Survey of antenatal women's knowledge of maternal and fetal risks of tobacco smoking and acceptability of nicotine replacement products in pregnancy.

A survey was carried out of 145 pregnant women in the third trimester in pregnancy to assess motivators to stop tobacco smoking and assess women's knowledge of fetal and maternal risk of smoking. In addition, the survey was to assess the acceptability of nicotine replacement products use in pregnancy. The findings of this survey show that pregnant women tend to know about the maternal risks of smoking but their knowledge is deficient about fetal risks. The knowledge of the association of cot death risk and tobacco smoking appears to be the greatest motivator to stop smoking. Overall, 74% wished to stop smoking in pregnancy and 68% would accept a nicotine replacement product.

Metabolic disposition and pharmacokinetics of [14C]-amprenavir, a human immunodeficiency virus type 1 (HIV-1) protease inhibitor, administered as a single oral dose to healthy male subjects.

The objective of this study was to determine the metabolic profile, routes of elimination, and total recovery of amprenavir and its metabolites after a single oral dose of [14C]-amprenavir. Six healthy male subjects each received a single oral 630 mg dose of amprenavir containing 95.76 microCi of [14C]-amprenavir in this Phase I mass balance study. The metabolic disposition of amprenavir was determined through analyses of radiocarbon in whole blood, plasma, urine, and stool samples, collected for a period of 10 to 17 days postdosing. Cerebral spinal fluid (CSF) sampling was conducted on day 1. The ratio of unchanged amprenavir AUC0-->infinity to plasma radiocarbon was 27%, suggesting that most of the radiocarbon was metabolites. The median total recovery of the administered dose of radiocarbon was 89% (range: 66%-93%), with 75% (range: 56%-80%) recovered in the feces and 14% (range: 10%-17%) in the urine. Most of the recovered radiocarbon in the feces and urine was excreted within 240 and 48 hours postdose, respectively. Of the 75% of the radiocarbon dose recovered in the feces, 62% was identified as a metabolite resulting from dioxidation of the tetrahydrofuran ring (GW549445X) and 32% as a metabolite resulting from subsequent oxidation of the p-aniline sulfonate group (GW549444X). Unchanged amprenavir was below the limit of quantitation in feces and urine. Therefore, approximately 94% of the dose excreted in the feces was accounted for by these two metabolites. Concentrations of radiocarbon in the CSF were below the limit of quantitation in 5 of 6 subjects sampled. In summary, oral amprenavir is extensively metabolized in humans, with concentrations of unchanged drug below the limits of quantitation in urine and feces. The majority (75%) of administered radiocarbon was excreted in feces.

Induction of P-glycoprotein and cytochrome P450 3A by HIV protease inhibitors.

P-Glycoprotein (Pgp) and cytochrome P450 3A (CYP3A) are important enzymes affecting the disposition of HIV protease inhibitors (HIV PIs). After multiple dosing experiments in rats, decreases in the plasma concentrations and area under plasma concentration-time curve (AUC) for HIV PIs have been observed. The purpose of these studies was to determine the changes in Pgp and CYP3A expression and HIV PI plasma exposure after multiple doses of HIV PIs. Male rats were orally dosed with an amprenavir prodrug (450 mg/kg/day amprenavir-equivalent) or nelfinavir (175 mg/kg/day) for 1 or 14 days. Relative to day 1, the C(max) and the AUC for amprenavir at day 14 were decreased by 33 and 51%, respectively. Similarly, the plasma concentration of nelfinavir at 1 h after the last dose (C(max)) was reduced by 52% after multiple doses. Compared with controls, dosing of amprenavir for 14 days increased intestinal Pgp and hepatic CYP3A protein levels by 59 and 151%, respectively, but did not alter intestinal CYP3A protein levels. In contrast, amprenavir treatment did not result in an increase in hepatic CYP3A activity. Nelfinavir treatment increased expression of intestinal Pgp and hepatic CYP3A levels by 83 and 85%, respectively, but not hepatic Pgp or intestinal CYP3A. HIV PIs also induced Pgp expression in the LS174T human intestinal cell line. These results indicate that HIV protease inhibitors induce both intestinal Pgp and hepatic CYP3A and suggest that induction of Pgp and CYP3A is a possible mechanism reducing drug exposure after multiple doses.

