these pharmacokinetic parameters, it appears that quail could be used as a model species to predict the appropriate OTC dosing regimen for small psittacine birds. A bootstrap procedure was also applied to these sparse data sets for both compartmental and noncompartmental analysis. The bootstrap procedure allowed for the calculation of variability of parameters; however, the estimates of the parameters were very similar to those calculated using the NPD and the data mean values.

Studies on itraconazole delivery and pharmacokinetics in mallard ducks (Anas platyrhynchos).

Avian aspergillosis is commonly treated with itraconazole (ITZ). This paper describes two studies using mallard ducks (Anas platyrhynchos). The first study evaluated in vivo release of ITZ from subcutaneously injected controlled-release gel formulations and the second study compared pharmacokinetic parameters for two ITZ oral suspensions. ITZ-A suspension was prepared by mixing contents of commercially available capsules with hydrochloric acid and orange juice. ITZ-B suspension was prepared by dispersing the complex of the drug with hydroxypropyl-beta-cyclodextrin in water. Concentrations of ITZ and its active metabolite, hydroxyitraconazole (OH-ITZ), in plasma and tissue samples were measured using high-performance liquid chromatography. In the second study, drug concentrations in plasma samples were also analyzed using a bioassay. After administration of two ITZ controlled-release formulations, plasma and tissue concentrations of ITZ and OH-ITZ were either very low (< or = 52 ng/mL) or undetectable. Exceptions included skin, subcutaneous fat, and muscle adjacent to the injection site. The drug from ITZ-A and ITZ-B suspensions was absorbed after oral administration. ITZ pharmacokinetic parameters for both suspensions in mallard ducks were similar and the bioassay successfully measured ITZ equivalents in plasma samples from ducks.

Meta-analysis of pharmacokinetic data of veterinary drugs using the Food Animal Residue Avoidance Databank: oxytetracycline and procaine penicillin G.

Investigators frequently face the quandary of how to interpret the often times disparate pharmacokinetic parameter values reported in the literature. Combining of data from multiple studies (meta-analysis) is a useful tool in pharmacokinetics. Few studies have explored the use of meta-analysis for veterinary species. Even fewer studies have explored the potential strengths and weaknesses of the various methods of performing a meta-analysis. Therefore, in this study we performed a meta-analysis for oxytetracycline (OTC) and procaine penicillin G (PPG) given intramuscularly to cattle. The analysis included 28 individual data sets from 18 published papers for PPG (288 data points), and 41 individual data sets from 25 published papers for OTC (489 data points). Three methods were used to calculate the parameters. The first was a simple statistical analysis of the parameter values reported in each paper. The second method was a standard Two-Stage Method (TSM) using the mean concentration vs. time data extracted from each paper. The third method was the use of nonlinear mixed effect modeling (NMEM) of the concentration vs. time data reported in the various papers, treating the mean data as if each set came from an individual animal. The results of this evaluation indicate that all three methods generate comparable mean parameter estimates for OTC and PPG. The only significant difference noted was for OTC absorption half-lives taken from the published literature, a difference attributable to the use of an alternative method of parameter calculation. The NMEM procedure offers the possibility of including covariates such as dose, age, and weight. In this study the covariates did not influence the derived parameters. A combination approach to meta-analysis of published mean data is recommended, where the TSM is the first step, followed by the NMEM approach.

Intravenous and subcutaneous pharmacokinetics of florfenicol in sheep.

Pharmacokinetic parameters of florfenicol were determined in 10 adult sheep (five wethers and five ewes) after a single 40 mg/kg intravenous (i.v.) dose, and three daily subcutaneous (s.c.) doses of 40 mg/kg of a commercial preparation (Nuflor((R))). The concentration of florfenicol in serum samples was assayed using a proprietary HPLC assay method, and pharmacokinetic parameters derived for individual animal data by each route using compartmental and noncompartmental approaches. Two animals (one male and one female) were excluded due to observed i.v. dosing problems, and a biexponential model was found to fit the i.v. data well for six of the other eight animals. Data from two males showed prolonged low concentrations of florfenicol in serum and were better fit by a three-compartment model. The mean +/- SD for the half-lives of the distribution and elimination phases for the six sheep best fit with a two-compartment model were 0.069 +/- 0.018 and 1.01 +/- 0.09 h respectively, and for the V(d(ss)) and clearances were 0.503 +/- 0.035 L/kg and 366 +/- 53 mL/h/kg respectively. The data collected during the s.c. multiple dose study were analyzed using noncompartmental methods only. The bioavailability (F%) after s.c. dosing was calculated in three ways to compare estimation methods as steady-state had not been reached and single dose s.c. data were not obtained past 24 h. Using the AUC(0--24) and AUC(0--> infinity ) from the first dose, the F% values averaged 27 and 40% respectively. Using the AUC(0--> infinity ) for all doses, the F% was 65%. Calculations of the mean time during which the serum concentration exceeded 0.5 and 1.0 microg/mL were 105 +/- 3.9 and 74.7 +/- 12.2 h respectively.

