RESUMO
Tafenoquine is being developed for radical cure and post-exposure prophylaxis of Plasmodium vivax malaria. In an open-label study, 1512 Australian Defence Force personnel received one of three tafenoquine 3 d regimens [400 mg once daily (od), 200 mg twice daily (bid), 200 mg od] or daily primaquine (22.5 mg) plus doxycycline (100 mg) over 14 d in Bougainville and in Timor-Leste for post-exposure prophylaxis. The relapse rate of subjects treated in Bougainville with tafenoquine (n=173) was 1.2% (200 mg bid x 3 d) and 2.3% (400 mg od x 3 d), while primaquine plus doxycycline (n=175) was 3.4%. For subjects treated in Timor-Leste with tafenoquine (n=636), the relapse rate was 4.9% (200 mg od x 3 d), 5.3% (200 mg bid x 3 d) and 11.0% (400 mg od x 3d), while primaquine plus doxycycline (n=289) was 10.0%. The most frequent adverse events reported across all groups were nausea, abdominal distress and diarrhoea. There was a dose-dependent reduction in adverse events with a reduced dose of tafenoquine, with the lowest dose (total 600 mg over 3 d) producing rates of adverse events equivalent to that of primaquine plus doxycycline. The much shorter dosing regimen of tafenoquine should increase compliance, which is often suboptimal with primaquine after leaving an endemic area. [Australian New Zealand Clinical Trials Registry Number 12607000588493].
Assuntos
Aminoquinolinas/administração & dosagem , Antimaláricos/administração & dosagem , Malária Vivax/tratamento farmacológico , Plasmodium vivax/efeitos dos fármacos , Primaquina/administração & dosagem , Adulto , Aminoquinolinas/efeitos adversos , Animais , Antimaláricos/efeitos adversos , Austrália , Relação Dose-Resposta a Droga , Feminino , Humanos , Malária Vivax/prevenção & controle , Masculino , Militares , Primaquina/efeitos adversos , Estatística como Assunto , Adulto JovemRESUMO
In an open-label sequential cohort study, we compared gastrointestinal (GI) disturbances and plasma tafenoquine concentrations after administration of single-dose (400mg daily x 3 days; n=76 males, 11 females) and split-dose (200 mg twice daily x 3 days; n=73 males, 13 females) tafenoquine regimens in healthy Australian Defence Force volunteers for post-exposure malaria prophylaxis. The female and male volunteers had comparable demographic characteristics (age, weight, height) in the single- and split-dose treatment groups. GI disturbances were generally mild and self-limiting for both groups. The frequency of nausea and abdominal distress was over two-fold higher in females than in males for both treatment groups. Reporting of GI disturbances in the single-dose group differed significantly between males and females, but this gender difference was not seen for the split-dose group. In those volunteers who experienced GI disturbances, the mean plasma tafenoquine concentrations 12 h after the last dose of tafenoquine were approximately 1.3-fold higher in females than in males (means+/-SD: 737+/-118 ng/ml vs. 581+/-113 ng/ml). These preliminary findings suggest that further studies are required in a larger number of females to determine whether there is a need to reduce the dose of tafenoquine to minimise GI disturbances in females.
Assuntos
Aminoquinolinas/efeitos adversos , Antimaláricos/efeitos adversos , Gastroenteropatias/induzido quimicamente , Malária Vivax/prevenção & controle , Adulto , Aminoquinolinas/sangue , Aminoquinolinas/uso terapêutico , Antimaláricos/sangue , Antimaláricos/uso terapêutico , Esquema de Medicação , Monitoramento de Medicamentos/métodos , Feminino , Gastroenteropatias/sangue , Humanos , Masculino , Náusea/sangue , Náusea/induzido quimicamente , Caracteres SexuaisRESUMO
No validated treatments exist for transmissible spongiform encephalopathies (TSEs or prion diseases) in humans or livestock. The search for TSE therapeutics is complicated by persistent uncertainties about the nature of mammalian prions and their pathogenic mechanisms. In pursuit of anti-TSE drugs, we and others have focused primarily on blocking conversion of normal prion protein, PrP(C), to the TSE-associated isoform, PrP(Sc). Recently developed high-throughput screens have hastened the identification of new inhibitors with strong in vivo anti-TSE activities such as porphyrins, phthalocyanines, and phosphorthioated oligonucleotides. New routes of administration have enhanced beneficial effects against established brain infections. Several different classes of TSE inhibitors share structural similarities, compete for the same site(s) on PrP(C), and induce the clustering and internalization of PrP(C) from the cell surface. These activities may represent a common mechanism of action for these anti-TSE compounds.
