An infrared spectroscopic method of quantitative analysis of dipeptides CH3CONHCHR1CONHR2 in solu... more An infrared spectroscopic method of quantitative analysis of dipeptides CH3CONHCHR1CONHR2 in solution is proposed. The application of the method to a series of dipeptides with different side chains shows that the amount of the “bent form” tends to become important when the side chain is large.
The aim of this study was to investigate which principal human cytochrome P450 (CYP450) enzymes a... more The aim of this study was to investigate which principal human cytochrome P450 (CYP450) enzymes are affected by artemisinin and to what degree the artemisinin derivatives differ with respect to their respective induction and inhibition capacity. Seventy-five healthy adults were randomized to receive therapeutic oral doses of artemisinin, dihydroartemisinin, arteether, artemether or artesunate for 5 days (days 1–5). A six-drug cocktail consisting of caffeine, coumarin, mephenytoin, metoprolol, chlorzoxazone and midazolam was administered orally on days −6, 1, 5 and 10 to assess the activities of CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Four-hour plasma concentrations of parent drugs and corresponding metabolites and 7-hydroxycoumarin urine concentrations were quantified by liquid chromatography-tandem mass spectrometry. The 1-hydroxymidazolam/midazolam 4-h plasma concentration ratio (CYP3A) was increased on day 5 by artemisinin [2.66-fold (98.75% CI: 2.10–3.36)], artemether [1.54 (1.14–2.09)] and dihydroartemisinin [1.25 (1.06–1.47)] compared with day −6. The S-4′-hydroxymephenytoin/S-mephenytoin ratio (CYP2C19) was increased on day 5 by artemisinin [1.69 (1.47–1.94)] and arteether [1.33 (1.15–1.55)] compared with day −6. The paraxanthine/caffeine ratio (CYP1A2) was decreased on day 1 after administration of artemisinin [0.27 (0.18–0.39)], arteether [0.70 (0.55–0.89)] and dihydroartemisinin [0.73 (0.59–0.90)] compared with day −6. The α-hydroxymetoprolol/metoprolol ratio (CYP2D6) was lower on day 1 compared with day −6 in the artemisinin [0.82 (0.70–0.96)] and dihydroartemisinin [0.83 (0.71–0.96)] groups, respectively. In the artemisinin-treated subjects this decrease was followed by a 1.34-fold (1.14–1.58) increase from day 1 to day 5. These results show that intake of artemisinin antimalarials affect the activities of several principal human drug metabolizing CYP450 enzymes. Even though not significant in all treatment groups, changes in the individual metrics were of the same direction for all the artemisinin drugs, suggesting a class effect that needs to be considered in the development of new artemisinin derivatives and combination treatments of malaria.
To compare parasite clearance times after oral and rectal administration of artemisinin in adults... more To compare parasite clearance times after oral and rectal administration of artemisinin in adults with uncomplicated malaria and to relate pharmacodynamics with artemisinin kinetics and to disclose any pharmacokinetic changes during treatment. Thirty male Vietnamese patients with falciparum malaria were randomized to treatment with 500 mg artemisinin daily by either the oral or rectal route of administration. Parasite densities in capillary blood were determined by microscopy every 4 to 6 hours. Artemisinin plasma concentrations on the first and last day of treatment were determined by HPLC and unbound fractions in plasma were determined by ultrafiltration. Mean parasite clearance times and 95% confidence intervals (95% CI) were 25 (95% CI, 16 to 33) and 29 (95% CI, 23 to 35) hours during oral and rectal treatment, respectively. The bioavailability after rectal relative to oral artemisinin was 30%. Artemisinin areas under the plasma concentration-time curve (AUC) on the fifth (last) day of oral or rectal treatment were 30% (95% CI, 4% to 56%) and 40% (95% CI, -6% to 91%), respectively, of those after the first dose. The fraction unbound in plasma was 15% (95% CI, 12% to 19%), increasing marginally during treatment. No relationship was found between main clinical end points and drug exposure, although indices for the rapidity of response onset were lower after oral treatment and correlated to unbound AUC values (rS = -0.7; p < 0.001). The similarity in parasite clearance times despite lower drug levels during rectal treatment suggests that initial oral doses may be unnecessarily high. The singular time dependency of artemisinin pharmacokinetics, attributed to autoinduction of drug elimination, has possible implications for combination chemotherapy. Decreasing artemisinin concentrations during treatment may partly explain recrudescences and increase the risk for resistance development.
