Safety, Tolerability and Pharmacokinetics of Icapamespib, a Selective Epichaperome Inhibitor, in Healthy Adults.

BACKGROUND: Neurodegenerative diseases are devastating conditions that most commonly affect individuals 65 years and older. Currently there are no effective treatments or cures for neurodegenerative diseases, and therapeutics that selectively target the underlying causes of these diseases are needed. Epichaperomes play a major role in the maintenance and progression of neuronal pathology. Inhibiting epichaperomes induces degradation of disease associated proteins and is a promising therapeutic approach to treat neurodegenerative diseases, in particular Alzheimer's Disease and amyotrophic lateral sclerosis. OBJECTIVES: This Phase 1 clinical study evaluated the safety, tolerability, pharmacokinetics, and bioavailability of icapamespib, a purine scaffold inhibitor of epichaperomes that is specific to epichaperomes, in healthy subjects. DESIGN: Double-blind, placebo-controlled dose escalating single ascending dose and multiple ascending doses and an unblinded two-period cross-over bioavailability study design. SETTING: Single site in the United States. PARTICIPANTS: Healthy men or women of 18 to 60 years of age, inclusive, for Part 1 (single ascending dose), ≥ 60 years of age for Part 2 (multiple ascending dose), or 18 to 49 years of age for Part 3 (bioavailability). TREATMENT: In the single ascending dose group, oral single doses (10, 20, and 30 mg icapamespib or placebo) were administered to healthy non-elderly subjects. In the multiple ascending dose group, multiple doses (20 and 30 mg icapamespib once daily for 7 days or placebo) were administered to healthy elderly subjects. In the bioavailability group, the bioavailability of once daily oral icapamespib solution and tablet was assessed in healthy non elderly subjects. MEASUREMENTS: Safety was evaluated based on assessments of treatment-emergent adverse events, physical examinations, clinical laboratory tests (hematology, clinical chemistry, and urinalysis), vital signs, and 12-lead electrocardiograms. Icapamespib concentration was evaluated in plasma and cerebrospinal fluid, the latter in Part 2 (multiple ascending dose) only. RESULTS: Forty-eight subjects in total were randomized and assessed for tolerability, pharmacokinetics, and bioavailability parameters as follows: 24 subjects in Part 1 (single ascending dose) with PU-AD 10 mg (n = 6), 20 mg (n = 6), 30 mg (n = 6), and placebo (n = 6); 16 subjects in Part 2 (multiple ascending dose) with icapamespib 20 mg (n = 6), 30 mg (n = 6), and placebo (n = 4); and 8 subjects in Part 3 (bioavailability) crossed-over between icapamespib 30 mg (tablet) and icapamespib 30 mg (oral solution). Single doses of icapamespib up to 30 mg and multiple doses of icapamespib up to 30 mg for 7 days were generally safe and well tolerated in healthy non-elderly and elderly subjects. Treatment-emergent adverse events were mild, with headache being the most common treatment-emergent adverse event. Mean icapamespib exposure (area under the curve) was dose-proportional over the dose range tested. The median time to maximum observed plasma concentration ranged from 1.00 to 2.00 h across single ascending dose, multiple ascending dose, and bioavailability groups; icapamespib exposure was 50% higher in elderly subjects compared with non-elderly subjects but was well tolerated. CONCLUSIONS: The study provides clinical evidence of the safety of icapamespib in healthy non elderly and elderly subjects and supports the advancement of icapamespib to Phase 2 evaluation in Alzheimer's Disease and other neurodegenerative diseases.

Population pharmacokinetic analysis of ropivacaine and its metabolite 2',6'-pipecoloxylidide from pooled data in neonates, infants, and children.

