CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease.

BACKGROUND: Sickle cell disease is caused by a defect in the ß-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny. METHODS: We performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin-immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1-2 clinical study to assess the safety and adverse-effect profile of OTQ923. RESULTS: In preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period. CONCLUSIONS: CRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.).

Randomised clinical trial: significant biochemical and colonic transit effects of the farnesoid X receptor agonist tropifexor in patients with primary bile acid diarrhoea.

BACKGROUND: In primary bile acid diarrhoea, feedback by farnesoid X receptor (FXR) and fibroblast growth hormone 19 (FGF19) on hepatic bile acid production is impaired. AIMS: To evaluate the safety, mechanisms and efficacy of negative feedback by FXR activation with tropifexor, a non-bile acid FXR agonist, in patients with primary bile acid diarrhoea. METHODS: In this double-blind, multicentre, randomised, cross-over study, patients received tropifexor 60 µg or placebo once daily for 14 days in each of two treatment periods. Primary objectives included tropifexor safety and tolerability, and on stool frequency and form. Other assessments included pharmacokinetic and pharmacodynamic measures, biochemical markers and gastrointestinal transit. RESULTS: Twenty patients (tropifexor 60 µg/placebo [N = 10]; placebo/tropifexor 60 µg [N = 10]) were enrolled. Adverse event rates were lower with tropifexor vs placebo (52.9% vs 73.7%). No patient had pruritus during tropifexor intake. There were no significant differences in stool frequency, stool form or loperamide use between treatments. Tropifexor increased FGF19 and decreased 7α-hydroxy-4-cholesten-3-one (C4) levels for up to 8 h. Plasma tropifexor concentrations peaked at 5 hours post-dose on days 1 and 12. At day 12, tropifexor caused reduction in peak total bile acid concentration (33%, P = 0.032) and exposure (36%, P = 0.005). Moreover, tropifexor showed a significant increase in ascending colon half-emptying time (P = 0.036). CONCLUSIONS: Tropifexor 60 µg once daily had acceptable safety and tolerability. Changes in FGF19 and C4 showed effective target engagement; however, higher doses may be required to observe stool frequency changes. Slowing of ascending colon emptying suggests therapeutic potential of tropifexor in patients with primary bile acid diarrhoea. ClinicalTrials.gov number: NCT02713243.

Relative bioavailability of diclofenac potassium from softgel capsule versus powder for oral solution and immediate-release tablet formulation.

The oral bioavailability of diclofenac potassium 50 mg administered as a soft gelatin capsule (softgel capsule), powder for oral solution (oral solution), and tablet was evaluated in a randomized, open-label, 3-period, 6-sequence crossover study in healthy adults. Plasma diclofenac concentrations were measured using a validated liquid chromatography-mass spectrometry/mass spectrometry method, and pharmacokinetic analysis was performed by noncompartmental methods. The median time to achieve peak plasma concentrations of diclofenac was 0.5, 0.25, and 0.75 hours with the softgel capsule, oral solution, and tablet formulations, respectively. The geometric mean ratio and associated 90%CI for AUCinf, and Cmax of the softgel capsule formulation relative to the oral solution formulation were 0.97 (0.95-1.00) and 0.85 (0.76-0.95), respectively. The geometric mean ratio and associated 90%CI for AUCinf and Cmax of the softgel capsule formulation relative to the tablet formulation were 1.04 (1.00-1.08) and 1.67 (1.43-1.96), respectively. In conclusion, the exposure (AUC) of diclofenac with the new diclofenac potassium softgel capsule formulation was comparable to that of the existing oral solution and tablet formulations. The peak plasma concentration of diclofenac from the new softgel capsule was 67% higher than the existing tablet formulation, whereas it was 15% lower in comparison with the oral solution formulation.

Evaluation of two novel tablet formulations of artemether-lumefantrine (Coartem) for bioequivalence in a randomized, open-label, two-period study.

