Characterization of false positive, contaminant-driven mutagenicity in impurities associated with the sotorasib drug substance.

Sotorasib (Lumakras™) is a first-in-class, non-genotoxic, small molecule inhibitor of KRAS G12C developed as an anticancer therapeutic for treatment of patients that have a high unmet medical need. Anticancer therapeutics are considered out of scope of ICH M7 guidance for control of mutagenic impurities; however, based on ICH S9 Q&A, mutagenicity assessments are needed for impurities that exceed the qualification threshold, consistent with ICH Q3A/B, and non-mutagenic drugs. Here, we carried out hybrid-based mutagenicity assessment of sotorasib drug substance (DS) impurities using in silico quantitative structure-activity relationship (QSAR) modelling and Ames tests (for in silico positive mutagens). We encountered contradictive mutagenicity results for 2 impurities (Beta-Chloride and PAC). PAC was negative initially by QSAR but positive in a GLP full plate Ames test and Beta-Chloride was positive by QSAR, negative in a non-GLP micro-Ames but positive in a GLP full plate Ames assay. Root cause analyses identified and characterized mutagenic contaminants, 3-chloropropionic acid in batches of Beta-Chloride and 3-chloropropionic acid and Chloro-PAC in batches of PAC, used in initial GLP full-plate Ames tests. Significant reduction of these contaminants in re-purified batches resulted in no induction of mutagenicity in follow-up GLP micro-Ames tests. In summary, root-cause analyses led to accurate mutagenicity assessment for sotorasib DS-associated impurities.

Threshold for Anaphylactoid Reaction to Polysorbate 80 in Canines.

Polysorbate 80 (PS80) functions as a dispersing agent or solubilizer in many pharmaceuticals, and as a stabilizer in biopharmaceuticals. Topical or parenteral administration of low doses of PS80 in biopharmaceuticals has been associated with mild allergic reactions, including local injection site reactions in humans. High doses of PS80, such as levels found in traditional Chinese herbal parenteral medicines, have been linked to systemic effects consistent with anaphylactoid-type reactions, which are characterized by the direct release of histamine from mast cells (degranulation). Nonclinical safety assessments of PS80 in vivo have mainly focused on canine model systems, a species established to be particularly sensitive to PS80. However, there is conflicting data about the dose and route of administration of PS80 required to elicit an anaphylactoid-type reaction in this model system. Therefore, studies using multiple dosing regimens in anesthetized and conscious dogs including a combination of cardiovascular data, clinical signs, and biomarkers of mast cell degranulation were conducted. An intravenous (IV) bolus of 1 mg/kg PS80 (0.25% w/v) elicited a positive anaphylactoid reaction including increased heart rate, hypotension, and clinical signs associated with anaphylactoid reactions (e.g., reddened muzzle). However, a full reaction was not observed with a subcutaneous (SC) injection of PS80 (0.25% w/v) up to 20 mg/kg and IV bolus or IV infusions up to 0.5 mg/kg. These data establish a threshold dose for eliciting an anaphylactoid reaction in canine which varies depending on the route of administration as well as the rate of PS80 infusion.

Polysorbate 80-Induced Anaphylactoid Reaction and the Effects on Cardiovascular Function: Dose Threshold and Species Comparison.

Polysorbate 80 (PS80) is commonly used in pre-clinical formulations. The dose threshold for cardiovascular (CV) changes and hypersensitivity reaction in the dog was assessed and compared to other species. PS80 was administered by intravenous (IV) bolus (.5, 1 mg/kg), IV infusion (.3, .5, 1, 3 mg/kg), subcutaneous (SC) injection (5, 10, 15 mg/kg) and oral gavage (10 mg/kg) to dogs with CV monitoring. Monkeys and minipigs received PS80 by IV infusion at 3 mg/kg. Plasma histamine concentration was measured following PS80 IV infusion and with diphenhydramine pre-treatment in dogs only. In dogs, PS80 was not associated with CV changes at doses up to 15 mg/kg SC and 10 mg/kg oral, but decreased blood pressure and increased heart rate with IV bolus at ≥ .5 mg/kg and IV infusion at ≥ 1.0 mg/kg and decreased body temperature with IV infusion at 3 mg/kg was observed. Transient edema and erythema were noted with all administration routes, in all three species including doses that were devoid of CV effects. In monkeys and minipigs, PS80 did not induce CV, cutaneous or histamine concentration changes. These results suggest that mild, transient skin changes occur following PS80 administration at doses that are not associated with CV effects in the dogs. In dogs, the cardiovascular effect threshold was <.5 mg/kg for IV bolus, .3 mg/kg for IV infusion, 15 mg/kg for SC injection, and 10 mg/kg for oral administration. Monkey and minipig were refractory to PS80-induced histamine release at 3 mg/kg by IV infusion over 15 minutes.

Nonclinical genotoxicity and carcinogenicity profile of apremilast, an oral selective inhibitor of PDE4.

Apremilast is an oral, selective small molecule inhibitor of phosphodiesterase-4 (PDE4) that has been approved for the treatment of active psoriatic arthritis, moderate to severe plaque psoriasis, and for patients with oral ulcers associated with Behçet's disease. Apremilast modulates the inflammatory cascade in cells by inhibiting PDE4, thus preventing the degradation of cyclic adenosine monophosphate, resulting in the upregulation of interleukin (IL)-10 and the downregulation of proinflammatory cytokines, including IL-23, interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα). Here, we evaluated the genotoxic and carcinogenic potential of apremilast using Good Laboratory Practice (GLP)-compliant in vitro and in vivo studies. Apremilast was not genotoxic in the genetic toxicology battery, as evaluated for mutagenicity in the Ames test up to concentrations of 5000 µg/plate, clastogenicity in cultured human peripheral blood lymphocytes up to concentrations of 700 ug/mL was in excess of the solubility limit in culture medium and not able to assess; and negative for the induction of micronuclei in the bone marrow micronucleus test in mice up to doses of 2000 mg/kg/day. Furthermore, apremilast did not increase the incidence of tumors in lifetime rat or mouse carcinogenicity studies up to the maximum tolerated dose. In summary, in non-clinical studies, apremilast is not genotoxic and is not carcinogenic.

Retinal Toxicity Induced by a Novel ß-secretase Inhibitor in the Sprague-Dawley Rat.

ß-Secretase 1 (BACE1) represents an attractive target for the treatment of Alzheimer's disease. In the course of development of a novel small molecule BACE1 inhibitor (AMG-8718), retinal thinning was observed in a 1-month toxicity study in the rat. To further understand the lesion, an investigational study was conducted whereby rats were treated daily with AMG-8718 for 1 month followed by a 2-month treatment-free phase. The earliest detectable change in the retina was an increase in autofluorescent granules in the retinal pigment epithelium (RPE) on day 5; however, there were no treatment-related light microscopic changes observed in the neuroretina and no changes observed by fundus autofluorescence or routine ophthalmoscopic examination after 28 days of dosing. Following 2 months of recovery, there was significant retinal thinning attributed to loss of photoreceptor nuclei from the outer nuclear layer. Electroretinographic changes were observed as early as day 14, before any microscopic evidence of photoreceptor loss. BACE1 knockout rats were generated and found to have normal retinal morphology indicating that the retinal toxicity induced by AMG-8718 was likely off-target. These results suggest that AMG-8718 impairs phagolysosomal function in the rat RPE, which leads to photoreceptor dysfunction and ultimately loss of photoreceptors.