Evaluation of Organic Anion Transporter 1A2-knock-in Mice as a Model of Human Blood-brain Barrier.

The present study aimed to establish a humanized mouse model with which to explore OATP1A2-mediated transcellular transport of drug substrates across the blood-brain barrier (BBB) and to evaluate the usefulness of the humanized mice in preclinical studies. Sulpiride, amisulpride, sultopride, and triptans were used as probes to discriminate OATP1A2 and Oatp1a4. We generated a mouse line humanized for OATP1A2 by introducing the coding region downstream of the Oatp1a4 promoter using the CRISPR/Cas9 technique. In the mice generated, OATP1A2 mRNA in the brain was increased corresponding to disappearance of Oatp1a4. OATP1A2 was localized on both the luminal and abluminal sides of the BBB. Unfortunately, study in vivo employing sulpiride, sumatriptan, and zolmitriptan as probes did not indicate any difference in their brain-to-plasma ratio between the control and humanized mice. Quantitative targeted absolute proteomic analysis of the BBB fraction from the humanized mice revealed that almost all analyzed transporters and membrane proteins were expressed at similar levels to those in control mice. The quantitative levels of OATP1A2 differed depending on the peptide quantified, which suggests that incomplete translation or posttranslational modification may occur. The blood-to-brain transport of zolmitriptan, determined by brain perfusion in situ, was 1.6-fold higher in the humanized mice than in the controls, whereas that of sulpiride was not significantly changed. To our knowledge, we established a mouse line humanized for a BBB uptake transporter for the first time. Unfortunately, because of limited impact, there is still room for improvement of the model system.

Investigation of Endogenous Compounds Applicable to Drug-Drug Interaction Studies Involving the Renal Organic Anion Transporters, OAT1 and OAT3, in Humans.

This study was a comprehensive analysis of metabolites in plasma and urine specimens from subjects who received probenecid, a potent inhibitor of renal organic anion transporters (OATs). Taurine and glycochenodeoxycholate sulfate (GCDCA-S) could be identified using authentic standards. Probenecid had no effect on the area under the plasma-concentration time curves of taurine and GCDCA-S, whereas it significantly inhibited their urinary excretion in a dose-dependent manner. Probenecid at 500, 750, and 1500 mg orally decreased the renal clearance (CLR) values of taurine and GCDCA-S by 45% and 60%, 59% and 79%, and 70% and 88%, respectively. The CLR values correlated strongly (r > 0.96) between the test compounds (benzylpenicillin, 6ß-hydroxycortisol, taurine, and GCDCA-S). Taurine and GCDCA-S were substrates of OAT1 and OAT3, with Km values of 379 ± 58 and 64.3 ± 3.9 µM, respectively. The Ki values of probenecid for the OAT1- and OAT3-mediated uptake of taurine and GCDCA-S (9.49 ± 1.27 and 7.40 ± 0.70 µM, respectively) were similar to those of their typical substrate drugs. The magnitude of the reduction in the CLR of taurine and GCDCA-S by probenecid could be reasonably explained using the geometric mean values of unbound probenecid concentration and Ki values. These results suggest that taurine and GCDCA-S can be used as probes for evaluating pharmacokinetic drug-drug interactions involving OAT1 and OAT3, respectively, in humans.

Population Pharmacokinetic and Pharmacodynamic Modeling of Fesoterodine in Pediatric Patients with Neurogenic Detrusor Overactivity.

BACKGROUND AND OBJECTIVE: Fesoterodine is a muscarinic receptor antagonist approved for the treatment of overactive bladder (OAB) in adults and neurogenic detrusor overactivity (NDO) in pediatric patients. This work aimed to characterize the population pharmacokinetics of 5-hydroxymethyl tolterodine (5-HMT, the active metabolite of fesoterodine) and its pharmacokinetic/pharmacodynamic relationship in pediatric patients with OAB or NDO following administration of fesoterodine. METHODS: 5-HMT plasma concentrations from 142 participants of age ≥ 6 years were analyzed, and a nonlinear mixed-effects model was developed. Weight-based simulations of 5-HMT exposure and maximum cystometric capacity (MCC) were conducted using the final models. RESULTS: A one-compartment model with first-order absorption and a lag time, which included the effects of body weight, sex, cytochrome (CYP) 2D6 metabolizer status and fesoterodine formulation on pharmacokinetic parameters, best described the 5-HMT pharmacokinetics. An Emax model described the exposure-response relationship adequately. The median maximum concentration at steady state for pediatric patients weighing 25-35 kg and receiving 8 mg once daily (QD) was estimated to be 2.45 times greater than that in adults receiving 8 mg QD. Furthermore, simulation results showed dosing with fesoterodine 4 mg QD to pediatric patients weighing 25-35 kg and 8 mg QD to pediatric patients weighing >35 kg would achieve adequate exposure to demonstrate a clinically meaningful change from baseline (CFB) MCC. CONCLUSIONS: Population models were developed for 5-HMT and MCC in pediatric patients. Weight-based simulations indicated that 4 mg QD for pediatric patients weighing 25-35 kg and 8 mg QD for those weighing > 35 kg provided similar exposures to those in adults following 8 mg QD and a clinically meaningful CFB MCC. CLINICAL TRIAL NUMBERS: NCT00857896, NCT01557244.

