Equivalence-by-Design: Targeting <Emphasis Type="BoldItalic">In Vivo</Emphasis> Drug Delivery Profile

In the United States (U.S.), drug products are considered therapeutically equivalent if they meet regulatory criteria of pharmaceutical equivalence and bioequivalence. These requirements can be traced back to 1977 when the U.S. Food and Drug Administration (FDA) published the regulations on bioavailability and bioequivalence. Over the years, to keep up with the advancement in science and technology, the FDA has been constantly updating the regulatory approaches to assessing and ensuring equivalence. In view of the recent growth in novel pharmaceutical dosage forms and delivery systems, this paper examines the current framework for documentation of therapeutic equivalence and explores the opportunities of further advancing equivalence methods for complex drug products. It is proposed that equivalence may be established by matching the in vivo drug delivery profile (iDDP) between drug products in comparison. This can be achieved by characterizing the iDDP of the reference formulation with application of an equivalence-by-design approach for pharmaceutical development. Critical variables can be identified to serve as in vitro markers or biomarkers for mapping the desired drug delivery profile in vivo. A multidisciplinary approach may be necessary to develop these markers for characterization of iDDPs. The opinions expressed in this article do not necessarily represent the views or policies of the U.S. Food and Drug Administration     

<Emphasis Type="Italic">N</Emphasis><Superscript>4</Superscript>,<Emphasis Type="Italic">N</Emphasis><Superscript>9</Superscript>-Dioleoyl Spermine Is a Novel Nonviral Lipopolyamine Vector for Plasmid DNA Formulation

Purpose To study the effect of synthesized N⁴,N⁹-dioleoyl spermine on DNA condensation and then measure its transfection efficiency in cell culture.Methods The lipopolyamine was synthesized from the naturally occurring polyamine spermine. The ability of this novel compound to condense DNA was studied using ethidium bromide fluorescence quenching and light scattering assays. Transfection efficiency was studied in primary skin cells (FEK4) and in an immortalized cancer cell line (HtTA), and compared with the commercially available transfection formulations Lipofectin and Lipofectamine.Results The synthesized N⁴,N⁹-dioleoyl spermine formula is efficient at condensing calf thymus and circular plasmid DNA and effectively transfects both primary skin cells and cancer cell lines at low charge ratios of (+/– ammonium/phosphate) 2.5.Conclusions N⁴,N⁹-Dioleoyl spermine condenses DNA and achieves high transfection levels in cultured cells.     

Synthesis and cytotoxic activities of 2-alkyl-2,3-dihydro-1<Emphasis Type="Italic">H</Emphasis>-2,6,9-triazacyclopenta[<Emphasis Type="Italic">b</Emphasis>]anthracene-5,10-diones

A series of 2-alkyl-2,3-dihydro-1H-2,6,9-triazacyclopenta[b]anthracene-5,10-diones (4a-f) were synthesized and their in vitro cytotoxic activities were evaluated against six human cancer cell lines (HCT15, SK-OV-3, A549, SNB19, MCF7 and MCF7/ADR). A number of compounds including 4c and 4d showed 2-180 times more potent cytotoxic activity than doxorubicin against all human cancer cell lines tested. Furthermore, these compounds retained considerable cytotoxic activity against the doxorubicin-resistant cell line MCF7/ADR, implying their therapeutic potential to treat doxorubicin-resistant tumors.     

One-Step Surface Modification of Poly(lactide-<Emphasis Type="BoldItalic">co</Emphasis>-glycolide) Microparticles with Heparin