Assuntos
Doenças Priônicas/tratamento farmacológico , Príons , Animais , Sítios de Ligação , Sistema Nervoso Central/patologia , Doenças Priônicas/prevenção & controle , Príons/metabolismo , Relação Estrutura-AtividadeRESUMO
AIMS: To describe the population pharmacokinetics of tafenoquine in healthy volunteers after receiving tafenoquine for malaria prophylaxis. METHODS: The population consisted of 135 male Thai soldiers (mean age 28.9 years; weight 60.3 kg). All soldiers were presumptively treated with artesunate for 3 days plus doxycycline for 7 days to remove any pre-existing malaria infections. After the treatment regime, 104 soldiers (drug group) received a loading dose of 400 mg tafenoquine base daily for 3 days followed by 400 mg tafenoquine monthly for 5 consecutive months. In the placebo group, 31 soldiers were infected with malaria during the study period. They were re-treated with artesunate for 3 days plus doxycycline for 7 days followed by a loading dose of 400 mg tafenoquine daily for 3 days and then 400 mg tafenoquine weekly for prophylaxis. Blood samples were randomly collected from each soldier on monthly and weekly prophylaxis. Plasma tafenoquine concentrations were measured by h.p.l.c. Population pharmacokinetic modelling was performed using NONMEM. RESULTS: A one-compartment model was found best to describe the pharmacokinetics of tafenoquine after oral administration. Age and weight influenced volume of distribution (V/F), and subjects who contracted malaria had higher clearance (CL/F), but none of these factors was considered to have sufficient impact to warrant change in dosing. The population estimates of the first-order absorption rate constant (Ka), CL/F and V/F were 0.694 h(-1), 3.20 l h(-1) and 1820 l, respectively. The intersubject variability in these parameters (coefficient of variation, CV%) was 61.2%, 25.3% and 14.8%, respectively. The absorption and elimination half-lives were 1.0 h and 16.4 days, respectively. The residual (unexplained) variability was 17.9%. CONCLUSIONS: The population pharmacokinetics of orally administered tafenoquine have been determined in Thai soldiers under field conditions. This information, together with its known potent antimalarial activity, portends well for the application of tafenoquine as a useful prophylactic drug or for short-term radical treatment of vivax malaria.
Assuntos
Aminoquinolinas/farmacocinética , Antimaláricos/farmacocinética , Artemisininas , Malária Vivax/prevenção & controle , Administração Oral , Adulto , Aminoquinolinas/sangue , Aminoquinolinas/química , Antimaláricos/administração & dosagem , Antimaláricos/sangue , Artesunato , Simulação por Computador , Doxiciclina/uso terapêutico , Meia-Vida , Humanos , Malária Vivax/tratamento farmacológico , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Militares , Modelos Biológicos , Variações Dependentes do Observador , Sesquiterpenos/uso terapêutico , TailândiaRESUMO
A simple, rapid, and accurate high-pressure liquid chromatographic method with fluorescence detection is described for the measurement of tafenoquine (TQ) (also known as WR 238605) from human plasma and venous and capillary blood. Tafenoquine was measured in plasma and venous blood following protein precipitation. Chromatographic separation was achieved using a Waters S5P Spherisorb phenyl analytical cartridge (150 mm x 4.6 mm I.D., 5 microm particle size) (Waters, Milford, MA, USA) and a mobile phase of 22 mM ammonium acetate, pH 4:acetonitrile (45:55, vol/vol). The flow rate was 1.5 mL/min and the retention times were approximately 3.5 min for WR VIIIAc (internal standard) and approximately 7.8 min for TQ. The interday and intraday coefficients of variation of TQ over a concentration range of 20-1000 ng/mL in plasma were < or =8.4% and in venous blood were < or =9.6%. The mean percent difference between added concentration and obtained concentration was 7.3% in plasma and 8.5% in venous blood over the corresponding concentration range. The limit of quantitation for both fluids was 10 ng/mL. Tafenoquine concentrations were comparable between capillary and venous blood with no significant difference between measurement in both biological fluids. The clinical application of the method was demonstrated by measuring plasma and whole blood concentrations of TQ from participants in a chemosuppression trial of the drug against malaria infections in Thailand.
