1-Methylnicotinamide protects against liver injury induced by concanavalin A via a prostacyclin-dependent mechanism: A possible involvement of IL-4 and TNF-α

We have recently demonstrated that concanavalin A (Con A)-induced hepatitis is associated with the release of endogenous 1-methylnicotinamide (MNA). Here we study the mechanism by which exogenous MNA alleviates Con A-induced liver inflammation and injury in vivo.The involvement of prostacyclin (PGI₂) in hepatoprotective action of MNA (30–100 mg kg^− 1; i.v.) was studied by the use of IP receptor antagonist RO3244794 (10 mg kg^− 1; p.o.) given prior to Con A (5–20 mg kg^− 1; i.v.). Liver damage was assessed by measurements of: liver specific transaminases in plasma (alanine aminotransferase; aspartate aminotransferase); cytokines release (IL-4, IFN-γ and TNF-α); liver histopathology; and 24 h survival rates. Additionally, the effect of a stable analog of prostacyclin (carbaprostacyclin) on IL-4, IFN-γ and TNF-α production by isolated spleen lymphocytes in response to Con A was analyzed.MNA diminished Con A-induced rise in liver specific transaminases, alleviated histopathological injury and improved 24 h survival rates, the latter effect in a degree comparable with the pretreatment of animals with dexamethasone (0.5 mg kg^− 1; i.p.). MNA inhibited also a rise in IL-4 and TNF-α concentration in plasma measured 2 h after Con A administration, while IFN-γ was less affected. The effects of MNA were reversed by pretreatment with IP antagonist RO3244794. In isolated spleen lymphocytes, carbaprostacyclin profoundly decreased production of IL-4, the effect on TNF-α was modest with no effect on IFN-γ production.In conclusion, MNA attenuated Con A-induced hepatitis by a prostacyclin-dependent mechanism involving the inhibition of lymphocytes-derived IL-4 and the inhibition of Kuppfer-cells derived TNF-α.     

Determination of lamivudine and zidovudine permeability using a different ex vivo method in Franz cells

IntroductionThe major processes that control the absorption of orally administered drugs are dissolution and gastrointestinal permeation. These processes depend on two main properties: solubility and permeability. Based on these characteristics, the Biopharmaceutical Classification System (BCS) was proposed as a tool to assist in biowaiver and bioavailability prediction of drugs.MethodsThe purpose of the present study was to evaluate the permeability of lamivudine (3TC) and zidovudine (AZT) using a different ex vivo method in Franz cells. A segment of jejunum was inserted in a Franz cells apparatus, in order to assess drug permeability in the apical–basolateral (A–B) and basolateral–apical (B–A) directions. Each drug was added to the donor chamber, collected from the acceptor chamber and analyzed by HPLC. Fluorescein (FLU) and metoprolol (METO) were used as low and high permeability markers, respectively.ResultsThe apparent permeability (P_app) results for the A–B direction were: P_{app FLU A–B} = 0.54 × 10^? 4 cm·s^? 1, P_{app METO A–B} = 7.99 × 10^? 4 cm·s^? 1, P_{app 3TC A–B} = 4.58 × 10^? 4 cm·s^? 1 and P_{app AZT A–B} = 5.34 × 10^? 4 cm·s^? 1. For the B–A direction, the P_app results were: P_{app FLU B–A} = 0.56 × 10^? 4 cm·s^? 1, P_{app METO B–A} = 0.25 × 10^? 4 cm·s^? 1, P_{app 3TC B–A} = 0.24 × 10^? 4 cm·s^? 1 and P_{app AZT B–A} = 0.19 × 10^? 4 cm·s^? 1.DiscussionFor the A–B direction, the P_app results of fluorescein and metoprolol show low and high permeability, respectively, indicating that the membranes were appropriate for permeability studies. For the A–B direction, the P_app results of 3TC and AZT suggest that these antiretroviral drugs have permeability values close to metoprolol. Nevertheless, for the B–A direction the P_app results do not suggest efflux mechanism for any of the drugs. Thereby, the different ex vivo methods using Franz cells can be successfully applied in drug permeability studies, in particular for drug biopharmaceutical classification.     

