RESUMO
Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network (the KLK activome) with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors enabling simultaneous orthogonal analysis of KLK2, KLK3, and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Importantly, we identifed a significant decoupling of KLK activity and abundance and suggest that KLK proteolysis should be considered as an additional parameter, along with the PSA blood test, for accurate PCa diagnosis and monitoring. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP), we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs, we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.
Assuntos
Antineoplásicos/farmacologia , Cumarínicos/farmacologia , Inibidores Enzimáticos/farmacologia , Calicreínas/antagonistas & inibidores , Antígeno Prostático Específico/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cumarínicos/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Calicreínas/metabolismo , Masculino , Estrutura Molecular , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
In the treatment of hookworm infections, pharmacotherapy has been only moderately successful and drug resistance is a threat. Therefore, novel treatment options including combination therapies should be considered, in which tribendimidine could play a role. Our aims were to (i) characterize the pharmacokinetics of tribendimidine's metabolites in adolescents receiving tribendimidine monotherapy or in combination with ivermectin or oxantel pamoate, (ii) evaluate possible drug-drug interactions (DDI), (iii) link exposure to response, and (iv) identify a treatment strategy associated with high efficacy, i.e., >90% cure rates (CRs), utilizing model-based simulations. A population pharmacokinetic model was developed for tribendimidine's primary and secondary metabolites, dADT and adADT, in 54 hookworm-positive adolescents, with combination therapy evaluated as a possible covariate. Subsequently, an exposure-response analysis was performed utilizing CRs as response markers. Simulations were performed to identify a treatment strategy to achieve >90% CRs. A two-compartmental model best described metabolite disposition. No pharmacokinetic DDI was identified with ivermectin or oxantel pamoate. All participants receiving tribendimidine plus ivermectin were cured. For the monotherapy arm and the arm including the combination with oxantel pamoate, Emax models adequately described the correlation between dADT exposure and probability of being cured, with required exposures to achieve 50% of maximum effect of 39.6 and 15.6 nmol/ml·h, respectively. Based on our simulations, an unrealistically high monotherapy tribendimidine dose would be necessary to achieve CRs of >90%, while combination therapy with ivermectin would meet this desired target product profile. Further clinical studies should be launched to develop this combination for the treatment of hookworm and other helminth infections.
Assuntos
Anti-Helmínticos , Infecções por Uncinaria , Adolescente , Ancylostomatoidea , Animais , Anti-Helmínticos/uso terapêutico , Infecções por Uncinaria/tratamento farmacológico , Humanos , Fenilenodiaminas/uso terapêuticoRESUMO
Tribendimidine has been successful in treating hookworm infections and may serve as an alternative to albendazole should resistance arise. Our aims were to (i) characterize the pharmacokinetics (PK) of tribendimidine's primary metabolite, deacetylated amidantel (dADT), and secondary metabolite, acetylated derivative of amidantel (adADT), in school-aged children and adolescents, (ii) link exposure to efficacy against hookworm, and (iii) evaluate whether tribendimidine pharmacotherapy in children could be further improved. First, a population PK model was developed based on dried-blood-spot samples collected from 155 school-aged children and adolescents with hookworm infections, following tribendimidine doses ranging from 100 to 400 mg. Second, an exposure-response analysis was conducted to link the active metabolite dADT to cure rates (CRs) and egg reduction rates (ERRs). Third, simulations were performed to identify a treatment strategy associated with >90% CRs. A two-compartmental model with transit compartments describing observed delay in absorption adequately described PK data of dADT and adADT. Allometric scaling was included to account for growth and development. The absorption rate was 56% lower with 200-mg tablets than with 50-mg tablets, while the extent of absorption remained unaffected. The identified Emax models linking dADT exposure to ERRs and CRs showed shallow curves, as increasing exposure led to marginal efficacy increase. Combination therapy should be considered, as a 12-fold-higher dose would be needed to achieve 95% ERRs and CRs >90% with tribendimidine alone. Further studies are warranted to evaluate safety of higher tribendimidine doses and combination therapies with other anthelmintic agents to improve treatment strategy for children with hookworm infection.
