Application of multivariate statistical procedures to identify transcription factors that correlate with MRP2, 3, and 4 mRNA in adult human livers.

Multidrug resistance-associated proteins 2-4 (MRP2-4) are membrane efflux transporters critical for the hepatic clearance of pharmaceuticals and endogenous chemicals. Little is known about the constitutive regulation of MRP2-4 mRNA in normal human liver. The purpose of this study was to identify transcription factors whose expression significantly correlates with MRP2-4 mRNA in human liver specimens. Ninety adult human livers were profiled for mRNA expression of MRP2-4 as well as aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPAR alpha) and gamma (gamma), liver X receptor alpha (LXR alpha), farnesoid X receptor (FXR), glucocorticoid receptor (GR), retinoid X receptor alpha (RXR alpha), hepatocyte nuclear factor 1 alpha (HNF1 alpha) and 4 alpha (4 alpha), and nuclear factor E2-related factor 2 (Nrf2) transcription factors. Using linear regression and stepwise selection of partial R(2)-values, CAR, HNF1 alpha, and PPAR alpha mRNA exhibited the greatest correlation with MRP2, 3, and 4, respectively. Interindividual variation in the expression of the identified transcription factors may account for the variability in constitutive mRNA levels of MRP2-4. The multivariate approach presented in this study should aid in predicting signalling pathways that participate either directly or indirectly in regulating hepatic drug disposition.

Pharmacokinetic parameters and mechanisms of inhibition of rat type 1 and 2 steroid 5alpha-reductases: determinants for different in vivo activities of GI198745 and finasteride in the rat.

The interaction of baculovirus expressed rat steroid 5alpha-reductase types 1 and 2 (r5AR1 and r5AR2) with 17beta-N-(2,5-bis(trifluoromethyl)phenyl)carbamoyl-4-aza-5alpha-androst-1-en-3-one (GI198745) was investigated at pH 7 and 37 degrees. This 5alpha-reductase inhibitor was found previously to be a time-dependent inhibitor of the two human 5alpha-reductase isozymes. In contrast, we demonstrate in the present study that although GI198745 is a potent time-dependent inhibitor of r5AR2, it is a classical rapid-equilibrium inhibitor of r5AR1. This type of behavior with human and rat 5alpha-reductases has been shown for the inhibitor 17beta-(N-tert-butylcarbamoyl)-4-aza-5alpha-androst-1-en-3-one (finasteride), a current therapy for benign prostatic hyperplasia. Inhibition of r5AR1 by GI198745 was competitive with testosterone and followed Michaelis-Menten kinetics with a K(i) value of 0.3 +/- 0.02 nM. Data for the inhibition of r5AR2 by GI198745 were consistent with a two-step mechanism, where K(i) is the dissociation constant for an initial enzyme-inhibitor complex and k(3) is the rate constant for the second slow step. The pseudo-bimolecular rate constant (k(3)/K(i)) for the association of GI198745 with r5AR2 was (2.0 +/- 0.4) x 10(7) M(-1) sec(-1). The high affinity of this inhibitor for r5AR2 was further demonstrated by the inability of the enzyme-inhibitor complex to dissociate after approximately 7 days of dialysis at 4 degrees. Both GI198745 and finasteride appear to inactivate r5AR2 by apparent irreversible modification, but are classical, reversible inhibitors of r5AR1. Therefore, we hypothesize that because of its pharmacokinetic parameters and increased potency against r5AR1, GI198745 is more effective than finasteride in preventing the growth of the rat prostate.

The pharmacokinetics and pharmacodynamics of GW395058, a peptide agonist of the thrombopoietin receptor, in the dog, a large-animal model of chemotherapy-induced thrombocytopenia.

GW395058, a PEGylated peptide agonist of the thrombopoietin receptor, stimulates megakaryocytopoiesis and has previously been shown to increase platelet counts in vivo. The pharmacokinetics and pharmacodynamics of GW395058 were characterized using a randomized, crossover study in a large-animal model (dog) of chemotherapy-induced thrombocytopenia. Nine beagle dogs received i.v. carboplatin (350 mg/m(2)) on day 0 and day 28. GW395058 (1.31 mg/kg) (n = 6) or vehicle control (n = 3) was administered on day 1 and day 29 either as an i.v. bolus or s.c. injection. After i.v. administration, peak concentrations of GW395058 occurred rapidly, while the half-life averaged approximately 56 h. Bioavailability (+/- standard deviation) of GW395058 given s.c. was 78.2% (20.9%). GW395058 (i.v. and s.c.) ameliorated the platelet nadir (p = 0.0086) and resulted in a shorter time to recovery compared to the control group. The mean nadir platelet counts following carboplatin administration were 197,000 cells/microl (80,000) for the i.v. GW395058-dose group, 183,000 cells/microl (72,000) for the s.c.-dose group and 71,000 cells/microl (38,000) for the vehicle-alone group. GW395058 reduced the thrombocytopenic effects of carboplatin in dogs. No GW395058-related adverse side effects were observed.