Assuntos
Aminoquinolinas/sangue , Antimaláricos/sangue , Cromatografia Líquida de Alta Pressão , Calibragem , HumanosRESUMO
Metabolism and disposition of most drugs used to treat malaria are substantially altered in malaria infection. Few data are available that specify effects of malaria infection on drug metabolism pathways in humans or animal model systems. In this report, studies were undertaken to determine the effect of Plasmodium berghei infection on cytochrome P-450 (CYP450) 2E1 and 3A2-mediated metabolism and enzyme expression in rat liver microsomes. Malaria infection (MAL) resulted in significant decreases in total cytochrome P-450 content (56%, P < 0.05) and NADPH cytochrome P-450 reductase activity (32%, P < 0.05) as compared to control (CON) rats. Chlorzoxazone 4-hydroxylase activity (CYP2E1-mediated) showed no significant difference between CON and MAL microsomes while testosterone 6-beta-hydroxylase activity (CYP3A2-mediated) was reduced by 41% (P < 0.05) in MAL. Enzyme kinetic studies and immunoblot analysis indicate that the loss of activity for CYP3A2 in malaria infection is due to significantly decreased CYP3A2 protein expression. The altered expression of CYP450s in malaria infection should be taken into account when treating patients with malaria in order to minimize drug-drug interactions or toxicity.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/metabolismo , Malária/metabolismo , Microssomos Hepáticos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Plasmodium berghei , Animais , Clorzoxazona/análise , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Densitometria , Immunoblotting , Técnicas In Vitro , Fígado/enzimologia , Malária/enzimologia , Masculino , Microssomos Hepáticos/enzimologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , Plasmodium berghei/fisiologia , Ratos , Ratos Sprague-Dawley , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Testosterona/análiseRESUMO
Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res. Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells. Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease-sensitive precursor, PrP-sen, after the latter reaches the cell surface. Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner. These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation.
Assuntos
Príons/análise , Animais , Técnicas de Cultura de Células/métodos , Sistema Livre de Células , Endopeptidases/metabolismo , Humanos , Métodos , Príons/metabolismoRESUMO
More than a million cattle infected with bovine spongiform encephalopathy (BSE) may have entered the human food chain. Fears that BSE might transmit to man were raised when atypical cases of Creutzfeldt-Jakob disease (CJD), a human transmissible spongiform encephalopathy (TSE), emerged in the UK. In BSE and other TSE diseases, the conversion of the protease-sensitive host prion protein (PrP-sen) to a protease-resistant isoform (PrP-res) is an important event in pathogenesis. Biological aspects of TSE diseases are reflected in the specificities of in vitro PrP conversion reactions. Here we show that there is a correlation between in vitro conversion efficiencies and known transmissibilities of BSE, sheep scrapie and CJD. On this basis, we used an in vitro system to gauge the potential transmissibility of scrapie and BSE to humans. We found limited conversion of human PrP-sen to PrP-res driven by PrP-res associated with both scrapie (PrP[Sc]) and BSE (PrP[BSE]). The efficiencies of these heterologous conversion reactions were similar but much lower than those of relevant homologous conversions. Thus the inherent ability of these infectious agents of BSE and scrapie to affect humans following equivalent exposure may be finite but similarly low.