Contribution of NO,ATP-sensitive K+ channels and prostaglandins to adenosine receptor agonists-induced relaxation of the rat tail artery

The mechanism of relaxation in the rat tail artery induced by the adenosine A1 receptor-selective agonist N⁶-cyclohexyladenosine (CHA, 10 nM–300 μM) and the adenosine A1/A_2a receptor agonist 5’-N-ethylcarboxamidoadenosine (NECA, 10 nM–300 μM) has been characterized. To do this, we used α₁-receptor agonist phenylephrine to evoke contraction (10 μM), and inhibitors of nitric oxide synthase (L-NAME, 10 μM), ATP-sensitive K⁺ channels (glibenclamide, 10 μM) and prostaglandin synthesis (indomethacin, 10 μM). CHA and NECA induced relaxation of rat-tail artery by 80% and 70% in a concentration-dependent manner, respectively. The relaxation effect of NECA was completely abolished in the presence of L-NAME, while glibenclamide and indomethacin prevented CHA-induced relaxation of the rat tail artery by approximately 25% and 40%, respectively. Our results indicate that nonspecific effects such nitric oxide and prostaglandins release or the activation of potassium channels significantly contributed to the effects of CHA and NECA.     

Pilot toxicokinetic study and absolute oral bioavailability of the Fusarium mycotoxin enniatin B1 in pigs

The aim of present study was to reveal the toxicokinetic properties and absolute oral bioavailability of enniatin B1 in pigs. Five pigs were administered this Fusarium mycotoxin per os and intravenously in a two-way cross-over design. The toxicokinetic profile fitted a two-compartmental model. Enniatin B1 is rapidly absorbed after oral administration (T_1/2a = 0.15 h, T_max = 0.24 h) and rapidly distributed and eliminated as well (T_1/2elα = 0.15 h; T_1/2elβ = 1.57 h). The absolute oral bioavailability is high (90.9%), indicating a clear systemic exposure. After intravenous administration, the mycotoxin is distributed and eliminated rapidly (T_1/2elα = 0.15 h; T_1/2elβ = 1.13 h), in accordance with oral administration.     

Angiotensin-(1–7) counteracts the effects of Ang II on vascular smooth muscle cells,vascular remodeling and hemorrhagic stroke: Role of the NFкB inflammatory pathway

Angiotensin (Ang)-(1–7) is a potential vasoprotective peptide. In the present study, we investigated its counteractive effects to Ang II on vascular smooth muscle cells (VSMCs) and intracerebral hemorrhagic stroke (ICH) through inflammatory mechanism. In in vitro experiments, human brain VSMCs (HBVSMCs) were treated with vehicle, Ang II, Ang II + Ang-(1–7), Ang II + A-779 or Ang II + Ang-(1–7) + A-779 (Mas receptor antagonist). HBVSMC proliferation, migration and apoptosis were determined by methyl thiazolyltetrazolium, wound healing assay and flow cytometry, respectively. In in vivo experiments, C57BL/6 mice were divided into vehicle, Ang II, Ang II + Ang-(1–7), Ang II + A-779 or Ang II + Ang-(1–7) + A-779 groups before they were subjected to collagenase-induced ICH or sham surgery. Hemorrhage volume and middle cerebral artery (MCA) remodeling were determined by histological analyses. Levels of NFκB, inhibitor of κBα (IκBα), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interleukin (IL-8) were measured by western blot or ELISA. We found that 1) Ang II increased HBVSMC migration, proliferation and apoptosis, and increased the blood pressure (BP), neurological deficit score, MCA remodeling and hemorrhage volume in ICH mice. 2) Ang-(1–7) counteracted these effects of Ang II, which was independent of BP, with the down-regulation of NFκB, up-regulation of IκBα, and decreased levels of TNF-α, MCP-1 and IL-8. 3) The beneficial effects of Ang-(1–7) could be abolished by A-779. In conclusion, Ang-(1–7) counteracts the effects of Ang II on ICH via modulating NFκB inflammation pathway in HBVSMCs and cerebral microvessels.     