BACKGROUND: The aim was to characterize ropivacaine and 2',6'-pipecoloxylidide (PPX) pharmacokinetics and factors affecting them in paediatric anaesthesia. METHODS: Population pharmacokinetics of ropivacaine and its active metabolite PPX were estimated after single and continuous ropivacaine blocks in 192 patients aged 0-12 yr from six pooled published studies. Unbound and total ropivacaine and PPX plasma concentration and PPX urinary excretion data were used for non-linear mixed-effects modelling by NONMEM. Covariates included age, body weight, gender, ethnic origin, ASA, site and method of administration, and total dose. RESULTS: One-compartment first-order pharmacokinetic models incorporating linear binding of ropivacaine and PPX to α(1)-acid glycoprotein were used. After accounting for the effect of body weight, clearance of unbound ropivacaine and PPX reached 41% and 89% of their mature values, respectively, at the age of 6 months. Ropivacaine half-life decreased with age from 13 h in the newborn to 3 h beyond 1 yr. PPX half-life differed from 19 h in the newborn to 8-11 h between 1 and 12 months to 17 h after 1 yr. Simulations indicate that for a single caudal block, the recommended dose could be increased by a factor of 2.9 (0-1 month group) and 6.3 (1-12 yr group) before the unbound plasma concentrations would cross the threshold for systemic toxicity. Corresponding factors for continuous epidural infusion are 1.8 and 4.9. CONCLUSIONS: Ropivacaine and PPX unbound clearance depends on body weight and age. The results support approved dose recommendations of ropivacaine for the paediatric population.

The genetics of isolated and syndromic clubfoot.

PURPOSE: Congenital clubfoot is a serious birth defect that affects nearly 0.1% of all births. Though there is strong evidence for a genetic basis of isolated clubfoot, aside from a handful of associations, much of the heritability remains unexplained. METHODS: By systematically examining the genes involved in syndromic clubfoot, we may find new candidate genes and pathways to investigate in isolated clubfoot. RESULTS: In addition to the expected enrichment of extracellular matrix and transforming growth factor beta (TGF-ß) signalling genes, we find many genes involved in syndromic clubfoot encode peroxisomal matrix proteins, as well as enzymes necessary for sulfation of proteoglycans, an important part of connective tissue. Further, the association of Filamin B with isolated clubfoot as well as syndromic clubfoot is an encouraging finding. CONCLUSION: We should examine these categories for enrichment in isolated clubfoot patients to increase our understanding of the underlying biology and pathophysiology of this deformity. Understanding the spectrum of syndromes that have clubfoot as a feature enables a better understanding of the underlying pathophysiology of the disorder and directs future genetic screening efforts toward certain genes and genetic pathways. LEVEL OF EVIDENCE: V.

Pharmacokinetics and safety of amprenavir and ritonavir following multiple-dose, co-administration to healthy volunteers.

OBJECTIVE: To evaluate the safety and pharmacokinetic interaction between amprenavir (APV) and ritonavir (RTV). METHODS: Three open-label, randomized, two-sequence, multiple-dose studies having the same design (7 days of APV or RTV alone followed by 7 days of both drugs together) used 450 or 900 mg APV with 100 or 300 mg RTV every 12 h with pharmacokinetic assessments on days 7 and 14. Safety was monitored as clinical adverse events (AEs) and laboratory abnormalities. RESULTS: Relative to APV alone, RTV co-administration resulted in a 3.3- to 4-fold and 10.84 to 14.25-fold increase in the geometric least-square (GLS) mean area under the plasma concentration--time curve (AUC(tau,ss)) and minimum concentration (C(min,ss)), respectively. APV 900 mg with RTV 100 mg resulted in a 2.09-fold and 6.85-fold increase in the GLS mean AUC(tau,ss) and C(min,ss), respectively. On day 14, the geometric mean (95% confidence interval) for 450 mg APV AUC(tau,ss) (micro x h/mL) was 23.49 (19.32--28.57) with 300 mg RTV and 35.42 (30.46--44.42) with 100 microg RTV, and for the 900 mg APV with 100 mg RTV 47.11 (39.47--61.24). The 450 mg APV C(min,ss) (microg/ml) were 1.32 (1.05--1.67) and 2.01 (1.70--2.61), and 2.47 (2.08--3.32) for 900 mg APV. The most common AEs were mild and included diarrhea, nausea/vomiting, oral parasthesias, and rash. The triglyceride and cholesterol increased significantly from RTV exposure. CONCLUSION: Adding RTV to APV resulted in clinically and statistically significant increases in APV AUC and C(min) with variable effects on maximum concentration. The two RTV doses had similar effects on APV but AEs were more frequent with 300 mg RTV.