Pharmacokinetics and hematological effects of the PEGylated thrombopoietin peptide mimetic GW395058 in rats and monkeys after intravenous or subcutaneous administration.

GW395058, a potent PEGylated peptide human thrombopoietin receptor (HuTPOr) agonist in vitro, is being evaluated for the treatment of thrombocytopenia. GW395058 shares no sequence homology with TPO. In this report the pharmacokinetics and hematological effects of GW395058 in rats and monkeys are described. Doses eliciting thrombocytosis in rodents (2 or 10 microg/kg s.c.) produced insufficient plasma concentration data for pharmacokinetic parameter estimate calculations. At higher i.v. doses in rats (500, 1,000 or 2,000 microg/kg) serum t1/2 (half-life) values were >20 h, and the area under the concentration time curve increased proportionally with dose. In cynomolgus monkeys GW395058 plasma t1/2 values ranged from 37 to 68 h after s.c. or i.v. dosing, and similar values were observed in rhesus monkeys following s.c. dosing. Rat platelet counts increased following 2 (1.6-fold) or 10 microg/kg (fourfold) s.c. doses. Cynomolgus and rhesus monkey platelet counts did not change significantly at comparable s.c. doses, but did increase slightly (

(-)-6-Aminocarbovir pharmacokinetics and relative carbovir exposure in rats.

The potential for the metabolic conversion of (-)-6-aminocarbovir to (-)-carbovir, a potent reverse transcriptase inhibitor effective against human immunodeficiency virus, has been examined in male Sprague-Dawley rats. Plasma (-)-6-aminocarbovir concentrations declined rapidly in a biphasic manner following an iv bolus dose of 20 mg/kg. The total systemic clearance was 5.4 liter/hr/kg and the terminal t1/2 was 0.35 hr. Following iv dosing, approximately half of the dose was excreted into the urine and comprised equivalent quantities of (-)-carbovir and (-)-6-aminocarbovir. Orally administered (-)-6-aminocarbovir was rapidly absorbed (tmax of 0.39 hr and Cmax of 4.96 micrograms/ml) following a 60 mg/kg dose. Following oral administration, 32% of the dose was eliminated in the urine, and comprised (-)-carbovir (75%) and (-)-6-aminocarbovir (25%). The oral bioavailability of (-)-6-aminocarbovir was 46% by plasma AUC comparison and 33% based on urinary excretion data. Exposure to (-)-carbovir was lower following (-)-6-aminocarbovir dosing than observed following (-)-Carbovir dosing, by both the oral and iv routes.

Reduced systemic drug exposure by combining intra-arterial chemotherapy with hemoperfusion of regional venous drainage.

Four patients with malignant cerebral gliomas received 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) into the internal carotid artery (ICA) while the ipsilateral jugular drainage was pumped extracorporeally through a hemoperfusion cartridge containing a nonionic adsorbant resin. Each patient received 220 mg/sq m BCNU, infused over 45 minutes through a toposcopic catheter positioned with the tip in the ICA beyond the origin of the ophthalmic artery. Jugular blood was pumped extracorporeally at 300 ml/min through a large-bore catheter in the jugular bulb. Plasma samples were obtained for BCNU measurement at frequent intervals from the right atrium. During a separate treatment, 6 weeks before or after the hemoperfusion treatment, the same dose of BCNU was infused into the ICA and atrial samples were obtained on a similar schedule. Hemoperfusion of the jugular blood during intracarotid infusion reduced the systemic exposure by 56% to 87% and increased total body clearance of BCNU by two- to eightfold. The calculated pharmacokinetic advantage (brain:body exposure ratio) was between 21 and 55:1 when the combined treatment was used.

Arterial drug infusion with extracorporeal removal. II. Internal carotid carmustine in the rhesus monkey.

During cancer chemotherapy by intra-arterial drug administration, systemic toxicity often limits the tolerable dose. We evaluated the pharmacokinetic advantage obtained by infusing carmustine (BCNU) into the internal carotid artery during BCNU removal from the blood from the perfused region by hemoperfusion. A hemoperfusion column (XR-010, Extracorporeal Medical Specialties) was shown to remove BCNU quantitatively from sheep blood flowing at 300 ml/minute when the drug was infused at 13 mg/minute for 30 minutes. Under general anesthesia, adult rhesus monkeys underwent catheterization of the internal carotid artery and placement of a catheter in the ipsilateral jugular vein at its junction with the sigmoid sinus. BCNU (10 mg/kg) was infused over 20 minutes while blood was pumped from the jugular vein through a small column and back into the inferior vena cava. The procedure reduced systemic exposure by 46%-84% compared with iv infusion of the same dose. Brain-to-systemic exposure ratios ranged from 18:1 to 87:1, depending on the pump flow rate and method of calculation. Hematopoietic toxicity was prevented. It is suggested that tumor exposure to BCNU comparable to that associated with very high tumor cell kill in vitro may be feasible with little or no systemic toxicity.

