CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Identification of cryptolepine metabolites in rat and human hepatocytes and metabolism and pharmacokinetics of cryptolepine in Sprague Dawley rats.

BACKGROUND: This study aims at characterizing the in vitro metabolism of cryptolepine using human and rat hepatocytes, identifying metabolites in rat plasma and urine after a single cryptolepine dose, and evaluating the single-dose oral and intravenous pharmacokinetics of cryptolepine in male Sprague Dawley (SD) rats. METHODS: The in vitro metabolic profiles of cryptolepine were determined by LC-MS/MS following incubation with rat and human hepatocytes. The in vivo metabolic profile of cryptolepine was determined in plasma and urine samples from Sprague Dawley rats following single-dose oral administration of cryptolepine. Pharmacokinetic parameters of cryptolepine were determined in plasma and urine from Sprague Dawley rats after single-dose intravenous and oral administration. RESULTS: Nine metabolites were identified in human and rat hepatocytes, resulting from metabolic pathways involving oxidation (M2-M9) and glucuronidation (M1, M2, M4, M8, M9). All human metabolites were found in rat hepatocyte incubations except glucuronide M1. Several metabolites (M2, M6, M9) were also identified in the urine and plasma of rats following oral administration of cryptolepine. Unchanged cryptolepine detected in urine was negligible. The Pharmacokinetic profile of cryptolepine showed a very high plasma clearance and volume of distribution (Vss) resulting in a moderate average plasma half-life of 4.5 h. Oral absorption was fast and plasma exposure and oral bioavailability were low. CONCLUSIONS: Cryptolepine metabolism is similar in rat and human in vitro with the exception of direct glucuronidation in human. Clearance in rat and human is likely to include a significant metabolic contribution, with proposed primary human metabolism pathways hydroxylation, dihydrodiol formation and glucuronidation. Cryptolepine showed extensive distribution with a moderate half-life.

Compound Property Optimization in Drug Discovery Using Quantitative Surface Sampling Micro Liquid Chromatography with Tandem Mass Spectrometry.

Surface sampling micro liquid chromatography tandem mass spectrometry (SSµLC-MS/MS) was explored as a quantitative tissue distribution technique for probing compound properties in drug discovery. A method was developed for creating standard curves using surrogate tissue sections from blank tissue homogenate spiked with compounds. The resulting standard curves showed good linearity and high sensitivity. The accuracy and precision of standards met acceptance criteria of ±30%. A new approach was proposed based on an experimental and mathematical method for tissue extraction efficiency evaluation by means of consecutively sampling a location on tissue twice by SSµLC-MS/MS. The observed extraction efficiency ranged from 69% to 82% with acceptable variation for the test compounds. Good agreement in extraction efficiency was observed between surrogate tissue sections and incurred tissue sections. This method was successfully applied to two case studies in which tissue distribution was instrumental in advancing project teams' understanding of compound properties.

A Comparison of LC-MS/MS and a Fully Integrated Autosampler/Solid-Phase Extraction System for the Analysis of Protein Binding Samples.

A new analysis approach was evaluated for measuring plasma protein binding (PPB) of small molecules using the Agilent RapidFire high-throughput system coupled with a Sciex API 4000 mass spectrometer (RF-MS/MS). Thirty-three proprietary and 12 literature compounds were subjected to rapid equilibrium dialysis (RED) and evaluated in parallel using RF-MS/MS at 16.4 s/sample and traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) at 3.5 min/sample, thus making the RF-MS/MS analysis over 12 times faster than LC-MS/MS. The high-throughput analysis method that was developed demonstrated excellent correlation with the traditional LC-MS/MS analysis method with an r(2) value of 0.96. The RF-MS/MS analysis method was implemented to increase sample throughput, decrease turnaround time for PPB data, and decrease time burden on existing LC-MS/MS instruments.

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

Structure-Activity Relationships, Pharmacokinetics, and in Vivo Activity of CYP11B2 and CYP11B1 Inhibitors.