Atovaquone inhibits the glucuronidation and increases the plasma concentrations of zidovudine.

The pharmacokinetic interaction between atovaquone, a 1,4-hydroxynaphthoquinone, and zidovudine was examined in an open, randomized, three-phase crossover study in 14 patients infected with human immunodeficiency virus. Atovaquone (750 mg every 12 hours) and zidovudine (200 mg every 8 hours) were given orally alone and in combination. Atovaquone significantly increased the area under the zidovudine concentration-time curve (AUC) (1.82 +/- 0.62 micrograms.hr/ml versus 2.39 +/- 0.68 micrograms.hr/ml; p < 0.05) and decreased the oral clearance of zidovudine (2029 +/- 666 ml/min versus 1512 +/- 464 ml/min; p < 0.05). In contrast, atovaquone tended to decrease the AUC of zidovudine-glucuronide (7.31 +/- 1.51 micrograms.hr/ml versus 6.89 +/- 1.42 micrograms.hr/ml; p < 0.1) and significantly decreased the ratio of AUC zidovudine-glucuronide/AUC zidovudine (4.48 +/- 1.94 versus 3.12 +/- 1.1; p < 0.05). The maximum concentration of zidovudine-glucuronide was significantly lowered by atovaquone (5.7 +/- 1.5 versus 4.57 +/- 0.97 micrograms/ml; p < 0.05). Zidovudine had no effect on the pharmacokinetic disposition of atovaquone. Atovaquone appears to increase the AUC of zidovudine by inhibiting the glucuronidation of zidovudine.

Metabolism, disposition, and kinetics of delta-9-tetrahydrocannabinol in men and women.

A comparative study was done in women and men of the effects of delta 9-tetrahydrocannabinol (delta 9-THC), intravenously or orally, on dynamic activity, metabolism, excretion, and kinetics. In general no differences between the two sexes were observed. delta 9-THC is converted by microsomal hydroxylation to 11-hydroxy-delta 9-THC (11-OH-delta 9-THC), which is both a key intermediate for further metabolism to 11-nor-delta 9-THC-9-carboxylic acid (11-nor-acid) by liver alcohol-dehydrogenase enzymes and a potent psychoactive metabolite. Major differences in the ratio of the concentration of 11-OH-delta 9-THC to that of delta 9-THC in plasma were found after intravenous dosing (ratio 1:10 to 20) compared with oral administration (ratio 0.5 to 1:1). The final metabolic products are the 11-nor-acids and the related, more polar acids. Urinary excretion of delta 9-THC is restricted to acidic nonconjugated and conjugated metabolites. After 72 hr mean cumulative urinary excretion, noted for both routes and for both sexes, ranged from 13% to 17% of the total dose. After 72 hr the cumulative fecal excretion for both sexes after intravenous administration ranged from 25% to 30%; after oral administration the range was 48% to 53%. Metabolites were found in the feces in large concentration in the nonconjugated form; concentrations of 11-OH-delta 9-THC were particularly noteworthy. Kinetics of delta 9-THC and metabolites were much the same for female and male subjects. For delta 9-THC, terminal-phase t1/2s for both sexes, irrespective of the route, ranged from 25 to 36 hr. A comparison of the results for AUC/dose (delta 9-THC) after oral dosing with comparable data from intravenous administration indicated bioavailability of the order of 10% to 20% for both sexes. After intravenous delta 9-THC, large apparent volumes of distribution were noted (about 10 l/kg for both sexes).

Three-dimensional quantitative structure-activity relationship study of nonsteroidal estrogen receptor ligands using the comparative molecular field analysis/cross-validated r2-guided region selection approach.

