Piperidine renin inhibitors: from leads to drug candidates.

Non-peptidomimetic renin inhibitors of the piperidine type represent a novel structural class of compounds potentially free of the drawbacks seen with peptidomimetic compounds so far. Synthetic optimization in two structural series focusing on improvement of potency, as well as on physicochemical properties and metabolic stability, has led to the identification of two candidate compounds 14 and 23. Both display potent and long-lasting blood pressure lowering effects in conscious sodium-depleted marmoset monkeys and double transgenic rats harboring both the human angiotensinogen and the human renin genes. In addition, 14 normalizes albuminuria and kidney tissue damage in these rats when given over a period of 4 weeks. These data suggest that treatment of chronic renal failure patients with a renin inhibitor might result in a significant improvement of the disease status.

The effect of warfarin on the pharmacokinetics and pharmacodynamics of napsagatran in healthy male volunteers.

OBJECTIVE: The effect of oral warfarin on the pharmacokinetics and pharmacodynamics of the synthetic direct thrombin inhibitor napsagatran was investigated. METHODS: In an open, randomised, two-way crossover study, 12 healthy male volunteers were infused napsagatran (80 micrograms/min) for 48 h. Each subject was administered 25 mg warfarin (Coumadin) at the start of the infusion in either the first or second treatment period. Sampling was performed regularly over the treatment period and 24 h thereafter for measurement of plasma levels of napsagatran, activated partial thromboplastin time (APTT) and prothrombin time (PT). RESULTS: The pharmacokinetic parameters of napsagatran were not significantly influenced by co-administration of warfarin. Napsagatran administration was followed by increases in APTT and PT. Co-administration of warfarin increased the AUEC (area under the effect curve) calculated for the period 0-48 h (corrected for baseline) for APTT by 45% (95% CI: 28-65%) and for PT by 438% (95% CI: 272-678%) compared to the treatment with napsagatran alone. CONCLUSION: Warfarin has no effect on the pharmacokinetics of napsagatran, but has a marked influence on the pharmacodynamic parameters (APTT, PT) of napsagatran. In clinical practice, this interaction between the two compounds should be taken into account. The PT cannot be used to monitor the effect of oral anticoagulants during the switch from this group of direct thrombin inhibitors to full oral anticoagulant therapy.

Pharmacokinetics of oral doxifluridine in patients with colorectal cancer.

AIMS AND BACKGROUND: Doxifluridine is a new fluoropyrimidine that has excellent absorption by the gastrointestinal tract when given orally. The aim of the study was to determine the disposition of doxifluridine and fluorouracil when the former is given orally for 5 days and to assess whether their pharmacokinetics are influenced by demographic or biologic parameters. METHODS AND STUDY DESIGN: Twenty colorectal cancer patients received levo-leucovorin, 25 mg orally on days 1-5, followed 2 hrs later by doxifluridine, 1200 mg/m2; the cycle was repeated every 10 days. Doxifluridine and fluorouracil levels were measured by reverse-phase high-performance liquid chromatography during the first cycle of therapy. The lowest dose given over the first 24 hrs was 1750 mg and the highest was 2500 mg. RESULTS: The distribution of doxifluridine parameters remained the same between days 1 and 5, with an AUC that ranged between 72.2 and 74.5 mmol h/l and a C max that remained in a narrow band of 67.1 to 68.3 mmol/l. In contrast, the variability of fluorouracil parameters increased from day 1 to day 5, with an AUC of respectively 5.46 and 7.52 mmol h/l and a C max that increased from 5.81 on day 1 to 7.34 mmol/l on day 5. A significant correlation between the AUC of doxifluridine and fluorouracil was found on day 1 and on day 5 (P < 0.001). None of the demographic or biologic parameters considered was significantly related to pharmacokinetic parameters. Fluorouracil levels remained low in comparison with levels measured after classical fluorouracil therapy, although detectable for a longer time. CONCLUSIONS: A large interpatient pharmacokinetic variability was observed without any significant correlation with the clinical parameters studied.

Interspecies pharmacokinetic comparisons and allometric scaling of napsagatran, a low molecular weight thrombin inhibitor.