Assuntos
Anti-Helmínticos , Infecções por Uncinaria , Adolescente , Albendazol/uso terapêutico , Ancylostomatoidea , Animais , Anti-Helmínticos/uso terapêutico , Criança , Infecções por Uncinaria/tratamento farmacológico , Humanos , Fenilenodiaminas , Resultado do TratamentoRESUMO
Soil-transmitted helminth (STH) infections still remain a major health problem in poor rural settings. The lack of efficacious drugs against all STH species raises interest in drug combinations. Drug-drug interactions (DDIs) are, however, of major concern, so careful in vitro and in vivo characterization is needed. The combination of tribendimidine with either ivermectin or oxantel pamoate targets a broad range of STHs and thus represents a promising treatment alternative. Drug-drug interactions, however, have not yet been investigated. Therefore, the effects of combinations of ivermectin, oxantel pamoate, and tribendimidine's active metabolite deacylated amidantel (dADT) on cytochrome P450 (CYP450) metabolism were evaluated, followed by a pharmacokinetic analysis of tribendimidine and ivermectin alone and in combination in healthy rats. Oxantel pamoate is only poorly absorbed and was therefore excluded from pharmacokinetic analysis. No evident effect was observed for tribendimidine-oxantel pamoate at the CYP450 metabolism level, whereas a combination of tribendimidine and ivermectin led to moderately increased CYP2D6 inhibition compared to ivermectin or tribendimidine alone. Coadministration of tribendimidine with ivermectin altered neither the time to maximum concentration of drug in plasma (Tmax) nor the elimination half-lives of dADT, the acetylated derivative of amidantel (adADT), and ivermectin. While the area under the concentration-versus-time curve (AUC) and maximum concentration of drug in plasma (Cmax) values of dADT, adADT, and ivermectin are reduced by coadministration, the change is insufficient to declare that a DDI has been detected. Further studies are necessary to understand the observed interaction of tribendimidine and ivermectin, which is not related to P450 metabolism, and its significance for the situation in humans.
Assuntos
Anti-Helmínticos/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Ivermectina/farmacocinética , Fenilenodiaminas/farmacocinética , Pamoato de Pirantel/análogos & derivados , Animais , Anti-Helmínticos/farmacologia , Área Sob a Curva , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Interações Medicamentosas , Quimioterapia Combinada , Helmintíase Animal/tratamento farmacológico , Helmintos/efeitos dos fármacos , Ivermectina/farmacologia , Masculino , Fenilenodiaminas/farmacologia , Pamoato de Pirantel/farmacocinética , Pamoato de Pirantel/farmacologia , RatosRESUMO
Albendazole is an effective anthelmintic intensively used for decades. However, profound pharmacokinetic (PK) characterization is missing in children, the population mostly affected by helminth infections. Blood microsampling would facilitate PK studies in pediatric populations but has not been applied to quantify albendazole's disposition. Quantification methods were developed and validated using liquid chromatography-tandem mass spectrometry to analyze albendazole and its metabolites albendazole sulfoxide and albendazole sulfone in wet samples (plasma and blood) and blood microsamples (dried-blood spots [DBS]; Mitra). The use of DBS was limited by a matrix effect and poor recovery, but the extraction efficiency was constant throughout the concentration range. Hookworm-infected adolescents were venous and capillary blood sampled posttreatment with 400 mg albendazole and 25 mg/kg oxantel pamoate. Similar half-life (t1/2 = â¼1.5 h), time to reach the maximum concentration (tmax = â¼2 h), and maximum concentration (Cmax = 12.5 to 26.5 ng/ml) of albendazole were observed in the four matrices. The metabolites reached Cmax after â¼4 h with a t1/2 of ca. 7 to 8 h. A statistically significant difference in albendazole sulfone's t1/2 as determined by using DBS and wet samples was detected. Cmax of albendazole sulfoxide (288 to 380 ng/ml) did not differ among the matrices, but higher Cmax of albendazole sulfone were obtained in the two microsampling devices (22 ng/ml) versus the wet matrices (14 ng/ml). In conclusion, time-concentration profiles and PK results of the four matrices were similar, and the direct comparison of the two microsampling devices indicates that Mitra extraction was more robust during validation and can be recommended for future albendazole PK studies.