A newly developed comparative molecular field analysis (CoMFA) technique, the cross-validated r2-guided region selection (CoMFA/q2-GRS) method, has been used to build a quantitative structure-activity relationship (3D-QSAR) for nonsteroidal estrogen receptor (ER) ligands. Ligands included in this study belong to a series of diethylstilbestrol (DES) and indenestrol analogues whose affinities for the mouse ER (mER) have been determined in our laboratory. The final model utilized 30 compounds and yielded a q2GRS (cross-validated r2, guided region selection) of 0.796, as compared to a q2 of 0.720 for conventional CoMFA, with a standard error of prediction of 0.594 at 3 principal components. This model was used to visualize steric and electrostatic features of the ligands that correspond with ER binding affinity. Results obtained from the CoMFA steric and electrostatic plots of this model have also been compared to information from the ER binding affinities of substituted estradiol analogues. This is in an effort to determine structural features of compounds in the CoMFA analysis that may correspond to those of the estradiol analogues and to further clarify the mode of binding of nonsteroidal ER ligands.

In vitro antiviral activity of 141W94 (VX-478) in combination with other antiretroviral agents.

141W94 (VX-478) is a novel HIV-1 protease inhibitor with an IC50 of 0.08 microM against HIV-1 (strain IIIB) and a mean IC50 of 0.012 microM against six HIV clinical isolates. 141W94 was synergistic on the basis of isobologram analysis with each of the following reverse transcriptase inhibitors: AZT, 935U83, 524W91, 1592U89 and ddl, 141W94 was also synergistic with saquinavir and additive with either indinavir or ritonavir. Resistance to 141W94 has been reported in vitro passage experiments. The binding of 141W94 to human alpha 1-acid glycoprotein was relatively weak (Kd = 4 microM) and the off-rate for the drug is very fast (> or = 100 s-1). Only a 2-fold reduction of in vitro antiviral activity was observed in the presence of 45% human plasma. No serious drug associated adverse experiences were reported in a Phase I placebo-controlled, single-dose escalation, pharmacokinetic and safety study. The average concentration of 141W94 at 8 and 12 h after single doses of 900 and 1200 mg, respectively, was in excess of 10 times the IC50. As 141W94 is synergistic with a variety of anti-HIV-1 agents and exhibits a unique cross resistance profile compared to other protease inhibitors, 141W94 is considered a good candidate for combination therapy.

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.

Bivariate assay for cleaved atrial natriuretic factor (ANF(101-105/106-126)) in sheep plasma.

A bivariate assay has been developed to measure both ANF, and cleaved ANF (ANF(101-105/106-126)) the primary product of endopeptidase-24.11 action on rat ANF(101-126). The assay employed two different antisera directed to the carboxy-terminal and ring regions of ANF to enable discrimination and measurement of intact endogenous ANF(99-126) and infused cleaved ANF (rat ANF(101-105/106-126)) in sheep plasma extracts. The assay which was validated by HPLC analysis of plasma extracts, had sufficient accuracy and precision to measure the metabolic clearance rate and half-life of cleaved ANF when infused in normal sheep.

Efficacy and pharmacokinetics of atovaquone and proguanil in children with multidrug-resistant Plasmodium falciparum malaria.

A trial was conducted in 32 Thai children with uncomplicated multidrug-resistant falciparum malaria to assess the efficacy, safety and pharmacokinetics of atovaquone and proguanil; plasma concentrations of atovaquone, proguanil and its metabolite, cycloguanil, were measured in a subset of 9 children. The children received atovaquone (17 mg/kg/d for 3 d) plus proguanil (7 mg/kg/d for 3 d). Twenty-six children who had only Plasmodium falciparum infection and remained in hospital for 28 d were assessed for drug efficacy. The combination regimen produced a cure rate of 100%. Parasite and fever clearance times were 47 h (range 8-75) and 50 h (range 7-111), respectively. Atovaquone and proguanil were rapidly absorbed, with median time to peak concentrations of 6 h (range 6-24) and 6 h (range 6-12), respectively. Peak concentrations of cycloguanil were achieved between 6 and 12 h (median 6) after administration of proguanil. Mean peak plasma concentration of atovaquone on day 3 was 5.1 micrograms/mL (SD = 2.1). The day 3 mean peak plasma concentration of proguanil was 306 ng/mL (SD = 108) compared with 44.3 ng/mL (SD = 27.3) for cycloguanil. Mean values for the AUC (area under plasma concentration-time curve) were 161.8 micrograms/mL.h (SD = 126.9) for atovaquone, 4646 ng/mL.h (SD = 1226) for proguanil, and 787 ng/mL.h (SD = 397) for cycloguanil. Terminal elimination half-lives of atovaquone, proguanil and cycloguanil were estimated as 31.8 h (SD = 8.9), 14.9 h (SD = 3.3) and 14.6 h (SD = 2.6), respectively. No major adverse effect was attributable to the study drugs. Atovaquone/proguanil combination is safe and highly effective, and should be especially valuable for treatment of multidrug-resistant falciparum malaria.