Half-life extension of the HIV-fusion inhibitor peptide TRI-1144 using a novel linker technology

We have previously developed a linker technology for half-life extension of peptides, proteins and small molecule drugs (1). The linkers undergo β-elimination reactions with predictable cleavage rates to release the native drug. Here we utilize this technology for half-life extension of the 38 amino acid HIV-1 fusion inhibitor TRI-1144. Conjugation of TRI-1144 to 40 kDa PEG by an appropriate β-eliminative linker and i.v. administration of the conjugate increased the in vivo half-life of the released peptide from 4 to 34 h in the rat, and the pharmacokinetic parameters were in excellent accord with a one-compartment model. From these data we simulated the pharmacokinetics of the PEG-TRI-1144 conjugate in humans, predicting a t_1/2,_β of 70 h for the released peptide, and that a serum concentration of 25 nM could be maintained by weekly doses of 8 μmol of the conjugate. Using a non-circulating carrier (2) similar simulations indicated a t_1/2,_β of 150 h for the peptide released from the conjugate and that dosing of only 1.8 μmol/week could maintain serum concentrations of TRI-1144 above 25 nM. Hence, releasable β-eliminative linkers provide significant half-life extension to TRI-1144 and would be expected to do likewise for related peptides.     

Interaction effects of Fusarium enniatins (A,A1, B and B1) combinations on in vitro cytotoxicity of Caco-2 cells

Foodstuff is usually contaminated by more than one mycotoxin, however toxicological data are lacking as regards the effects in combinations compared to their individual effect. This study investigated the in vitro effects of enniatins (ENs) A, A₁, B and B₁, alone and in combinations, on Caco-2 cells viability by MTT assay after 24 h of exposure. Cells were treated with concentrations ranging from 0.9 to 15.0 μM, individually and in combination of two, three and four mycotoxins. Dose–response curves were generated for each mycotoxin and the isobologram method was used to determine the interactive effects of tested mixtures. Tested ENs produced significant cytotoxic effects both individually and in combination in a dose-dependent manner. IC₅₀ values obtained for all individually tested mycotoxins ranged from 1.3 to >15 μM. In ENs combination tests, synergistic effect in Caco-2 viability are observed for EN B + EN A₁, EN B₁ + EN A₁ and EN A + EN A₁ + EN B (CI = 0.33–0.52). All other combinations showed additive effect at medium and high affected fraction with exception of lower fraction affected and the EN B + EN B₁ mixture that produced antagonistic effect (CI = 1.76–10.36). The use of combination index-isobole method could help to better understand the potential interaction between co-occurring mycotoxins and may contribute to their risk assessment.     

Aflatoxin B1 modulates the insulin-like growth factor-2 dependent signaling axis

Although aflatoxin B₁ (AFB₁) is known as a mycotoxin that induces hepatocellular carcinoma (HCC), its effects on HCC cells have not been sufficiently investigated.The HCC cell lines HepG2, Huh-6, Huh-7, and PLC were cultured (5 × 10⁵ cells/ml) and various concentrations of AFB₁ were added. The expression levels of the α-fetoprotein (AFP), insulin-like growth factor-2 (IGF-2), and insulin-like growth factor-1 receptor (IGF-1R) genes in each sample were determined by real-time PCR, with the following results:(1) The level of AFP expression in HepG2 increased at 5–50 ng/ml of AFB₁ in a dose-dependent manner. The AFP expression level in Huh-6 increased at 0.01–5 ng/ml of AFB₁ in a dose-dependent manner and decreased to half controls level at 50 ng/ml of AFB₁. The AFP expression level in Huh-7 decreased to one-third the original level at 0.5–50 ng/ml of AFB₁. The AFP expression level in PLC decreased at 0–0.5 ng/ml of AFB₁ in a dose-dependent manner, and decreased to one-third at concentrations of AFB₁ between 0.5 and 50 ng/ml.(2) The IGF-2 and IGF-1R expression levels in Huh-6 increased more than 10-fold at 0.5–5 ng/ml of AFB₁, but decreased to half at 50 ng/ml of AFB₁. The IGF-2 and IGF-1R expression levels in other cell lines increased in a dose-dependent manner.AFB₁ induced translations of IGF-2 and IGF-1R and cell proliferation: When 50 ng/ml AFB₁ was administrated, cell numbers were 2.0-, 1.7-, and 1.5-fold higher than those of controls after 3 days of culture in HepG2, Huh-7, and PLC, respectively. Particularly, in Huh-6, it increased 2.5-fold higher than those of controls following 5 ng/ml AFB₁ administration. The ratio of fold-change phospho-IGF-1R in all cell lines that were treated with AFB₁, increased 1.1–1.5-fold.These results indicate that AFB₁ may enhance HCC cell proliferation through an IGF-2-dependent signal axis, although it remains to be investigated whether those effects are associated with human hepatocarcinogenesis resulting from AFB₁ exposure.     

