Pharmacokinetics, mass balance, tissue distribution, metabolism, and excretion of [14C]aficamten following single oral dose administration to rats.

The pharmacokinetics, metabolism, excretion, mass balance, and tissue distribution of [14C]aficamten were evaluated following oral administration of an 8 mg/kg dose in Sprague Dawley rats and in a quantitative whole-body autoradiography study in Long Evans rats.[14C]Aficamten accounted for ∼80% and a hydroxylated metabolite (M1) accounted for ∼12% of total radioactivity in plasma over 48-h (AUC0-48). Plasma tmax was 4-h and the t1/2 of total plasma radioactivity was 5.8-h.Tissues showing highest Cmax exposures were myocardium and semitendinosus muscle.Most [14C]aficamten-derived radioactivity was excreted within 48-h post-administration. Mean cumulative recovery in urine and faeces over 168-h was 8.3% and 90.7%, respectively.In urine and bile, unchanged aficamten was detected at <0.1 and <0.2% of dose, respectively; however, based on total radioactivity excreted in urine (8.0%) and bile (51.7%), approximately 60% of dose was absorbed.[14C]Aficamten was metabolised by hydroxylation with subsequent glucuronidation where the most abundant metabolite recovered in bile was M5 (35.2%), the oxygen-linked glucuronide of hydroxylated aficamten (M1a). The major metabolite detected in faeces was a 1,2,4-oxadiazole moiety ring-cleaved metabolite (M18, 35.3%), shown to be formed from the metabolism of M5 in incubations with rat intestinal contents solution.

Bioaccumulation and in vivo fate of toxic benzylalkyldimethylammonium chloride in rats via the radiotracer analysis.

Benzylalkyldimethylammonium chloride (BAC), a quaternary ammonium compound (QAC), is utilized in industrial and biomedical applications for antimicrobial purposes. Since the coronavirus disease (COVID-19) outbreak, various types of BAC-containing household chemicals have been produced. BACs have several adverse effects; however, their biological uptake, translocation, and excretion in animal models (essential for better understanding in vivo behavior and toxicological impact) remain unclear. In this study, we performed the first biodistribution and whole-body imaging studies of BAC in male Sprague Dawley rats, using two different administration routes. Quantitative whole-body autoradiography (QWBA) data obtained for intranasal 14C-labeled BAC ([14C]C12-BAC) exposure showed substantial uptake values for the respiratory organs (e.g. 346 ng g-1 of lung at 3 h post administration) and the radiotracer was transported to other internal organs. The amount of radiotracer in the heart, adrenal gland, and pancreas were 198, 1410, and 186 ng g-1 tissue respectively at 168 h following exposure. Autoradiograms obtained after intravenous injection also showed high accumulation and slow excretion in these organs. The cumulative excretion analysis revealed that approximately 6.4% of the administered radioactivity remained in rats after a week. The results indicated that continuous inhalation exposure to BAC leads to potential toxic effects in extrapulmonary organs and the respiratory tract. Thus, the radiolabeling method utilized may help assess various synthetic QACs in living subjects.

Assessment of the Pharmacokinetics, Disposition, and Duration of Action of the Tumour-Targeting Peptide CEND-1.

CEND-1 (iRGD) is a bifunctional cyclic peptide that can modulate the solid tumour microenvironment, enhancing the delivery and therapeutic index of co-administered anti-cancer agents. This study explored CEND-1's pharmacokinetic (PK) properties pre-clinically and clinically, and assessed CEND-1 distribution, tumour selectivity and duration of action in pre-clinical tumour models. Its PK properties were assessed after intravenous infusion of CEND-1 at various doses in animals (mice, rats, dogs and monkeys) and patients with metastatic pancreatic cancer. To assess tissue disposition, [3H]-CEND-1 radioligand was administered intravenously to mice bearing orthotopic 4T1 mammary carcinoma, followed by tissue measurement using quantitative whole-body autoradiography or quantitative radioactivity analysis. The duration of the tumour-penetrating effect of CEND-1 was evaluated by assessing tumour accumulation of Evans blue and gadolinium-based contrast agents in hepatocellular carcinoma (HCC) mouse models. The plasma half-life was approximately 25 min in mice and 2 h in patients following intravenous administration of CEND-1. [3H]-CEND-1 localised to the tumour and several healthy tissues shortly after administration but was cleared from most healthy tissues by 3 h. Despite the rapid systemic clearance, tumours retained significant [3H]-CEND-1 several hours post-administration. In mice with HCC, the tumour penetration activity remained elevated for at least 24 h after the injection of a single dose of CEND-1. These results indicate a favourable in vivo PK profile of CEND-1 and a specific and sustained tumour homing and tumour penetrability. Taken together, these data suggest that even single injections of CEND-1 may elicit long-lasting tumour PK improvements for co-administered anti-cancer agents.

Biodistribution and respiratory toxicity of chloromethylisothiazolinone/methylisothiazolinone following intranasal and intratracheal administration.

