Rapid in vivo evaluation system for cholestasis-related genes in mice with humanized bile acid profiles.

BACKGROUND: Pediatric cholestatic liver diseases (Ped-CLD) comprise many ultrarare disorders with a genetic basis. Pharmacologic therapy for severe cases of Ped-CLD has not been established. Species differences in bile acid (BA) metabolism between humans and rodents contribute to the lack of phenocopy of patients with Ped-CLD in rodents and hinder the development of therapeutic strategies. We aimed to establish an efficient in vivo system to understand BA-related pathogenesis, such as Ped-CLD. METHODS: We generated mice that express spCas9 specifically in the liver (L-Cas9Tg/Tg [liver-specific Cas9Tg/Tg] mice) and designed recombinant adeno-associated virus serotype 8 encoding small-guide RNA (AAV8 sgRNA) targeting Abcc2, Abcb11, and Cyp2c70. In humans, ABCC2 and ABCB11 deficiencies cause constitutional hyperbilirubinemia and most severe Ped-CLD, respectively. Cyp2c70 encodes an enzyme responsible for the rodent-specific BA profile. Six-week-old L-Cas9Tg/Tg mice were injected with this AAV8 sgRNA and subjected to biochemical and histological analysis. RESULTS: Fourteen days after the injection with AAV8 sgRNA targeting Abcc2, L-Cas9Tg/Tg mice exhibited jaundice and phenocopied patients with ABCC2 deficiency. L-Cas9Tg/Tg mice injected with AAV8 sgRNA targeting Abcb11 showed hepatomegaly and cholestasis without histological evidence of liver injury. Compared to Abcb11 alone, simultaneous injection of AAV8 sgRNA for Abcb11 and Cyp2c70 humanized the BA profile and caused higher transaminase levels and parenchymal necrosis, resembling phenotypes with ABCB11 deficiency. CONCLUSIONS: This study provides proof of concept for efficient in vivo assessment of cholestasis-related genes in humanized bile acid profiles. Our platform offers a more time- and cost-effective alternative to conventional genetically engineered mice, increasing our understanding of BA-related pathogenesis such as Ped-CLD and expanding the potential for translational research.

Influence of food on pharmacokinetics and pharmacodynamics of 4-phenylbutyrate in patients with urea cycle disorders.

Urea cycle disorders (UCDs), inborn errors of hepatocyte metabolism, cause hyperammonemia and lead to neurocognitive deficits, coma, and even death. Sodium 4-phenylbutyrate (NaPB), a standard adjunctive therapy for UCDs, generates an alternative pathway of nitrogen deposition through glutamine consumption. Administration during or immediately after a meal is the approved usage of NaPB. However, we previously found that preprandial oral administration enhanced its potency in healthy adults and pediatric patients with intrahepatic cholestasis. The present study evaluated the effect of food on the pharmacokinetics and pharmacodynamics of NaPB in five patients with UCDs. Following an overnight fast, NaPB was administered orally at 75 mg/kg/dose (high dose, HD) or 25 mg/kg/dose (low dose, LD) either 15 min before or immediately after breakfast. Each patient was treated with these four treatment regimens with NaPB. With either dose, pre-breakfast administration rather than post-breakfast administration significantly increased plasma PB levels and decreased plasma glutamine availability. Pre-breakfast LD administration resulted in a greater attenuation in plasma glutamine availability than post-breakfast HD administration. Plasma levels of branched-chain amino acids decreased to the same extent in all tested regimens. No severe adverse events occurred during this study. In conclusion, preprandial oral administration of NaPB maximized systemic exposure of PB and thereby its efficacy on glutamine consumption in patients with UCDs.

A randomized trial to examine the impact of food on pharmacokinetics of 4-phenylbutyrate and change in amino acid availability after a single oral administration of sodium 4-phenylbutyrarte in healthy volunteers.