Feasibility of using half-life multipliers to estimate extended withdrawal intervals following the extralabel use of drugs in food-producing animals.

Under the Animal Medicinal Drug Use Clarification Act of 1994, veterinarians are legally allowed to use drugs in food-producing animals in an extralabel manner. This could potentially lead to violative residues in food of animal origin. It is therefore essential that an appropriately extended withdrawal interval be established. Ideally, these extended withdrawal intervals should be calculated on the basis of the tissue half-life of the drug in the target animal. However, these data are not readily available for all drugs of extralabel use in food-producing animals. For this reason, the use of a half-life multiplier has been proposed as a simple alternative method to estimate the effective tissue half-life of a drug. Extended withdrawal intervals, estimated using various half-life multipliers, were compared with the withdrawal intervals calculated using actual tissue half-lives. For the group of drugs investigated, a half-life multiplier of 5 resulted in estimates of extended withdrawal intervals that were potentially inadequate to prevent violative tissue residues for drugs that had relatively long tissue half-lives, high tolerances, or both. This is possibly because fewer half-lives are required for these drugs to reach the target tissue concentrations following administration at label doses. Use of a smaller half-life multiplier (in this case 3) is therefore suggested to ensure that extended withdrawal intervals are adequate to prevent violative tissue residues.

Pharmacokinetics of ceftiofur in llamas and alpacas.

Pharmacokinetic studies of antibiotics in South American camelids are uncommon, therefore drugs are often administered to llamas and alpacas based on dosages established in other domestic species. The disposition of ceftiofur sodium was studied in llamas following intramuscular administration and in alpacas following intravenous and intramuscular administration. Eleven adult llamas were given ceftiofur sodium by intramuscular injection. Each animal received either a standard dose of 2.2 mg/kg or an allometrically scaled dose ranging from 2.62 to 2.99 mg/kg in a crossover design. Ten adult alpacas were given ceftiofur sodium by intravenous and intramuscular injections. Each animal received a standard dosage of 1 mg/kg or an allometrically scaled dose ranging from 1.27 to 1.44 mg/kg i.v., and 1.31-1.51 mg/kg i.m. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, and 72 h after administration of the ceftiofur. Pharmacokinetic parameters of ceftiofur in llamas and alpacas were similar following i.m. dosing at both dose levels. The only differences noted were in the total AUC between dose levels, but the AUC/dose values were not different. A sequence effect was noted in the alpaca data, which resulted in lower AUCs for the second dose when the i.v. dose was given first, and with higher AUCs for the second dose when the i.m. dose was given first. Overall, ceftiofur pharmacokinetics in llamas and alpacas are similar, and also very similar to reported parameters for sheep and goats.

Disposition of cyclosporine after intravenous and multi-dose oral administration in cats.

The aim of this study was to evaluate the disposition of cyclosporine after intravenous (i.v.) and oral administration and to evaluate single sampling times for therapeutic monitoring of cyclosporine drug concentrations in cats. Six adult male cats (clinically intact) were used. Two treatments consisting of a single i.v. cyclosporine (1 mg/kg) and multiple oral cyclosporine (3 mg/kg b.i.d p.o. for 2 weeks) doses. Whole blood cyclosporine concentrations were measured at fixed times by high performance liquid chromatography and pharmacokinetic values were calculated. Mean values for the i.v. data included AUC (7413 ng/mL.h), t1/2 distribution and elimination (0.705 and 9.7 h, respectively), Cmax (1513 ng/mL), and Vd(ss) (1.71 L/kg). Mean values for the oral data included AUC (6243 ng/mL.h), t1/2 of absorption and elimination (0.227 and 8.19 h, respectively), and Cmax (480.0 ng/mL). Bioavailability of orally administered cyclosporine was 29 and 25% on days 7 and 14 respectively. Whole blood comment cyclosporine concentration 2 h after administration (C2) better correlated with AUC on days 7 and 14 than trough plasma concentration (C12). The rate of oral cyclosporine absorption was less than expected and there was substantial individual variation. Therapeutic drug monitoring strategies for cyclosporine in cats should be re-evaluated.

A physiologically based pharmacokinetic model for oxytetracycline residues in sheep.

