Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.

Potential Obstacles in the Acquisition of Oral Anticancer Medications.

PURPOSE: To determine the amount of time elapsed between prescriber order and patient receiving oral anticancer medication. PATIENTS AND METHODS: Adult patients with a diagnosis of cancer were prospectively identified in three outpatient oncology clinics when oral anticancer agents were prescribed during a 4-month observation period. For each patient, time to obtain medication was analyzed by the following time points: date of prescription, date of submission to insurance, date prior authorization was obtained, date financial assistance was received, date prescription was processed by pharmacy, and date patient received medication. Out-of-pocket cost and time spent by clinic staff to facilitate the medication acquisition process were recorded. RESULTS: Thirty-four patients were prescribed oral anticancer medication during the data collection period. For the 27 patients who were eligible for the primary end point, medication acquisition required a median of 10 days (range, 3 to 28 days). Overall, the rate-limiting step for medication acquisition was processing by the pharmacy, with a median of 6 days (range, 1 to 27 days). Most patients' prescription insurance plan covered a portion of medication cost, and the majority of patients considered their out-of-pocket expense to be affordable. Clinic staff spent a median of 2 hours per prescription to facilitate medication acquisition. CONCLUSION: Patients may encounter process barriers in acquiring oral therapy, particularly because of pharmacy processing time, as well as high copays. Time to treatment initiation may have implications for patients' clinical outcomes. Adequate staff with dedicated time to facilitate this process should be used in the ambulatory oncology practice setting.

Ocular toxicity of AUY922 in pigmented and albino rats.

AUY922, a heat shock protein 90 inhibitor is associated with ocular adverse events (AEs). To provide a better understanding of ocular AEs in patients, 4 investigative studies were performed in a step-wise approach to assess retinal structure and function in pigmented (Brown Norway) and albino (Wistar) rats. In rats administered 30mg/kg of AUY922, the AUC0-24h and Cmax are comparable to that in patients at 70mg/m(2). AUY922 at ≥30mg/kg was poorly tolerated by rats with morbidity or mortality generally after the third weekly treatment. Electroretinography (ERG) changes were observed at doses ≥30mg/kg. The ERG changes were dose dependent, consistent with an effect on the photoreceptors, and fully reversible. The ERG effects could not be minimized by decreasing the Cmax while maintaining AUC. Histopathological changes were seen mainly when rats were administered AUY922 at 100mg/kg. The 2-hour infusion of AUY922 at 100mg/kg caused disorganization of the outer segment photoreceptor morphology in male Brown Norway rats; the severity of the disorganization increased with the number of administrations, but was reversible during a 4-week posttreatment period. There was no major difference in ocular response between Brown Norway and Wistar rats. No changes in serum iron levels, and no changes in rhodopsin, PDE6α, ß-transducin concentrations, or retinal pigment epithelium-specific protein RPE65 expression were observed after single and multiple infusions of AUY922 at 100mg/kg compared to vehicle-treated controls. AUY922 retinal toxicity in rats recapitulates and further characterizes that reported in patients and is shown to be reversible, while a precise molecular mechanism for the effect was not determined.

Preclinical evaluation of potential nilotinib cardiotoxicity.

In vitro, concentrations ≥ 10 µM of nilotinib were needed to induce markers of cytotoxicity, apoptosis, and endoplasmic reticulum stress in both neonatal rat ventricular myocytes, a putative target tissue, and non-target heart fibroblasts, indicating a lack of cardiomyocyte-specific nilotinib toxicity in vitro. In rats, oral nilotinib treatment at 80 mg/kg for 4 weeks induced increased heart weight; however, this was not associated with relevant histopathological changes or effects on heart function. Thus, nilotinib at and above clinically relevant concentrations (4.27 µM) did not induce overt cardiovascular pathologies or heart failure in vitro or in vivo under study conditions.

Imatinib does not induce cardiotoxicity at clinically relevant concentrations in preclinical studies.

Cytotoxic concentrations of imatinib mesylate (10-50 microM) were required to trigger markers of apoptosis and endoplasmic reticulum stress response in neonatal rat ventricular myocytes and fibroblasts, with no significant differences observed between c-Abl silenced and nonsilenced cells. In mice, oral or intraperitoneal imatinib treatment did not induce cardiovascular pathology or heart failure. In rats, high doses of oral imatinib did result in some cardiac hypertrophy. Multi-organ toxicities may have increased the cardiac workload and contributed to the cardiac hypertrophy observed in rats only. These data suggest that imatinib is not cardiotoxic at clinically relevant concentrations (5 microM).

