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1.
J Pharmacol Exp Ther ; 2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30361237

RESUMO

Plasma pyridoxic acid (PDA) and homovanillic acid (HVA) were recently identified as novel endogenous biomarkers of OAT1/3 function in monkeys. Consequently, this clinical study assessed the dynamic changes and utility of plasma PDA and HVA as an initial evaluation of OAT1/3 inhibition in early-phase drug development. The study was designed as a single dose randomized, three-phase, crossover study; 14 Indian healthy volunteers received probenecid (PROB) (1,000 mg orally) alone, furosemide (FSM) (40 mg orally) alone, or FSM 1 h after receiving PROB (40 mg and 1,000 mg orally) on Days 1, 8, and 15, respectively. PDA and HVA plasma concentrations remained stable over time in the prestudy and FSM groups. Administration of PROB significantly increased AUC of PDA by 3.1-fold (dosed alone; p < 0.05), and 3.2-fold (coadministered with FSM; p < 0.01), as compared with the prestudy and FSM groups, respectively. The corresponding increase in HVA AUC was 1.8-fold (p > 0.05) and 2.1-fold (p < 0.05), respectively. The increases in PDA AUC are similar to those in FSM AUC whereas those of HVA are smaller (3.1-3.2 and 1.8-2.1 versus 3.3, respectively). PDA and HVA CLR were decreased by PROB to smaller extents compared to FSM (0.35-0.37 and 0.67-0.73 versus 0.23, respectively). These data demonstrate that plasma PDA is a promising endogenous biomarker for OAT1/3 function and its plasma exposure respond in a similar fashion to FSM upon OAT1/3 inhibition by PROB. The magnitude and variability of response in PDA AUC and CLR between subjects is more favourable relative to HVA.

2.
Pharmaceutics ; 10(3)2018 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-30096834

RESUMO

Previously we reported that coproporphyrin-I (CP-I) is an optimal probe substrate for multidrug resistance-associated protein 2 (MRP2), and stimulation of MRP2-mediated transport is probe substrate-dependent. In the present investigation, we assessed if the in vitro stimulation is physiologically relevant. Similar to human MRP2 transport, CP-I was transported by rat Mrp2 in a typical Michaelis-Menten kinetics with apparent Km and Vmax values of 15 ± 6 µM and 161 ± 20 pmol/min/mg protein, respectively. In vivo Mrp2 functions were monitored by biliary and renal secretion of CP-I and its isomer CP-III, in bile-duct cannulated rats before and after treatment with mitoxantrone, progesterone, and verapamil. These compounds stimulated Mrp2-mediated CP-I transport in vitro. No significant increase in biliary or renal clearances, as well as in the cumulative amount of CP-I or CP-III eliminated in bile, were detected following treatment with the in vitro stimulators, indicating an in vitro to in vivo disconnect. In presence of 10 µM bilirubin, the in vitro stimulation was suppressed. We concluded that the in vitro stimulation of CP-I transport mediated by Mrp2 is not translatable in vivo, and proposed that the presence of endogenous compounds such as bilirubin in the liver may contribute to the in vitro to in vivo disconnect.

3.
Xenobiotica ; : 1-9, 2018 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-29898636

RESUMO

1-Aminobenzotriazole (ABT) is a mechanism-based inactivator of major cytochrome P450 (CYP) enzymes, which is used in multiple mechanistic studies. The purpose was to evaluate the effect of 2 and 16-h pretreatment regimens of ABT on the exposures of triazolam in rat. Another objective was to evaluate the effect of ABT on gastric emptying of acetaminophen. Plasma area under the curve (AUC) of triazolam was increased by 101-fold and 81-fold for the rats pretreated with ABT at 2 and 16 h, respectively, compared to control rats. Time to reach maximum concentration was 0.3, 4.8 and 3.7 h in control, 2 and 16-h pretreatment animals, respectively. In the case of acetaminophen, where Tmax was not delayed, the mean absorption time (MAT) in control, 2 and 16 h ABT pretreatment groups were 0.3, 4.6 and 2.9 h, respectively, suggesting delayed absorption. This hypothesis was further supported by GastroPlusTM simulation. In summary, extent of triazolam absorption was increased to a similar extent with both 2 and 16 h ABT pretreatment regimens, suggesting that either of the regimen can be used to increase parent exposures in rat. With ABT pretreatment, delayed absorption of triazolam and acetaminophen was observed, as suggested by delay in Tmax and MAT, respectively.