Purpose The aim of this study was to modify the surface of poly(lactide-co-glycolide) (PLGA) microparticles with heparin. The heparin-coated PLGA may enhance blood and tissue compatibility of PLGA devices and provide a novel approach to deliver growth factors. Materials and Methods A one-step method using heparin to replace traditional emulsifiers (e.g., PVA) during emulsion-solvent evaporation process was employed to surface-entrap heparin in PLGA microspheres. The emulsifying activity of heparin was modified via varying counter ion form, including univalent (Na⁺, K⁺, Li⁺, and ) and divalent (Ca²⁺, Mg²⁺, Ba²⁺, and Zn²⁺) cations, and complexation with amino acids (Arg, Lys, Leu, Val, Gly and Glu). Surface accessible and total heparin loading were determined by a modified toluidine blue assay and elemental analysis, respectively. Results Heparin bound with univalent counter ions and amino acids exhibited emulsifying activity to varying degrees, whereas divalent heparin salts tended to cause complete aggregation of the PLGA o/w emulsion. Increasing pH (≥7.4) of hardening medium enhanced heparin adsorption and significantly stabilized the PLGA o/w emulsion. The initial surface density of heparin on the PLGA microspheres prepared using univalent heparin salts was around 8–33 mg/m². Surface associated heparin desorbed quickly; potassium heparin showed the best retention, with ~0.2 and 0.1 mg/m² detected on PLGA microsphere surface following 1- and 14-day incubation in PBST at 37°C, respectively. Conclusions PLGA microparticles were successfully surface-modified with heparin. Univalent salts and amino acid complexes of heparin, as effective emulsifiers, can become surface-immobilized in PLGA microspheres.     

Characterisation of (<Emphasis Type="Italic">R</Emphasis>/<Emphasis Type="Italic">S</Emphasis>)-propafenone and its metabolites as substrates and inhibitors of P-glycoprotein

Digoxin is a drug with a narrow therapeutic index, which is a substrate of the ATP-dependent efflux pump P-glycoprotein. Increased or decreased digoxin plasma concentrations occur in humans due to the inhibition or induction of this drug transporter in organs with excretory function such as small intestine, liver and kidney. It is well known that serum concentrations of digoxin increase considerably in humans if propafenone is given simultaneously. However, it has not been investigated in detail whether propafenone and its metabolites are substrates and/or inhibitors of human P-glycoprotein. The aim of this study, therefore, was to investigate the P-glycoprotein-mediated transport and inhibition properties of propafenone and its major metabolites 5-hydroxypropafenone and N-desalkylpropafenone in Caco-2 cell monolayers. Inhibition of P-glycoprotein-mediated transport by propafenone and its metabolites was determined using digoxin as a P-glycoprotein substrate. No polarised transport was observed for propafenone and N-desalkylpropafenone in Caco-2 cell monolayers. However, 5-hydroxypropafenone translocation was significantly greater from basal-to-apical compared with apical-to-basal (P_app basal–apical vs. P_app apical–basal, 10.21±2.63×10^–6 vs. 4.34±1.84×10^–6 cm/s; P<0.01). Moreover, propafenone, 5-hydroxypropafenone and N-desalkylpropafenone inhibited P-glycoprotein-mediated digoxin transport with IC₅₀ values of 6.8, 19.9, and 21.3 M, respectively. In summary, whereas propafenone and N-desalkylpropafenone are not substrates of P-glycoprotein, 5-hydroxypropafenone is translocated by human P-glycoprotein across cell monolayers. In addition, propafenone and its two major metabolites 5-hydroxypropafenone and N-desalkylpropafenone are inhibitors of human P-glycoprotein and therefore contribute to the digoxin–propafenone interaction observed in humans.     

Construction of a Functional Transporter Analysis System Using <Emphasis Type="BoldItalic">MDR1</Emphasis> Knockdown Caco-2 Cells

Purpose The efflux transporter, P-glycoprotein (P-gp), located in the brush-border membrane of intestinal absorptive cells, reduces the bioavailability of a wide range of orally administered drugs. Using P-gp inhibitors in transport experiments in Caco-2 cell monolayers is widely accepted as an efficient way to estimate the contribution of P-gp to the intestinal absorption of drugs. However, there still remain some arguments that the inhibitors might affect the function of other proteins. Multidrug resistance 1 gene (MDR1) specifically inhibited Caco-2 cells were constructed, therefore, as a better in vitro evaluation system of intestinal drug absorption.Methods The effective sites of RNAi were selected using siRNA libraries and single siRNAs and MDR1 stable knockdown Caco-2 cells were constructed using a tRNA^val-shRNA expression vector.Results In siRNA stably expressed Caco-2 cells, the expression level of MDR1 was reduced at mRNA and protein levels. Transcellular transport studies using digoxin revealed that the P-gp function was suppressed completely, similar to that in verapamil-treated cells.Conclusions MDR1 stable knockdown Caco-2 cells were successfully constructed by RNAi technology. This will consequently allow the development of a selection system for candidate drugs with improved absorption properties.     