The objective of this work was to assess the pharmacokinetics of napsagatran, a low molecular weight thrombin inhibitor, after intravenous administration in a variety of laboratory animals, and prospectively to help design the first pharmacokinetic studies in man. Napsagatran is actively excreted into the bile and urine of various species and pronounced species-differences in its pharmacokinetics are observed. It is, therefore, an interesting compound to use in tests of the limitations of presently available inter-species scaling methods. The present data suggest that allometric exponent values which are consistent with the values expected for physiological processes and small organic molecules are not necessarily associated with successful predictions in man when active transport processes are involved in the disposition of the compounds. For example, compared with the values observed in man, the clearance (CL), non-renal clearance (CL(nr)) and the volume of distribution at steady state (Vd(ss)) were over-predicted by 3-, 7- and 2-fold, respectively, by use of allometry. Of the species tested, the cynomolgus monkey seemed to be the most useful for predicting kinetics in man when the approach based on concentration-time transformations was used. Thus, for half-life (t(1/2)), CL and Vd(ss), the observed mean values of 1.7 h, 459 mL min(-1) and 24 L kg(-1) in man were very close to the values predicted from the cynomolgus monkey (1.7 h, 652 mL min(-1) and 22 L kg(-1), respectively). The results show that there are large inter-species differences for kidney and liver excretion of napsagatran. This is probably because of the involvement of active transport processes, which compromised the kinetic extrapolation from animal to man, although a more thorough investigation of the transporters involved in the disposition of napsagatran is necessary to enable better understanding of the species differences observed.

Antimalarial activity of the bisquinoline trans-N1,N2-bis (7-chloroquinolin-4-yl)cyclohexane-1,2-diamine: comparison of two stereoisomers and detailed evaluation of the S,S enantiomer, Ro 47-7737.

The S,S enantiomer of the bisquinoline trans-N1,N2-bis(7-chloroquinolin-4-yl)cyclohexane-1,2-diamine, Ro 47-7737, is significantly more potent against chloroquine-resistant Plasmodium falciparum than the R,R enantiomer and the previously described racemate. Both the enantiomers and the racemate are more potent inhibitors of heme polymerization than chloroquine, and their activities are probably mediated by inhibition of this parasite-specific process. The S,S enantiomer, Ro 47-7737, was studied in more detail and proved to be a potent antimalarial in the treatment of P. vivax ex vivo and P. berghei in vivo. Its suppression of P. berghei growth in a mouse model (50% effective dose, 2.3 mg/kg of body weight) was equal to that of chloroquine and mefloquine, and Ro 47-7737 was found to be more potent than these two drugs in the Rane test, in which the curative effect of a single dose is monitored. The dose at which 50% of animals were permanently cured (34 mg/kg) was markedly superior to those of chloroquine (285 mg/kg) and mefloquine (> 250 mg/kg). When administered orally at 50 mg/kg, Ro 47-7737 also showed a faster clearance of parasites than either chloroquine or mefloquine, and unlike the other two compounds, Ro 47-7737 showed no recrudescence. In a study to compare prophylactic efficacies of oral doses of 50 mg/kg, Ro 47-7737 provided protection for 14 days compared to 3 days for mefloquine and 1 day for chloroquine. The good curative and prophylactic properties of the compound can be explained in part by its long terminal half-life. The ability to generate parasite resistance to Ro 47-7737 was also assessed. With a rodent model, resistance could be generated over eight passages. This rate of resistance generation is comparable to that of mefloquine, which has proved to be an effective antimalarial for many years. Toxicity liabilities, however, ruled out this compound as a candidate for drug development.

4-aminoquinoline analogs of chloroquine with shortened side chains retain activity against chloroquine-resistant Plasmodium falciparum.

We have synthesized several 4-aminoquinolines with shortened side chains that retain activity against chloroquine-resistant isolates of Plasmodium falciparum malaria (W. Hofheinz, C. Jaquet, and S. Jolidon, European patent 94116281.0, June 1995). We report here an assessment of the activities of four selected compounds containing ethyl, propyl, and isopropyl side chains. Reasonable in vitro activity (50% inhibitory concentration, < 100 nM) against chloroquine-resistant P. falciparum strains was consistently observed, and the compounds performed well in a variety of plasmodium berghei animal models. However, some potential drawbacks of these compounds became evident upon in-depth testing. In vitro analysis of more than 70 isolates of P. falciparum and studies with a mouse in vivo model suggested a degree of cross-resistance with chloroquine. In addition, pharmacokinetic analysis demonstrated the formation of N-dealkylated metabolites of these compounds. These metabolites are similarly active against chloroquine-susceptible strains but are much less active against chloroquine-resistant strains. Thus, the clinical dosing required for these compounds would probably be greater for chloroquine-resistant strains than for chloroquine-susceptible strains. The clinical potential of these compounds is discussed within the context of chloroquine's low therapeutic ratio and toxicity.

Doxifluridine and leucovorin: an oral treatment combination in advanced colorectal cancer.