Eight healthy male, Vietnamese subjects were administered 1 x 250, 2 x 250, and 4 x 250 mg artemi... more Eight healthy male, Vietnamese subjects were administered 1 x 250, 2 x 250, and 4 x 250 mg artemisinin capsules in a cross-over design with randomized sequence with a 7-day washout period between administrations. The inter-individual variability in artemisinin pharmacokinetics was large with parameter coefficient of variation (CV) typically between 50-70%. The parameter with the smallest variability was the elimination half-life (CV approximately equal to 30-40%). Analysis of variance indicated also a large intra-subject variability. (CV, or = 24%) for the dose-normalized area under the plasma concentration-time curve (AUC/dose). The pharmacokinetic results suggested artemisinin to be subject to high pre-systemic extraction. Artemisinin half-life could not predict the extent of in vivo exposure to the drug, there being no correlation between half-life and oral clearance. Artemisinin oral plasma clearance was about 400 L h-1 exhibiting a slight decrease with dose, although the effect was weak. Thus results from studies using different artemisinin doses may, within the studied dose range, be compared without the complication of disproportionate changes in drug exposure with varying dose levels. Half-lives appeared to increase with dose. An observed period effect in the analysis of variance was tentatively associated with time-dependency in artemisinin pharmacokinetics. There was a high correlation between artemisinin plasma concentrations determined at various time-points after drug administration and the AUCs after the 500 and 1000 mg doses, but less so after the 250 mg dose. This may show a tentative approach to assess the systemic exposure of the patients to artemisinin from the determination of artemisinin plasma concentrations in one or two plasma samples only. Artemisinin was well tolerated with no apparent dose or time dependent effects on blood pressure, heart rate or body temperature.
This study investigated whether time-dependent artemisinin pharmacokinetics correlated to CYP3A4 ... more This study investigated whether time-dependent artemisinin pharmacokinetics correlated to CYP3A4 or CYP2C19 activity in vivo. Artemisinin (two oral doses per day of 250 mg) was given to nine healthy Vietnamese subjects for 7 days (day 1 to day 7). Single 20 mg doses of omeprazole were given orally on day -7, day 1, and day 7. Single doses of artemisinin and omeprazole were given in combination on day 14 after a 6-day washout period. The pharmacokinetics of artemisinin, omeprazole, hydroxyomeprazole, and omeprazole sulfone were evaluated on days -7, 1, 7, and 14. On the same days urine was collected for the determination of 6beta-hydroxycortisol and cortisol excretion. Areas under plasma concentration-time curves (AUC) for artemisinin and omeprazole decreased on day 7 to 20% (95% confidence intervals, 13%, 28%) and 35% (25%, 46%), respectively, compared with values on day 1. AUC ratios for hydroxyomeprazole/omeprazole increased 2.2-fold (1.7, 2.7) on day 7 compared with values on day 1. All values were normalized at day 14. There were no significant changes in the omeprazole sulfone/omeprazole ratio or in the 6beta-hydroxycortisol/cortisol ratio between the study days. In one subject found to have poor CYP2C19 metabolization, the elimination of omeprazole increased after artemisinin exposure, with no change in the hydroxyomeprazole/omeprazole AUC ratio. Artemisinin did not alter CYP3A4 activity, whereas an increase in CYP2C19 activity was observed. The increased elimination of omeprazole in both poor and extensive CYP2C19 metabolizers suggests artemisinin induces both CYP2C19 and another enzyme.
Some experimental data are given on the infrared spectra between 3300 and 3500 cm−1 of dilute sol... more Some experimental data are given on the infrared spectra between 3300 and 3500 cm−1 of dilute solutions in carbon tetrachloride of three types of model compounds: CH3−CONH-CH(R1)-CONH(R2), (I); CH3-CON(CH3)-CH(R1)-CONH(R2), (II) and CH3-CONH-CH(R1)-CON(R2)2, (III). In studying the N-H stretching bands, it was found that there are two types of intramolecular hydrogen bonds in these molecules; these result in two different cyclized conformations, C5 and C7, which contain respectively, five and seven atoms in the ring. By using model substances I, II, and III, in which the nitrogen atoms are unequally substituted, it is possible to identify the N-H stretching bands which are to be ascribed to the N-H oscillators included in the two different chelated conformations. It is found also that the stretching frequency of a free N-H oscillator depends upon the substituent on the nitrogen atom. Thus, it is possible to observe, with some of the model compounds I, four different absorption bands located at 3340, 3420, 3440, and 3460 cm−1. The first two are ascribed to the N-H oscillators included in the Hbonds which lock the C7 and C5 conformations; the last two correspond to free N-H which differ with the substituent on the nitrogen atom.
