S. Peter Hong

Effects of tyrosine kinase inhibitors on rat isolated heart function and protein biomarkers indicative of toxicity

INTRODUCTION Cardiac toxicity, manifested as diminished contractility, ischemic heart disease, and heart failure is a major issue in drug safety. Concerns revolve around targeted drugs (TKIs) where contractility effects were not anticipated. The ability to predict cardiac toxicity early would help to de-risk drugs in development and prepare physicians to manage risk in the clinic. Issues with current preclinical studies include insufficient testing with informative, translatable models, and predictive biomarkers. The isolated heart model is amenable to multiple assessments which can be combined with current technologies to assess toxicity on a multi-scale level. METHODS Rat isolated heart model was used to assess changes in left ventricular (LV) contractility and protein biomarkers BNP, IL6, TNFα, and cardiac troponins T (TnT) and I (TnI). Responses were assessed during perfusion with modified Henseleit Krebs (MHK), and 20 min concentration escalations of verapamil, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), isoproterenol, or 20 min escalations bracketing clinical blood concentrations of sunitinib, sorafenib, and erlotinib. LV parameters and effluent for biomarkers were collected before and during escalating drug concentrations. RESULTS Verapamil reduced inotropy with no change in biomarkers, FCCP and isoproterenol reduced and increased heart function respectively and increased TnT and TNFα. Erlotinib had no significant effects on function or biomarkers. Sunitinib diminished function, increased TNFα at 0.1 μM, and increased TnT at higher concentrations. Sorafenib dose dependently increased TNFα beginning at 0.1 μM, reducing contractility and flow rate at 0.6 μM. DISCUSSION The ex-vivo assay is a sensitive and predictive model for assessing changes in heart function and biomarkers of toxicity and injury. This assay demonstrates the potential for sunitinib and sorafenib to cause cardiac toxicity in humans. Also, TNFα appears to be a biomarker in the heart prior to injury. Due to its versatility, the isolated heart assay has potential to fill gaps in cardiac safety testing early in drug development.

Toxicokinetics of α-thujone following intravenous and gavage administration of α-thujone or α- and β-thujone mixture in male and female F344/N rats and B6C3F1 mice

Plants containing thujone have widespread use and hence have significant human exposure. α-Thujone caused seizures in rodents following gavage administration. We investigated the toxicokinetics of α-thujone in male and female F344/N rats and B6C3F1 mice following intravenous and gavage administration of α-thujone or a mixture of α- and β-thujone (which will be referred to as α,β-thujone). Absorption of α-thujone following gavage administration was rapid without any dose-, species-, sex- or test article-related effect. Absolute bioavailability of α-thujone following administration of α-thujone or α,β-thujone was generally higher in rats than in mice. In rats, females had higher bioavailability than males following administration of either test article although a sex difference was not observed in mice. Cmax and AUC∞ increased greater than proportional to the dose in female rats following administration of α-thujone and in male and female mice following administration of α,β-thujone suggesting possible saturation of elimination kinetics with increasing dose. Dose-adjusted AUC∞ for male and female rats was 5- to 15-fold and 3- to 24-fold higher than mice counterparts following administration of α-thujone and α,β-thujone, respectively (p-value<0.0001 for all comparisons). Following both intravenous and gavage administration, α-thujone was distributed to the brains of rats and mice with females, in general, having higher brain:plasma ratios than males. These data are in support of the observed toxicity of α-thujone and α,β-thujone where females were more sensitive than males of both species to α-thujone-induced neurotoxicity. In general there was no difference in toxicokinetics between test articles when normalized to α-thujone concentration.