Antiglaucoma drug GLC756 and its effect on cellular cAMP and tumor necrosis factor alpha release in vitro of activated human monocytic leukemia cells.

PURPOSE: GLC756, a putative antiglaucoma drug with dopamine D(2) agonist and D(1) antagonist properties, significantly decreases tumor necrosis factor alpha (TNF-alpha) levels in lipopolysaccharide (LPS)-induced rats. The present study describes the effects of GLC756 on cellular adenosine 3', 5'-cyclic monophosphate (cAMP) in relation to TNF-alpha production on LPS-stimulated human acute monocytic leukemia cells. METHODS: A human peripheral blood acute monocytic leukemia cell line (THP-1) was activated via LPS. THP-1 cells were incubated with GLC756 or betamethasone (positive control) at concentrations of 1, 10, and 30 microM. The TNF-alpha concentration in supernatant and cAMP levels in cellular extract were measured by enzyme-linked immunosorbent assay 0,1, 2.5, 4.5, 7, and 24 h post-activation. RESULTS: Compared with LPS controls, both GLC756 at 30 muM and betamethasone at > or =1 microM had a significant inhibitory effect on TNF-alpha release from THP-1 cells 2.5 to 24 h post-activation. Parallel to the TNF-alpha decrease, GLC756 induced significant increases of cellular cAMP 2.5 and 7 h post-activation. Betamethasone had no effect on the cellular cAMP level. CONCLUSION: Intracellular signaling pathway leading to inhibition of the production of the proinflammatory cytokine TNF-alpha after GLC756 treatment might be mediated through the second messenger cAMP.

Effects of latanoprost, timolol and GLC756, a novel dopamine D(2) agonist and D(1) antagonist on LTC(4) release after rat mast cell activation.

PURPOSE: Mast cells participate in ocular allergic inflammation by releasing biologically active mediators. Leukotrienes are released from activated mast cells via an IgE-dependent mechanism, and play a crucial role in ocular allergic inflammation. In this study, the effect of three topical antiglaucoma drugs, that is, latanoprost, timolol and GLC756, a novel dopamine D(2) agonist and D(1) antagonist, on leukotriene C(4) (LTC(4)) release after rat mast cell activation was examined. METHODS: A rat basophilic leukaemia RBL-2H3 mast cell line was activated via IgE/anti-IgE. Rat mast cells were incubated with latanoprost, timolol, or GLC756 at concentrations of 0.1, 1, 10 and 30 microM. LTC(4) concentration in supernatant was assessed 5 h post activation by EIA. RESULTS: Compared with controls, timolol showed no relevant effect on LTC(4) release, 5 h after mast cell activation. Latanoprost and GLC756, in contrast, revealed an inhibitory effect on LTC(4) release, which was dose-related and statistically significant at the concentrations of 10 and 30 microM. CONCLUSION: The results of this study suggest that timolol has no significant influence on LTC(4) release from activated mast cells. By contrast, latanoprost and GLC756 inhibited LTC(4) release, suggesting a possible anti-inflammatory effect on ocular allergic inflammatory processes in topical glaucoma medication.

Effect of GLC756, a novel mixed dopamine D1 receptor antagonist and dopamine D2 receptor agonist, on TNF-alpha release in vitro from activated rat mast cells.

Tumor necrosis factor-alpha (TNF-alpha) is released from activated mast cells via an IgE-dependent mechanisms, and plays a crucial role in ocular allergic inflammation. This study examined the influence of three antiglaucoma drugs differing in their chemical structure and pharmacological profile (i.e. latanoprost, timolol, GLC756) on TNF-alpha release from activated rat mast cells. A rat basophilic leukemia mast cell line (RBL-2H3) was activated via IgE/anti-IgE. Rat mast cells were incubated with latanoprost, timolol, GLC756 or betamethasone (positive control) at concentrations of 0.1, 1, 10 and 30 microM. TNF-alpha concentration in supernatant was measured by ELISA 5 h post-activation. Compared to controls, the prostaglandin derivative latanoprost and the beta-blocker timolol in the concentration range 0.1-30 microM, had no significant effect on TNF-alpha release from rat mast cells measured 5h after activation. By contrast, the dopaminergic drug GLC756 compared to controls in the concentration range 1-30 microM significantly inhibited TNF-alpha release from activated rat mast cells in a concentration-dependent manner. The positive control betamethasone inhibited TNF-alpha release almost completely at all concentrations tested. In conclusion, the results of this study suggest that latanoprost and timolol do not reduce inflammation triggered by activated mast cells. By contrast, the dopaminergic drug GLC756 inhibited TNF-alpha release from activated mast cells, suggesting an palliative potential of dopaminergic compounds on allergic conjunctivitis in topical glaucoma medication.