A physiologically based pharmacokinetic model (PBPK) for oxytetracycline (OTC) residues in sheep was developed using previously published data from a combined serum pharmacokinetic and tissue residue study [Craigmill et al. (2000) J. Vet. Pharmacol. Ther.23, 345]. Physiological parameters for organ weights and tissue blood flows were obtained from the literature. The tissue/serum partition coefficients for OTC were estimated from the serum and tissue residue data obtained at slaughter. The model was developed to include all of the tissues for which residue data were available (serum, kidney, liver, fat, muscle and injection site), and all of the remaining tissues were combined into a slowly perfused compartment with low permeability. Total body clearance of OTC calculated in the previous study was used as the starting value for clearance in the PBPK model, with the kidney being the only eliminating organ. The model was built using ACSL (Advanced Continuous Simulation Language) Graphic Modeler, and the model was fit to the serum and tissue data using the ACSL Math/Optimizer software (AEgis Technologies Group, Inc., Huntsville, AL, USA). A sensitivity analysis was also performed to determine which parameters had the greatest effect on the goodness of fit. Numerous strategies were tested to model the injection site, and a model providing a biexponential absorption of the drug from the injection bolus gave the best fit to the experimental data. The model was validated using the clearance parameters calculated from the traditional pharmacokinetic model for each individual animal in the PBPK model. This simple PBPK model well predicted OTC residues in sheep tissues after intramuscular dosing with a long-acting preparation and may find use for other species and other veterinary drugs.

Serum pharmacokinetics and tissue and milk residues of oxytetracycline in goats following a single intramuscular injection of a long-acting preparation and milk residues following a single subcutaneous injection.

Separate groups of goats were used to determine drug depletion patterns in serum (n=10), tissue (n=20) and milk (n=8) following a single intramuscular (i.m.) dose of 20 mg/kg of a long-acting oxytetracycline (OTC) formulation (Liquamycin LA-200). Milk residues were also determined following a subcutaneous (s.c.) administration of the same product at the same dose. Serum samples were taken for 24 h post-treatment and tissues (fat, liver, kidney, muscle and injection site) collected at 4, 7, 14, 21 and 28 days following injection. Milk from lactating goats was collected every 12 h for 8 days following both the i.m. and s.c. treatments utilizing an intervening 5-week washout period. Residues in serum and tissue were measured using a microbial inhibition assay, while milk residues were measured using both a microbial inhibition assay and a validated HPLC method. The serum pharmacokinetic parameters of OTC in goats were determined, with a mean AUC=67.4 microg h/mL, mean terminal half-life=14.4 h, and apparent clearance=0.33 L/kg h. Tissue half-lives could not be determined with confidence because the collection times provided only two points at which residues could be measured for most tissues. Oxytetracycline residues in all goat tissue samples measured less then cattle tissue tolerance by 96 h postdosing. One-compartment model describing milk depletion data for i.m. and s.c. dosing had terminal slope half-lives of 20.1 and 36.1 h, respectively. By 96 h post-treatment none of the milk samples contained OTC residues in excess of the cattle milk tolerance (0.3 p.p.m.). For both milk and tissue, the upper-bound 99% confidence intervals for the samples taken from goats 96 h postdosing were lower than approved cow milk and tissue tolerances.

Comparison of the in vitro antiproliferative effects of five immunosuppressive drugs on lymphocytes in whole blood from cats.

OBJECTIVE: To compare the in vitro immunosuppressive effects of cyclosporine and 4 novel immunosuppressive drugs on lymphocytes in whole blood collected from healthy cats. SAMPLE POPULATION: Whole blood samples collected from 10 healthy adult domestic shorthair cats. PROCEDURE: Mitogen-stimulated lymphocyte proliferation in whole blood incubated with and without various concentrations of cyclosporine, tacrolimus, sirolimus, mycophenolic acid (MPA), or A771726 was measured by use of [3H]thymidine incorporation. Drug concentrations that resulted in a 50% inhibition of mitogen-induced proliferation (IC50) were calculated. Lymphocyte viability was determined by use of the trypan blue dye exclusion method. RESULTS: An obvious dose-response relationship for the antiproliferative effects of each drug was detected. Mean IC50 determined with concanavalin A was 46 nM for cyclosporine, 9 nM for tacrolimus, 12 nM for sirolimus, 16 nM for MPA, and 30 mM for A771726, whereas with pokeweed mitogen, mean IC50 was 33 nM for cyclosporine, 5 nM for tacrolimus, 15 nM for sirolimus, 14 nM for mycophenolic acid, and 25 mM for A771726. Mitogen-stimulated and nonstimulated lymphocytes remained viable, regardless of drug evaluated. CONCLUSIONS AND CLINICAL RELEVANCE: Tacrolimus, sirolimus, MPA, and A771726 inhibited in vitro mitogen-stimulated proliferation of feline lymphocytes in a dose-dependent manner. These novel immunosuppressive drugs may be useful for management of immune-mediated inflammatory diseases and prevention and treatment of rejection in cats that undergo organ transplantation.