BACKGROUND: Artemether-lumefantrine (Coartem; AL) is a standard of care for malaria treatment as an oral six-dose regimen, given twice daily over three days with one to four tablets (20/120 mg) per dose, depending on patient body weight. In order to reduce the pill burden at each dose and potentially enhance compliance, two novel fixed-dose tablet formulations (80/480 mg and 60/360 mg) have been developed and tested in this study for bioequivalence with their respective number of standard tablets. METHODS: A randomized, open-label, two-period, single-dose, within formulation crossover bioequivalence study comparing artemether and lumefantrine exposure between the novel 80/480 mg tablet and four standard tablets, and the novel 60/360 mg tablet and three standard tablets, was conducted in 120 healthy subjects under fed conditions. Artemether, dihydroartemisinin, and lumefantrine were measured in plasma by HPLC/UPLC-MS/MS. Pharmacokinetic (PK) parameters were determined by non-compartmental analyses. RESULTS: Adjusted geometric mean AUClast for artemether were 345 and 364 ng·h/mL (geometric mean ratio (GMR) 0.95; 90% CI 0.89-1.01) and for lumefantrine were 219 and 218 µg·h/mL (GMR 1.00; 90% CI 0.93-1.08) for 80/480 mg tablet versus four standard tablets, respectively. Corresponding Cmax for artemether were 96.8 and 99.7 ng/mL (GMR 0.97; 90% CI 0.89-1.06) and for lumefantrine were 8.42 and 8.71 µg/mL (GMR 0.97; 90% CI 0.89-1.05). For the 60/360 mg tablet versus three standard tablets, adjusted geometric mean AUClast for artemether were 235 and 231 ng·h/mL (GMR 1.02; 90% CI 0.94-1.10), and for lumefantrine were 160 and 180 µg·h/mL (GMR 0.89; 90% CI 0.83-0.96), respectively. Corresponding Cmax for artemether were 75.5 and 71.5 ng/mL (GMR 1.06; 90% CI 0.95-1.18), and for lumefantrine were 6.64 and 7.61 µg/mL (GMR 0.87; 90% CI 0.81-0.94), respectively. GMR for Cmax and AUClast for artemether and lumefantrine for all primary comparisons were within the bioequivalence acceptance criteria (0.80-1.25). In addition, secondary PK parameters also met bioequivalence criterion. CONCLUSION: Both of the novel artemether-lumefantrine tablet formulations evaluated are bioequivalent to their respective standard Coartem tablet doses. These novel formulations are easy to administer and may improve adherence in the treatment of uncomplicated malaria caused by Plasmodium falciparum. CLINICAL TRIAL REGISTRATION NUMBER: CTRI/2011/12/002256.

Evaluation of pharmacokinetic interactions between amlodipine, valsartan, and hydrochlorothiazide in patients with hypertension.

The steady-state pharmacokinetic (PK) interaction potential between amlodipine (10 mg), valsartan (320 mg), and hydrochlorothiazide (HCTZ; 25 mg) was evaluated in patients with hypertension in a multicenter, multiple-dose, open-label, 4-cohort, parallel-group study. Eligible patients were randomly allocated to the dual combination of valsartan + HCTZ, amlodipine + valsartan, or amlodipine + HCTZ and nonrandomly allotted to amlodipine + valsartan + HCTZ triple combination treatment. After 6 days of treatment with a half-maximal dose of different combinations, patients were up-titrated to the maximal drug doses from day 7 through day 17. PK parameters of corresponding analytes from the triple- and dual-treatment groups were estimated on day 17 and compared. Safety and tolerability of all treatments was assessed. The C ( ssmax ) and AUC(0-τ) values of amlodipine or HCTZ remained unaffected when administered with valsartan + HCTZ or valsartan + amlodipine, respectively. On the other hand, valsartan exposure increased by 10% to 25% when coadministered with HCTZ and amlodipine, which is not considered clinically relevant. In conclusion, there were no clinically relevant PK interactions with amlodipine, valsartan, and HCTZ triple combination compared with the corresponding dual combinations. All treatments were safe and well tolerated.