Prolonged, but not diminished, zidovudine absorption induced by a high-fat breakfast.

STUDY OBJECTIVE: To determine the effect of a high-fat breakfast on single-dose, zidovudine (ZDV) pharmacokinetics. DESIGN: Open-label, randomized, crossover study. PATIENTS: Eighteen asymptomatic subjects (12 men, 6 women) infected with the human immunodeficiency virus (mean CD4 cell counts of 512 +/- 178/mm3). INTERVENTIONS: Subjects received single 100-mg oral doses of ZDV as follows: after an 8-hour fast (treatment A), with a high-fat breakfast (treatment B), and 3 hours after a high-fat breakfast (treatment C). MEASUREMENTS AND MAIN RESULTS: The high-fat breakfast significantly reduced the mean (coefficient of variation) maximum plasma concentration (Cmax) from 806 (55%) ng/ml with treatment A to 341 (47%) and 424 (42%) ng/ml with treatments B and C, respectively. The time to Cmax was significantly prolonged from 0.68 (30%) hours with treatment A to 1.7 (54%) and 1.3 (42%) hours with treatments B and C, respectively. Area under the plasma ZDV concentration-time curve (AUC) was not statistically different across the study treatments. Men had significantly lower (35%) renal clearances of both ZDV and its glucuronide metabolite than women. CONCLUSIONS: When ZDV was given either with or 3 hours after a high-fat breakfast, its absorption was prolonged and Cmax was reduced relative to fasting. However, systemic exposure, as indicated by AUC, was unchanged.

Pharmacokinetic interaction between ketoconazole and amprenavir after single doses in healthy men.

STUDY OBJECTIVE: To determine the effects of coadministration of amprenavir and ketoconazole on the pharmacokinetics of both drugs, and to assess the utility of the erythromycin breath test (ERMBT) to predict and explain these effects. DESIGN: Open-label, randomized, balanced, single-dose, three-period crossover study. SETTING: University research center. SUBJECTS: Twelve healthy men. INTERVENTION: Subjects received amprenavir 1200 mg, ketoconazole 400 mg, and amprenavir 1200 mg plus ketoconazole 400 mg. Each treatment was separated by 14 days. MEASUREMENTS AND MAIN RESULTS: Serial plasma samples for amprenavir and ketoconazole concentrations were measured by high-performance liquid chromatography. Coadministration of the drugs increased amprenavir area under the curve extrapolated to infinity (AUCinfinity) by 31% and reduced its maximum concentration (Cmax) by 16%. Amprenavir increased the AUCinfinity of ketoconazole by 44% and increased the drug's half-life and Cmax by 23% and 19%, respectively. Both agents resulted in substantial inhibition of ERMBT. CONCLUSION: Coadministration of ketoconazole and amprenavir results in a statistically significant increase in AUC for both agents, but the changes are not likely to be clinically important.

Atovaquone suspension in HIV-infected volunteers: pharmacokinetics, pharmacodynamics, and TMP-SMX interaction study.