A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian s... more A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.
Objective To investigate the pharmacokinetic properties of piperaquine after repeated oral admini... more Objective To investigate the pharmacokinetic properties of piperaquine after repeated oral administration of the antimalarial combination CV8 in healthy subjects. Methods Twelve healthy fasted Vietnamese males were administered four tablets CV8 (320 mg piperaquine phosphate, 32 mg dihydroartemisinin, 5 mg primaquine phosphate, 90 mg trimethoprim) on day 1, followed by two tablets every 24th hour, for a total of 3 days. Blood samples were frequently drawn on days 1 and 3 and sparsely drawn until day 29. Samples were analyzed for piperaquine using solid phase extraction followed by high-performance liquid chromatography. Population pharmacokinetic parameter estimates were obtained by nonlinear mixed effects modeling of the observed data using NONMEM. Results A two-compartment disposition model with an absorption lag time described the observed piperaquine concentrations. Absorption profiles were found to be irregular with double or multiple peaks. A dual pathway first-order absorption model improved the goodness of fit. Piperaquine pharmacokinetics were characterized by a large volume of distribution and a terminal half-life of several days. Estimates [95% confidence interval (CI)] of CL/F, Vss/F and t½z were found to be 56.4 (29–84) l/h, 6,000 (3,500–8,500) l and 11.7 (8.3–15.7) days, respectively. Conclusion Piperaquine pharmacokinetics after repeated oral doses were characterized by multiple concentration peaks and multiphasic disposition, resulting in a long terminal half-life. Sustained exposure to the drug after treatment should be taken into account when designing future clinical studies, e.g. duration of follow-up, and may also drive resistance development in areas of high malaria transmission.
An infrared spectroscopic method of quantitative analysis of dipeptides CH3CONHCHR1CONHR2 in solu... more An infrared spectroscopic method of quantitative analysis of dipeptides CH3CONHCHR1CONHR2 in solution is proposed. The application of the method to a series of dipeptides with different side chains shows that the amount of the “bent form” tends to become important when the side chain is large.
The aim of this study was to investigate which principal human cytochrome P450 (CYP450) enzymes a... more The aim of this study was to investigate which principal human cytochrome P450 (CYP450) enzymes are affected by artemisinin and to what degree the artemisinin derivatives differ with respect to their respective induction and inhibition capacity. Seventy-five healthy adults were randomized to receive therapeutic oral doses of artemisinin, dihydroartemisinin, arteether, artemether or artesunate for 5 days (days 1–5). A six-drug cocktail consisting of caffeine, coumarin, mephenytoin, metoprolol, chlorzoxazone and midazolam was administered orally on days −6, 1, 5 and 10 to assess the activities of CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Four-hour plasma concentrations of parent drugs and corresponding metabolites and 7-hydroxycoumarin urine concentrations were quantified by liquid chromatography-tandem mass spectrometry. The 1-hydroxymidazolam/midazolam 4-h plasma concentration ratio (CYP3A) was increased on day 5 by artemisinin [2.66-fold (98.75% CI: 2.10–3.36)], artemether [1.54 (1.14–2.09)] and dihydroartemisinin [1.25 (1.06–1.47)] compared with day −6. The S-4′-hydroxymephenytoin/S-mephenytoin ratio (CYP2C19) was increased on day 5 by artemisinin [1.69 (1.47–1.94)] and arteether [1.33 (1.15–1.55)] compared with day −6. The paraxanthine/caffeine ratio (CYP1A2) was decreased on day 1 after administration of artemisinin [0.27 (0.18–0.39)], arteether [0.70 (0.55–0.89)] and dihydroartemisinin [0.73 (0.59–0.90)] compared with day −6. The α-hydroxymetoprolol/metoprolol ratio (CYP2D6) was lower on day 1 compared with day −6 in the artemisinin [0.82 (0.70–0.96)] and dihydroartemisinin [0.83 (0.71–0.96)] groups, respectively. In the artemisinin-treated subjects this decrease was followed by a 1.34-fold (1.14–1.58) increase from day 1 to day 5. These results show that intake of artemisinin antimalarials affect the activities of several principal human drug metabolizing CYP450 enzymes. Even though not significant in all treatment groups, changes in the individual metrics were of the same direction for all the artemisinin drugs, suggesting a class effect that needs to be considered in the development of new artemisinin derivatives and combination treatments of malaria.
