RESUMO
Drug induced phospholipidosis (PLD) may be observed in the preclinical phase of drug development and pose strategic questions. As lysosomes have a central role in pathogenesis of PLD, assessment of lysosomal concentrations is important for understanding the pharmacokinetic basis of PLD manifestation and forecast of potential clinical appearance. Herein we present a systematic approach to provide insight into tissue-specific PLD by evaluation of unbound intracellular and lysosomal (reflecting acidic organelles) concentrations of two structurally related diprotic amines, GRT1 and GRT2. Their intratissue distribution was assessed using brain and lung slice assays. GRT1 induced PLD both in vitro and in vivo. GRT1 showed a high intracellular accumulation that was more pronounced in the lung, but did not cause cerebral PLD due to its effective efflux at the blood-brain barrier. Compared to GRT1, GRT2 revealed higher interstitial fluid concentrations in lung and brain, but more than 30-fold lower lysosomal trapping capacity. No signs of PLD were seen with GRT2. The different profile of GRT2 relative to GRT1 is due to a structural change resulting in a reduced basicity of one amino group. Hence, by distinct chemical modifications, undesired lysosomal trapping can be separated from desired drug delivery into different organs. In summary, assessment of intracellular unbound concentrations was instrumental in delineating the intercompound and intertissue differences in PLD induction in vivo and could be applied for identification of potential lysosomotropic compounds in drug development.
Assuntos
Diaminas/farmacologia , Lipidoses/induzido quimicamente , Modelos Biológicos , Animais , Encéfalo/metabolismo , Química Farmacêutica , Líquido Extracelular/metabolismo , Feminino , Células Hep G2 , Humanos , Pulmão/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Modelos Animais , Modelos Químicos , Fosfolipídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Distribuição TecidualRESUMO
Intestinal barrier is a first line of defense that prevents entry of various harmful substances from the lumen into the systemic environment. Impaired barrier function with consequent translocation of harmful substances into systemic circulation ("leaky gut") is a central theme in many gastrointestinal, autoimmune, mental, and metabolic diseases. Probiotics have emerged as a promising strategy to maintain intestinal integrity and address "leaky gut". Using in silico, in vitro and avian in vivo analyses, we previously showed that two novel L. reuteri strains, PTA-126787 (L. reuteri 3630) and PTA-126788 (L. reuteri 3632), isolated from broiler chickens possess favorable safety profiles. Consistent with a recent study, here we show that L. reuteri 3630 and 3632 are phylogenetically similar to human L. reuteri strains. Daily administration of high doses of L. reuteri 3630 and 3632 to Sprague Dawley rats for 28 days was found to be safe with no adverse effects. More importantly, administration of L. reuteri 3630 and 3632 significantly reduced markers associated with alcohol-induced leaky gut, by downregulating inflammatory cytokines and upregulating anti-inflammatory cytokines in an alcohol model of leaky gut in mice. While L. reuteri 3630 cells and supernatant showed no activation, L. reuteri 3632 cells but not supernatant showed activation of AhR, a key transcription factor that regulates gut and immune homeostasis. L. reuteri 3630 is creamish white in morphology typical of Lactobacillus species and L. reuteri 3632 displays a unique orange pigmentation, which was stable even after passaging for 480 generations. We identified a rare polyketide biosynthetic gene cluster in L. reuteri 3632 that likely encodes for the orange-pigmented secondary metabolite. Similar to L. reuteri 3632 cells, the purified orange metabolite activated AhR. All together, these data provide evidence on the phylogenetic relatedness, safety, efficacy, and one of the likely mechanisms of action of L. reuteri 3630 and 3632 for potential probiotic applications to address "leaky gut" and associated pathologies in humans.
Assuntos
Homeostase , Limosilactobacillus reuteri , Probióticos , Ratos Sprague-Dawley , Animais , Limosilactobacillus reuteri/metabolismo , Ratos , Galinhas/microbiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos , Etanol/metabolismo , Humanos , Masculino , Modelos Animais de Doenças , Microbioma Gastrointestinal/efeitos dos fármacosRESUMO
The metabolite of several amide anaesthetics, 2,6-xylidine, is a possible human (Group 2B) carcinogen and induced nasal tumours in rats after dietary administration. However, published papers on the genotoxicity of 2,6-xylidine in vitro have given inconsistent results. It has been proposed that the genotoxicity of 2,6-xylidine is dependent on its metabolism to a key metabolite dimethylphenyl N-hydroxylamine (DMHA), which would then be further converted to form a reactive nitrenium ion by phase 2 (mainly acetylation) metabolism. In order to study whether the inconsistent results could be explained by different systems having different potential for DMHA to be formed and to induce genotoxicity in vitro, we have tested 2,6-xylidine in conventional Ames bacteria, and strains engineered to overexpress acetyltransferase, in the presence of different concentrations of induced rat liver and human liver S9. All tests gave consistently negative results. The formation of DMHA by induced rat liver S9 and human S9 was clearly shown to occur, and to be concentration- and time-dependent. The potential inhibitory effects of the solvent DMSO were also studied, but it was clearly not responsible for the negative results with 2,6-xylidine. Thus, whatever is the mode of action of 2,6-xylidine carcinogenicity in rodents, it has proven impossible to detect mutagenic effects in Ames tests with numerous variations of metabolic conditions, or even using acetyltransferase overexpressing strains of bacteria.