Accelerating Global Deletion of the Abnormal Toxicity Test for vaccines and biologicals. Planning common next steps. A workshop Report.

This online workshop Accelerating Global Deletion of the Abnormal Toxicity Test for vaccines and biologicals. Planning common next steps was organized on October 14th, 2021, by the Animal Free Safety Assessment Collaboration (AFSA), the Humane Society International (HSI), the European Federation of Pharmaceutical Industries and Associations (EFPIA), in collaboration with the International Alliance of Biological Standardization (IABS). The workshop saw a participation of over a hundred representatives from international organizations, pharmaceutical industries and associations, and regulatory authorities of 28 countries. Participants reported on country- and region-specific regulatory requirements and, where present, on the perspectives on the waiving and elimination of the Abnormal Toxicity Test. With AFSA, HSI, EFPIA and IABS representatives as facilitators, the participants also discussed specific country/global actions to further secure the deletion of ATT from all regulatory requirements worldwide.

Identification of 2-[2-(4-tert-Butylphenyl)ethyl]-N-[4-(3-cyclopentylpropyl)-2-fluorophenyl]-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide as an Orally Active MGAT2 Inhibitor.

We previously reported 2-[2-(4-tert-butylphenyl)ethyl]-N-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide 2 as on orally available monoacylglycerol acyltransferase 2 (MGAT2) inhibitor which exhibited an in vivo efficacy at an oral dose of 100 mg/kg in a mouse oral lipid tolerance test. Further optimization of compound 2 to improve the intrinsic potency culminated in the identification of compound 11. Compound 11 showed a >50-fold lower IC50 against human MGAT2 enzyme than 2. Oral administration of 11 at a dose of 3 mg/kg in the oral lipid tolerance test resulted in significant suppression of triglyceride synthesis.

Identification of 2-[2-(4-tert-butylphenyl)ethyl]-N-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (29) as an orally available MGAT2 inhibitor.

MGAT2 (monoacylglycerol acyltransferase 2) is expected to be an attractive target for the drug treatment of obesity, diabetes, and other disease. We describe our exploration and structure-activity relationship (SAR) study of 2,3-dihydro-1H-isoindole-5-sulfonamide derivatives. In this study, we identified 29 as an orally available inhibitor of MGAT2 through optimization especially in terms of solubility. This compound exhibited moderate potency in the enzyme inhibitory assay (IC50 = 1522 nM) and significant suppression of fat absorption (57% inhibition) in mice oral lipid tolerance test.

Molecular design to enhance the penetration into the retina via ocular instillation.

To investigate the molecular design of drugs that have good penetration into the retina from anterior segment of the eye via ocular instillation, we optimized the length of methoxyethylene glycol chain (mEG) in the P3 region of an oral bioavailable calpain inhibitor SNJ-1945 (2) as a model compound. Modulation of the mEG length led to the optimal balance between hydrophilicity and lipophilicity and provided the compound with higher retinal exposure via ocular instillation. Incorporation of a mEG moiety would be a useful and convenient approach to attain high intraocular penetration.

Molecular design for enhancement of ocular penetration.

Over the past two decades, many oral drugs have been designed in consideration of physicochemical properties to attain optimal pharmacokinetic properties. This strategy significantly reduced attrition in drug development owing to inadequate pharmacokinetics during the last decade. On the other hand, most ophthalmic drugs are generated from reformulation of other therapeutic dosage forms. Therefore, the modification of formulations has been used mainly as the approach to improve ocular pharmacokinetics. However, to maximize ocular pharmacokinetic properties, a specific molecular design for ocular drug is preferable. Passive diffusion of drugs across the cornea membranes requires appropriate lipophilicity and aqueous solubility. Improvement of such physicochemical properties has been achieved by structure optimization or prodrug approaches. This review discusses the current knowledge about ophthalmic drugs adapted from systemic drugs and molecular design for ocular drugs. I propose the approaches for molecular design to obtain the optimal ocular penetration into anterior segment based on published studies to date.