GLC756 decreases TNF-alpha via an alpha2 and beta2 adrenoceptor related mechanism.

GLC756, a polyvalent anti-glaucoma drug showed in an endotoxin-induced-uveitis model (EIU) in rats a significant tumor necrosis factor-alpha (TNF-alpha) decrease in serum, indicating an additional anti-inflammatory potential of this compound. The receptors on which GLC756 binds (D1, D2, D4, alpha-1, alpha-2, 5-HT1A, 5-HT2C, 5-HT1D, 5-HT2 A, beta-1, and beta-2) were suggested to play a role. In order to identify a receptor type mediating the TNF-alpha lowering response, GLC756 was combined with various counteracting compounds (CP). For EIU, 8-week-old Lewis rats were intravenously injected at 160 microg lipopolysaccharide (LPS) from Salmonella typhimurium. Before EIU-induction animals received either one of the CP's or GLC756 alone, or GLC756 in combination with one of the CP's. TNF-alpha was determined in serum 2h post EIU-induction. Treatment with CP's alone indicated that agonistic effects on beta-2 adrenoceptors and antagonistic effects on alpha-2, 5-HT1A and 5-HT1D receptors resulted in statistically significant decreased TNF-alpha levels in comparison to the LPS-control group. In combination with GLC756, the counteracting CP's domitor (alpha-2 adrenoceptor agonist) and ICI 118551 (beta-2 adrenoceptor antagonist) inhibited completely the TNF-alpha decreasing effect of GLC756. Counteracting the 5-HT1A receptor with the 5-HT1A agonist 8-OH-DPAT could not prevent the TNF-alpha decreasing effect of GLC756. In conclusion, the antagonistic effect on alpha-2 adrenoceptors and the agonistic effect on beta-2 adrenoceptors were identified as mechanism for the TNF-alpha decreasing effect of GLC756.

The minipig in dermatotoxicology: methods and challenges.

In view of more morphological and physiological similarities between human and porcine skin than for other laboratory animal species, the minipig is a preferred model to evaluate the safety profile of dermally applied xenobiotics. Different methods of dermal administration and examples of non-invasive and invasive investigations during the in-life phase of the studies are described. Routine and special post-mortem examinations in dermal studies are presented to assess responses to the topical treatment of minipig skin. Challenges in dermal minipig studies are discussed with respect to animal welfare and husbandry, test formulations, application methods and different types of investigations. One of the most significant issues for dermal minipig studies is the extensive measures required to prevent cross-contamination of blood and tissue samples taken to monitor local and systemic exposure to the test item.

Effects of antiglaucoma drugs timolol and GLC756, a novel dopamine D2 agonist and D1 antagonist, on endotoxin-induced-uveitis in rats.

Antiglaucoma drugs with anti-inflammatory properties may be of particular value for the long-term treatment of glaucoma since they may reduce the risk for treatment-related inflammatory processes in outer compartments of the eye. The purpose of this study was, to evaluate the effect of systemic and topical administration of GLC756, a novel mixed dopamine D(2) receptor agonist and D(1) receptor antagonist which lowered intraocular pressure in man, and timolol on endotoxin-induced-uveitis (EIU) in rats. For EIU, 8-week-old Lewis rats received an intravenous injection of 160 microg lipopolysaccharide (LPS) from Salmonella typhimurium. GLC756, timolol, or betamethasone, as positive control, were administered either topically (0.4, 0.5, and 0.1%, respectively, 16-times 20 microl eye drops during 48 hr) or systemically (1 mg kg(-1) subcutaneously for 5 days). Protein content, released TNF-alpha, the number of cells as well as cells expressing TNF-alpha were determined in aqueous humor 48 hr after LPS-injection and served as parameters for inflammation. LPS induced an increase of protein content, infiltrating cells and cells expressing TNF-alpha in the aqueous humor. Topical and systemic administration of GLC756 and betamethasone, almost completely suppressed the increase of protein content and betamethasone in addition also suppressed the number of cells in aqueous humor. In conclusion, the almost complete suppression of LPS-induced protein increase in aqueous humor by GLC756 suggests an additional anti-inflammatory potential of dopaminergic compounds in glaucoma treatment. Timolol did not show any effect on EIU in rats.