Human Safety and Pharmacokinetic Study of Intramuscular Midazolam Administered by Autoinjector

Midazolam in an autoinjector was evaluated in an open‐label dose escalation study involving 39 healthy participants. Safety and pharmacokinetic parameters were determined for doses ranging from 5 to 30 mg. No serious adverse events were noted during the study. Two participants (30 mg) experienced changes in their electrocardiogram (trigeminy and prolongation of QRS complex) that met the criteria for dose‐limiting adverse events. No significant respiratory depression was noted during the study. The midazolam doses studied exhibited a median tmax of 0.5 hours with a geometric mean terminal elimination half‐life value of 4.1 hours (range, 2.9–4.5 hours). The extent of systemic exposure, assessed by area under the curve (AUC) and maximum concentration (Cmax), tended to increase proportionally with increasing doses from 5 to 30 mg; however, for the male 30‐mg group, there was evidence of a larger than proportional increase in AUC.

Toxicokinetics of Isoeugenol in F344 rats and B6C3F1 mice

Abstract 1. Isoeugenol (IEG) has been tested for toxicity and carcinogenicity due to high potential for human exposure and the structural resemblance to known carcinogenic allylbenzenes. In order to support the interpretation of toxicity and carcinogenecity study outcomes, a toxicokinetic study was performed in which both sexes of F344 rats and B6C3F1 mice were given IEG as a single intravenous (IV) or gavage administration. 2. Following IV administration, IEG was rapidly eliminated from systemic circulation in both species and sexes. Gavage administration revealed a rapid absorption of IEG with tmax values ≤20 min for both species and sexes. In rats, AUC increased in a greater than dose-proportional manner and Clapp values decreased with increasing dose in both sexes suggesting saturation of IEG metabolism. On the other hand, Clapp values in male mice increased with increasing dose suggesting induction of IEG metabolism although this was not evident in the females. 3. Absolute bioavailability was greater in female rats (19%) than male rats (10%) (p < 0.0001), but was not different between the sexes for mice (28% males; 31% females) (p = 0.2437). The collective toxicokinetic data supported that low bioavailability following administration of IEG was the result of extensive first-pass metabolism.

Toxicokinetics of methyleugenol in F344 rats and B6C3F1 mice

1. Methyleugenol (MEG) has been used as a flavouring agent in food, as a fragrance in cosmetic products, and as an insect attractant. MEG was carcinogenic in both rats and mice following gavage administration. In this study we investigated plasma toxicokinetics of MEG in F344 rats and B6C3F1 mice of both sexes following single gavage (37, 75, or 150 mg/kg) and intravenous (IV) (37 mg/kg) administration. 2. Following IV administration, MEG was rapidly distributed and cleared from the systemic circulation in both species and sexes. Absorption of MEG was rapid following gavage administration with secondary peaks in the plasma MEG concentration-versus-time profiles. Cmax and AUCT increased and the clearance decreased greater than proportional to the dose in rats and mice of both sexes. In general, rats had higher internal exposure to MEG than mice. 3. The results for AUCT and clearance suggest that perhaps the metabolism of MEG is saturated at higher doses tested in this study. Absolute bioavailability following gavage administration of 37 mg/kg was low in both rats (~4%) and mice (7–9%) of both sexes indicating extensive first-pass metabolism. There was no sex difference in plasma toxicokinetics of MEG following gavage administration both in rats and mice.

Efficacy and Pharmacokinetic/Pharmacodynamic Study of 1,1′-Methylenebis{4-[(hydroxyimino)methyl] pyridinium} Dimethanesulfonate in Guinea Pigs and Rhesus Macaques Exposed to Cyclosarin