Exploration of orally available calpain inhibitors 2: peptidyl hemiacetal derivatives.

We previously reported a potent calpain inhibitor 1 (SJA6017, N-(4-fluorophenyl)-l-valyl-l-leucinal), which displayed relatively low oral bioavailability (BA). Replacing the metabolically labile aldehyde moiety of 1with more chemically stable warheads, such as a cyclic hemiacetal, hydrazone, and alpha-ketoamide, provided the inhibitors with improved in vitro metabolic stability. Cyclic hemiacetal 2 was the most stable of these compounds. The optimization of 2 led to hemiacetal 8 (SNJ-1715) which exhibited high potency, good aqueous solubility, excellent oral BA, and prolonged plasma half-life in rats. Furthermore, 8 showed neuroprotective efficacy via oral administration in a rat retinal ischemia model.

Exploration of orally available calpain inhibitors. Part 3: Dipeptidyl alpha-ketoamide derivatives containing pyridine moiety.

Calpain-mediated proteolysis has been implicated as a major process in neuronal cell death including retinal neurological degeneration. The previously reported calpain inhibitor SJA6017 (1) showed oral efficacy in a retinal pharmacological model, but its oral bioavailability was low due to the metabolic lability and low water-solubility. The purpose of present study was to identify good orally bioavailable calpain inhibitors. A series of water-soluble dipeptidyl alpha-ketoamides containing a pyridine moiety at P3 were designed, synthesized, and evaluated for their oral bioavailability and retinal penetration. Introduction of a pyridineethanol moiety provided the potent alpha-ketoamide inhibitor 8 with good oral bioavailability. Compound 8 showed about 12-fold higher retinal AUC than 1.

Retinal penetration of calpain inhibitors in rats after oral administration.

Calpain-mediated proteolysis has been involved in neuronal cell death of retinal neurological degeneration. An aldehyde-based calpain inhibitor, SJA6017 (1), was effective following oral administration in a rat retinal ischemia model but had low oral bioavailability. The aim of this study was to identify calpain inhibitors with good retinal penetration after oral dosing. The orally bioavailable inhibitors, hemiacetal 3 (SNJ-1715), amphipathic ketoamide 5 (SNJ-1945), and pyridine ketoamide 6 (SNJ-2008), were evaluated for their retinal pharmacokinetic (PK) profiles. The retinal drug exposure of these inhibitors was more than tenfold higher than 1. Among these compounds, 5 exhibited the most favorable retinal PK properties, such as good penetration and long half-life. Comparisons of 5 and the structurally related ketoamide 6 suggested that the presence of a methoxy diethylene glycol moiety resulted in the inhibitor with high penetration into the retina and the sustained high retinal levels. Ketoamide 5 was selected as the development candidate for the treatment of retinal diseases.

Exploration of orally available calpain inhibitors: peptidyl alpha-ketoamides containing an amphiphile at P3 site.

A novel series of dipeptidyl alpha-ketoamide derivatives with amphiphile was designed and synthesized as water-soluble calpain inhibitors. The introduction of amphiphiles at the P3 site increased water solubility without loss of membrane permeability and provided the oral available inhibitors. Extension of the ethylene glycol chain at the P3 site led to an improvement in persistence of plasma levels. In particular, introduction of a combination of a diethylene glycol methyl ether moiety at the P3 site, a phenylalanine residue at the P1 site and a cyclopropyl moiety at the P' site was the most effective modification for an increase in plasma drug exposure.

An HPLC method to evaluate purity of a steroidal drug, loteprednol etabonate.

