Pharmacological Inhibition of MALT1 Protease Leads to a Progressive IPEX-Like Pathology.

Genetic disruption or short-term pharmacological inhibition of MALT1 protease is effective in several preclinical models of autoimmunity and B cell malignancies. Despite these protective effects, the severe reduction in regulatory T cells (Tregs) and the associated IPEX-like pathology occurring upon congenital disruption of the MALT1 protease in mice has raised concerns about the long-term safety of MALT1 inhibition. Here we describe the results of a series of toxicology studies in rat and dog species using MLT-943, a novel potent and selective MALT1 protease inhibitor. While MLT-943 effectively prevented T cell-dependent B cell immune responses and reduced joint inflammation in the collagen-induced arthritis rat pharmacology model, in both preclinical species, pharmacological inhibition of MALT1 was associated with a rapid and dose-dependent reduction in Tregs and resulted in the progressive appearance of immune abnormalities and clinical signs of an IPEX-like pathology. At the 13-week time point, rats displayed severe intestinal inflammation associated with mast cell activation, high serum IgE levels, systemic T cell activation and mononuclear cell infiltration in multiple tissues. Importantly, using thymectomized rats we demonstrated that MALT1 protease inhibition affects peripheral Treg frequency independently of effects on thymic Treg output and development. Our data confirm the therapeutic potential of MALT1 protease inhibitors but highlight the safety risks and challenges to consider before potential application of such inhibitors into the clinic.

Deferasirox reduces iron overload in a murine model of juvenile hemochromatosis.

Mutations in hemojuvelin (HJV) cause severe juvenile hemochromatosis, characterized by iron loading of the heart, liver, and pancreas. Knockout (KO) mice lacking HJV (Hjv-/-) spontaneously load with dietary iron and, therefore, present a model for hereditary hemochromatosis (HH). In HH, iron chelation may be considered in noncandidates for phlebotomy. We examined the effects of deferasirox, an oral chelator, in Hjv-/- mice. Hepatic, cardiac, splenic, and pancreatic iron were determined by measuring elemental iron and scoring histological sections. Heart and liver iron levels were also determined repeatedly by quantitative R2* magnetic resonance imaging (MRI). The time course of iron loading without intervention was followed from Week 8 of age (study start) to Week 20, when once-daily (5x/week) deferasirox was administered, to Week 28. At 8 weeks, liver iron of KO mice was already markedly elevated versus wild-type mice (P<0.001) and reached a plateau around Week 14. In contrast, Week 8 cardiac and pancreatic iron levels were similar in both KO and wild-type mice and, compared with the liver, showed a delayed but massive iron loading up to Week 20. Contrary to the liver, heart, and pancreas, the KO mice spleen had lower iron content versus wild-type mice. In Hjv-/- mice, liver and heart iron burden was effectively reduced with deferasirox 100 mg/kg (P<0.05). Although deferasirox was less efficacious at this dose in the pancreas, over the observed time period, a clear trend toward reduced organ iron load was noted. There was no noticeable effect of deferasirox upon splenic iron in Hjv-/- mice. Quantitative R2* MRI demonstrated the ability to assess iron concentrations in the liver and myocardial muscle accurately and repetitively. Hepatic (R=0.86; P=3.2*10(-12)) and delayed myocardial (R=0.81; P=2.9*10(-10)) iron accumulation could be followed noninvasively with high agreement to invasive methods.

Evaluation of sparfloxacin distribution by mass spectrometry imaging in a phototoxicity model.

Mass spectrometry imaging (MSI) was applied to samples from mouse skin and from a human in vitro 3D skin model in order to assess its suitability in the context of photosafety evaluation. MSI proved to be a suitable method for the detection of the model compound sparfloxacin in biological tissues following systemic administration (oral gavage, 100 mg/kg) and subsequent exposure to simulated sunlight. In the human in vitro 3D skin model, a concentration-dependent increase as well as an irradiation-dependent decrease of sparfloxacin was observed. The MSI data on samples from mouse skin showed high signals of sparfloxacin 8 h after dosing. In contrast, animals irradiated with simulated sunlight showed significantly lower signals for sparfloxacin starting already at 1 h postirradiation, with no measurable intensity at the later time points (3 h and 6 h), suggesting a time- and irradiation-dependent degradation of sparfloxacin. The acquisition resolution of 100 µm proved to be adequate for the visualization of the distribution of sparfloxacin in the gross ear tissue samples, but distinct skin compartments were unable to be resolved. The label-free detection of intact sparfloxacin was only the first step in an attempt to gain a deeper understanding of the phototoxic processes. Further work is needed to identify the degradation products of sparfloxacin implicated in the observed inflammatory processes in order to better understand the origin and the mechanism of the phototoxic reaction.

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).

Detection of photogenotoxicity in skin and eye in rat with the photo comet assay.

Photosensitizing drugs increase the sensitivity of the skin and the eye toward normally harmless sunlight conditions and are known to enhance the induction of skin tumors or severe injuries to the eye. The photogenotoxicity of five common drugs (sparfloxacin, dacarbazine, chlorpromazine and 8-methoxypsoralen, promazine) was investigated in the skin as well as in the retina and cornea of Wistar rats. The compounds were administered once orally by gavage and the resulting DNA damage was analyzed in the newly developed in vivo photo comet assay. All drugs except of promazine were clearly photogenotoxic in the skin. In the cornea sparfloxacin and dacarbazine induced an increased DNA damage following irradiation. A photogenotoxic effect in the retina was observed by sparfloxacin, which is the only compound tested that absorbs wavelengths reaching the retina. The drug concentration analysis revealed that the compounds were distributed into plasma, skin and eye at concentrations, which were photogenotoxic in vitro. Additionally, histopathological analysis showed no relevant alterations or inductions of necrosis, apoptosis or inflammation in the skin or eye. In conclusion, we confirmed the photogenotoxic potential of compounds from different chemical classes in the skin. Moreover, it is the first time that photogenotoxicity has been detected in the retina and cornea in an in vivo study. Based on our results it is concluded that the photo comet assay in rat is an easy and reliable method to elucidate drug induced photogenotoxicity under conditions, which are relevant to human exposure.