Antimicrobial phenolic derivatives from<Emphasis Type="Italic">Dendranthema zawadskii</Emphasis> var.<Emphasis Type="Italic">latilobum</Emphasis> kitamura (Asteraceae)

A phytochemical study on the root ofDendranthema zawadskii var.latilobum Kitamura, using a series of silica gel column chromatography and reversed phase C-18 HPLC chromatography, led to the isolation of (1S, 2S)-1, 2, 3-trihydroxy-1-(3, 4-methylenedioxyphenyl)propane (1), 4-methoxycinnamic acid (2), acacetin (3) and caffeic acid methyl ester (4). The structures of these compounds were determined using spectroscopic analyses (UV, IR, HRTOFMS and NMR), with comparison of their spectral data with previously reported values. Compound1 was isolated for the first time, with compounds2 and4 from this plant reported for the first time. The antibacterial and antifungal activities of the isolated compounds were measured using the disc diffusion method. Also, their cytotoxicities against the cancer cell lines, A549, B16F1 and SK-Mel-2, and brine shrimp lethalities were evaluated.     

A Novel Method for Imaging <Emphasis Type="BoldItalic">In Vivo</Emphasis> Degradation of Poly(L-Glutamic Acid), a Biodegradable Drug Carrier

Purpose To develop an L-PG-based imaging probe suitable for assessing the degradation of L-PG in vivo. Materials and Methods Conjugates of L-PG and a near-infrared fluorescence (NIRF) dye, NIR813, were characterized with regard to quenching efficiency and degradability by cathepsin B (CB) and other proteases. The kinetics of L-PG-NIR813’s degradation and its degradation in orthotopic human U87/TGL glioma in nude mice after intravenous injection was assessed using NIRF optical imaging (n = 3). Results The fluorescence signal from L-PG-NIR813 was efficiently quenched and activated at NIR813 loadings of 8–10%. Upon exposure to CB, the fluorescence intensity of L-PG-NIR813 increased 10-fold. L-PG-NIR813 was also degraded by another cysteine protease cathepsin L, but not by MMP-2, cathepsin E, cathepsin D, and plasmin. A selective CB inhibitor blocked the fluorescence activation. After intravenous injection, the degradation of L-PG-NIR813 was visualized primarily in the liver, which peaked at 4 h postinjection. Activation of L-PG-NIR813 but not D-PG-NIR813 was clearly seen in U87/TGL tumors. Conclusion Our results indicate that L-PG-NIR813 may be used to monitor the in vivo degradation of L-PG-based polymeric drugs, and that this agent may prove useful in noninvasive imaging of protease activity, particularly that of cysteine proteases.     

Enhanced liver targeting by synthesis of<Emphasis Type="Italic">N</Emphasis><Subscript>1</Subscript>-stearyl-5-Fu and incorporation into solid lipid nanoparticles

To enhance the liver targeting and reduce the side effects of 5-fluorouracil (5-Fu), it was acylated by stearyl chloride to obtain N1-stearyl-5-Fu (5-FuS). The chemical structure of the prodrug was confirmed by Nuclear Magnetic Resonance and Infrared Spectrometry. 5-FuS was incorporated into solid lipid nanoparticles (SLN), which were prepared by the physical agglomeration method. The mean diameter of 5-FuS-SLN was 240.19 nm and the drug loading was 20.53%. The release characteristics in vitro of 5-FuS-SLN were fitted to the first-order pharmacokinetic model. Compared with 5-Fu injection, a study on the distribution of 5-FuS-SLN in mice showed that 5-FuS-SLN could double 5-Fu concentration in mice livers. The main pharmacokinetic parameters of 5-FuS-SLN in rabbits is shown as follows: Vd=0.04336 L/kg, T(1/2) beta=1.2834 h, CL=0.1632 L/h. In conclusion, 5-FuS-SLN has significant liver targeting properties. The employment of a prodrug to enhance drug liposoluble properties and the preparation method presented in this paper, seem to be an alternative strategy to the traditional colloidal delivery system.     