Validation of an analytical method for impurities and degradation products in an active pharmaceutical ingredient is important to assessment of quality and safety in a new pharmaceutical product. In the present study, a high-performance liquid chromatographic method was validated to evaluate purity of loteprednol etabonate (LE). LE and its four related substances, major process impurities and degradation products (PJ-90, PJ-91, LE-11-keto and LE-methyl ester) were well resolved using a phenyl-stationary phase under isocratic conditions. Two photo-degradation products were identified as chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-5alpha-methyl-2-oxo-19-norandrosta-1(10),3-diene-17beta-carboxylate and chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-1-methyl-3-oxo-6(5-->10alpha)-abeo-19-norandrosta-1,4-diene-17beta-carboxylate. A photo-degradation product, chloromethyl 1beta,11beta-epoxy-17alpha-ethoxycarbonyloxy-2-oxo-10alpha-androsta-4-ene-17beta-carboxylate, was not abundant by ultraviolet detector. The risk depending on only ultraviolet detection should be noted. Calibration curves for PJ-90, PJ-91, LE-11-keto and LE-methyl ester showed linearity over the range of 0.05-2.0% levels in LE with correlation coefficient of 0.999. Accuracy (n = 3) at the concentration of 0.5% level in LE for PJ-90, PJ-91, LE-11-keto and LE-methyl ester were 2.0, 2.0, 2.3 and 2.0%, respectively. Intra-day repeatability (n = 6) at the concentration of 0.5% level in LE for PJ-90, PJ-91, LE-11-keto and LE-methyl ester were 1.4, 1.4, 1.8 and 1.4%, respectively. The lower limits of detection for PJ-90, PJ-91, LE-11-keto and LE-methyl ester were 0.002, 0.001, 0.004 and 0.003% levels in LE, respectively.

Isolation and structure elucidation of the major photodegradation products of loteprednol etabonate.

Photodegradation of loteprednol etabonate (5), a steroid anti-inflammatory drug, in the solid state, in aqueous suspension, and in aqueous acetonitrile solution has been investigated. Analysis by HPLC showed that the profile of photodegradation products in the solid state was qualitatively similar to that in the aqueous suspension, although the profile in the aqueous acetonitrile solution was considerably different. The major photodegradation products were isolated from the aqueous suspension and the aqueous acetonitrile solution by using preparative reversed-phase HPLC and their structures were elucidated on the basis of spectroscopic data. Photolysis in the solid state and in aqueous suspension yielded three rearrangement products, chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-5alpha-methyl-2-oxo-19-norandrosta-1(10),3-diene-17beta-carboxylate (8), chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-1-methyl-3-oxo-6(5-->10alpha)-abeo-19-norandrosta-1,4-diene-17beta-carboxylate (9), and chloromethyl 1beta,11beta-epoxy-17alpha-ethoxycarbonyloxy-2-oxo-10alpha-androsta-4-ene-17beta-carboxylate (10). In aqueous acetonitrile solution, 10 was the major product, however, 8 and 9 were not obtained. Pathways for the formation of these compounds from loteprednol etabonate (5) are proposed.

Novel 6-hydroxy-3-morpholinones as cornea permeable calpain inhibitors.

A novel series of 6-hydroxy-3-morpholinones, in which the functional aldehyde and the hydroxy group of P(2) site form a cyclic hemiacetal, was identified as calpain inhibitors. The placement of isobutyl group at the 2-position of the 3-morpholinone was the most effective modification for inhibiting micro- and m-calpains. Substitutions of benzyl at the 5-position in the S-configuration had virtually no effect on inhibitory activity. Several compounds showed appreciable selectivity for calpains over cathepsin B. NMR experiments demonstrated that the representative 6-hydroxy-3-morpholinone 10a (SNJ-1757) was more stable to nucleophilic attack than the corresponding aldehyde inhibitor 24. Furthermore, 6-hydroxy-3-morpholinone 10a proved to have better corneal permeability than aldehyde inhibitor 24 in an in vitro experiment.

Synthesis and PDF inhibitory activities of novel benzothiazolylidenehydroxamic acid derivatives.

A novel series of benzothiazolylidenehydroxamic acid derivatives has been designed and synthesized as PDF inhibitors. Some of this novel class of PDF inhibitors exhibited micromolar order enzyme inhibitory activity and antibacterial activity.