4.
Curr Drug Metab ; 18(8): 757-768, 2017 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-28738769

RESUMO

BACKGROUND: Drug-Drug Interactions (DDI) by modulation of drug transporters or drug metabolizing enzymes are common in multi-drug therapy. DDI potential of any new drug is assessed by conducting separate clinical studies using relevant probe substrates, which involves additional resource and cost. Recently, several endogenous compounds have been evaluated as substrates of transporters and enzymes that could be assessed as part of early clinical trials along with the assessment of drug pharmacokinetics, pharmacodynamics and safety studies. This enables an early readout on potential DDIs avoiding or minimally delaying the conduct of definitive DDI studies until later in clinical development. METHOD: This review describes various endogenous biomarkers reported for drug transporters and metabolizing enzymes with their advantages and limitations. CONCLUSION: Furthermore, the authors describe strategies to adopt while exploring a new endogenous biomarker, and factors to be considered in selection of biomarkers with the current challenges and opportunities.


Assuntos
Biomarcadores/metabolismo , Interações de Medicamentos , Proteínas de Membrana Transportadoras/metabolismo , Animais , Transporte Biológico , Humanos
5.
J Cent Nerv Syst Dis ; 9: 1179573517693596, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28469522

RESUMO

The role of uptake transporter (organic anion-transporting polypeptide [Oatp]) in the disposition of a P-glycoprotein (P-gp) substrate (digoxin) at the barriers of central nervous system, namely, the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and brain-cerebrospinal fluid barrier (BCSFB), was studied using rat as a preclinical species. In vivo chemical inhibition of P-gp and Oatp was achieved using elacridar and rifampicin, respectively. Our findings show that (1) digoxin had a low brain-to-plasma concentration ratio (B/P) (0.07) in rat; (2) in the presence of elacridar, the B/P of digoxin increased by about 12-fold; (3) rifampicin administration alone did not change the digoxin B/P significantly when compared with digoxin B/P alone; (4) rifampicin administration along with elacridar resulted only in 6-fold increase in the B/P of digoxin; (5) similar fold changes and trends were seen with the spinal cord-to-plasma concentration ratio of digoxin, indicating the similarity between BBB and the BSCB; and (6) unlike BBB and BSCB, the presence of rifampicin further increased the cerebrospinal fluid-to-plasma concentration ratio (CSF/P) for digoxin, suggesting a differential orientation of the uptake transporters at the BCSFB (CSF to blood) compared with the BBB (blood to brain). The observations for digoxin uptake, at least at the BBB and the BSCB, advocate the importance of uptake transporters (Oatps). However, the activity of such uptake transporters became evident only after inhibition of the efflux transporter (P-gp).

6.
Drug Metab Dispos ; 45(6): 604-611, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28325716

RESUMO

Inside-out-oriented membrane vesicles are useful tools to investigate whether a compound can be an inhibitor of efflux transporters such as multidrug resistance-associated protein 2 (MRP2). However, because of technical limitations of substrate diffusion and low dynamic uptake windows for interacting drugs used in the clinic, estradiol-17ß-glucuronide (E17ßG) remains the probe substrate that is frequently used in MRP2 inhibition assays. Here we recapitulated the sigmoidal kinetics of MRP2-mediated transport of E17ßG, with apparent Michaelis-Menten constant (Km) and Vmax values of 170 ±17 µM and 1447 ± 137 pmol/mg protein/min, respectively. The Hill coefficient (2.05 ± 0.1) suggests multiple substrate binding sites for E17ßG transport with cooperative interactions. Using E17ßG as a probe substrate, 51 of 97 compounds tested (53%) showed up to 6-fold stimulatory effects. Here, we demonstrate for the first time that coproporphyrin-I (CP-I) is a MRP2 substrate in membrane vesicles. The uptake of CP-I followed a hyperbolic relationship, adequately described by the standard Michaelis-Menten equation (apparent Km and Vmax values were 7.7 ± 0.7 µM and 48 ± 11 pmol/mg protein/min, respectively), suggesting the involvement of a single binding site. Of the 47 compounds tested, 30 compounds were inhibitors of human MRP2 and 8 compounds (17%) stimulated MRP2-mediated CP-I transport. The stimulators were found to share the basic backbone structure of the physiologic steroids, which suggests a potential in vivo relevance of in vitro stimulation of MRP2 transport. We concluded that CP-I could be an alternative in vitro probe substrate replacing E17ßG for appreciating MRP2 interactions while minimizing potential false-negative results for MRP2 inhibition due to stimulatory effects.