Assuntos
Encefalopatia Espongiforme Bovina/transmissão , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Scrapie/transmissão , Zoonoses/transmissão , Animais , Bovinos , Sistema Livre de Células , Síndrome de Creutzfeldt-Jakob/transmissão , Cricetinae , Suscetibilidade a Doenças , Endopeptidase K/metabolismo , Humanos , Mesocricetus , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas PrPC/genética , Príons/metabolismo , Ovinos , Especificidade da Espécie , Células Tumorais CultivadasRESUMO
Denaturation studies with guanidine HCl (GdnHCl) were performed to test the relationship between scrapie infectivity and properties of scrapie-associated prion protein (PrP(Sc)). Large GdnHCl-induced reductions in infectivity were associated with the irreversible elimination of both the proteinase K resistance and apparent self-propagating converting activity of PrP(Sc). In intermediate GdnHCl concentrations that stimulate converting activity and partially disaggregate PrP(Sc), both scrapie infectivity and converting activity were associated with residual partially protease-resistant multimers of PrP(Sc).
Assuntos
Guanidinas/farmacologia , Proteínas PrPSc/toxicidade , Animais , Cricetinae , Endopeptidase K/farmacologia , Guanidina , Mesocricetus , Proteínas PrPSc/efeitos dos fármacos , Desnaturação ProteicaRESUMO
The conversion of the normal form of prion protein (PrPC) to a disease-specific form (PrPSc) is a central event in scrapie and other transmissible spongiform encephalopathies. PrPSc is distinguished from PrPC by its insolubility and its resistance to proteolysis. PrPSc is also capable of converting 35S-PrPC, in vitro, into a form which is indistinguishable from PrPSc with respect to its protease-sensitivity. Both the "converting activity" and the protease-resistance of isolated hamster PrPSc can be at least partially eliminated by denaturation and recovered by renaturation, provided that the concentration of denaturant does not exceed a threshhold. This study was undertaken in order to localize the regions of native PrPSc structure that must remain intact to allow refolding. Proteinase K was used to digest exposed, denatured PrPSc sequences, and the residual fragments were characterized using anti-PrP antibodies directed toward four PrP epitopes. A 16-kDa fragment marked by an epitope within residues 143-156 remained protease-resistant under conditions which at least partially unfolded epitopes within residues 90-115 and 217-232. However, dilution of denaturant restored protease-resistance to these epitopes. This reversible unfolding was observed with both purified PrPSc and PrPSc in crude brain homogenates. Size fractionation of partially GdnHCl-solubilized PrPSc revealed that only the insoluble aggregates retained the ability to refold, consistent with the hypothesis that native PrPSc is an ordered aggregate. When the threshold denaturant concentration was exceeded, both protease-resistance of the 16-kDa C-terminal domain and converting activity were irreversibly destroyed. These results suggest that the in vitro converting activity requires ordered, protease-resistant PrPSc aggregates and that a critical aspect of the PrPSc structure is the folding of a particularly stable approximately 16-kDa C-terminal domain.
Assuntos
Proteínas PrPSc/química , Animais , Cricetinae , Endopeptidase K , Guanidina , Guanidinas/farmacologia , Técnicas In Vitro , Mesocricetus , Estrutura Molecular , Peso Molecular , Proteína PrP 27-30/química , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica , Dobramento de Proteína , Scrapie/etiologia , Scrapie/metabolismoRESUMO
INTRODUCTION: Scrapie infection instigates the in vivo conversion of normal, protease-sensitive prion protein (PrPC) into a protease-resistant form (PrPSc) by an unknown mechanism. In vitro studies have indicated that PrPSc can induce this conversion, consistent with proposals that PrPSc itself might be the infectious scrapie agent. Using this cell-free model of the PrPC to PrPSc conversion, we have studied the dependence of conversion on reactant concentration, and the properties of the PrPSc-derived species that has converting activity. RESULTS: The cell-free conversion of 35S PrPC to the proteinase K-resistant form was dependent on the reaction time and initial concentrations of PrPSc (above an apparent minimum threshold concentration) and 35S PrPC. Analysis of the physical size of the converting activity indicated that detectable converting activity was associated only with aggregates. Under mildly chaotropic conditions, which partially disaggregated PrPSc and enhanced the converting activity, the active species were heterogeneous in size, but larger than either effectively solubilized PrP or molecular weight standards of approximately 2000 kDa. CONCLUSIONS: The entity responsible for the converting activity was many times larger than a soluble PrP monomer and required a threshold concentration of PrPSc. These results are consistent with a nucleated polymerization mechanism of PrPSc formation and inconsistent with a heterodimer mechanism.
