Various V-J rearrangement efficiencies shape the mouse λ B cell repertoire

The diversity of the B cell repertoire of C_x knockout mice is limited by the expression of four λ light chain types. Among the spleen B cells, λ1 is expressed by the majority (58%) of cells, and λ3 by the minority (8%), while λ2(V2) and λ2(Vx) are expressed in intermediate quantities (18% and 16%, respectively). To assess the influence of mechanistic pressures on the λ subtype distribution, the proportions of the different λ rearrangements were determined in various B cell subpopulations divided on the basis of the λ subtype expressed, and the VλJλ junction sequences were studied at different steps of B cell differentiation (pre-B, immature and mature B cells). The data show that (1) the ratio of productive/non-productive VJ junctions is determined by the nature of the λ segments that are rearranged as can be observed in the pre-B cells, (2) V1-J1 non-productive rearrangements are often found in the λ1-negative B cells in the periphery, and (3) V1J3 junctions are often non-productive regardless of the nature of the cells analyzed. Our results, therefore, suggest that a strong probability of initiating a V1-J1 rearrangement and a weak probability of giving a productive V1J3 junction are responsible for the λ1 dominance and the λ3 under-expression, respectively. The intermediate proportion of λ2(V2) subtype is most likely due to a probability of obtaining a productive joint that is better than that for V1J3 and a probability of initiating a rearrangement that is lower than that for V1J1. However, the λ2(Vx) cell proportion cannot be determined only by these parameters.     

Pharmacokinetics of 1 -(2-Deoxy-2-fluoro-β-L-arabino-furanosyl)-5-methyluracil (L-FMAU) in Rats

Purpose. The objective of this study was to characterize the pharmacokinetics of 1 -(2-deoxy-2-fluoro--L-arabinofuranosyl)-5-methyluracil (L-FMAU), a nucleoside analogue with potent activity against the hepatitis B virus and the Epstein-Barr virus, in rats. Methods. Three doses of L-FMAU were administered intravenously (10, 25, and 50 mg/kg) to rats, and L-FMAU concentrations in plasma and urine were measured by HPLC. Pharmacokinetic parameters were generated by using area-moment analysis. Results. There were no significant differences in the pharmacokinetic parameters between the three doses ( < 0.05). Thus, the disposition of L-FMAU was linear over the dosage of 10 to 50 mg/kg. Plasma concentrations of L-FMAU declined rapidly with a terminal phase half-life of 1.33 ± 0.45 h (mean ± SD). Total clearance of L-FMAU was moderate, averaging 1.15 ± 0.28 L/h/kg. The fraction of compound excreted unchanged in urine was 0.59 ± 0.13. No glucuronide metabolite was found in the urine. The steady-state volume of distribution was 1.12 ± 0.26 L/kg indicating intracellular distribution of the compound. The fraction of L-FMAU bound to plasma proteins was approximately 15% and was independent of nucleoside concentration. Conclusions. The pharmacokinetics of L-FMAU in rats were independent of dose over the dosage range of 10 to 50 mg/kg.     

Hybrid Pharmacokinetic Models to Describe Anti-HIV Nucleoside Brain Disposition Following Parent and Prodrug Administration in Mice

Brain delivery of active anti-HIV compounds is important for successful treatment of the AIDS patient. As an initial step in predicting human brain drug concentrations, hybrid pharmacokinetic models were developed to characterize the disposition of anti-HIV nucleosides following parent and prodrug administrations in mice. Mouse data were obtained following intravenous administration of 3-azido-2,3-dideoxyuridine (AZddU or AZDU), 3-azido-3-deoxythymidine (AZT), and their dihydropyridine prodrugs (AZddU-DHP and AZT-DHP). Exponential equations were fitted to the serum concentration–time data for each species, including the pyridinium ion moieties, and subsequently used in differential mass balance equations describing the brain dynamics of each compound. Model parameters for the mass balance equations were estimated by various techniques, including the utilization of in vitro data. In general, model-predicted brain concentrations agreed with the observed data. Similar data in larger animals will permit scale-up of the current model to predict human brain drug concentrations.     

Investigation of mezlocillin disposition with a porcine model

The suitability of the pig as an animal model for mezlocillin disposition was assessed. Serum, urine, and bile were collected after the administration of 50, 100 and 200 mg kg-1 mezlocillin to pigs and drug pharmacokinetics were characterized. Mezlocillin concentrations in biological fluids were determined by HPLC and free mezlocillin was determined by ultrafiltration. The pharmacokinetics of mezlocillin appeared to be independent of dose over the dosage range studied. Total clearance, renal clearance, and biliary clearance were 0.18 (0.05) 1 h-1 kg-1, 0.13 (0.03) 1 h-1 kg-1, and 0.07 (0.02) 1 h-1 kg-1, respectively. The steady-state volume of distribution was 0.29 (0.08) 1 kg-1. The pharmacokinetic parameters determined in the porcine model are similar to those reported for health human volunteers. Therefore, this model appears suitable for the study of mezlocillin disposition, and may be applied to the study of other agents that are appreciably biliary excreted.     