PURPOSE: This study was designed to test the activity and feasibility of an all-oral regimen of levo-leucovorin and doxifluridine (dFUR) in the treatment of advanced colorectal cancer and to establish whether the pharmacokinetics of dFUR and fluorouracil (FU) are affected by demographic and/or biologic parameters. MATERIALS AND METHODS: One hundred eight patients with histologically proven colorectal cancer received orally administered levo-leucovorin 25 mg followed 2 hours later by dFUR 1,200 mg/m2 on days 1 to 5, with the cycle being repeated every 10 days. RESULTS: Among 62 previously untreated patients, two complete responses (CRs) and 18 partial responses (PRs) were observed (overall response rate, 32%; 95% confidence interval, 21% to 45%). The median response duration was 4 months (range, 2 to 13) and the median survival time, 14 months. Among 46 pretreated patients, there were three CRs and three PRs (response rate, 13%; 95% confidence interval, 5% to 26%). In this group of patients, the median response duration was 4 months (range, 1 to 12) and the median survival time, 12 months. No toxic deaths were observed. The only World Health Organization (WHO) grade 3 to 4 side effect was diarrhea (32 patients). CONCLUSION: This regimen is active in previously untreated colorectal cancer patients and combines good compliance with safety. Limited but definite efficacy was also detected in the patients previously treated with FU, which suggests incomplete cross-resistance between the two drugs. The pharmacokinetic results suggest that the conversion rate of dFUR to FU increases between days 1 and 5, but that FU levels remain low in comparison to those measured after classical FU therapy. Under the experimental conditions used in this study, the interpatient variability of pharmacokinetic parameters remains largely unexplained by the tested variables.

Factors affecting the establishment and maintenance of embryogenic callus and suspension cultures of wheat (Triticum aestivum L.).

Improved suspension cell culture systems are needed to facilitate the application of recombinant DNA technology for wheat germplasm enhancement. This study evaluated three wheat (Triticum aestivum L.) cultivars, and the effects of medium basal salts, 2,4-D, sucrose, and L-proline concentrations on the establishment of rapidly growing and highly embryogenic callus and suspension cultures. Percent embryogenic calli was visually estimated and verified with light and scanning electron microscopy. The most highly embryogenic callus was produced by cultivar Bobwhite on medium with MS basal salts, 5.6 µ M 2,4-D, 58 mM sucrose, and zero proline. The suspension cultures that produced the greatest number of regenerated plants utilized callus tissue produced on solid medium with MS basal salts, 87 mM sucrose, 9 µM 2,4-D, and no proline.

Animal pharmacokinetics and metabolism of the new antimalarial Ro 42-1611 (arteflene).

Ro 42-1611 (arteflene) is a new synthetic structural analogue of yingzhaosu, a Chinese traditional herbal drug, now under development for treatment of malaria. The in vivo activity of arteflene in a mouse animal model was 4-5 fold higher after parenteral than after oral administration. Pharmacokinetics of the drug were investigated in mice, rats, dogs, marmosets and cynomolgus monkeys. Plasma concentrations of arteflene were determined using a specific HPLC-UV method; the limit of quantification was 45 ng/ml using 0.5 ml plasma. The oral bioavailability was very low and variable (0.6% in mice, 4-5% in rats, 2.5 +/- 1% in dogs, < or = 0.5% in marmosets and < 0.5% in cynomolgus) as expected from the high metabolic clearance and the relative short apparent half-life (1.4-4.7 h). However, a metabolite (MA) was observed in plasma of all species indicating that drug was absorbed but underwent extensive first-pass metabolism. MA was also detected in samples of human plasma, collected during an oral tolerability study in healthy volunteers. After incubation of 14C-arteflene with liver microsomes of mice, rats, dogs and humans, the same major metabolite was detected and both samples were identical to Ro 47-6936 which was chemically synthesized as a reference compound. The in vitro activity of Ro 47-6936 was tested against Plasmodium falciparum and found to be about 1/4 that of the parent drug. Therefore, this metabolite makes a significant contribution to the biological activity in vivo, partially explaining the high activity of arteflene after oral administration in spite of its low bioavailability. Moreover, comparison of the metabolic patterns from human, rat and dog microsomes indicated that the dog is an appropriate species for toxicological evaluations.

Cloning and expression of genes required for coronamic Acid (2-ethyl-1-aminocyclopropane 1-carboxylic Acid), an intermediate in the biosynthesis of the phytotoxin coronatine.

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.

Percutaneous absorption of amorolfine following a single topical application of an amorolfine cream formulation.