Fesoterodine is a muscarinic receptor antagonist approved for the treatment of overactive bladder (OAB) in adults and neurogenic detrusor overactivity (NDO) in pediatric patients in the US. Population pharmacokinetic and pharmacokinetic/pharmacodynamic models were developed for 5-HMT based on data from two pediatric clinical trials that included 142 patients of age ≥ 6 years with OAB or NDO. Weight-based simulations of 5-HMT exposure and maximum cystometric capacity were conducted using the final models to examine the impact of covariates on 5-HMT exposure and the exposure­response profile. The results of these simulations indicate that 4 mg QD for pediatric patients weighing 25­35 kg and 8 mg QD for those weighing > 35 kg provide similar exposures to those in adults following 8 mg QD.

[Development of in vivo drug sensing system with needle-type diamond microelectrode].

Continuous and real-time measurement of local concentrations of systemically administered drugs in vivo must be crucial for pharmacological studies. Nevertheless, conventional methods require considerable samples quantity and have poor sampling rates. Additionally, they cannot determine how drug kinetics correlates with target function over time. Here, we describe a system with two different sensors. One is a needle-type microsensor composed of boron-doped diamond with a tip of ~40 µm in diameter, and the other is a glass microelectrode. We first tested bumetanide. This diuretic can induce deafness. In the guinea-pig cochlea injected intravenously with bumetanide, the changes of the drug concentration and the extracellular potential underlying hearing were simultaneously measured in real time. We further examined an antiepileptic drug lamotrigine in the rat brain, and tracked its kinetics and at the same time the local field potentials representing neuronal activity. The action of the anticancer reagent doxorubicin was also monitored in the cochlea. This microsensing system may be applied to analyze pharmacokinetics and pharmacodynamics of various drugs at local sites in vivo, and contribute to promoting the pharmacological researches.

Central Histamine Boosts Perirhinal Cortex Activity and Restores Forgotten Object Memories.

BACKGROUND: A method that promotes the retrieval of lost long-term memories has not been well established. Histamine in the central nervous system is implicated in learning and memory, and treatment with antihistamines impairs learning and memory. Because histamine H3 receptor inverse agonists upregulate histamine release, the inverse agonists may enhance learning and memory. However, whether the inverse agonists promote the retrieval of forgotten long-term memory has not yet been determined. METHODS: Here, we employed multidisciplinary methods, including mouse behavior, calcium imaging, and chemogenetic manipulation, to examine whether and how the histamine H3 receptor inverse agonists, thioperamide and betahistine, promote the retrieval of a forgotten long-term object memory in mice. In addition, we conducted a randomized double-blind, placebo-controlled crossover trial in healthy adult participants to investigate whether betahistine treatment promotes memory retrieval in humans. RESULTS: The treatment of H3 receptor inverse agonists induced the recall of forgotten memories even 1 week and 1 month after training in mice. The memory recovery was mediated by the disinhibition of histamine release in the perirhinal cortex, which activated the histamine H2 receptor. Histamine depolarized perirhinal cortex neurons, enhanced their spontaneous activity, and facilitated the reactivation of behaviorally activated neuronal ensembles. A human clinical trial revealed that treatment of H3 receptor inverse agonists is specifically more effective for items that are more difficult to remember and subjects with poorer performance. CONCLUSIONS: These results highlight a novel interaction between the central histamine signaling and memory engrams.

A microsensing system for the in vivo real-time detection of local drug kinetics.

Real-time recording of the kinetics of systemically administered drugs in in vivo microenvironments may accelerate the development of effective medical therapies. However, conventional methods require considerable analyte quantities, have low sampling rates and do not address how drug kinetics correlate with target function over time. Here, we describe the development and application of a drug-sensing system consisting of a glass microelectrode and a microsensor composed of boron-doped diamond with a tip of around 40 µm in diameter. We show that, in the guinea pig cochlea, the system can measure-simultaneously and in real time-changes in the concentration of bumetanide (a diuretic that is ototoxic but applicable to epilepsy treatment) and the endocochlear potential underlying hearing. In the rat brain, we tracked the kinetics of the drug and the local field potentials representing neuronal activity. We also show that the actions of the antiepileptic drug lamotrigine and the anticancer reagent doxorubicin can be monitored in vivo. Our microsensing system offers the potential to detect pharmacological and physiological responses that might otherwise remain undetected.