A variety of isothiazolinone-containing small molecules have been registered and used as chemical additives in many household products. However, their biodistribution and potential harmful effects on human health, especially respiratory effects, were not yet identified in sufficient detail. The purpose of this study was to investigate whether a biocide comprising a mixture of chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT) could reach the lungs and induce lung injury when exposure occurs by two administration routes involving the respiratory tract: intratracheal and intranasal instillation. To investigate the biodistribution of CMIT/MIT, we quantified the uptake of 14C-labeled CMIT/MIT in experimental animals for up to seven days after intratracheal and intranasal instillation. In the toxicity study, lung injury was assessed in mice using total inflammatory cell count in bronchoalveolar lavage fluid (BALF) and lung histopathology. The results of the biodistribution study indicated that CMIT/MIT were rapidly distributed throughout the respiratory tract. Using quantitative whole-body autoradiogram analysis, we confirmed that following intranasal exposure, CMIT/MIT reached the lungs via the respiratory tract (nose-trachea-lung). After 5 min post intratracheal and intranasal instillation, the amount of radiotracer ([14C]CMIT/MIT) in the lungs was 2720 ng g-1 and 752 ng g-1 tissue, respectively, and lung damage was observed. A higher amount of the radiotracer resulted in higher toxicity. Both intratracheal and intranasal instillation of CMIT/MIT increased inflammatory cell counts in the BALF and induced injuries in the alveoli. The frequency and the severity scores of injuries caused by intratracheal instillation were approximately-four to five times higher than those induced by intranasal instillation. Therefore, we concluded that CMIT/MIT could reach the lungs following nasal and intratracheal exposure and cause lung injuries, and the extent of injury was dependent on the exposure dose.

Siponimod (BAF312) penetrates, distributes, and acts in the central nervous system: Preclinical insights.

BACKGROUND: Siponimod (BAF312), a selective S1P1/S1P5 agonist, reduces disability progression in secondary progressive MS. Recent observations suggest it could act via S1P1/S1P5-dependent anti-inflammatory and pro-myelination effects on CNS-resident cells. OBJECTIVE: Generate preclinical evidence confirming siponimod's CNS penetration and activity. METHODS: Siponimod's CNS penetration and distribution was explored in rodents and non-human primates (NHPs) using: Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS), quantitative whole-body autoradiography (QWBA) using 14C-radiolabeled siponimod or non-invasive single-photon emission CT (SPECT) with a validated 123I-radiolabeled siponimod analog. Functional CNS activity was investigated by S1P1 receptor quantification in brain homogenates. RESULTS: In mice/rats, siponimod treatments achieved dose-dependent efficacy and dose-proportional increase in drug blood levels, with mean brain/blood drug-exposure ratio (Brain/BloodDER) of 6-7. Efficacy in rat brain tissues was revealed by a dose-dependent reduction in brain S1P1 levels. QWBA distribution analysis in rats indicated that [14C]siponimod related radioactivity could readily penetrate CNS, with particularly high uptakes in white matter of cerebellum, corpus callosum, and medulla oblongata versus lower exposures in other areas such as olfactory bulb. SPECT monitoring in NHPs revealed CNS distribution with a brain/bloodDER of ∼6, as in rodents. CONCLUSION: Findings demonstrate siponimod's CNS penetration and distribution across species, with high translational potential to human.

Simple methodology for ensuring the precision of measuring radioactivity at low concentrations in very small tissues using quantitative whole-body autoradiography.

Quantitative whole-body autoradiography (QWBA) is largely used to evaluate tissue distribution of small molecule drugs. In QWBA, radioactivity is measured as the intensity obtained from the autoradiogram. It is known that lower intensity per a region of interest (ROI) or smaller size of ROI increases the variability of intensity. In fact, as some tissues are very small (e.g., the choroidea), ensuring reliability on the intensity for measuring radioactivity in these tissues is difficult in case of under- or over-estimation of radioactivity concentration owing to their variation of low radioactivity intensity of ROI. We thus analyzed the relationships between the size, intensity, and precision of ROI to determine the statistically significant lower limit of quantification (LLOQ) in very small tissues. To investigate the difference in correlation between the radiation source (commercial planar radiation standard [com-ST] and self-made radiation standard [self-ST] consisting of radioactive compounds and matrices), apparatus, or setting environment of the apparatus, correlation analysis was conducted under various conditions. Our results revealed that LLOQ can be calculated by simply using the correlation equation because a common relationship was observed between self-ST, which is used in QWBA, and com-ST. This methodology was thus considered valuable for ensuring LLOQ determination in QWBA.

Quantitative biodistribution of biotherapeutics at whole body, organ and cellular levels by autoradiography.