Concentration-response relationship of the α7 nicotinic acetylcholine receptor agonist FRM-17874 across multiple in vitro and in vivo assays

Pharmacological activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) may improve cognition in schizophrenia and Alzheimer’s disease. The present studies describe an integrated pharmacological analysis of the effects of FRM-17874, an analogue of encenicline, on α7 nAChRs in vitro and in behavioral and neurophysiological assays relevant to cognitive function. FRM-17874 demonstrated high affinity binding to human α7 nAChRs, displacing [³H]-methyllacaconitine (K_i = 4.3 nM). In Xenopus laevis oocytes expressing human α7 nAChRs, FRM-17874 acted as an agonist, evoking inward currents with an EC₅₀ of 0.42 μM. Lower concentrations of FRM-17874 (0.01–3 nM) elicited no detectable current, but primed receptors to respond to sub-maximal concentrations of acetylcholine. FRM-17874 improved novel object recognition in rats, and enhanced memory acquisition and reversal learning in the mouse water T-maze. Neurophysiological correlates of cognitive effects of drug treatment, such as synaptic transmission, long-term potentiation, and hippocampal theta oscillation were also evaluated. Modulation of synaptic transmission and plasticity was observed in rat hippocampal slices at concentrations of 3.2 and 5 nM. FRM-17874 showed a dose-dependent facilitation of stimulation-induced hippocampal theta oscillation in mice and rats. The FRM-17874 unbound brain concentration–response relationship for increased theta oscillation power was similar in both species, exhibited a biphasic pattern peaking around 3 nM, and overlapped with active doses and exposures observed in cognition assays. In summary, behavioral and neurophysiological assays indicate a bell-shaped effective concentration range and this report represents the first attempt to explain the concentration–response function of α7 nAChR-mediated pro-cognitive effects in terms of receptor pharmacology.     

Characterisation of the roles of ABCB1, ABCC1, ABCC2 and ABCG2 in the transport and pharmacokinetics of actinomycin D in vitro and in vivo

Actinomycin D plays a key role in the successful treatment of Wilms tumour. However, associated liver toxicities remain a drawback to potentially curative treatment. We have used MDCKII cells over-expressing ABCB1, ABCC1, ABCC2 and ABCG2, alongside knockout mouse models to characterise actinomycin D transport and its impact on pharmacokinetics. Growth inhibition, intracellular accumulation and cellular efflux assays were utilised. A 59-fold difference in GI₅₀ was observed between MDCKII-WT and MDCKII-ABCB1 cells (12.7 nM vs. 745 nM, p < 0.0001). Reduced sensitivity was also seen in MDCKII-ABCC1 and ABCC2 cells (GI₅₀ 25.7 and 40.4 nM respectively, p < 0.0001). Lower intracellular accumulation of actinomycin D was observed in MDCKII-ABCB1 cells as compared to MDCKII-WT (0.98 nM vs. 0.1 nM, p < 0.0001), which was reversed upon ABCB1 inhibition. Lower accumulation was also seen in MDCKII-ABCC1 and ABCC2 cells. Actinomycin D efflux over 2 h was most pronounced in MDCKII-ABCB1 cells, with 5.5-fold lower intracellular levels compared to WT. In vivo studies showed that actinomycin D plasma concentrations were significantly higher in Abcb1a/1b^?/? as compared to WT mice following administration of 0.5 mg/kg actinomycin D (AUC_0–6 h 242 vs. 152 μg/L h respectively). While comparable actinomycin D concentrations were observed in the kidneys and livers of Abcb1a/1b^?/? and Abcc2^?/? mice, concentrations in the brain were significantly higher at 6 h following drug administration in Abcb1a/1b^?/? mice compared to WT. Results confirm actinomycin D as a substrate for ABCB1, ABCC1 and ABCC2, and indicate that Abcb1a/1b and Abcc2 can influence the in vivo disposition of actinomycin D. These data have implications for ongoing clinical pharmacology trials involving children treated with actinomycin D.     