Age dependency of cerebral P-gp function measured with (<Emphasis Type="Italic">R</Emphasis>)-[<Superscript>11</Superscript>C]verapamil and PET

Purpose  The aim of this study was to assess the influence of age on the functional activity of the multidrug efflux transporter P-glycoprotein (P-gp) at the human blood-brain barrier. Methods  Seven young (mean age: 27 ± 4 years) and six elderly (mean age: 69 ± 9 years) healthy volunteers underwent dynamic (R)-[¹¹C]verapamil (VPM) positron emission tomography (PET) scans and arterial blood sampling. Parametric distribution volume (DV) images were generated using Logan linearisation, and age groups were compared with statistical parametric mapping (SPM). Brain regions that SPM analysis had shown to be most affected by age were analysed by a region of interest (ROI)-based approach using a maximum probability brain atlas, before and after partial volume correction (PVC). Results  SPM analysis revealed significant clusters of DV increases in cerebellum, temporal and frontal lobe of elderly compared to younger subjects. In the ROI-based analysis, elderly subjects showed significant DV increases in amygdala (+30%), insula (+26%) and cerebellum (+25%) before PVC, and in insula (+33%) after PVC. Conclusions  Increased VPM DV values in the brains of elderly subjects suggest a decrease in cerebral P-gp function with increasing age.     

2,2-dimethyl-2<Emphasis Type="Italic">H</Emphasis>-pyran-derived alkaloids I. Practical synthesis of acronycine and benzo[<Emphasis Type="Italic">b</Emphasis>]acronycine and their biological properties

The 2,2-dimethyl-2H-pyran-derived alkaloids acronycine and its demethylated congeners were prepared in three steps from anthranilic acid and phloroglucinol. The phenylboronic acid-mediated interamolecular cyclization reaction of 1,3-dihydroxyacridone and 3-methylbut-2-enal was employed as a key step, which was also applied to the synthesis of related cytotoxic benzo[b]acronycine. Inhibitory activities of the compounds prepared on topoisomerase I and II as well as their cytotoxicities were evaluated. Cytotoxicity of 2 is closely related to the strong inhibitory activity against topo II at 20 microM level.     

Prediction of the Effects of Genetic Polymorphism on the Pharmacokinetics of CYP2C9 Substrates from <Emphasis Type="BoldItalic">In Vitro</Emphasis> Data

Purpose  The *2 and *3 alleles of CYP2C9, with decreased enzymatic activity, are highly polymorphic and contribute to inter-individual differences in pharmacotherapy of CYP2C9 substrates. Here, we sought for a simplified theoretical method to predict the pharmacokinetic changes with minimal in vivo data. Methods  The changes in clearances of CYP2C9 substrates in subjects with these alleles were quantitatively estimated by parameters from literature data: intrinsic metabolic clearance and the enzyme expression level of mutated CYP2C9, contribution of CYP2C9 to the CYP-mediated clearance (f _m2C9), and the contribution of the dominant metabolic pathways to the total clearance (f _h). To validate the accuracy of our prediction, the changes were compared to reported in vivo values. Results  Sufficient data were available for nine substrates: celecoxib, diclofenac, S-flurbiprofen, losartan, S-phenprocoumon, phenytoin, tolbutamide, torsemide, and S-warfarin. These predicted values, either using the intrinsic clearance specific to each substrate, or the averaged values (*2: 0.66, *3: 0.13, (ratio to *1)), correlated well with observed values (r ² = 0.812, 0.786, respectively). Conclusions  This theoretical method well estimated the quantitative changes in pharmacokinetics of CYP2C9 substrates in subjects with mutated alleles of CYP2C9. This can be applied to drug development even from the early clinical phases. Makiko Kusama and Kazuya Maeda contributed equally to this work.     