Second generation model for prednisolone pharmacodynamics in the rat

An improved model describing receptor/gene-mediated pharmacodynamics of prednisolone is presented which consists of seven differential equations. Data for plasma prednisolone concentrations, free hepatic glucocorticoid receptors, and hepatic tyrosine aminotransferase activity (TAT) following low (5 mg/kg) and high (50 mg/kg) doses of prednisolone are used to quantitate the kinetics and dynamics of this synthetic steroid in the rat. In contrast to the earlier model, the newer model provides for a coupling and simultaneous fitting of receptor and TAT data and is able to describe the recycling of receptors between cytosol and nucleus and the return of cytosolic receptors to baseline following glucocorticoid elimination. A numerical technique to determine the efficiency of TAT induction based on area under the curve calculations is presented, which supports the hypothesis that nonlinear dose-response effects are due to dose and time-dependent receptor depletion in the cytosol. Simulations are presented to examine the major determinants of corticosteroid effects and to compare the effects of single-and multiple-dose regimens in maximizing drug effects.Supported in part by Grant 24211 from the National Institutes of General Medical Sciences, National Institutes of Health.     

Dose-dependent pharmacokinetics of prednisolone in normal and adrenalectomized rats

The pharmacokinetics of prednisolone after 5- and 50-mg/kg doses given as the sodium succinate salt was examined in normal and adrenalectomized rats. Prednisolone, prednisone, and corticosterone concentrations in plasma were determined by HPLC and free prednisolone measured by equilibrium dialysis. Prednisolone sodium succinate was rapidly and completely hydrolyzed to prednisolone as indicated by the absence of the ester from plasma within 5 min after intravenous injection. Prednisolone was rapidly metabolized to prednisone, while corticosterone concentrations in normal rats declined rapidly and were undetectable by 1 hr. Adrenalectomy had no effect on the disposition and protein binding of prednisolone. Dose, however, had a marked effect on prednisolone pharmacokinetics, with mean plasma clearance decreasing from 6.18 to 3.07 L/h per kg and mean steady-state volume of distribution decreasing from 2.14 to 1.05 L/kg from the lower to higher steroid dose. Half-life (0.50 hr) and mean residence time (0.35 hr) were unaffected by dose. Prednisolone plasma protein binding was nonlinear due to saturation of transcortin binding. Changes in pharmacokinetic parameters were not related to the nonlinear plasma binding, but were more likely caused by saturation of elimination pathways and tissue binding sites.     

Pharmacokinetics of bis(t-butyl-SATE)-AZTMP, a bispivaloylthioethyl prodrug for intracellular delivery of zidovudine monophosphate, in mice

The pharmacokinetics of a bispivaloylthioethyl prodrug of zidovudine monophosphate (AZTMP), bis(t-butyl-SATE)-AZTMP, and intracellular conversion of the prodrug to AZTMP were characterized following intravenous (i.v.) and oral (p.o.) administration of the prodrug to mice. Concentrations of bis(t-butyl-SATE)-AZTMP, AZTMP and zidovudine (AZT) in blood, red blood cells, plasma, brain and lymph nodes were determined by HPLC. Following i.v. administration of bis(t-butyl-SATE)-AZTMP, concentrations of the prodrug declined rapidly with low levels of the prodrug detected until 4 h. Both bis(t-butyl-SATE)-AZTMP and AZTMP were detected in brain 3 min after dosing. AZTMP was found in both plasma and peripheral red blood cells, peaking at approximately 30 min and remaining detectable until 2 h. No AZTMP was detected in lymph nodes. Compared to the pharmacokinetics of AZT following its i.v. administration, i.v. administration of bis(t-butyl-SATE)-AZTMP produced lower peak concentrations of AZT in plasma, peripheral red blood cells, brain and lymph nodes. However, terminal half-lives of AZT were significantly prolonged following administration of the prodrug. Following p.o. administration of bis(t-butyl-SATE)-AZTMP, neither the prodrug nor AZTMP were detectable in whole blood. The conversion of AZT from bis(t-butyl-SATE)-AZTMP in plasma and peripheral red blood cells following p.o administration was 12.1% of that following i.v. administration of the prodrug. Bis(t-butyl-SATE)-AZTMP demonstrated promising potential for intracellular delivery of AZTMP. The prodrug also prolonged the retention of AZT in mice, and particularly increased delivery of AZT to the lymphatic and central nervous systems.     