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 (

Role of P-glycoprotein on the CNS disposition of amprenavir (141W94), an HIV protease inhibitor.

PURPOSE: To determine the role of P-glycoprotein (Pgp) on the CNS penetration of the HIV protease inhibitor (PI) amprenavir (141W94) and to test the hypothesis that co-administration of a second HIV PI (ritonavir) could enhance amprenavir's brain penetration in vivo. METHODS: Pgp-mediated efflux was investigated in vitro with Caco-2 cells and in vivo by whole-body autoradiography (WBA). "Genetic" mdr1a/1b double knockout mice, "chemical" Pgp knockout mice generated by administration of the Pgp inhibitor GF120918, and mice pretreated with ritonavir were used in WBA studies to investigate the effects of Pgp modulation on the CNS penetration of amprenavir. RESULTS: Amprenavir, indinavir, ritonavir, and saquinavir had 2- to 23-fold higher transport rates from the basolateral to apical direction than from the apical to basolateral direction across Caco-2 monolayers. Incubation with GF120918 negated this difference, suggesting that the efflux was Pgp-mediated. WBA studies demonstrated a 13- and 27-fold increase in the brain and a 3.3-fold increase in the CSF concentrations of amprenavir in mice pretreated with GF120918 and in mdr1a/1b double knockout mice. In contrast, pretreatment with ritonavir did not alter the CNS exposure of amprenavir. CONCLUSIONS: These results provide evidence that amprenavir and other HIV PIs are Pgp substrates and that co-administration of a specific Pgp inhibitor will enhance amprenavir's CNS penetration in vivo. These results will have an important therapeutic impact in the treatment of AIDS dementia.

Atovaquone and proguanil hydrochloride: a review of nonclinical studies.

BACKGROUND: Safe and effective antimalarial drugs are needed for treatment and prophylaxis of malaria. The combination of atovaquone and proguanil hydrochloride is a new antimalarial drug combination that has recently become available in many countries. METHODS: Data were reviewed from nonclinical studies evaluating the microbiology, secondary pharmacology, pharmacokinetics, and toxicology of atovaquone and proguanil hydrochloride. RESULTS: Atovaquone is highly active against asexual erythrocytic stages of Plasmodium falciparum in vitro (IC50 0.7-6 nM) and in animal models. Proguanil per se has only weak antimalarial activity in vitro (IC50 2.4-19 microM), and its effectiveness depends on the active metabolite cycloguanil (IC50 0.5-2.5 nM). The combination of atovaquone and proguanil is synergistic in vitro. Both drugs also have activity against gametocytes and pre-erythrocytic (hepatic) stages of malaria parasites. Atovaquone is a ubiquinone antagonist that inhibits mitochondrial electron transport and collapses mitochondrial membrane potential. The proguanil metabolite cycloguanil is a dihydrofolate reductase inhibitor, but the mode of action of proguanil is unknown. In screening evaluations of secondary pharmacology, neither atovaquone nor proguanil had activity that adversely affected gastrointestinal, cardiovascular, or central or autonomic nervous system functions at clinically relevant concentrations. After oral administration, atovaquone exposure is extensive in rats but limited in dogs, while proguanil and cycloguanil exposure is extensive in dogs but limited in rats. In both species, toxicity was related to proguanil exposure, the principal manifestations being salivation, emesis, and loss of body weight. Neither atovaquone nor proguanil was teratogenic or mutagenic. An increased incidence of hepatic adenomas and adenocarcinomas was seen in mice, but not rats, after lifetime exposure to atovaquone, and appears to be related to species-specific differences in hepatic enzymatic activity. No additional toxicity was evident in animals treated with the combination of atovaquone and proguanil hydrochloride compared to those treated with either drug alone. CONCLUSION: Nonclinical studies of atovaquone and proguanil hydrochloride supported the clinical development of this combination for treatment and prophylaxis of malaria.