Assuntos
Albendazol/análogos & derivados , Albendazol/farmacocinética , Anti-Helmínticos/farmacocinética , Infecções por Uncinaria/sangue , Plasma/química , Adolescente , Albendazol/sangue , Albendazol/uso terapêutico , Ancylostomatoidea/efeitos dos fármacos , Animais , Anti-Helmínticos/sangue , Anti-Helmínticos/uso terapêutico , Cromatografia Líquida/métodos , Teste em Amostras de Sangue Seco/métodos , Infecções por Uncinaria/tratamento farmacológico , Infecções por Uncinaria/parasitologia , Humanos , Masculino , Pamoato de Pirantel/análogos & derivados , Pamoato de Pirantel/farmacocinética , Pamoato de Pirantel/uso terapêutico , Espectrometria de Massas em Tandem/métodosRESUMO
There is a growing consensus to include preschool-aged children in preventive chemotherapy programs with praziquantel to improve schistosomiasis control. However, pharmacokinetic data, crucial to establish a safe and effective dose for this age group, are sparse. The objective of this study was to establish and compare the pharmacokinetic parameters of praziquantel in preschool- and school-aged children with schistosomiasis. Two pharmacokinetic trials in school- and preschool-aged children infected with Schistosoma mansoni or S. haematobium were conducted in Côte d'Ivoire. Dried blood spot samples were taken from 492 children at 10 time points following a single oral dose of 20, 40, or 60 mg/kg of body weight of praziquantel and analyzed using liquid chromatography-mass spectrometry. Noncompartmental analysis (NCA) was performed to obtain the pharmacokinetic parameters of R-praziquantel (RPZQ), S-praziquantel (SPZQ), and R-trans-4-hydroxy-praziquantel. No significant differences in pharmacokinetic parameters between species-specific infections were observed. While pharmacokinetic parameters differed significantly between age groups for S. mansoni, this trend was not observed with S. haematobium Neither the area under the curve (AUC) nor the maximal blood concentration (Cmax) presented clear dose proportionality for R- and SPZQ. Logistic regression indicated a relationship between the RPZQ AUC and Cmax and the probability of cure. Praziquantel is subject to complex metabolic processes following erratic absorption. While the results of NCA are a very informative base for a better understanding of the drug, a more targeted approach in the form of population modeling is needed to quantify the factors influencing metabolic processes and draw conclusions.
Assuntos
Praziquantel/farmacocinética , Schistosoma haematobium/patogenicidade , Schistosoma mansoni/patogenicidade , Esquistossomose mansoni/sangue , Adolescente , Animais , Criança , Pré-Escolar , Cromatografia Líquida , Humanos , Praziquantel/uso terapêutico , Esquistossomose mansoni/tratamento farmacológico , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Helminthiases are very prevalent worldwide, yet their treatment and control rely on a handful of drugs. Emodepside, a marketed broad-spectrum veterinary anthelminthic with a unique mechanism of action, undergoing development for onchocerciasis is an interesting anthelmintic drug candidate. We tested the in vitro and in vivo activity of emodepside on nematode species that serve as models for human soil-transmitted helminth infection as well as on schistosomes. METHODS: In vitro viability assays were performed over a time course of 72 hours for Trichuris muris, Necator americanus, Ancylostoma ceylanicum, Heligmosomoides polygyrus, Strongyloides ratti, Schistosoma mansoni and Schistosoma haematobium. The drug effect was determined by the survival rate for the larvae and by phenotypical scores for the adult worms. Additionally, mice infected with T. muris and hamsters harboring hookworm infection (N. americanus or A. ceylanicum) were administered orally with emodepside at doses ranging from 1.25 to 75 mg/kg. Expelled worms in the feces were counted until 3 days post-drug intake and worms residing in the intestines were collected and counted after dissection. RESULTS: After 24 hours, emodepside was very active in vitro against both larval and adult stages of the nematodes T. muris, A. ceylanicum, N. americanus, H. polygyrus and S. ratti (IC50 < 4 µM). The good in vitro activity was confirmed in vivo. Hamsters infected with the hookworms were cured when administered orally with 2.5 mg/kg of the drug. Emodepside was also highly active in vivo against T. muris (ED50 = 1.2 mg/kg). Emodepside was moderately active on schistosomula in vitro (IC50 < 8 µM) 24 h post-drug incubation and its activity on adult S. mansoni and S. haematobium was low (IC50: 30-50 µM). CONCLUSIONS: Emodepside is highly active against a broad range of nematode species both in vitro and in vivo. The development of emodepside for treating soil-transmitted helminth infections should be pursued.