Cardiovascular effects of the adenosine A<Subscript>1</Subscript> receptor agonist<Emphasis Type="Italic"> N</Emphasis><Superscript>6</Superscript>-cyclopentyladenosine (CPA) decisive for its therapeutic efficacy in sarin poisoning

Mortality and occurrence of cholinergic symptoms upon sarin intoxication (144 µg/kg s.c., ~2×LD₅₀) in rats is completely prevented by treatment with the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA, 2 mg/kg i.m.). Previously, we have shown that CPA treatment altered the distribution of sarin into the brain, presumably through its cardiovascular side effects. Therefore, the objective of the present study was to evaluate the contribution of the cardiodepressant effects of CPA to its therapeutic efficacy against sarin intoxication. Intramuscular treatment of rats with 0.5 and 2.0 mg/kg CPA 1 min after sarin poisoning attenuated most cholinergic symptoms and prevented mortality, which seemed to be directly associated with an immediate strong and long-lasting bradycardia and hypotension caused by CPA. Treatment with lower doses of CPA (0.1 and 0.05 mg/kg i.m.) caused similar levels of bradycardia and hypotension, albeit a few minutes later than at the higher doses of CPA. Upon sarin intoxication, this was correlated with increased incidence of cholinergic symptoms and decreased survival rates. Pretreatment with the peripheral adenosine A₁ receptor antagonist 8-p-sulphophenyltheophylline (8-PST, 20 mg/kg i.p.) counteracted the cardiodepressant effects of 0.05 mg/kg CPA almost completely, thereby nearly abolishing its therapeutic efficacy against sarin poisoning. In conclusion, the present results strongly indicate that bradycardia and hypotension induced by the peripheral adenosine A₁ receptor play a prominent role in the therapeutic efficacy of CPA in cases of sarin poisoning.     

Human experimental exposure study on the uptake and urinary elimination of <Emphasis Type="Italic">N</Emphasis>-methyl-2-pyrrolidone (NMP) during simulated workplace conditions

A human experimental study was carried out with 16 volunteers to examine the elimination of N-methyl-2-pyrrolidone (NMP) after exposure to the solvent under simulated workplace conditions. The NMP concentrations were 10, 40 and 80 mg/m³ for 2 × 4 h with an exposure-free interval of 30 min. Additionally, a peak exposure scenario (25 mg/m³ baseline, 160 mg/m³ peaks for 4 × 15 min, time-weighted average: 72 mg/m³) was tested. The influence of physical activity on the uptake and elimination of NMP was studied under otherwise identical exposure conditions but involving moderate workload on a bicycle ergometer (75 W for 6 × 10 min). The peak times and biological half-lives of urinary NMP and its main metabolites 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) in urine were analysed as well as the interrelationships between exposure and biomarkers. All analytes showed a close correlation between their post-shift peak concentrations and airborne NMP. An exposure to the current German workplace limit value of 80 mg/m³ under resting conditions resulted in urinary peak concentrations of 2,400 μg/L NMP, 117 mg/g creatinine 5-HNMP and 32 mg/g creatinine 2-HMSI (workload conditions: 3,400 μg/L NMP, 150 mg/g creatinine 5-HNMP, 44 mg/g creatinine 2-HMSI). Moderate workload enhanced the total uptake of NMP by approximately one third. Differences between the estimated and the observed total amount of urinary metabolites point to a significant contribution of dermal absorption on the uptake of NMP. This aspect, together with the influence of physical workload, should be considered for the evaluation of a biological limit value for NMP.     

Role of a Novel Excipient Poly(Ethylene Glycol)-<Emphasis Type="BoldItalic">b</Emphasis>-Poly(<Emphasis Type="SmallCaps">L</Emphasis>-Histidine) in Retention of Physical Stability of Insulin in Aqueous Solutions