CYP2B6 haplotype and biological factors responsible for hepatotoxicity in HIV-infected patients receiving efavirenz-based antiretroviral therapy

Data on the pharmacogenetic markers of CYP2B6 and biological factors associated with hepatotoxicity in HIV-infected patients receiving an efavirenz-based antiretroviral therapy (ART) regimen are very limited. A total of 134 HIV-infected Thai adults were prospectively enrolled to receive a once-daily regimen of efavirenz 600 mg/tenofovir/lamivudine. Seven single nucleotide polymorphisms (SNPs) within CYP2B6 were genotyped using real-time PCR. At 12 weeks after ART, plasma efavirenz concentrations at 12 h after dosing were measured. The mean ± standard deviation patient age was 37 ± 8 years, and 77.6% were male. The median (IQR) CD4 count was 43 cells/mm³ (17–105 cells/mm³). Eighteen patients (13.4%) had positive anti-HCV and 5 patients (3.7%) had positive HBsAg. The frequencies of heterozygous/homozygous mutants of each SNP were 64C>T (11%), 499C>G (0%), 516G>T (55%), 785A>G (63%), 1375A>G (0%), 1459C>T (3%) and 21563C>T (62%). The three most frequent haplotypes identified included *1/*6 (40.3%), *1/*1 (34.3%) and *6/*6 (8.2%). The median (IQR) plasma efavirenz concentration was 2.3 mg/L (1.4–3.7 mg/L). At 24 weeks, median (IQR) serum ALP was 98 mg/dL (73–133 mg/dL) and direct bilirubin was 0.11 mg/dL (0.10–0.19 mg/dL). The proportion of grade 1 and grade 2 elevated serum ALP was 12.7% and 1.5%, respectively. By multivariate analysis, factors associated with high ALP, total bilirubin and direct bilirubin included CYP2B6 haplotype *6/*6, high serum ALP at Week 0 and positive anti-HCV (all P < 0.05). In summary, HIV-infected patients with the pharmacogenetic marker ‘CYP2B6 haplotype *6/*6’ may have increased susceptibility to hepatotoxicity with efavirenz-based ART.     

Zn2+, derived from cell preparation,partly attenuates Ca2+-dependent cell death induced by A23187, calcium ionophore,in rat thymocytes

A23187, a calcium ionophore, is used to induce Ca²⁺-dependent cell death by increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i) under in vitro condition. Since this ionophore also increases membrane permeability of metal divalent cations such as Zn²⁺ and Fe²⁺ rather than Ca²⁺, trace metal cations in cell suspension may affect Ca²⁺-dependent cell death induced by A23187. Therefore, the effects of chelators for divalent metal cations, EDTA and TPEN, on the A23187-induced cytotoxicity were cytometrically examined in rat thymocytes. The cytotoxicity of A23187 was attenuated by 1 mM EDTA while it was augmented by 50 μM EDTA and 10 μM TPEN. These changes were statistically significant. The A23187-induced increase in Fluo-3 fluorescence intensity, a parameter for [Ca²⁺]_i, was significantly reduced by 1 mM EDTA while it was not the case for 50 μM EDTA and 10 μM TPEN. The intensity of FluoZin-3 fluorescence, a parameter for [Zn²⁺]_i, increased by A23187 was respectively reduced by 50 μM EDTA and 10 μM TPEN. It is suggested that the attenuation of A23187-induced cytotoxicity by 1 mM EDTA is due to the chelation of extracellular Ca²⁺ and Zn²⁺ while the augmentation by 50 μM ETDA or 10 μM TPEN is due to the chelation of extracellular Zn²⁺. The Tyrode’s solution without thymocytes contained 32.4 nM of zinc while it was 216.9 nM in the cell suspension. In conclusion, trace Zn²⁺, derived from cell preparation, partly attenuates the Ca²⁺-dependent cell death induced by A23187.     