To compare parasite clearance times after oral and rectal administration of artemisinin in adults... more To compare parasite clearance times after oral and rectal administration of artemisinin in adults with uncomplicated malaria and to relate pharmacodynamics with artemisinin kinetics and to disclose any pharmacokinetic changes during treatment. Thirty male Vietnamese patients with falciparum malaria were randomized to treatment with 500 mg artemisinin daily by either the oral or rectal route of administration. Parasite densities in capillary blood were determined by microscopy every 4 to 6 hours. Artemisinin plasma concentrations on the first and last day of treatment were determined by HPLC and unbound fractions in plasma were determined by ultrafiltration. Mean parasite clearance times and 95% confidence intervals (95% CI) were 25 (95% CI, 16 to 33) and 29 (95% CI, 23 to 35) hours during oral and rectal treatment, respectively. The bioavailability after rectal relative to oral artemisinin was 30%. Artemisinin areas under the plasma concentration-time curve (AUC) on the fifth (last) day of oral or rectal treatment were 30% (95% CI, 4% to 56%) and 40% (95% CI, -6% to 91%), respectively, of those after the first dose. The fraction unbound in plasma was 15% (95% CI, 12% to 19%), increasing marginally during treatment. No relationship was found between main clinical end points and drug exposure, although indices for the rapidity of response onset were lower after oral treatment and correlated to unbound AUC values (rS = -0.7; p < 0.001). The similarity in parasite clearance times despite lower drug levels during rectal treatment suggests that initial oral doses may be unnecessarily high. The singular time dependency of artemisinin pharmacokinetics, attributed to autoinduction of drug elimination, has possible implications for combination chemotherapy. Decreasing artemisinin concentrations during treatment may partly explain recrudescences and increase the risk for resistance development.
Eight healthy male, Vietnamese subjects were administered 1 x 250, 2 x 250, and 4 x 250 mg artemi... more Eight healthy male, Vietnamese subjects were administered 1 x 250, 2 x 250, and 4 x 250 mg artemisinin capsules in a cross-over design with randomized sequence with a 7-day washout period between administrations. The inter-individual variability in artemisinin pharmacokinetics was large with parameter coefficient of variation (CV) typically between 50-70%. The parameter with the smallest variability was the elimination half-life (CV approximately equal to 30-40%). Analysis of variance indicated also a large intra-subject variability. (CV, or = 24%) for the dose-normalized area under the plasma concentration-time curve (AUC/dose). The pharmacokinetic results suggested artemisinin to be subject to high pre-systemic extraction. Artemisinin half-life could not predict the extent of in vivo exposure to the drug, there being no correlation between half-life and oral clearance. Artemisinin oral plasma clearance was about 400 L h-1 exhibiting a slight decrease with dose, although the effect was weak. Thus results from studies using different artemisinin doses may, within the studied dose range, be compared without the complication of disproportionate changes in drug exposure with varying dose levels. Half-lives appeared to increase with dose. An observed period effect in the analysis of variance was tentatively associated with time-dependency in artemisinin pharmacokinetics. There was a high correlation between artemisinin plasma concentrations determined at various time-points after drug administration and the AUCs after the 500 and 1000 mg doses, but less so after the 250 mg dose. This may show a tentative approach to assess the systemic exposure of the patients to artemisinin from the determination of artemisinin plasma concentrations in one or two plasma samples only. Artemisinin was well tolerated with no apparent dose or time dependent effects on blood pressure, heart rate or body temperature.