Male Hartley guinea pigs and male rhesus macaques were used to determine an efficacious dose of 1,1′-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate (MMB4 DMS) that would result in 80% survival, 24 hours following a single exposure to cyclosarin (GF). The pharmacokinetic/pharmacodynamic relationship between acetylcholinesterase activity and MMB4 plasma concentrations relative to survival was evaluated. Guinea pigs and non-human primates (NHPs) were concurrently administered MMB4 DMS (guinea pigs: 0, 10, 30, or 40 mg/kg, intramuscular [IM] and NHPs: 0.1, 1, 5, 10, or 20 mg/kg, IM), atropine, and diazepam following a 3 × median lethal dose (LD50) GF challenge. Clinical observations were evaluated using a quality-of-life (QOL) scoring system. All GF-exposed animals exhibited typical signs of nerve agent poisoning immediately following challenge. In guinea pigs, 24-hour survival was 0%, 50%, 90%, and 90% for 0, 10, 30, and 40 mg/kg MMB4 DMS groups, respectively. In addition, nearly all animals surviving to 24 hours were clinically normal, with many in the 30 and 40 mg/kg MMB4 DMS dose group observed as normal by 4 hours post-challenge. In NHPs, survival was 100% for all treatment groups, with all animals noted as clinically normal by 48 hours. Following treatment with atropine/MMB4 DMS/diazepam, NHPs exhibited dose- and temporal-related decreases in incidence and duration of the clinical signs of toxicity. The QOL scores improved with increasing MMB4 DMS dose in both species. The estimated ED80s were 25.5 mg/kg MMB4 DMS (human equivalent dose [HED] of 5.5 mg/kg) and ≤0.1 mg/kg (HED of 0.03 mg/kg) in guinea pigs and NHPs, respectively.

Comparative Toxicokinetics of MMB4 DMS in Rats, Rabbits, Dogs, and Monkeys Following Single and Repeated Intramuscular Administration:

1,1′-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Time courses of MMB4 concentrations in plasma were characterized following 7-day repeated intramuscular (IM) administrations of MMB4 DMS to male and female Sprague-Dawley rats, New Zealand White rabbits, beagle dogs (single dose only), and rhesus monkeys at drug dose levels used in earlier toxicology studies. In general, there were no significant differences in MMB4 toxicokinetic (TK) parameters between males and females for all the species tested in these studies. After a single IM administration to rats, rabbits, dogs, and monkeys, MMB4 DMS was rapidly absorbed, resulting in average T max values ranging from 5 to 30 minutes. Although C max values did not increase dose proportionally, the overall exposure to MMB4 in these preclinical species, as indicated by area under the curve (AUC) extrapolated to the infinity (AUC∞) values, increased in an approximately dose-proportional manner. The MMB4 DMS was extensively absorbed into the systemic circulation after IM administration as demonstrated by greater than 80% absolute bioavailability values for rats, rabbits, and dogs. Repeated administrations of MMB4 DMS for 7 days did not overtly alter TK parameters for MMB4 in rats, rabbits, and monkeys (150 and 300 mg/kg/d dose groups only). However, C max and AUC values decreased in monkeys given 450 and 600 mg/kg IM doses of MMB4 DMS following repeated administrations for 7 days. Based on the TK results obtained from the current study and published investigations, it was found that the apparent volume of distribution and clearance values were similar among various preclinical species, except for the rat.

Pharmacokinetics of MMB4 DMS in Rats, Rabbits, and Dogs Following a Single IV Administration

Organophosphorus (OP) nerve agents pose tremendous threats to both military and civilian populations. The substance 1,1′-methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) is being developed as a replacement for the currently fielded 2-pyridine aldoxime, or pralidoxime (2-PAM) as a treatment for OP nerve agent–induced toxicity. The present study characterized pharmacokinetic (PK) profiles of MMB4 in male and female Sprague-Dawley rats, New Zealand White rabbits, and beagle dogs given a single intravenous (IV) administration of MMB4 dimethanesulfonate (DMS) at 55, 25, and 15 mg/kg dose, respectively. The plasma MMB4 concentration versus time profiles were biphasic for all species tested and fit a 2-compartment model with first-order elimination. There were no overt sex-related differences in the calculated PK parameters. For the rat, rabbit, and dog, the average systemic exposure parameters predicted Cmax (µg/mL) and AUC∞ (µg·h/mL) were 273 and 71.0, 115 and 48.1, and 87.4 and 39.6; the average volume of distribution (mL/kg) values to the central and peripheral compartments were 207 and 143, 242 and 172, and 198 and 213; and the average elimination half-life (hour) and clearance (mL/h/kg) values were 0.18 and 778, 0.29 and 577, and 0.32 and 430, respectively, when the PK parameters for males and females were combined. The current study revealed a similarity in the volume of distribution to the central compartment for MMB4 among the 3 species tested while demonstrating species-related differences in the elimination half-life and clearance of MMB4.