STUDY OBJECTIVE: To evaluate the pharmacokinetics and safety of atovaquone suspension in volunteers infected with the human immunodeficiency virus ((HIV). DESIGN: Open-label, nonrandomized study. SETTING: Two clinical research centers. PATIENTS: Twenty-two HIV-infected volunteers with a median CD4 cell count of 37 cells/mm3. INTERVENTIONS: Patients received atovaquone suspension fasting or fed for 2-week periods with crossover at dosages of 500 mg/day, and randomization to fasting or fed at dosages of 750 and 1000 mg/day. A subset of patients also received 750 mg twice/day with food, and a subset of those who received 1000 mg/day fasting also received 1000 mg with food. During a long-term dosing phase, a subset of subjects were evaluated for an interaction between atovaquone and trimethoprim-sulfamethoxazole (TMP-SMX). MEASUREMENTS AND MAIN RESULTS: Average steady-state atovaquone concentrations at 500 mg were 6.7 +/- 3.2 microg/ml fasted and 11.3 +/- 5.0 microg/ml with food; at 750 mg, 9.9 +/- 7.1 microg/ml fasted and 12.5 +/- 5.9 microg/ml with food; at 1000 mg, 9.7 +/- 4.3 microg/ml fasted and 13.6 +/- 5.0 microg/ml with food; and at 1500 mg, 21.1 +/- 5.0 microg/ml with food. Thus, plasma concentrations were not proportional to dose. Concomitant food ingestion resulted in a 1.3- to 1.7-fold increase in values. Average steady-state concentrations were less than 10 microg/ml in 21% and more than 15 microg/ml in 36% of patients at 1000 mg/day with food; at 750 mg twice/day, all five patients had levels above 15 microg/ml. Atovaquone suspension was well tolerated; diarrhea, nausea, fatigue, and rash were the most common adverse events. Concomitant administration of TMP-SMX did not change atovaquone concentrations and resulted in small decreases in concentrations of TMP (16%) and SMX (10%). CONCLUSION: Plasma concentrations are significantly higher when atovaquone suspension is administered with food compared with fasting. Total doses of 1500 mg/day are likely to achieve concentrations effective for prophylaxis of Pneumocystis carinii pneumonia.

Separation of indenestrol A and B isomers and enantiomers by high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) methods have been developed for the separation of substituted indenestrol A and B isomers on different columns. The isomers were separated by normal-phase liquid chromatography with a silica gel column. Enantiomers of these compounds were separated by chiral HPLC and the most successful separations were achieved with a Chiralcel OJ column.

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. X. Heterozygous effects of multilocus deletion mutations of genotype ad-3A or ad-3B.

Previous studies on X-ray-induced irreparable adenine-3 mutations (designated [ad-3]IR), induced in heterokaryon 12 of Neurospora crassa, demonstrated that they were not recessive and exhibited heterozygous effects in terms of markedly reduced linear growth rates (de Serres, 1965). Complementation tests with a series of tester strains carrying multilocus deletion mutations in the ad-3 and immediately adjacent genetic regions demonstrated that X-ray-induced irreparable mutations map, in the main part, as a series of overlapping multilocus deletion mutations that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential function) between ad-3A and ad-3B (de Serres, 1968, 1989). Further studies (de Serres and Miller, 1988) have shown that the heterozygous effects of multilocus deletion mutations in the ad-3 region can be modified genetically and biochemically. In the present paper, the heterozygous effects of X-ray-induced multilocus deletion mutations of genotype ad-3A or ad-3B, induced in heterokaryon 12 (Webber and de Serres, 1965; de Serres, 1988, 1989), have been determined. These data show that 57.7% (15/26) of X-ray-induced multilocus deletion mutations covering the ad-3A locus have heterozygous effects, in terms of reduced linear growth rates, in forced dikaryons with a gene/point mutant at the ad-3B locus and 80.0% (20/25) in forced dikaryons with a multilocus deletion mutation covering the ad-3B locus. In addition, 35.1% (20/57) of X-ray-induced multilocus deletion mutations covering the ad-3B locus have heterozygous effects in forced dikaryons with a gene/point mutant at the ad-3A locus, and 100.0% (35/35) in forced dikaryons with a multilocus deletion mutation covering the ad-3A locus. These results demonstrate that the dominant or recessive characteristics of X-ray-induced specific-locus mutations resulting from multilocus deletion mutations are allele specific.

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. XI. Heterozygous effects of gene/point mutations of genotype ad-3A or ad-3B.