Assuntos
Anti-Helmínticos/farmacologia , Depsipeptídeos/farmacologia , Nematoides/efeitos dos fármacos , Schistosomatidae/efeitos dos fármacos , Administração Oral , Animais , Anti-Helmínticos/uso terapêutico , Cricetinae , Depsipeptídeos/uso terapêutico , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Fezes/parasitologia , Feminino , Infecções por Uncinaria/tratamento farmacológico , Humanos , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Necator americanus/efeitos dos fármacos , Schistosoma mansoni/efeitos dos fármacos , Trichuris/efeitos dos fármacosRESUMO
Pharmacokinetic (PK) studies with paediatric populations are increasing in importance for drug development. However, conventional PK sampling methods are characterised by invasiveness and low patient adherence, unsuitable for use with sensitive population, such as children. Mitra™ is a novel volumetric absorptive micro-sampling device, which offers an alternative to the dried blood spotting (DBS) technique, a current popular sampling technique within PK studies. We tested Mitra™ for the first time in the framework of a randomised controlled trial in rural Côte d'Ivoire. Thirty-five school-aged children, infected with Schistosoma haematobium, were sampled with both DBS and Mitra™, at 10 time points after treatment with praziquantel (PZQ). An extraction method for PZQ from Mitra™ was developed, optimised and validated. Analytes, namely R- and S-praziquantel (R-/SPZQ) and the main human metabolite, R-trans-4-OH-praziquantel, were measured using liquid chromatography-tandem mass spectrometry and the results were compared with Bland-Altman analysis to determine agreement between matrices. PK parameters, such as maximal plasma concentration and area under the concentration-time curve, were estimated using non-compartmental analysis. While we observed strong positive correlation (R2â¯>â¯0.98) and agreement between both matrices within the calibration line and quality control samples, Mitra™ revealed higher concentrations of all the analytes in the majority of patients' samples compared to DBS sampling, namely 63% samples for RPZQ, 49% for SPZQ and 78% for the metabolite were overestimated. While T1/2 and Tmax were in agreement between both matrices, area under the curve and maximal blood concentration were up to 2× higher for Mitra™ samples, with Pâ¯<â¯0.005 for all parameters except Cmax of SPZQ, which was not significantly different between the two matrices. The reasons for the higher PZQ concentrations, more pronounced in incurred Mitra™ samples compared to spiked samples, are yet to be fully explored. Mitra™ appears superior to DBS in terms of simplicity and practicality however labelling issues and the high price of Mitra™ are difficult to overlook.
Assuntos
Anti-Helmínticos/análise , Coleta de Amostras Sanguíneas/instrumentação , Praziquantel/análise , Esquistossomose Urinária/tratamento farmacológico , Absorção Fisico-Química , Fatores Etários , Animais , Anti-Helmínticos/farmacocinética , Anti-Helmínticos/uso terapêutico , Coleta de Amostras Sanguíneas/economia , Coleta de Amostras Sanguíneas/métodos , Criança , Cromatografia Líquida de Alta Pressão/métodos , Côte d'Ivoire , Teste em Amostras de Sangue Seco/economia , Teste em Amostras de Sangue Seco/instrumentação , Teste em Amostras de Sangue Seco/métodos , Feminino , Hematócrito , Humanos , Masculino , Praziquantel/farmacocinética , Praziquantel/uso terapêutico , População Rural , Schistosoma haematobium/isolamento & purificação , Esquistossomose Urinária/sangue , Espectrometria de Massas em Tandem/métodosRESUMO
Millions of people are treated with anthelmintics to control soil-transmitted helminth infections; yet, drug distribution in the plasma and gastrointestinal tract compartments and the pathway of drug uptake into gastrointestinal nematodes responsible for the pharmacological effect are unknown. We assessed the distribution and uptake of albendazole, albendazole sulfoxide, albendazole sulfone in the hookworm Heligmosomoides polygyrus in vitro and in vivo as well as the distribution and uptake of albendazole, mebendazole, and oxantel pamoate in the whipworm Trichuris muris in vitro and in vivo. Oral and intraperitoneal treatments (100 mg/kg) were studied. Drug quantities in helminths and host compartments (stomach, the contents and mucosa of the small and large intestine, and the plasma) were determined using HPLC-UV/vis and anthelmintic activities were recorded using phenotypic readout. The influence of 1-aminobenzotriazole (ABT), an irreversible and unspecific cytochrome P450 inhibitor, on albendazole disposition in mice harboring H. polygyrus was evaluated. In vivo, albendazole was found in quantities up to 10 nmol per ten H. polygyrus and up to 31 nmol per ten T. muris. ABT did not change the levels of albendazole or its metabolites in the plasma of mice harboring H. polygyrus or in H. polygyrus, whereas drug levels in the gastrointestinal tract of host mice doubled. Mebendazole and oxantel pamoate quantities per ten T. muris were as high as 21 nmol and 34 nmol, respectively. Albendazole revealed a very dynamic distribution and high rate of metabolism, hence, H. polygyrus and T. muris are exposed to albendazole and both metabolites via multiple pathways. Diffusion through the cuticle seems to be the crucial pathway of oxantel pamoate uptake into T. muris, and likely also for mebendazole. No relationship between concentrations measured in helminths and concentrations in plasma, intestinal content and mucosa of mice, or drug efficacy was noted for any of the drugs studied.