Purpose This study is to investigate whether poly(ethylene glycol) (PEG)-b-poly(L-histidine) [PEG-polyHis] can reduce aggregation of insulin in aqueous solutions on agitation by forming ionic complexes. Materials and Methods Insulin aggregation on agitation was monitored spectrophotometrically and by fibrillation studies with a dye Thioflavin T. Pluronic F-127 as a control and PEG-polyHis as a novel multifunctional excipient were added to prevent destabilization of insulin. Conformation of insulin was evaluated in a circular dichroism (CD) study. Results Ionic interactions between insulin and PEG-polyHis were induced in the pH range: 5.5–6.5. pH 5.5 was selected for further evaluation based on particle size/zeta potential studies. Ionic complexation with PEG-polyHis is more effective at pH 5.5 in stabilizing insulin (75% of insulin retained versus 0% with no excipient) than Pluronic F-127 (42% retained). PEG-polyHis guards against insulin aggregation in non-complexing pH conditions (pH 7.4), 64% insulin retained versus 58% with F-127 and 0% with no excipient) pointing to the potential role played by PEG in modulation of insulin surface adsorption. Rate of fibrillation was higher for plain insulin compared with addition of PEG-polyHis and Pluronic F-127 at both pH. Conclusions Understanding and manipulation of such polyelectrolyte-protein complexation will likely play a role in protein stabilization.     

Total synthesis of norneolignans from<Emphasis Type="Italic">Krameria</Emphasis> species

The total synthesis of nomeolignans isolated from Krameria species, 2-aryl-5-(E)-propenylbenzofurans (5, 11), is described. The key step involves the one-pot reaction for 2-arylbenzofurans (2, 7) from 4-hydroxyphenylacetone with 4'-acetoxy-2-chloro-2-(methylthio)acetophenone (1) and 2-chloro-2-methylthio-(2',4',6'-trimethoxy)acetophenone (6) under Friedel-Crafts reactionconditions.     

Dose-escalation study of isoniazid in healthy volunteers with the rapid acetylator genotype of arylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase 2

Objective Genetic polymorphisms of arylamine N-acetyltransferase 2 (NAT2) result in large interindividual differences in the plasma concentration of isoniazid (INH). We hypothesized that the internationally recommended dosage should be increased for patients with two active NAT2 alleles (RA type) in order to achieve appropriate antituberculous efficiency; however, the pharmacokinetic effects of the dose increase have not been fully addressed. To estimate an optimal dosage for RA-type patients, we conducted a dose escalation study in healthy male volunteers carrying NAT2*4/*4. Methods Oral doses of 300 mg, 600 mg, and 900 mg of INH were administered to eight RA-type subjects, whereas 300 mg was administered to eight IA-type subjects with one active allele (NAT2*4). The pharmacokinetic parameters were estimated from plasma INH concentrations for 24 h postdose. Results The ratio of the mean area under the plasma-concentration time curve (AUC) was not proportional to the doses (1:2.6:5.0 for 300:600:900-mg dose) in parallel to the plasma concentration at 1 h (C₁) and 2 h (C₂) after administration. Compared with the IA-type group given 300 mg, the RA-type group had lower pharmacokinetic parameters at 300 mg (AUC, 66%; C₁, 72%; C₂, 61%), but higher parameters at 600 mg (AUC, 175%; C₁, 196%; C₂, 170%). Plasma concentrations of the IA-type group were within the therapeutic range. An optimal INH dose was calculated as 430 mg (7.2 mg/kg) for RA-type subjects to obtain an AUC comparable with that in IA-type subjects dosed with 300 mg. Conclusion In RA-type subjects, the pharmacokinetic parameters appeared to lack linearity with the increased dose of INH. We propose that the proper daily dose for RA-type patients is 1.5-times higher than that currently recommended.     

Antioxidant principles of<Emphasis Type="Italic">Nelumbo nucifera</Emphasis> stamens

In our ongoing study to identity antioxidants from natural sources, the antioxidant activity of Nelumbo nucifera stamens was evaluated for their potential to scavenge stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, inhibit total reactive oxygen species (ROS) generation, in kidney homogenatas using 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA), and scavenge authentic peroxynitrites (ONOO-). A methanol (MeOH) extract of the stamens of N. nucifera showed strong antioxidant activity in the ONOO- system, and marginal activity in the DPPH and total ROS systems, so were therefore fractionated with several organic solvents, such as dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and n-butanol (n-BuOH). The EtOAc soluble fraction, which exhibited strong antioxidant activity in all the model systems tested, was further purified by repeated silica gel and Sephadex LH-20 column chromatographies. Seven known flavonoids [kaempferol (1), kaempferol 3-O-beta-D-glucuronopyranosyl methylester (2), kaempferol 3-O-beta-D-glucopyranoside (3), kaempferol 3-O-beta-D-galactopyranoside (4), myricetin 3',5'-dimethylether 3-O-beta-D-glucopyranoside (5), kaempferol 3-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (6) and kaempferol 3-O-beta-D-glucuronopyranoside (7)], along with beta-sitosterol glucopyranoside (8), were isolated. Compound 1 possessed good activities in all the model systems tested. Compounds 2 and 7 showed scavenging activities in the DPPH and ONOO- tests, while compounds 3 and 4 were only active in the ONOO- test. Conversely, compound 8 showed no activities in any of the model systems tested.     