In vivo acetylcholinesterase reactivation in male guinea pigs and rhesus macaques following cyclosarin exposure and treatment with 1,1'-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate.

Acetylcholinesterase (AChE) reactivation studies were conducted in guinea pigs (GPs) and nonhuman primates (NHPs) to determine the 1,1′-methylenebis{4-[(hydroxyimino)methyl] pyridinium} dimethanesulfonate (MMB4 DMS) dose that reactivated at least 20% of blood AChE within 15 minutes following cyclosarin (GF) dosing (used as the criterion for efficacy). Male GPs and male rhesus macaques (NHPs) were pretreated with atropine 15 minutes prior to GF administration (1 × median lethal dose [LD50]) and MMB4 DMS 15 minutes following GF administration. The GP survival was 5 of 8, 8 of 8, 8 of 8, and 6 of 8 for the 0.75, 3.0, 6.0, or 12.0 mg/kg MMB4 DMS treatment groups, respectively. In NHPs, survival was 6 of 6 at 0.5, 1.2, 3.0, or 9.3 mg/kg MMB4 DMS, respectively, 24 hours post-challenge, with the majority of animals noted as clinically normal by 24 hours. Pharmacokinetic/pharmacodynamic modeling revealed that 1.8 mg/kg in GPs or 0.013 mg/kg in NHPs would result in an average 20% reactivation; human equivalent doses were calculated as 0.39 mg/kg (based on GP data) and 0.004 mg/kg (based on NHP data). The model suggested that MMB4 plasma concentrations of 1000 ng/mL and AChE reactivation of 80% would be most effective. Although a 0.5 mg/kg MMB4 DMS dose in NHPs resulted in 100% survival and an average of 78% AChE reactivation, adverse effects associated with GF administration were still observed 24 hours post-challenge (tremors, mydriasis, and weakness were observed in 3 of 6 animals). In comparison, 6 of 6 animals treated with 1.2 mg/kg MMB4 DMS were observed as clinically normal 24 hours post-challenge.

Evaluations of In Vitro Metabolism, Drug–Drug Interactions Mediated by Reversible and Time-Dependent Inhibition of CYPs, and Plasma Protein Binding of MMB4 DMS

1,1′-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Drug metabolism and plasma protein binding for MMB4 DMS were examined using various techniques and a wide range of species. When 14C-MMB4 DMS was incubated in liver microsomes, 4-pyridine aldoxime (4-PA) and an additional metabolite were detected in all species tested. Identity of the additional metabolite was postulated to be isonicotinic acid (INA) based on liquid chromatography with a tandem mass spectrometry analysis, which was confirmed by comparison with authentic INA. Formation of INA was dependent on species, with the highest level found in monkey liver microsomes. The MMB4 DMS exhibited reversible inhibition in a concentration-dependent manner toward cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human liver microsomes showing the highest inhibition for CYP2D6. Human recombinant CYPs were used to evaluate inhibitory curves more adequately and determine detailed kinetic constants for reversible inhibition and potential time-dependent inhibition (TDI). The MMB4 DMS exhibited reversible inhibition toward human-recombinant CYP2D6 with an inhibition constant (K i) value of 66.6 µmol/L. Based on the k inact/K I values, MMB4 DMS was found to exhibit the most potent TDI toward CYP2D6. The MMB4 DMS at 5 different concentrations was incubated in plasma for 5 hours using an equilibrium dialysis device. For all species tested, there were no concentration-dependent changes in plasma protein binding, ranging from 10% to 17%. These results suggest that MMB4 was not extensively bound to plasma protein, and there were no overt species-related differences in the extent of MMB4 bound to plasma protein.