In an open-label, parallel-group, randomized study percutaneous absorption of 14C-labelled amorolfine incorporated into a cream formulation was assessed in healthy male volunteers (n = 12). A single dose of 0.5 g of the 0.25% cream formulation was applied to 100 cm2 of intact (n = 6) and stripped (n = 6) skin for 24 h using occlusive dressing. The remaining drug was removed and the treated skin area of both groups was stripped with adhesive tape. Total urine and faeces were collected in portions up to 3 weeks after the experiment and blood samples were taken at intervals for 3 weeks. Radioactivity was measured in the skin strippings and in the urine, faeces and plasma samples. The intact drug was assessed in the plasma samples. Using mass balance techniques it could be shown that a mean of 92% (range: 84-101%) of the applied radioactivity could be recovered. Small differences in the absorption and elimination of the radioactivity were observed between the two groups but they were not statistically significant (alpha = 0.05). Therefore data from the two groups were pooled. Elimination of drug and drug-related material from the body was very slow. During the 3-week collection period, a mean of 7% (range: 3.8-10.2%) of the dose was excreted in urine and faeces. Another 0.9-3.3% of the dose was retained in the upper layers of the skin as shown by the skin strippings after treatment. Levels of radioactivity and of intact drug in plasma were below the detection limit of 0.5 ng-equiv./ml, respectively. Present data suggest that mean percutaneous absorption of amorolfine following topical application of the 0.25% cream formulation should not exceed 8-10% of the dose applied.

Genetic analysis of a grass dwarf mutation induced by wheat callus culture.

Genetic variation induced by passage through tissue culture (somaclonal variation) has been characterized for many agronomic traits of wheat. The study presented here was conducted to genetically and phenotypically characterize a mutation influencing plant height that was induced by wheat callus culture. Dwarf plants were identified in the progeny of a tall plant regenerated from immature embryo-derived callus tissue of the hard red winter wheat 'TAM 105'. The dwarfs are significantly shorter, later in heading, and have a greater number of tillers, fewer seeds per spike, lower grain yield per plant, and lower floret fertility than 'TAM 105'. The dwarfs also exhibit branching at the aerial nodes when grown under cool temperatures (<20°C) and short daylengths (<12h). We hypothesize that a single, partially dominant gene which acts in a complementary manner with the grass-dwarf gene D1 is responsible for this phenotype. Based on phenotype and the dominance relationship between mutant and wild-type alleles, we hypothesize that the mutation is a new allele at either the D2 or D4 grass-dwarfism locus. The utilization of genotypes lacking any of the grass-dwarfism alleles would greatly reduce the chance of recovering these undesirable genotypes by mutations arising during tissue culture. It is also important to recognize the grass-dwarf phenotype. If transgenic plants, somatic hybrids, or regenerants from in vitro selection strategies have a grass-dwarf phenotype, they can be induced to enter reproductive development by long daylengths (>14 h) and high temperatures (>26°C).

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography.

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.

Simultaneous determination of pyrimethamine and mefloquine in human plasma by high-performance liquid chromatography with ultraviolet detection.

A rapid, sensitive and selective method was developed for the simultaneous determination of pyrimethamine and mefloquine, two of the active ingredients of Fansimef, in human plasma. The procedure involved extraction of the compounds and the internal standard nitrazepam from basified plasma with dichloromethane and chromatography on a C18 column (microBondapak, 300 X 3.9 mm I.D.) with acetonitrile-phosphate buffer as the mobile phase and UV detection at 222 nm. The limit of quantification was 10 ng/ml for both substances, using a 1-ml plasma specimen. The mean inter-assay precision was 2.8% for pyrimethamine and 4.7% for mefloquine up to 800 ng/ml. The practicability of the method was demonstrated by the analysis of more than 1200 plasma samples from several pharmacokinetic studies involving single-dose administration of Fansimef to both patients and volunteers.

Stereochemical consequences of dynamic gearing in substituted bis(9-triptycyl) methanes and related molecules.

Empirical force-field calculations show that bis(9-triptycyl)methane has a C(s) ground state and that the securely meshed triptycyl groups undergo nearly unhindered cogwheel rotation through a C(2) transition state, with an activation energy of approximately 1.0 kcal mol(-1). These features lead to note-worthy stereochemical attributes in appropriately substituted derivatives and analogs. Bis(9-triptycyl)carbinol has been prepared as an example of a compound that is chemically achiral, even though all conformations are chiral under the constraint of gear meshing. It is shown that, under the same constraint, ring substitution of either compound (or analogous systems) may lead to residual stereoisomerism. A permutational analysis of chemical isomers and isomerizations for every possible type of substitution pattern gives an enumeration of residual stereoisomers under the operation of various gearing and nongearing modes and provides information essential for a choice among mechanistic alternatives for a given isomer count.