CRISPR-Cas9 In Vivo Gene Editing of KLKB1 for Hereditary Angioedema.

BACKGROUND: Hereditary angioedema is a rare genetic disease that leads to severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1), with the goal of lifelong control of angioedema attacks after a single dose. METHODS: In this phase 1 dose-escalation portion of a combined phase 1-2 trial of NTLA-2002 in adults with hereditary angioedema, we administered NTLA-2002 at a single dose of 25 mg, 50 mg, or 75 mg. The primary end points were the safety and side-effect profile of NTLA-2002 therapy. Secondary and exploratory end points included pharmacokinetics, pharmacodynamics, and clinical efficacy determined on the basis of investigator-confirmed angioedema attacks. RESULTS: Three patients received 25 mg of NTLA-2002, four received 50 mg, and three received 75 mg. At all dose levels, the most common adverse events were infusion-related reactions and fatigue. No dose-limiting toxic effects, serious adverse events, grade 3 or higher adverse events, or clinically important laboratory findings were observed after the administration of NTLA-2002. Dose-dependent reductions in the total plasma kallikrein protein level were observed between baseline and the latest assessment, with a mean percentage change of -67% in the 25-mg group, -84% in the 50-mg group, and -95% in the 75-mg group. The mean percentage change in the number of angioedema attacks per month between baseline and weeks 1 through 16 (primary observation period) was -91% in the 25-mg group, -97% in the 50-mg group, and -80% in the 75-mg group. Among all the patients, the mean percentage change in the number of angioedema attacks per month from baseline through the latest assessment was -95%. CONCLUSIONS: In this small study, a single dose of NTLA-2002 led to robust, dose-dependent, and durable reductions in total plasma kallikrein levels, and no severe adverse events were observed. In exploratory analyses, reductions in the number of angioedema attacks per month were observed at all dose levels. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830.).

Fampridine is a Substrate and Inhibitor of Human OCT2, but not of Human MATE1, or MATE2K.

PURPOSE: The renal clearance of fampridine (Fampyra®, or Ampyra®) significantly exceeds the glomerular filtration rate, suggesting active renal secretion is likely the major elimination pathway. The goal of this study was to identify the renal transporters that are involved in the renal active secretion, and elucidate the active renal secretion mechanism of fampridine. METHODS: The uptake of fampridine to HEK-293 cells overexpressing human OCT2, MATE1 or MATE2K was determined in the absence and presence of Cimetidine, the prototypical inhibitor of the transporters. The inhibition potential of fampridine on the renal transporters was evaluated by determining the uptake of TEA and Metformin, the probe substrates of the transporters of OCT2 and MATEs, respectively, in the absence or presence of fampridine. RESULTS: Significant time- and concentration-dependent uptake of fampridine by human OCT2 was observed. The Km and Vmax were determined as 51.0 ± 17.1 µM and 1107 ± 136 pmole/min/106 cells, respectively. Fampridine also inhibited OCT2 mediated uptake of Metformin with estimated IC50 of 66.8 µM. In contrast, there was not significant uptake of fampridine by human MATE1 or MATE2K, and fampridine did not inhibit MATE1 or MATE2K mediated uptake of TEA. CONCLUSION: The studies indicated fampridine is a substrate and inhibitor of OCT2, but not MATE1 or MATE2K. Results from the study suggested the active renal secretion of fampridine is mediated by human OCT2 but not MATE1 or MATE2K. To our knowledge, fampridine is the first reported substrate specific to OCT2 but not to MATE1 or MATE2K.

Organic anion transporting polypeptide (OATP)-mediated transport of coproporphyrins I and III.

1. Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are polyspecific transporters that mediate the transport of organic acids into hepatocytes. Inactivating mutations of both OATP1B1 and OATP1B3 alleles lead to Rotor syndrome, a disease characterized by coproporphyrinuria, an elevated urinary excretion of coproporphyrins I and III. It was hypothesized that transport of coproporphyrins I and III was mediated by OATP1B1 and OATP1B3. 2. This hypothesis was tested using cells transfected with OATP1B1 and OATP1B3. OATP1B-mediated transport of coproporphyrin was time-dependent and concentration-dependent. OATP1B1-mediated transport of coproporphyrins I and III (Km = 0.13 and 0.22 µM, respectively), as did OATP1B3 (Km = 3.25 and 4.61 µM, respectively). The OATP1B-mediated transport of each coproporphyrin was inhibited by rifampicin. 3. The specificity of coproporphyrin transport was also investigated where OATP2B1 demonstrated meaningful transport of coproporphyrin III (Km = 0.31 µM), while OCT1, OCT2, OAT1, OAT3 and NTCP were negative for coproporphyrin transport. 4. The identification of coproporphyrins as OATP substrates in vitro more clearly defines the role of OATPs in the hepatic disposition and renal excretion of coproporphyrins I and III and provides compelling evidence for future in vivo exploration of coproporphyrins as biomarkers of OATP activity.

Design and synthesis of lactam-thiophene carboxylic acids as potent hepatitis C virus polymerase inhibitors.

Herein we report the successful incorporation of a lactam as an amide replacement in the design of hepatitis C virus NS5B Site II thiophene carboxylic acid inhibitors. Optimizing potency in a replicon assay and minimizing potential risk for CYP3A4 induction led to the discovery of inhibitor 22a. This lead compound has a favorable pharmacokinetic profile in rats and dogs.

Assessment of biliary clearance in early drug discovery using sandwich-cultured hepatocyte model.

It is challenging to predict biliary clearance (CL(b) ) for new chemical entities (NCEs) in early drug discovery. Although sandwich-cultured hepatocyte (SCH) model has offered a valuable tool for characterizing hepatobiliary disposition and drug-drug interaction potential of NCEs, no comprehensive study was reported to project in vivo biliary clearance (in vivo CL(b,observed) ) potential using in vitro SCH model during the drug discovery stage. In this study, the CL(b) of 110 discovery compounds was evaluated using rat SCH model. Parallel artificial membrane permeability assay, Caco-2, and rat liver microsomes were employed in parallel to explore the interplay of biliary excretion with cellular permeability and liver metabolism. Selected compounds were further tested in bile-duct-cannulated rats, confirming the value of the SCH model for ranking and predicting in vivo CL(b,observed) during drug discovery. For compounds with extremely low passive permeability and metabolism, rat SCH may underestimate in vivo CL(b,observed) . The combination of passive permeability, metabolic intrinsic clearance, and the SCH model could serve as an initial screening platform for biliary excretion potential as well as a means for improving compound liabilities and properties. A preliminary evaluation strategy was proposed to highlight biliary excretion risk evaluation during the drug discovery process.