Previous studies on X-ray-induced irreparable adenine-3 mutants (designated ad-3IR), induced in heterokaryon 12 of Neurospora crassa, showed that they were not recessive, and that they demonstrated heterozygous effects in terms of markedly reduced linear growth rates as compared with a wild-type control (de Serres, 1965, 1988). Homology tests on X-ray-induced irreparable mutants showed that they map, in the main part, as a series of overlapping multilocus deletions that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential, function) between ad-3A and ad-3B (de Serres, 1969, 1989a). Studies on a larger sample of X-ray-induced multilocus deletion mutations of genotype (ad-3A)IR or (ad-3B)IR (de Serres et al., 1992) demonstrated that heterozygous effects are allele specific and that there was no correlation with genotype, radiation dose or complementation map position. Furthermore, the heterozygous effects of multilocus deletions in the ad-3 region can be modified genetically and biochemically (de Serres and Miller, 1988). In the present paper, the heterozygous effects of X-ray-induced gene/point mutations of genotype ad-3AR or ad-3BR, induced in heterokaryon 12 (Webber and de Serres, 1965; de Serres, 1988, 1989a), were determined. The studies presented in this paper show that 8.1% (3/37) of X-ray-induced ad-3AR mutations exhibit heterozygous effects in terms of reduced linear growth rates in forced dikaryons with a gene/point mutant at the ad-3B locus, and 10.8% (4/37) in forced dikaryons with a multilocus deletion mutation covering the ad-3B locus. In addition, 24.3% (9/37) of ad-3AR mutations exhibit heterozygous effects in terms of enhanced linear growth rates in forced dikaryons with a gene/point mutant at the ad-3B locus. Similar studies with X-ray-induced ad-3BR mutations showed that 54.9% (28/51) exhibit heterozygous effects in terms of reduced growth rates in forced dikaryons with a gene/point mutant at the ad-3A locus and 100.0% (48/48) in forced dikaryons with a multilocus deletion covering the ad-3A locus. These studies have also shown that about a 13-fold higher percentage of X-ray-induced multiple-locus mutations of genotype ad-3AR + RLCL have heterozygous effects resulting in reduced growth rates than X-ray-induced single-locus mutations of genotype ad-3AR. The overall data base on X-ray-induced ad-3 gene/point mutations in the present studies demonstrates that heterozygous effects are allele specific, genotype specific, and locus specific.(ABSTRACT TRUNCATED AT 400 WORDS)

ENU mutagenesis in the mouse electrophoretic specific-locus test, 1. Dose-response relationship of electrophoretically-detected mutations arising from mouse spermatogonia treated with ethylnitrosourea.

The mouse electrophoretic specific-locus test for induced germ-cell mutations, was used to determine the response of spermatogonial stem cells to a series of doses of the germ cell mutagen N-ethyl-N-nitrosourea (ENU). Male DBA/2J and C57B1/6J mice were treated with doses of 50, 100, 200 or 250 mg/kg ENU and their progeny screened for electrophoretically-detectable mutations at 32 separate loci. As expected, increasing doses of ENU led to increasing mutant frequencies. The differences in mutant frequencies between treated DBA/2J and C57B1/6J males were not statistically significant.

Metabolism, disposition and excretion of [14C]melamine in male Fischer 344 rats.

The metabolism, excretion and disposition of melamine were determined after administration of a single oral dose of 0.025 mCi (0.38 mg) [14C]melamine to adult male Fischer 344 rats. Within the first 24 hr, 90% of the administered dose was excreted in the urine. Negligible radioactivity appeared in breath and faeces. There was little difference in blood, liver or plasma concentrations of 14C, suggesting that melamine distributes in body water. The only organs showing radioactivity levels much higher than plasma were the kidney and bladder. The bladder level was by far the highest, a finding probably due either to back diffusion from urine or to contamination of bladder tissue with urine. Virtually no residual radioactivity was observed in tissues examined at 24 hr or later. The elimination-phase half-life calculated from plasma data, 2.7 hr, was in good agreement with the urinary-excretion half-life of 3.0 hr. The renal clearance of melamine was 2.5 ml/min. Radioactivity in plasma or urine co-chromatographed with that of the dosing solution, indicating that melamine is not metabolized in the male Fischer 344 rat.