This study investigated whether time-dependent artemisinin pharmacokinetics correlated to CYP3A4 ... more This study investigated whether time-dependent artemisinin pharmacokinetics correlated to CYP3A4 or CYP2C19 activity in vivo. Artemisinin (two oral doses per day of 250 mg) was given to nine healthy Vietnamese subjects for 7 days (day 1 to day 7). Single 20 mg doses of omeprazole were given orally on day -7, day 1, and day 7. Single doses of artemisinin and omeprazole were given in combination on day 14 after a 6-day washout period. The pharmacokinetics of artemisinin, omeprazole, hydroxyomeprazole, and omeprazole sulfone were evaluated on days -7, 1, 7, and 14. On the same days urine was collected for the determination of 6beta-hydroxycortisol and cortisol excretion. Areas under plasma concentration-time curves (AUC) for artemisinin and omeprazole decreased on day 7 to 20% (95% confidence intervals, 13%, 28%) and 35% (25%, 46%), respectively, compared with values on day 1. AUC ratios for hydroxyomeprazole/omeprazole increased 2.2-fold (1.7, 2.7) on day 7 compared with values on day 1. All values were normalized at day 14. There were no significant changes in the omeprazole sulfone/omeprazole ratio or in the 6beta-hydroxycortisol/cortisol ratio between the study days. In one subject found to have poor CYP2C19 metabolization, the elimination of omeprazole increased after artemisinin exposure, with no change in the hydroxyomeprazole/omeprazole AUC ratio. Artemisinin did not alter CYP3A4 activity, whereas an increase in CYP2C19 activity was observed. The increased elimination of omeprazole in both poor and extensive CYP2C19 metabolizers suggests artemisinin induces both CYP2C19 and another enzyme.
Some experimental data are given on the infrared spectra between 3300 and 3500 cm−1 of dilute sol... more Some experimental data are given on the infrared spectra between 3300 and 3500 cm−1 of dilute solutions in carbon tetrachloride of three types of model compounds: CH3−CONH-CH(R1)-CONH(R2), (I); CH3-CON(CH3)-CH(R1)-CONH(R2), (II) and CH3-CONH-CH(R1)-CON(R2)2, (III). In studying the N-H stretching bands, it was found that there are two types of intramolecular hydrogen bonds in these molecules; these result in two different cyclized conformations, C5 and C7, which contain respectively, five and seven atoms in the ring. By using model substances I, II, and III, in which the nitrogen atoms are unequally substituted, it is possible to identify the N-H stretching bands which are to be ascribed to the N-H oscillators included in the two different chelated conformations. It is found also that the stretching frequency of a free N-H oscillator depends upon the substituent on the nitrogen atom. Thus, it is possible to observe, with some of the model compounds I, four different absorption bands located at 3340, 3420, 3440, and 3460 cm−1. The first two are ascribed to the N-H oscillators included in the Hbonds which lock the C7 and C5 conformations; the last two correspond to free N-H which differ with the substituent on the nitrogen atom.
A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian s... more A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.
Objective To investigate the pharmacokinetic properties of piperaquine after repeated oral admini... more Objective To investigate the pharmacokinetic properties of piperaquine after repeated oral administration of the antimalarial combination CV8 in healthy subjects. Methods Twelve healthy fasted Vietnamese males were administered four tablets CV8 (320 mg piperaquine phosphate, 32 mg dihydroartemisinin, 5 mg primaquine phosphate, 90 mg trimethoprim) on day 1, followed by two tablets every 24th hour, for a total of 3 days. Blood samples were frequently drawn on days 1 and 3 and sparsely drawn until day 29. Samples were analyzed for piperaquine using solid phase extraction followed by high-performance liquid chromatography. Population pharmacokinetic parameter estimates were obtained by nonlinear mixed effects modeling of the observed data using NONMEM. Results A two-compartment disposition model with an absorption lag time described the observed piperaquine concentrations. Absorption profiles were found to be irregular with double or multiple peaks. A dual pathway first-order absorption model improved the goodness of fit. Piperaquine pharmacokinetics were characterized by a large volume of distribution and a terminal half-life of several days. Estimates [95% confidence interval (CI)] of CL/F, Vss/F and t½z were found to be 56.4 (29–84) l/h, 6,000 (3,500–8,500) l and 11.7 (8.3–15.7) days, respectively. Conclusion Piperaquine pharmacokinetics after repeated oral doses were characterized by multiple concentration peaks and multiphasic disposition, resulting in a long terminal half-life. Sustained exposure to the drug after treatment should be taken into account when designing future clinical studies, e.g. duration of follow-up, and may also drive resistance development in areas of high malaria transmission.
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