Costimulatory receptors in jawed vertebrates: conserved CD28, odd CTLA4 and multiple BTLAs

CD28 family of costimulatory receptors is comprised of molecules with a single V-type extracellular Ig domain, a transmembrane and an intracytoplasmic region with signaling motifs. CD28 and cytotoxic T lymphocyte antigen-4 (CTLA4) homologs have been recently identified in rainbow trout. Other sequences similar to mammalian CD28 family members have now been identified using teleost, Xenopus and chicken databases. CD28- and CTLA4 homologs were found in all vertebrate classes whereas inducible costimulatory signal (ICOS) was restricted to tetrapods, and programmed cell death-1 (PD1) was limited to mammals and chicken. Multiple B and T Lymphocyte Attenuator (BTLA) sequences were found in teleosts, but not in Xenopus or in avian genomes. The intron/exon structure of btlas was different from that of cd28 and other members of the family. The Ig domain encoded in all the btla genes has features of the C-type structure, which suggests that BTLA does not belong to the CD28 family. The genomic localization of these genes in vertebrate genomes supports the split between the BTLA and CD28 families.     

Pharmacokinetics of 3''-fluoro-3''-deoxythymidine and 3''-deoxy-2'',3''-didehydrothymidine in rhesus monkeys.

3'-Fluoro-3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine are nucleoside analogs which inhibit human and simian immunodeficiency virus in vitro. The pharmacokinetic properties of these compounds in rhesus monkeys after intravenous, oral, and subcutaneous administration of the drug were compared. Half-lives, total clearances, and steady-state volumes of distribution of the two drugs were determined. The half-lives for the drugs by the different routes were between 0.58 and 1.4 h. Oral bioavailability of 3'-deoxy-2',3'-didehydrothymidine was incomplete, with an average of 42% +/- 15% of the dose reaching the systemic circulation. Absorption of 3'-fluoro-3'-deoxythymidine after oral administration was variable, with bioavailability ranging from 21 to 95%. Bioavailability after subcutaneous administration ranged from 59 to 77% for 3'-deoxy-2',3'-didehydrothymidine and from 52 to 59% for 3'-fluoro-3'-deoxythymidine. The ratio of concentrations in cerebrospinal fluid and serum for the drugs was about 0.15 at 1 h after drug administration and was independent of the route of administration, suggesting that a nucleoside carrier-mediated process is involved in the transport of these compounds to the central nervous system. Because of the similar metabolism of nucleoside analogs in monkeys and humans, the potential glucuronide formation was assessed. Whereas the glucuronide of 3'-fluoro-3'-deoxythymidine was readily detected in urine, the amount of 3'-deoxy-2',3'-didehydrothymidine glucuronidated was small or not detectable in one-half of the urine samples. Pharmacokinetic parameters for the two drugs were similar to each other and analogous to those for 3'-azido-3'-deoxythymidine in monkeys, suggesting that the same dose and scheduling of the drug can be used for all three compounds in prophylactic and therapeutic efficacy drug studies in rhesus monkeys.     

Effects of dose and sex on the pharmacokinetics of piroxicam in the rat

The effects of dose and sex on the pharmacokinetics of piroxicam were studied in the rat. Piroxicam was administered intravenously at doses of 0.50 and 5.0 mg kg-1 to male and female rats. Plasma drug concentrations were determined by a highly sensitive high-performance liquid chromatographic technique. Non-compartmental pharmacokinetic parameters were calculated by area/moment analysis. A prolonged terminal half-life averaging 13.3 h in male rats and 40.8 h in female rats was observed. Dose had no effect on the disposition of piroxicam. The sex of the rat, however, had a marked effect on piroxicam pharmacokinetics, with mean total clearance differing three-fold from 0.0184 l h-1 kg-1 in male rats to 0.00622 l h-1 kg-1 in female rats. The free fraction of piroxicam in serum was greater in male rats than in female rats owing to a higher association constant for piroxicam binding to female rat serum proteins. Free piroxicam clearance differed approximately two-fold with mean values of 0.764 l h-1 kg-1 and 0.418 l h-1 kg-1 in male and female rats, respectively. Thus, protein binding partially explained the sex-dependent disposition of piroxicam. However, sex-dependent metabolism of the drug also appears to be a major determinant of sex-related differences in piroxicam pharmacokinetics. Steady-state volume of distribution was unaffected by sex. Half-life and mean residence time were three-fold greater in female rats owing to the three-fold lower clearance value compared to male rats.