LAV694, a new antiproliferative agent showing improved skin tolerability vs. clinical standards for the treatment of actinic keratosis.

The skin tolerability of the tubulin polymerisation inhibitor LAV694 was compared to that of 5% 5-fluorouracil (5-FU) and 0.5% podophyllotoxin in vitro using a human reconstructed epidermis (HRE), and in vivo using minipigs. Topical treatment of HRE for 1 or 3 days with a 0.2, 0.6 or 1% LAV694 cream or the placebo showed no signs of irritation in terms of morphology, cell viability (lactate dehydrogenase leakage) or interleukin-8 mRNA expression and release. 5-FU increased interleukin-8 production and induced morphological signs of irritation. The substances were also applied under occlusion to the back of two minipigs, twice daily, for 9 days to allow intraindividual comparison of skin effects and tolerability. Skin reactions were monitored by visual scoring, chromometry, pro-inflammatory activity, cell cycle and apoptosis by RT-PCR, laser scanning cytometry and histopathological examination of biopsies. Application of podophyllotoxin and 5-FU had to be stopped on days 4 and 8, respectively, due to severe skin lesions. LAV694 (1%) induced only moderate skin reddening after 9 days. 5-FU and podophyllotoxin, but not LAV694, increased mRNA expression of pro-inflammatory cytokines. LAV694 arrested keratinocytes in the M phase of the cell cycle and apoptosis was detected histologically in the basal layer. LAV694 increased the expression of pro-apoptotic genes in both experimental models. In conclusion, LAV694 selectively induced apoptosis, rather than necrosis, of growth-arrested keratinocytes, thus avoiding the occurrence of extensive inflammation. This resulted in an improved skin tolerability in comparison with 5-FU and podophyllotoxin.

Effects of epithelial growth factor receptor (EGFR) kinase inhibitors on genetically reconstituted mouse mammary glands.

UNLABELLED: The aim of the study was to determine the effects of a specific epithelial growth factor Receptor kinase inhibitor (EGFR-KI) and Taxol on tumor growth in a novel tumor model. MATERIAL & METHODS: A genetically engineered tumor model which uses "transgenic" organs in immune competent mice was used. NeuT-transfected immortalized HC11 epithelial cells and primary mouse mammary epithelial cells have been transplanted into the gland-free mammary fat pad of female BALB/c mice. Mammary tumors developed after a latency period of three to four weeks. The mice were thereafter daily orally treated over a 19 or 22-day period with 0, 38, 75, 2 x 75 mg/kg body weight (b.w.) EGFR-KI (n: 7-9 per group) or intravenously with 10 mg/kg b.w. Taxol. After necropsy the histopathological evaluation of the tumors was performed in a coded manner. The proliferation activity of tumor cells was analyzed by laser scanning cytometry (LSC) using anti-Ki67-antibodies. RESULTS: Oral Treatment with EGFR-KI in this transgenic organ model showed clear antitumor efficacy in a dose-dependent manner in the range between 38 and 75 mg/kg b.w. This antiproliferative effect appears to be minimally increased at 75 mg/kg/day twice per day. For all treatments a strong correlation between the biological behavior of the tumor, histopathology and cell proliferation could be established. In contrast, treatment with Taxol showed no significant reduction of tumor growth or cell proliferation in this model. This new transgenic organ model comprising histopathological evaluation and cell proliferation analysis appears to be a suitable test system for drug candidates that affect specific biochemical pathways. It may have greater predictive nature for clinical effects in humans as compared to conventional tumor models because of its c-erb B2 gene overexpression.

Combination of low-dose valsartan and enalapril improves endothelial dysfunction and coronary reserve in Nomega-nitro-L-arginine methyl ester-treated spontaneously hypertensive rats.