Preparation and characterisation of novel chlorothiazide potassium solid-state salt forms: Intermolecular self assembly suprastructures

Chlorothiazide (CTZ) is a poorly soluble diuretic agent. The aim of the present work was to produce and characterise a potassium salt form of chlorothiazide which has the potential advantages of improved aqueous solubility and potassium supplementation. A number of novel potassium salt forms of CTZ (CTZK) were prepared: CTZK monohydrate (form I), CTZK dihydrate (form II), anhydrous CTZK (form III), CTZK monohydrate hemiethanolate (form IV) and a desolvate of CTZK monohydrate hemiethanolate (form V). These salt forms were characterised by thermal analysis, PXRD, NMR, elemental analysis, FTIR, Karl Fisher titrimetry, ICP-MS and GC–MS. The ethanol-free CTZK forms were also characterised by dynamic vapour sorption analysis (DVS). CTZK form I was stable (in the DVS) over the range 0–60% RH. The dihydrate form of the salt was stable (in the DVS) over a broader range of relative humidities, 10–90% RH at 25 °C. CTZK form II was less hygroscopic at high humidities (70–90% RH) than the previously reported CTZNa dihydrate. Single crystal X-ray analysis indicated that chlorothiazide potassium, crystallised from water or water/acetone mixture, formed a dihydrated polymeric-like intermolecular self-assembly (ISA) suprastructure. The ISA coordination was determined to be: (CTZ)₃·K·(H₂O)₂(CTZ)₂·(H₂O)₂·K·(CTZ)₃ (monoclinic, space group: C2/c, single crystal cell parameters: a = 18.328(4) Å, b = 7.3662(16) Å, c = 19.993(5) Å, α = 90°, β = 99.729(3)°, γ = 90°). When CTZK was crystallised from ethanol, a monohydrate hemiethanolate ISA was formed, described as (CTZ)₃·K·CTZ·(H₂O)₂·CTZ·K·(CTZ)₂ (triclinic, space group: P-1, single crystal cell parameters: a = 7.078(3) Å, b = 9.842(5) Å, c = 21.994(11) Å, α = 87.522(13)°, β = 84.064(14)°, γ = 78.822(12)°). The aqueous solubility of CTZK dihydrate, was determined to be 78.71 ± 1.82 mg/ml, approximately 400-fold higher than chlorothiazide, indicating a biopharmaceutical advantage associated with the potassium salt form.     

CD4+ B220+ TCRγδ+ T cells produce IL-17 in lupus-prone MRL/lpr mice

Systemic lupus erythematosus is an autoimmune disease with comprehensive immune cell disorders. Recent studies suggested that pro-inflammatory cytokine IL-17 plays important role in lupus, leaving the cellular sources and their pathogenic and physiologic characters largely unknown. In the current study, by using lupus-prone MRL/lpr mice, we demonstrated that Th17 response prevails in lupus disease regarding significantly accumulated serum IL-17, increased IL-17-producing splenocytes, and elevated phospho-STAT3 in CD4⁺ T cells. Intracellular staining revealed that unusual CD4⁺ B220⁺ T cells are major IL-17-producing cells, whereas conventional CD4⁺ B220⁻ T cells are major IFN-γ-producing cells. Subsequent studies showed that CD4⁺ B220⁺ cells contains both αβ and γδ T cells in the spleen and thymus of MRL/lpr mice. Further study showed that around 60% of γδ T cells in MRL/lpr mice co-express both B220 and CD4 on their surface, and are the major RORγt⁺ cells in MRL/lpr mice. Finally, CD4⁺ B220⁺ T cells alone do not proliferate, but could enhance the proliferation and IFN-γ-production of conventional CD4⁺ B220⁻ T cells. Our findings suggest the pathogenic role of unusual CD4⁺ B220⁺ T cells in lupus disease in MRL/lpr mice according to their IL-17-producing ability and stimulatory function for conventional CD4⁺ B220⁻ T cells.     