The influence of dose and <Emphasis Type="Italic">N</Emphasis>-acetyltransferase-2 (NAT2) genotype and phenotype on the pharmacokinetics and pharmacodynamics of isoniazid

Objective This study evaluated the pharmacokinetics of isoniazid (INH) associated with optimal early bactericidal activity (EBA), defined as 90% of the maximum EBA (EBA₉₀) and the influence of N-acetyltransferase-2 (NAT2) subtype on the ability of pulmonary tuberculosis (PTB) patients to reach the identified pharmacokinetic values after INH doses ranging from 0.2 to 10–12 mg/kg body weight. Methods INH serum concentrations and NAT2 subtype were determined during four studies of PTB patients in three of whom the EBA of INH was determined. The relationship of EBA to area under the curve (AUC) and 2-h serum concentrations was examined by exponential regression and fitted curves estimated the and 2-h serum concentrations at which EBA₉₀ was reached. Results EBA₉₀ was reached at an of 10.52 μg/ml per hour and 2-h serum concentrations of 2.19 μg/ml. An of 10.52 μg/ml per hour was reached by all 66 patients receiving a 10–12 mg/kg INH dose and all 21 receiving 6 mg/kg, except 1 of 10 (10%) homozygous fast (FF) acetylators; however, at 5 mg/kg, 4 of 12 (33%) FF and 26 of 27 (96%) heterozygous fast (FS), but all 21 homozygous slow (SS) acetylators did so; and 1 of 3 (33%) FF, 2 of 6 (33%) FS, but all 4 SS acetylators at dose 3 mg/kg. An INH 2-h serum concentration of 2.19 μg/ml was reached by all 66 patients receiving 10–12 mg/kg and all 21 receiving 6 mg/kg, except for 2 (20%) FF acetylators at a dose of 5 mg/kg; however, only 3 (25%) of 12 FF acetylators, but 26 (96%) of 27 FS acetylators, and all 21 SS acetylators reached this concentration; and at a dose of 3 mg/kg, 1 (33%) of 3 FF acetylators, 2 (33%) of 6 FF, but all 4 SS acetylators. Conclusions At a 6 mg/kg dose, all except a minority of FF NAT2 acetylators, achieve an INH and 2-h INH serum concentrations associated with EBA₉₀, as did all 4 SS acetylators receiving 3 mg/kg. Any dose reduction below 6 mg/kg body weight will tend to disadvantage a significant proportion of faster acetylators, but, conversely, SS acetylators require only a 3 mg/kg dose to achieve a satisfactory exposure to INH.     

Platelet anti-aggregating activities of bupleurumin from the aerial parts of<Emphasis Type="Italic">Bupleurum falcatum</Emphasis>

An aryltetralin lactone, (-)-(1S,2R,3R)-1-(3',4'-methylenedioxyphenyl)-3-(hydroxymethyl)-6,7-dimethoxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid lactone, was isolated from the methanol extract of the aerial parts of Bupleurum falcatum and named bupleurumin. The structure of the compound was determined on the basis of chemical and spectroscopic methods, including two-dimensional NMR spectrometry (e.g., 1H- and 13C-NMR, homo- and heteronuclear COSY, HMBC). Bupleurumin showed an 8-fold potent inhibitory effect (IC50: 47.5 uM) compared to that of acetylsalicylic acid (ASA, IC50 : 420 uM) on collagen induced platelet aggregation and comparable effects as ASA on arachidonic acid-induced platelet aggregation.