Effects of aerosolized synthetic surfactant, atovaquone, and the combination of these on murine Pneumocystis carinii pneumonia.

An immunosuppressed rat model was used to determine the pharmacokinetics of aerosolized atovaquone (administered with and without a synthetic surfactant) and to evaluate the efficacy of inhaled atovaquone in the prevention and treatment of Pneumocystis carinii pneumonia (PCP). After a single dose by aerosol, mean peak concentrations of atovaquone averaged 52 microg/mL in plasma and 31 microg/g in lungs of rats infected with P. carinii. When atovaquone was combined with surfactant, mean peak concentrations of 94 microg/mL in plasma and 51 microg/g in lung were achieved. Aerosolized synthetic surfactant alone significantly increased survival of rats with PCP and, when combined with atovaquone, increased plasma and lung concentrations of the drug and eradication of the organism.

The pharmacokinetics of 1954U89, 1,3-diamino-7-(1-ethylpropyl)-8-methyl-7H-pyrrolo-(3,2-f)quinazoline, in dogs and rats after intravenous and oral administration.

1954U89, 1,3-diamino-7-(1-ethylpropyl)-8-methyl-7H-pyrrolo-(3, 2-f)quinazoline, is a potent, lipid-soluble inhibitor of dihydrofolate reductase. The pharmacokinetics and bioavailability of 1954U89 were examined in male beagle dogs and male CD rats. Dogs received single intravenous (2.5 mg kg-1) and oral (5.0 mg kg-1) doses of 1954U89 with and without successive administration of calcium leucovorin. Single intravenous (5.0 mg kg-1) and oral (10 mg kg-1) doses of [1,3-14C2]1954U89 were administered to rats. Plasma concentrations of total radiocarbon were determined by scintillation counting, and intact 1954U89 was measured by HPLC. The mean plasma half-life was 3.2 +/- 0.62 and 4.2 +/- 0.68 h after intravenous and oral administration, respectively, to dogs. The pooled plasma half-life after intravenous administration to rats averaged 1.2 h; a reliable plasma half-life value after oral administration could not be determined. Mean total-body clearance was 2.4 +/- 0.39 and 4.5 +/- 1.1 L h-1 kg-1 after intravenous and oral administration, respectively, to dogs, and averaged 12 and 77 L h-1 kg-1 after intravenous and oral administration, respectively, to rats. Neither clearance nor bioavailability of 1954U89 in dogs was affected significantly by administration of calcium leucovorin. Absolute bioavailability was 54 +/- 12% in dogs and 16% in rats.

A robotics-based liquid chromatographic assay for the measurement of atovaquone in plasma.

A precise and specific robotics-based liquid chromatographic (LC) method for measuring atovaquone concentrations in plasma was developed and validated, and the method was compared with an existing manual LC method. The compound was isolated from plasma by liquid-liquid extraction, separated by reversed-phase LC, and quantitated against an internal standard with UV detection. Least-squares linear regression with 1/concentration2 weighting was used as the calibration model. The range of the calibration curve for the assay under routine conditions was 0.25-50 micrograms ml-1. No endogenous interferences with the compound or the internal standard were noted in either untreated human plasma or in plasma from patients enrolled in Phase III clinical trials of atovaquone. The accuracy of the assay (determined as the percent bias) ranged from -4.8% to -9.4% in the validation runs. The intra- and interassay precisions (determined as the relative standard deviation) were less than 6.8% and 6.4%, respectively. The contribution of an internal standard on assay accuracy and precision also was examined. Interassay variability was marginally improved by the incorporation of an internal standard to the assay; accuracy and intra-assay precision were essentially unchanged. A paired t-test between estimates of atovaquone concentrations in healthy volunteer and HIV + patient human plasma samples assayed by the automated and manual methods demonstrated no significant difference (p = 0.31) between the values determined by each method.