CYP11B2, the aldosterone synthase, and CYP11B1, the cortisol synthase, are two highly homologous enzymes implicated in a range of cardiovascular and metabolic diseases. We have previously reported the discovery of LCI699, a dual CYP11B2 and CYP11B1 inhibitor that has provided clinical validation for the lowering of plasma aldosterone as a viable approach to modulate blood pressure in humans, as well normalization of urinary cortisol in Cushing's disease patients. We now report novel series of aldosterone synthase inhibitors with single-digit nanomolar cellular potency and excellent physicochemical properties. Structure-activity relationships and optimization of their oral bioavailability are presented. An illustration of the impact of the age of preclinical models on pharmacokinetic properties is also highlighted. Similar biochemical potency was generally observed against CYP11B2 and CYP11B1, although emerging structure-selectivity relationships were noted leading to more CYP11B1-selective analogs.

Discovery of LFF571: an investigational agent for Clostridium difficile infection.

Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death. Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of the natural product and previous development candidates (2, 3) and improving antibacterial activity. Structure-activity relationships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of magnitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for treating C. difficile infection.

Estudo experimental da eficácia de uma órtese caseira de baixo custo para lombalgia em grávidas. / Experimental study of the effectiveness of a ortes caseira low cost to lombalgia in pregnant women.

Objetivo: verificar a eficácia de uma órtese caseira de baixo custo para lombalgia em grávidas.Método: estudo experimental com oito grávidas que fizeram uso da órtese caseira durante um mês,avaliando a dor lombar através de um questionário qualitativo e da E.V.A. Resultados: A media doaparecimento da dor teve media de 5 meses, 100% das grávidas apresentaram melhora significativa daE.V.A. depois do uso da órtese. Conclusões: Apesar de ser uma queixa freqüente das grávidas, alombalgia no período gestacional é muito pouco explorada. E essas dores influenciam de modonegativo na qualidade de vida das grávidas, e também na qualidade do sono, na disposição física, nodesempenho no trabalho, na vida social, na atividade doméstica e no lazer. E provamos que é possívelproduzir uma órtese de baixo custo com eficiência muito satisfatória, igual, ou melhor, a órtese jácomercializada, devido os pequenos furos feitos na parte da frente, que vão servir para circulação dear. Expondo uma vantagem de circulação de ar, e o velcro preso somente de um lado da órtese permitique ela seja facilmente colocada pela própria gestante

Objective: To verify the effectiveness of a stent homemade low cost for low back pain in pregnantwomen. Method: an experimental study with eight pregnant women who used orthesis home for amonth, assessing pain a questionnaire, and EVA Results: The mean onset of pain rating was 5 months,100% of pregnant women showed significant improvement in EVA after bracing. Conclusions:Despite being a frequent complaint of pregnant women, low back pain during pregnancy is very littleexplored. And those pains affect negatively the quality of life of pregnant women, and also the qualityof sleep, physical layout, performance at work, social life, domestic activity and leisure. And we provethat it is possible to produce a low cost orthosis with very satisfactory performance, like, or better, thestent already committed, because the small holes made in the front, which will be used for aircirculation. Exposing an advantage of air circulation, and the Velcro stuck on only one side of thestent allowed it to be easily placed by the very pregnant

1-amino-4-benzylphthalazines as orally bioavailable smoothened antagonists with antitumor activity.

Abnormal activation of the Hedgehog (Hh) signaling pathway has been linked to several types of human cancers, and the development of small-molecule inhibitors of this pathway represents a promising route toward novel anticancer therapeutics. A cell-based screen performed in our laboratories identified a new class of Hh pathway inhibitors, 1-amino-4-benzylphthalazines, that act via antagonism of the Smoothened receptor. A variety of analogues were synthesized and their structure-activity relationships determined. This optimization resulted in the discovery of high affinity Smoothened antagonists, one of which was further profiled in vivo. This compound displayed a good pharmacokinetic profile and also afforded tumor regression in a genetic mouse model of medulloblastoma.