Urea cycle disorders (UCDs), inborn errors of hepatocyte metabolism, result in the systemic accumulation of ammonia to toxic levels. Sodium 4-phenylbutyrate (NaPB), a standard therapy for UCDs for over 20 years, generates an alternative pathway of nitrogen deposition through glutamine consumption. Administration during or immediately after a meal is the accepted use of NaPB. However, this regimen is not based on clinical evidence. Here, an open-label, single-dose, five-period crossover study was conducted in healthy adults to investigate the effect of food on the pharmacokinetics of NaPB and determine any subsequent change in amino acid availability. Twenty subjects were randomized to one of four treatment groups. Following an overnight fast, NaPB was administered orally at 4.3 g/m2 (high dose, HD) or 1.4 g/m2 (low dose, LD) either 30 min before or just after breakfast. At both doses, compared with post-breakfast administration, pre-breakfast administration significantly increased systemic exposure of PB and decreased plasma glutamine availability. Pre-breakfast LD administration attenuated plasma glutamine availability to the same extent as post-breakfast HD administration. Regardless of the regimen, plasma levels of branched-chain amino acids (BCAA) were decreased below baseline in a dose-dependent manner. In conclusion, preprandial oral administration of NaPB maximized systemic exposure of the drug and thereby its potency to consume plasma glutamine. This finding may improve poor medication compliance because of the issues with odor, taste, and pill burden of NaPB and reduce the risk of BCAA deficiency in NaPB therapy.

Modeling Human Bile Acid Transport and Synthesis in Stem Cell-Derived Hepatocytes with a Patient-Specific Mutation.

The bile salt export pump (BSEP) is responsible for the export of bile acid from hepatocytes. Impaired transcellular transport of bile acids in hepatocytes with mutations in BSEP causes cholestasis. Compensatory mechanisms to regulate the intracellular bile acid concentration in human hepatocytes with BSEP deficiency remain unclear. To define pathways that prevent cytotoxic accumulation of bile acid in hepatocytes, we developed a human induced pluripotent stem cell-based model of isogenic BSEP-deficient hepatocytes in a Transwell culture system. Induced hepatocytes (i-Heps) exhibited defects in the apical export of bile acids but maintained a low intracellular bile acid concentration by inducing basolateral export. Modeling the autoregulation of bile acids on hepatocytes, we found that BSEP-deficient i-Heps suppressed de novo bile acid synthesis using the FXR pathway via basolateral uptake and export without apical export. These observations inform the development of therapeutic targets to reduce the overall bile acid pool in patients with BSEP deficiency.

Effect of food on the pharmacokinetics and therapeutic efficacy of 4-phenylbutyrate in progressive familial intrahepatic cholestasis.

Progressive familial intrahepatic cholestasis (PFIC), a rare inherited disorder, progresses to liver failure in childhood. We have shown that sodium 4-phenylbutyrate (NaPB), a drug approved for urea cycle disorders (UCDs), has beneficial effects in PFIC. However, there is little evidence to determine an optimal regimen for NaPB therapy. Herein, a multicenter, open-label, single-dose study was performed to investigate the influence of meal timing on the pharmacokinetics of NaPB. NaPB (150 mg/kg) was administered orally 30 min before, just before, and just after breakfast following overnight fasting. Seven pediatric PFIC patients were enrolled and six completed the study. Compared with postprandial administration, an approved regimen for UCDs, preprandial administration significantly increased the peak plasma concentration and area under the plasma concentration-time curve of 4-phenylbutyrate by 2.5-fold (95% confidential interval (CI), 2.0-3.0;P = 0.003) and 2.4-fold (95% CI, 1.7-3.2;P = 0.005). The observational study over 3 years in two PFIC patients showed that preprandial, but not prandial or postprandial, oral treatment with 500 mg/kg/day NaPB improved liver function tests and clinical symptoms and suppressed the fibrosis progression. No adverse events were observed. Preprandial oral administration of NaPB was needed to maximize its potency in PFIC patients.