High-performance liquid chromatographic assay for the measurement of atovaquone in plasma.

A rapid and efficient isocratic high-performance liquid chromatographic assay for the measurement of atovaquone in plasma has been developed and validated. The drug was extracted from plasma with organic solvents, assayed on a C1 column with a mobile phase of methanol-0.1% acetic acid (70:30, v/v), and detected by ultraviolet absorbance at 254 nm. Recovery of atovaquone from plasma was greater than 85%. Intra- and inter-assay variability were less than 8%, and the average accuracy of the assay (expressed as % bias) ranged from -7.4 to + 2.2%. The upper and lower limits of quantitation were 100 and 0.25 microgram/ml, respectively. Measurement of atovaquone in spiked plasma control samples during routine runs of clinical trial samples confirmed the reliability of the assay.

The disposition and metabolism of [14C]piritrexim in dogs after intravenous and oral administration.

The disposition of [14C]piritrexim ([14C]PTX) in male dogs after iv and po doses of 1.8 mg/kg was examined. After either route of administration, greater than 90% of the dose was recovered in the exreta within 72 hr; approximately 20% was recovered in urine and 70% in feces. [14C]PTX was extensively metabolized by dogs; unchanged drug accounted for less than 15% of the dose in the excreta. The O-demethylated metabolites, 2'- and 5'-demethyl PTX, the glucuronide conjugate of 2'-demethyl PTX, and the sulfate conjugate of 5'-demethyl PTX were the major metabolites. Unchanged drug accounted for a large proportion of the drug-related radiocarbon in plasma. The average plasma half-life of PTX after iv administration was 2.6 +/- 0.3 hr, and the average total body clearance was 0.33 +/- 0.13 liter/hr/kg. After po administration, peak plasma concentrations of 0.9 +/- 0.3 micrograms/ml occurred about 1.1 hr after the dose; the absolute oral bioavailability of PTX was 0.63 +/- 0.14. Because the O-demethyl metabolites were active dihydrofolate reductase inhibitors, 2'- and 5'-demethyl PTX were synthesized, and the pharmacokinetics and bioavailability of these compounds in dogs after iv and po administration (5 mg/kg) were examined. The plasma concentration-time data for both compounds after iv doses were described by a two-compartment model, with t1/2 beta = 1.3 and 0.8 hr for the 2'- and 5'- demethyl compounds, respectively. Neither compound showed significant advantages over PTX in terms of pharmacokinetics or bioavailability.

The disposition and metabolism of [14C]piritrexim in rats after intravenous and oral administration.

The disposition of [14C]piritrexim in male rats after iv (5 and 10 mg/kg) and po (5, 10, and 20 mg/kg) doses was studied. After an iv dose of 10 mg/kg, rats excreted an average of 57% of the dose in feces and 32% in urine; after a po dose of 10 mg/kg, 84% of the dose was excreted in feces and 9% in urine. After iv doses, the elimination of unchanged drug from plasma was first order, with a t1/2 of 0.6 hr; at any time point, unchanged drug accounted for less than 50% of the total radiocarbon in the plasma. Oral bioavailability of unchanged drug was less than 5%. O-Demethylation and subsequent conjugation were the main pathways of metabolism; the demethyl metabolites of piritrexim were potent inhibitors of dihydrofolate reductase and were cytotoxic to cells in culture. Concentrations of radiocarbon were highest in liver 24 hr after an iv dose, but less than 1% of the radiocarbon was unchanged drug. Concentrations of radiocarbon in liver after po doses were approximately 40% of those attained after equivalent iv doses.

Disposition, metabolism, and excretion of the anticancer agent crisnatol in the rat.