Assuntos
Endopeptidases/metabolismo , Príons/metabolismo , Príons/farmacologia , Scrapie/metabolismo , Animais , Western Blotting , Sistema Livre de Células , Eletroforese em Gel de Poliacrilamida , Endopeptidases/isolamento & purificação , Cinética , Peso Molecular , Príons/isolamento & purificação , Radioisótopos de Enxofre , Ultracentrifugação , UltrafiltraçãoRESUMO
Amyloids are a class of noncrystalline, yet ordered, protein aggregates. A new approach was used to provide the initial structural data on an amyloid fibril--comprising a peptide (beta 34-42) from the C-terminus of the beta-amyloid protein--based on measurement of intramolecular 13C-13C distances and 13C chemical shifts by solid-state 13C NMR and individual amide absorption frequencies by isotope-edited infrared spectroscopy. Intermolecular orientation and alignment within the amyloid sheet was determined by fitting models to observed intermolecular 13C-13C couplings. Although the structural model we present is defined to relatively low resolution, it nevertheless shows a pleated antiparallel beta-sheet characterized by a specific intermolecular alignment.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Doença de Alzheimer/etiologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Biblioteca Gênica , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação ProteicaRESUMO
The infectious agents causing scrapie and other transmissible spongiform encephalopathies have been postulated to consist solely of the protease-resistant form of prion protein (PrPSc). One unprecedented requirement of the protein-only model is that the 'inheritance' of pathogen strain differences must be mediated by stable variations in PrPSc structure, rather than mutations in an agent-specific nucleic acid. Strain differences in PrPSc structure have been described for the hyper (HY) and drowsy (DY) strains of hamster transmissible mink encephalopathy (TME), a scrapie-like disease originating in mink. Although HY and DY PrPSc are both post-translationally derived from the precursor prion protein (PrPC) they are cleaved at different amino-terminal sites by proteinase K (ref. 8). Here we investigate whether this strain-specific property of PrPSc is transmitted to PrPC during formation of new PrPSc. PrPSc from the HY and DY TME strains converted the protease-sensitive PrPC into two distinct sets of protease-resistant PrP products in a cell-free system. These data provide evidence that self-propagation of PrPSc polymers with distinct three-dimensional structures could be the molecular basis of scrapie strains.
Assuntos
Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Príons/metabolismo , Animais , Cetilpiridínio/farmacologia , Cricetinae , Técnicas de Cultura , Endopeptidase K , Guanidina , Guanidinas/farmacologia , Mesocricetus , Proteínas PrPC/química , Proteínas PrPSc/química , Doenças Priônicas/metabolismo , Príons/classificação , Serina Endopeptidases/metabolismo , Especificidade da EspécieRESUMO
Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrPc) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to protease-resistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction. The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPc and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.
Assuntos
Endopeptidases/metabolismo , Príons/metabolismo , Scrapie/metabolismo , Animais , Cricetinae , Resistência a Medicamentos , Glicosilação , Camundongos , Camundongos Endogâmicos , Modelos Estruturais , Príons/química , Príons/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Especificidade da Espécie , Tunicamicina/farmacologiaRESUMO
The infectious agent (or 'prion') of the transmissible spongiform encephalopathies (TSEs) such as scrapie resembles a virus in that it replicates in vivo and has distinct strains, but it was postulated long ago to contain only protein. More recently, PrPSc, a pathogenic, scrapie-associated form of the host-encoded prion protein (PrP), was identified as a possible primary TSE agent protein. PrPSc is defined biochemically by its insolubility and resistance to proteases and is derived post-translationally from normal, protease-sensitive PrP (PrPc). The conversion seems to involve conformational change rather than covalent modification. However, the conversion mechanism and the relationship of PrPSc formation to TSE agent replication remain unclear. Here we report the conversion of PrPc to protease-resistant forms similar to PrPSc in a cell-free system composed of substantially purified constituents. This conversion was selective and required the presence of preexisting PrPSc, providing direct evidence that PrPSc derives from specific PrPc-PrPSc interactions.