Assuntos
Coproporfirinas/farmacologia , Corantes Fluorescentes/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Sítios de Ligação/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/metabolismo , Humanos , Cinética , Proteínas de Membrana Transportadoras/metabolismo
7.
J Immunol ; 198(3): 1308-1319, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28003376

RESUMO

The serine/threonine kinase IL-1R-associated kinase (IRAK)4 is a critical regulator of innate immunity. We have identified BMS-986126, a potent, highly selective inhibitor of IRAK4 kinase activity that demonstrates equipotent activity against multiple MyD88-dependent responses both in vitro and in vivo. BMS-986126 failed to inhibit assays downstream of MyD88-independent receptors, including the TNF receptor and TLR3. Very little activity was seen downstream of TLR4, which can also activate an MyD88-independent pathway. In mice, the compound inhibited cytokine production induced by injection of several different TLR agonists, including those for TLR2, TLR7, and TLR9. The compound also significantly suppressed skin inflammation induced by topical administration of the TLR7 agonist imiquimod. BMS-986126 demonstrated robust activity in the MRL/lpr and NZB/NZW models of lupus, inhibiting multiple pathogenic responses. In the MRL/lpr model, robust activity was observed with the combination of suboptimal doses of BMS-986126 and prednisolone, suggesting the potential for steroid sparing activity. BMS-986126 also demonstrated synergy with prednisolone in assays of TLR7- and TLR9-induced IFN target gene expression using human PBMCs. Lastly, BMS-986126 inhibited TLR7- and TLR9-dependent responses using cells derived from lupus patients, suggesting that inhibition of IRAK4 has the potential for therapeutic benefit in treating lupus.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Prednisolona/uso terapêutico , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/fisiologia , Receptor 7 Toll-Like/fisiologia , Receptor Toll-Like 9/fisiologia
8.
Xenobiotica ; 44(12): 1108-16, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24947446

RESUMO

1. The effect of age and obesity on the pharmacokinetics (PK), hepatic blood flow (HBF) and liver metabolism of 10 compounds was determined in rats. The animals fed a high-fat diet were defined as the diet-induced obese (DIO) group, while the animals that were aged similar to the DIO rats but not fed with high-fat diet were called the age-matched (AM) group. 2. The clearance (CL) values of high CL compounds (CL > 50 mL/min/kg, namely propranolol, diazepam, phenytoin, ethinylestradiol, lorcaserin and fenfluramine) decreased significantly (1.5- to 6-fold) in DIO and AM rats as compared to lean rats, while there was no clear trend for change in CL for the low-to-moderate CL compounds (CL < 50 mL/min/kg, namely atenolol, chlorzoxazone, vancomycin and sibutramine). Hepatocytes incubations revealed a change in half life (t1/2) only for phenytoin. The body weight normalized liver weights and HBF of AM and DIO rats were found to be 2- to 3-fold lower than in lean rats. 3. Our findings suggest that age, and diet to a lesser extent, can reduce HBF and body normalized liver weights and, hence, also reduce CL values for high CL compounds in rats.


Assuntos
Envelhecimento/fisiologia , Fígado/irrigação sanguínea , Fígado/metabolismo , Obesidade/metabolismo , Animais , Células Cultivadas , Gorduras na Dieta/efeitos adversos , Meia-Vida , Hepatócitos/metabolismo , Obesidade/etiologia , Ratos , Ratos Sprague-Dawley
9.
Mol Pharm ; 11(2): 477-85, 2014 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-24380373

RESUMO

The unbound concentration in plasma drives the transport of the drug into the brain, and the unbound drug concentration in the central nervous system (CNS) drives the interaction with the target eliciting the pharmacological effect. Delivery of the drug to the CNS is a challenge because of the unique neurovascular unit, which restricts the passage of drugs into the brain. The efflux transporters [especially P-glycoprotein (P-gp)] present at the blood-brain barrier (BBB) act as one of the major detractors for keeping drugs outside the CNS. The cerebrospinal fluid (CSF) drug concentration has been used as a surrogate for unbound brain concentrations and has proven to be a good indicator to relate to CNS activity. Herein, we have established a serial CSF sampling technique in rats, which allowed CSF sampling from a single animal and reduced the number of animals required, as well as the interanimal variance associated with a composite/terminal study design. Concentrations in the CSF sampled from the cisterna magna serially from the same rat were compared with the concentrations obtained from discrete CSF sampling and with brain concentrations. The serial CSF sampling technique was also authenticated by ensuring no change in the barrier without any indication of damage caused by the repeated puncture of cisterna magna. This technique was corroborated using three passively permeable compounds (carbamazepine, theophylline, and propranolol), three P-gp substrates (quinidine, verapamil, and digoxin), and one l-amino acid uptake transporter substrate (gabapentin). The P-gp substrates were also used in separate studies with the P-gp inhibitor elacridar to assess the effect on CSF concentration versus brain concentration on P-gp inhibition. The CSF concentration and unbound brain concentration were comparable (within 3-fold) for all compounds, including P-gp substrates even in the presence of elacridar. Therefore, this technique can prove to be beneficial for predicting the unbound drug concentrations in the brain from the CSF concentrations and reduce the cost incurred in preclinical animal models. Chemical inhibition by elacridar and prediction of the brain unbound concentrations from the serial CSF sampling of P-gp substrates in the rat may be an attractive alternative to the use of genetically knocked out rodents.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/líquido cefalorraquidiano , Química Encefálica , Proteínas do Líquido Cefalorraquidiano/análise , Subfamília B de Transportador de Cassetes de Ligação de ATP/sangue , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Líquido Cefalorraquidiano/química , Limite de Detecção , Masculino , Ratos , Ratos Sprague-Dawley
10.
Curr Drug Metab ; 14(3): 324-40, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23167621