Disposition and metabolism of crisnatol (14C-labeled), a novel antitumor agent, was examined after po and iv administration to rats. After both routes of drug administration, there was rapid elimination of the administered radioactivity in the urine (6-12% of the dose) and feces (81-92% of the dose). The drug appeared to be rapidly absorbed after oral dose and there was substantial "first-pass" metabolism. Analysis of the excreta indicated extensive metabolism of crisnatol by the rat, with the intact compound being the major radiolabeled component in the feces (17-20% of dose). Intact drug was not present in urine. Biotransformation of crisnatol by the rat mainly involves oxidation and conjugation pathways. Hydroxylation and dihydrodiol formation in the chrysene ring and oxidation of the propanediol side chain resulted in the formation of the three major fecal metabolites. The principal metabolite in the urine was also a dihydrodiol. Concentrations of intact drug in each tissue assayed exceeded those in plasma, and in the lungs the tissue/plasma ratio approached 300 and 82 at 2 hr after iv and po doses, respectively.

Failure of trimethoprim-sulfamethoxazole therapy in experimental enterococcal endocarditis.

To assess the potential efficacy of trimethoprim-sulfamethoxazole (TMP-SMX) against serious enterococcal infections, we used a rat enterococcal endocarditis model comparing TMP-SMX therapy (500 mg of TMP plus 2,500 mg of SMX per kg of body weight per day given every 8 h by intragastric gavage) with intravenous ampicillin therapy (1,000 mg/kg per day). Despite concentrations of active drug in serum well in excess of the MIC and MBC, the mean residual vegetation bacterial titer in TMP-SMX-treated rats was similar to that in untreated controls (8.4 +/- 1.1 versus 8.6 +/- 1.3 log10 CFU/g) and significantly higher than that in the ampicillin-treated group (3.6 +/- 1.5 log10 CFU/g; P less than or equal to 0.001). This demonstrates discordance between in vitro activity and in vivo efficacy of TMP-SMX in serious enterococcal infection.

High-performance liquid chromatographic assay for the simultaneous measurement of trimethoprim and sulfamethoxazole in plasma or urine.

Procedures for the simultaneous determination of trimethoprim (TMP) and sulfamethoxazole (SMX) in plasma or urine are reported. The drugs are extracted from plasma or urine by a single solid-phase extraction and quantitated by high-performance liquid chromatography. Both drugs are analyzed in the same chromatographic run. Intra- and interassay variability are less than 10% for both compounds, and the recovery and precision of TMP measurement are unaffected by concurrent SMX concentrations. Limits of quantitation for TMP and SMX in plasma were 0.02 and 0.21 microgram/ml, respectively. In urine, the limit of quantitation for both drugs was 1.0 microgram/ml. Metabolites of TMP and SMX did not interfere with the assay. Pharmacokinetic parameters from volunteers given two formulations of co-trimoxazole in a crossover comparison study are reported.

A phase I clinical and pharmacological study of weekly intravenous infusions of piritrexim (BW301U).

Thirty-eight patients with advanced resistant cancers were enrolled on this study of piritrexim (PTX; BW 301U) administered intravenously weekly for 4 weeks. Of 50 courses of treatment begun, 39 evaluable 4-week courses of the drug were completed by this group of patients. Dosages ranged from 44 to 530 mg/m2/week. One patient at each dosage level received an initial weekly dose of PTX in oral form accompanied by pharmacokinetic blood sampling after the oral dose and also after a subsequent intravenous dose. Toxicities included mild nausea and vomiting, and moderate to severe peripheral vein phlebitis. Anemia and thrombocytopenia were the dominant hematological toxicities. One patient with pulmonary metastases from malignant fibrous histiocytoma experienced a 12-week partial response to PTX treatment at a dosage of 400 mg/m2/week. Pharmacokinetic analysis of plasma for PTX concentrations was accomplished utilizing a competitive protein binding assay. The estimated total body clearance ranged from 136 to 173 ml/min/1.73 m2. Mean terminal half-life after intravenous administration was 5.61 +/- 2.38 h (S.D.), and after oral administration was 5.72 +/- 2.04 h. Mean systemic bioavailability after oral administration was 75 +/- 56%.