Quantitation of testolactone and 4,5-dihydrotestolactone in plasma and urine using high-performance liquid chromatography.

A rapid, sensitive, and selective assay is described for the quantitation of both testolactone and its recently identified metabolite, 4,5-dihydrotestolactone, in plasma and urine using high-performance liquid chromatography. The procedure includes a methylene chloride extraction prior to chromatography and quantitation using peak height ratios (ultraviolet absorbance detection, 242 nm) of testolactone and 4,5-dihydrotestolactone to the internal standard, testosterone. A sensitivity of 20 ng/ml for both testolactone and 4,5-dihydrotestolactone is easily achieved using only 0.5 ml of sample. Mean recoveries for testolactone and its metabolite are 95.0% and 81.8%, respectively, and the mean coefficient of variation of the procedure is 3.5% for the drug and 7.1% for the metabolite. This method is currently being used to study the pharmacokinetics of testolactone and 4,5-dihydrotestolactone in male patients. A steady-state plasma concentration versus time profile from a representative patient is included.

Rapid GC method for quantitation of nifedipine in serum using electron capture detection.

For studying the pharmacokinetics of nifedipine after single doses, a rapid, specific, reliable gas chromatographic assay procedure for nifedipine in serum is developed. Using a single-step solvent extraction or a bonded-phase column extraction and electron capture detection, an assay sensitivity of 10 to 25 ng/ml can be achieved using 0.25 ml of serum. The assay quantitates intact nifedipine and separates it reproducibly from its nitro- and nitrosopyridine derivatives.

Systemic bioavailability and pharmacokinetics of methylprednisolone in patients with rheumatoid arthritis following 'high-dose' pulse administration.

The absolute and relative bioavailability of two methylprednisolone formulations (capsules and suspension) was determined along with its pharmacokinetics in four arthritic female patients, following an unconventional high-dose pulse of 1 g. Plasma concentrations of the drug were measured by a sensitive and specific high-performance liquid chromatographic (HPLC) procedure. The disposition of methylprednisolone from plasma following intravenous (i.v.) infusion of its succinate ester appeared monoexponential with a mean half-life of 2.4 h and an apparent volume of distribution (Vd) of 50 l (0.87 l/kg). The total body clearance (Cl) averaged 15.12 l/h. Absolute bioavailability was assessed by comparing the areas under the plasma concentration time curves (normalized to dose) following oral administration of capsule or suspension with those of intravenous administration. No significant difference (p greater than 0.2) was observed when systemic availability (f, expressed in per cent) following administration of drug in capsule (f = 49.35 per cent) was compared with that obtained following the administration of drug in a suspension (f = 58.26 per cent). The difference in the observed and predicted f may be due to incomplete absorption, hepatic and/or extrahepatic metabolism of methylprednisolone. Subjective evaluation showed no side effects of this high-dose pulse therapy in any of the patients.

Quantitation of 6-mercaptopurine in biologic fluids using high-performance liquid chromatography: a selective and novel procedure.

A simple, selective, sensitive and rapid procedure is described for the quantitation of 6-mercaptopurine (6-MP) in biological fluids. A sensitivity of at least 5 ng/ml is easily achieved in plasma on a reversed-phase octadecylsilane (C18) column using a high-performance liquid chromatography system following an initial protein precipitation and a clean-up step. Mean extractability of the drug from plasma following this procedure is greater than 98% and the overall coefficient of variation for the assay is below 6%. Plasma levels of 6-MP were measured in a rhesus monkey for 12 h following an intravenous administration of a single bolus dose (4 mg/kg) of 6-MP.

Kinetics of chlorambucil hydrolysis using high-pressure liquid chromatography.

A stability-specific high-pressure liquid chromatographic (HPLC) method was developed to assay intact chlorambucil (I) in the presence of its hydrolytic decomposition products. The HPLC method was used to follow the degradation kinetics of I over pH 1.0-10.0 in the presence of various buffers with and without added chloride ion. In the absence of chloride ion, the hydrolysis of I followed first-order kinetics and the pH rate profile showed a sharp inflection around pH 2.5 attributable to the ionization of the nitrogen mustard and a shallower inflection around pH 5.0 attributable to the ionization of the carboxylic group. The rate was pH independent over pH 6.0-10.0 and independent of buffer species in the absence of chloride ion. IN the presence of chloride ion, the kinetics of I hydrolysis was still first order. However, the degradation half-life at a particular pH and buffer concentration increased linearly with chloride concentration. Kinetic evidence is presented to show that the mechanism of chloride stabilization involves the attack of chloride ion on the unstable cyclic ethyleneimmonium intermediate to give back I. Implications of the kinetic data obtained on the fate of orally administered I are discussed.

Antimicrobial activity of neomycin C against Staphylococcus epidermidis.

In vitro studies indicate that neomycin C is 50% as active as neomycin B against Staphylococcus epidermidis (ATCC 12228).