In vitro activity of tigecycline in Plasmodium falciparum culture-adapted strains and clinical isolates from Gabon

Emerging drug resistance in Plasmodium falciparum and its rapid spread in endemic countries have made the quest for new antimalarials a research priority. Tetracycline and its derivatives are well-established compounds for combination with faster-acting drugs in the current practice of malaria treatment. Tigecycline is the first marketed derivative of a new class of tetracycline antibiotics. Its altered structure leads to enhanced activity against bacteria and may also be associated with improved antimalarial activity. Using the histidine-rich protein 2 (HRP2) drug sensitivity assay, we determined the geometric mean 50% inhibitory concentrations (IC₅₀) of tigecycline in culture-adapted strains as well as in 23 clinical P. falciparum isolates from Lambaréné, Gabon. These values were compared with other tetracyclines as well as with clindamycin. Assays with 3 days and 6 days of incubation were evaluated to explore the impact of delayed parasite death on drug activity. IC₅₀ values in clinical isolates after 6 days of incubation were 160.0 nM [95% confidence interval (CI) 114.6–223.4 nM] for tigecycline, 739.4 nM (445.9–1226.1 nM) for doxycycline and 9.2 nM (95% CI 6.6–12.9 nM) for clindamycin. Tigecycline was found to act faster against plasmodia than any of the other antibiotics tested. This study demonstrates the potential of tetracycline derivatives in the development of improved antimalarials.     

Human Skin is Permselective for the Small,Monovalent Cations Sodium and Potassium but not for Nickel and Chromium

The molar conductance of excised human skin (Λ_skin) immersed in electrolyte solutions comprising four cationic (Na⁺, K⁺, Ni^2 +, and Cr^3 +) and five anionic (Cl^?, NO₃^?, SO₄^2 ?, CrO₄^2 ?, and Cr₂O₇^2 ?) species was determined as a function of concentration in Franz diffusion cells. Cation transport numbers for four of these electrolytes were measured in Franz cells by the electromotive force method. Parallel experiments were conducted in solutions alone to establish the validity of the technique. Molar conductance decreased with increasing concentration, following the Kohlrausch law, over a 4–12-fold concentration range. Molar conductance and cation transport values at infinite dilution were extrapolated from these data and used to estimate ionic conductances at infinite dilution. These values were subsequently used to calculate limiting ion mobilities and diffusivities in solution and skin. Results for skin showed the expected increase in cation permselectivity for monovalent cations and a 40–110-fold reduction in effective diffusivities with respect to those in solution. However, Ni^2 + and Cr^3 + were relatively less mobile in skin than in solution. Salt diffusivities calculated from ionic mobilities in skin provided a partial explanation for the difference in allergenic potency of NiCl₂compared with NiSO₄ and Cr^3 + versus Cr^6 + salts.     

Antidepressant-like effects of 071031B,a novel serotonin and norepinephrine reuptake inhibitor

SNRIs (serotonin and norepinephrine reuptake inhibitors) have been proposed to exert increased therapeutic efficacy or be faster acting compared to commonly used antidepressants. In this study, we performed in vitro binding and uptake assays and in vivo behavioral tests to assess the pharmacological properties and antidepressant-like efficacy of the compound 071031B; we also performed cytotoxicity tests using HepG2 cells and SH-SY5Y cells to predict the toxicity of 071031B. In vitro, 071031B had high affinity for both serotonin transporters and norepinephrine transporters prepared from rat cortex tissue (K_i=2.68 and 1.09 nM, respectively) and recombinant cells (K_i=1.57 and 0.36 nM, respectively). Moreover, 071031B also potently inhibited the uptake of serotonin (5-HT) and norepinephrine (NE) into rat cortical synaptosomes (K_i=1.99 and 1.09 nM, respectively) and recombinant cells (K_i=3.23 and 0.79 nM, respectively). In vivo, acute administration of 071031B dose-dependently reduced the immobility time in the tail suspension test in mice and the forced swimming test in mice and rats with higher efficacy than duloxetine and showed no stimulatory effect on the locomotor activity. Chronic 071031B treatment (5 or 10 mg/kg) significantly reversed depressive-like behaviors in chronically stressed rats, including reduced sucrose preference, decreased locomotor activity, and prolonged latency to begin eating. Furthermore, 071031B also exhibited lower cytotoxicity in HepG2 cells and SH-SY5Y cells in vitro than duloxetine. These findings suggest that 071031B is a novel, balanced serotonin and norepinephrine reuptake inhibitor, with more potent antidepressant effects and lower hepatotoxicity and neurotoxicity in vitro than duloxetine.     