Combination of nonhypotensive doses of valsartan and enalapril markedly improved survival (+87%) compared with untreated animals (37%) in spontaneously hypertensive rats (SHRs) with endothelial dysfunction. However, the combination had no effect on kidney function, and proteinuria persisted over the 12 weeks of the study. It was hypothesized that the greater survival was due to improvement in endothelial function or coronary vasculature despite blockade of nitric oxide synthase and high blood pressure. Therefore, endothelial function was evaluated in isolated aortic ring and maximal coronary blood flow was studied in isolated perfused SHR hearts (20-24 weeks) treated with -nitro-l-arginine methyl ester (L-NAME) (50 mg/l) for 4 weeks. The animals received vehicle, valsartan 5 mg/kg/d, enalapril 1 mg/kg/d, valsartan 50 mg/kg/d, or the combination valsartan 5 mg/kg/d with enalapril 1 mg/kg/d in drinking water. Normotensive Wistar-Kyoto (WKY) rats were used as control. Blood pressure was measured by telemetry. Histopathology was performed on heart, kidney (hematoxylin-eosin), and aorta (Masson trichrome). L-NAME elevated blood pressure by 50 mm Hg after vehicle (199 +/- 5 mm Hg). Valsartan 50 mg/kg/d completely abolished this increase (150 +/- 4 mm Hg) whereas the valsartan-enalapril combination synergistically decreased blood pressure (-37 mm Hg at 162 +/- 7 mm Hg) compared with monotherapy (valsartan 5 mg/kg/d -10 mm Hg; enalapril 1 mg/kg/d -15 mm Hg). All treatments improved the histopathology, most markedly in those receiving the valsartan-enalapril combination. The severity mean grades for lesions were 2.1, 1.9, 1.7, 1.1, and 0.9 in vehicle-treated SHRs, enalapril 1 mg/kg/d, valsartan 5 mg/kg/d, valsartan 5 or 50 mg/kg/d, and the valsartan-enalapril combination, respectively, compared with 0.02 in WKY rats. Acetylcholine-induced relaxation was significantly greater in treated SHRs than after vehicle (-40% at 0.1 mmol acetylcholine) but the combination induced the maximal relaxation (-85%). The ratio of maximal tension induced by serotonin in rings with and without endothelium was 1.4 and 1.3 in vehicle and valsartan 5 mg/kg/d-treated rats whereas it did not differ from 1 in WKY rats and all other treated groups. The cardiac hypertrophy (+27%) was prevented by valsartan 50 mg/kg/d and the valsartan-enalapril combination. Coronary reserve was significantly increased by valsartan 50 mg/kg/d (+85% at 7.8 +/- 0.7 ml/min/g) and the valsartan-enalapril combination (+42% at 6.0 +/- 0.4 ml/min/g) compared with 4.2 +/- 0.5 (vehicle). This was not different of 8.8 +/- 0.5 (WKYs). Despite the maintenance of a high blood pressure, low-dose valsartan-enalapril significantly improved endothelial function and histopathology and increased coronary reserve in SHRs chronically receiving L-NAME.

Laser technologies in toxicopathology.

One of the main concepts in toxicology and risk assessment is the identification of compounds with the least toxicity, gaining increased understanding of the underlying mechanisms of efficacy and toxicity so as to accelerate the early selection of compounds for development. For this purpose, "cutting-edge" technologies, such as flow cytometry (FC), laser scanning cytometry (LSC) and confocal laser scanning microscopy (CLSM), have proved to be valuable tools. FC, LSC and CLSM have been successfully applied in a wide range of areas within toxicology and research including genetics, reproduction, dermatology, pathology and target organ toxicity. The scope of this paper is to give a short overview of the usefulness of the different laser applications. Specific examples of the impact of these technologies will be presented or can be found in the references. Flow cytometry methods have been successfully applied in immunophenotyping, micronuclei scoring, polyploidy determination, apoptosis and cell cycle evaluation, cell proliferation and quantification. A three-parameter FC method for the analysis of testicular toxicity has also been established as an alternative to traditional histopathological methods. This method allows a large number of cells to be analysed in a short time and provides quantitative values to evaluate testicular damage in the rat. Laser scanning cytometry has been used in our unit for rat blood cell immunophenotyping, tumor proliferation, apoptosis and cell cycle analysis on minipig and rat skin and cardiac cells identification. The wide range of applications that can be applied with the LSC shows the enormous potential of this technology in research and development. Confocal laser scanning microscope was used in our laboratory, in collaboration with the research department, to investigate the mechanisms underlying hepatic lesions found in dogs, to detect fibrinogen influx into rat lung, to explore the mechanism of eye toxicity and to quantify dopaminergic fibers in brain sections. Integrating these technologies within discovery pathology allowed us to understand disease processes with respect to their development and subsequent consequences. It contributes to descriptive pathologic diagnostic and allows a productive interaction with research and development. These technologies offer a range of novel applications and have been shown to be useful tools in terms of specificity, sensitivity, reliability, rapidity and quantification. Expertise in cutting-edge technologies, pathology and cell and molecular biology is essential to a successful and flexible interaction across all therapeutic areas in drug discovery.