Competitive protein binding assay for piritrexim.

A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with [125l]methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found. published HPLC method was found.

Modulation of hematoporphyrin derivative-sensitized phototherapy with corynebacterium parvum in murine transitional cell carcinoma.

The interaction of photodynamic therapy (PDT) with hematoporphyrin derivative (Hpd) and immunotherapy with Corynebacterium parvum (CP) was studied in a murine transitional cell carcinoma (MBT-2) model. C3H/He mice were transplanted subcutaneously in the hind limb with 2.5 X 10(5) tumor cells. One day after transplantation, mice were randomized into groups to receive saline (control), PDT, CP 25 micrograms, CP 250 micrograms, CP 25 micrograms + PDT, and CP 250 micrograms + PDT. PDT was administered by intraperitoneal (IP) injection of Hpd (12.5 micrograms/g body weight), followed twenty-four hours later by photoirradiation. CP was given intralesionally at the same time as IP injection of Hpd (24 hours before photoirradiation). A low dose of CP (25 micrograms) was shown to enhance the effect of PDT while PDT reduced the benefit obtained with high dose of CP (250 micrograms). In a second series of experiments, CP (250 micrograms) treatment after photoirradiation was shown to give significantly greater benefit than CP treatment before photoirradiation. The study thus indicates that the effectiveness of combined immunophototherapy is dependent on the sequence of the combination and its intricate relationship with the dosage of CP. The enhancement of PDT by low dose of CP in this model suggests the usefulness of this combined immunophototherapy in enhancing tumor control and in lessening deleterious side effects.

Phagocytic and natural killer cytotoxic responses of murine transitional cell carcinoma to postsurgical immunochemotherapy.

Postsurgical immunochemotherapy with Corynebacterium parvum (CP) and cis-diamminedichloroplatinum (II) (CDDP) was evaluated in mice with transitional cell carcinoma (MBT-2). C3H/He mice were transplanted subcutaneously in the hind limb with 5 x 10(5) tumor cells. Ten to 14 days later when the tumor reached a diameter of five to seven mm., it was surgically removed. Mice were then randomized into four groups to receive a total of three treatments on days 1, 3 and 5 after surgery: 1) saline (control group); 2) CP, 250 micrograms. into the surgical site; 3) CDDP, 5 micrograms./gm. body weight intraperitoneally; and 4) combined CP and CDDP. Recurrence of tumor occurred in 70%, 52%, 55% and 28% of mice receiving surgery only, CP, CDDP, and combined CP and CDDP respectively. In the second part of the experiment, phagocytic activity using chemiluminescence assay and natural killer (NK) activity using chromium-51 release assay were determined with cells from the peritoneum, spleen and inguinal lymph nodes. CP or CDDP alone enhanced the phagocytic and NK activity. The most significant enhancement was obtained with cells from the inguinal lymph nodes of mice receiving combined CP and CDDP, the group with the lowest tumor recurrence. These results suggest that combination of CP and CDDP may be useful in control of postsurgical recurrence of bladder cancer.

Effect of the calcium entry blocker verapamil on renal ischemia.

The ability of the calcium entry blocker verapamil to ameliorate the effects of renal ischemia was studied in ten sheep. Postanesthesia, bilateral cutaneous ureterostomies were placed in each sheep to facilitate urine collection and analysis. Both kidneys were made ischemic for one hour by occluding each renal artery. However, immediately before occlusion of the right renal artery, 0.05 mg/kg of verapamil was injected into the artery. Comparison of urinary creatinine excretion and urine volume for 72 h after reversal of ischemia demonstrated that those kidneys pretreated with verapamil had greater functional preservation (p less than .05). In this study, verapamil appeared to provide protection against renal damage after an ischemic insult.