Extractive spectrophotometric assay of cyclizine in a pharmaceutical formulation and biological fluids

Two highly sensitive and simple spectrophotometric methods were developed to quantitate the drug cyclizine (CYC) in its pure form and in a pharmaceutical formulation. The two methods involved ion-associate formation reactions (method A) with mono-acid azo dyes, i.e., sudan (I) and sudan (II), as well as ion-pair reactions (method B) with bi-azo dyes, i.e., sudan (III), sudan (IV) and sudan red 7B (V). The reactions were extracted with chloroform, and the extraction products were quantitatively measured at 480, 550, 500, 530 and 570 nm using reagents I–V, respectively. The reaction conditions were monitored and optimised. The Beer plots for reagents I–V showed linear relationships for the concentrations of 4.2–52.0, 5.4–96.0, 3.5–43.0, 4.4–80.0 and 0.6–18.0 μg mL^?1, respectively, with molar absorptivities of 2.2 × 10⁴, 4.1 × 10⁴, 3.6 × 10⁴, 2.5 × 10⁴ and 1.3 × 10⁴ L mol^?1 cm^?1, respectively. Sandell sensitivities and detection limits were calculated and analysed. The implementation of the two methods to the analysis of a commercial tablet (Valoid) succeeded, and the recovery study suggested that there was no interference from common excipients in the tablet. Regarding the accuracy and precision of the methods, a statistical comparison of the results was performed using Student’s t-test and the F-test at the 95% confidence level. The accuracy and precision of the proposed methods were not significantly different.     

A new and simple HPLC method for determination of etamsylate in human plasma and its application to pharmacokinetic study in healthy adult male volunteers

A new and simple HPLC assay method was developed and validated for the determination of etamsylate in human plasma. After protein precipitation with 6% perchloric acid, satisfactory separation was achieved on a HyPURITY C₁₈ column (250 mm × 4.6 mm, 5 μm) using a mobile phase comprising 20 mM sodium dihydrogen phosphate-2 hydrate (pH was adjusted to 3.5 by phosphoric acid) and acetonitrile at a ratio of 95:5 v/v. The elution was isocratic at ambient temperature with a flow rate of 0.75 ml/min. Allopurinol was used as internal standard. The calibration curve was linear over the range from 0.25 to 20 μg/ml (r² = 0.999). The limit of quantification for etamsylate in plasma was 0.25 μg/ml. The within day coefficient of variance (%CV) ranged from 3.9% to 10.2%, whereas the between-day %CV ranged from 3.1% to 8.7%. The assay method has been successfully used to estimate the pharmacokinetics of etamsylate after oral administration of a 500 mg tablet under fasting conditions to 24 healthy Egyptian human male volunteers. Various pharmacokinetic parameters including AUC_0–t, AUC_0–∞, C_max, T_max, t_1/2, MRT, Cl/F, and V_d/F were determined from plasma concentration–time profile of etamsylate.     

P2X1 receptor-mediated pressor responses in the anesthetized mouse

P2X₁ receptors and adrenoceptors are mainly responsible for vasoconstriction in a variety of blood vessels. However, previous studies have shown that α,β-methylene adenosine 5′-triphosphate (α,β-MeATP), a stable analogue of ATP, can induce both pressor and depressor responses in laboratory animals. In this study, the effects of increasing intravenous doses of α,β-MeATP and noradrenaline (NA) (0–30 nmol/kg) administered at 20 min intervals on systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure in groups of anesthetized mice (n=6) were compared. Both α,β-MeATP and NA caused transient, dose-dependent increases in SBP, DBP and MBP but the effect of α,β-MeATP was more rapid and significantly larger at doses of 10 and 30 nmol/kg (P<0.01). At the dose of 30 nmol/kg, α,β-MeATP increased SBP, DBP and MBP by 65.8±7.0, 65.7±5.0 and 65.7±5.5 mmHg, respectively, compared to increases of 36.8±4.6, 33.3±4.9 and 34.5±4.7 mmHg, respectively, produced by NA. These results indicate P2X₁ receptors play an important role in BP regulation although purinergic vasoconstriction alone may not explain the more potent pressor response to α,β-MeATP in the anesthetized mouse.