RESUMO

In a preclinical setting, plasma or whole tissue drug concentrations are often correlated with pharmacodynamics, although according to the free drug hypothesis, unbound drug concentration should be more pharmacologically relevant. Alternatively, blood concentrations may be a good surrogate for tissue concentration for passively permeable compounds. However, for a large number of compounds that are substrates for uptake and/or efflux transporters expressed at the tissue level, significant discrepancies are expected between unbound concentrations in blood and those in tissues. Consequently, attempts have been made to measure tissue unbound drug concentrations using tissue homogenates, slices and microdialysis. Mathematical expressions for calculating the rate and extent of drug distribution into tissues have also been established. For example, a ratio of unbound concentration in the tissue to that in plasma (K(p,uu)) is the best indicator of the extent of tissue distribution. Despite these technical advances, however, very few examples demonstrate a focus on tissue unbound drug concentrations in a preclinical setting. This review will illustrate various techniques to estimate tissue unbound drug concentrations, relevant parameters to calculate the rate and extent of tissue distribution and different factors affecting tissue unbound concentration. The review will also highlight various examples from the literature where tissue unbound drug concentrations have demonstrated a superior correlation with efficacy. The impact of tissue unbound drug concentrations on the projection of human efficacious dose is also discussed.


Assuntos
Descoberta de Drogas , Farmacocinética , Animais , Humanos , Preparações Farmacêuticas/metabolismo , Distribuição Tecidual
11.
Eur J Drug Metab Pharmacokinet ; 37(1): 23-30, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21948266

RESUMO

Pharmacokinetic (PK) studies in mice usually require discrete and parallel blood sampling owing to a restriction on the volume of blood that can be withdrawn. This results in dosing large number of animals and generating composite PK profile. To reduce the number of animals and generate individual animal PK profiles, we developed a serial sampling technique via tail vein bleeding in mice, in which only 20-30 µL blood was withdrawn per time point. Due to the small blood volume, a dried-blood spot (DBS) technique was applied for sample processing. The utility of this technique was demonstrated using three test compounds (amodiaquine, chloroquine and chlorthalidone), with varying degrees of blood-to-plasma partition ratios. The PK studies were carried out in male Balb/c mouse weighing 25-30 g. The compounds were administered intravenously via the saphenous vein. Blood was collected by composite (retro-orbital plexus) or serial (tail vein bleeding) sampling techniques at different time points. Blood samples were processed as blood lysate or DBS. Blood or plasma samples were analyzed by sensitive and rapid UPLC-MS/MS methods. The blood concentrations (both from blood lysate and DBS) obtained from serial sampling matched with those from composite sampling. The ratio of blood AUC to plasma AUC correlated well with the in vitro blood-to-plasma partition ratio of the compounds. The systemic clearance and volume of distribution at steady state calculated from blood or plasma AUCs were in proportion to the respective AUCs. Our results indicated that the serial sampling technique would reduce the number of animals and also compound usage, as well as improve the quality of pharmacokinetic data. Also, the serial sampling technique does not require the use of anesthesia and allows estimation of inter-animal variability in PK. A small volume serial sampling is possible due to the availability of the DBS technique.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Cromatografia Líquida de Alta Pressão/métodos , Teste em Amostras de Sangue Seco/métodos , Espectrometria de Massas em Tandem/métodos , Amodiaquina/farmacocinética , Animais , Área Sob a Curva , Cloroquina/farmacocinética , Clortalidona/farmacocinética , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sensibilidade e Especificidade , Fatores de Tempo , Distribuição Tecidual
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