AIM: Tools for mapping and quantifying monoclonal antibody (mAb) and peptide biotherapeutics distribumtion were evaluated by comparing data from three independent methods conducted at the whole body, organ or tissue, and cellular levels. MATERIALS & METHODS: [3H]-mAb1 and [3H]-peptide A were administered intravenously to rats followed by quantitative whole-body autoradiography, kidney macro-autoradiography and micro-autoradiography. RESULTS: [3H]-mAb1 and [3H]-peptide A concentrations were measured in anatomical regions ranging from whole body to whole organ to sub-organ level, such as the kidney glomerulus, with increasing resolution. The tissue/blood [3H]-mAb1 concentrations in selected kidney microenvironments were comparable among the three quantitative methods. CONCLUSION: Quantitative whole-body autoradiography, tissue macro-autoradiography and micro-autoradiography all provide useful tools for quantifying the concentrations of biotherapeutics at different anatomical levels in tissues, facilitating better predictions of efficacy and toxicity.

Tissue Distribution and Elimination of Isavuconazole following Single and Repeat Oral-Dose Administration of Isavuconazonium Sulfate to Rats.

Quantitative whole-body autoradiography was used to assess the distribution and tissue penetration of isavuconazole in rats following single and repeated oral-dose administration of radiolabeled isavuconazonium sulfate, the prodrug of isavuconazole. Following a single-dose administration of radiolabeled isavuconazonium sulfate (labeled on the active moiety), radioactivity was detectable within 1 h postdose in 56 of 65 tissue/fluid specimens. The highest maximum concentrations (Cmax) were observed in bile and liver (66.6 and 24.7 µg eq/g, respectively). The lowest Cmax values were in bone and eye lens (0.070 and 0.077 µg eq/g, respectively). By 144 h postdose, radioactivity was undetectable in all tissues/fluids except liver (undetectable at 336 h) and adrenal gland tissues (undetectable at 672 h). Following daily administration for up to 21 days, 1-h-postdose Cmax values were the highest on or before day 14 in all except seven tissues/fluids, of which only rectum mucosa and small intestine mucosa had Cmax values >25% higher than all other 1-h-postdose values. For 24-h-postdose Cmax values, only large intestine, large intestine mucosa, and urine had the highest Cmax values at day 21. The penetration of single oral doses of unlabeled isavuconazole (25 mg/kg of body weight isavuconazonium sulfate) and voriconazole (50 mg/kg) into rat brain (assessed using liquid chromatography-tandem mass spectrometry) was also compared. Brain concentration/plasma concentration ratios reached approximately 1.8:1 and 2:1, respectively. These data suggest that isavuconazole penetrates most tissues rapidly, reaches a steady state in most or all tissues/fluids within 14 days, does not accumulate in tissues/fluids over time, and achieves potentially efficacious concentrations in the brain.

Metabolic disposition of the anti-cancer agent [(14)C]laromustine in male rats.

1. Laromustine (VNP40101M, also known as Cloretazine) is a novel sulfonylhydrazine alkylating (anticancer) agent. This article describes the use of quantitative whole-body autoradiography (QWBA) and mass balance to study the tissue distribution, the excretion mass balance and pharmacokinetics after intravenous administration of [(14)C]VNP40101M to rats. A single 10 mg/kg IV bolus dose of [(14)C]VNP40101M was given to rats. 2. The recovery of radioactivity from the Group 1 animals over a 7-day period was an average of 92.1% of the administered dose, which was accounted for in the excreta and carcass. Most of the radioactivity was eliminated within 48 h via urine (48%), with less excreted in feces (5%) and expired air accounted for (11%). The plasma half-life of [(14)C]laromustine was approximately 62 min and the peak plasma concentration (Cmax) averaged 8.3 µg/mL. 3. The QWBA study indicated that the drug-derived radioactivity was widely distributed to tissues through 7 days post-dose after a single 10 mg/kg IV bolus dose of [(14)C]VNP40101M to male pigmented Long-Evans rats. The maximum concentrations were observed at 0.5 or 1 h post-dose for majority tissues (28 of 42). The highest concentrations of radioactivity were found in the small intestine contents at 0.5 h (112.137 µg equiv/g), urinary bladder contents at 3 h (89.636 µg equiv/g) and probably reflect excretion of drug and metabolites. The highest concentrations in specific organs were found in the renal cortex at 1 h (28.582 µg equiv/g), small intestine at 3 h (16.946 µg equiv/g), Harderian gland at 3 h (12.332 µg equiv/g) and pancreas at 3 h (12.635 µg equiv/g). Concentrations in the cerebrum (1.978 µg equiv/g), cerebellum (2.109 µg equiv/g), medulla (1.797 µg equiv/g) and spinal cord (1.510 µg equiv/g) were maximal at 0.5 h post-dose and persisted for 7 days. 4. The predicted total body and target organ exposures for humans given a single 100 µCi IV dose of [(14)C]VNP40101M were well within the medical guidelines for maximum radioactivity exposures in human subjects.

A peripherally restricted P2Y12 receptor antagonist altered rat tumor incidences with no human relevance: Mode of action consistent with dopamine agonism.

BACKGROUND: Ticagrelor is an orally available, direct acting and reversible P2Y12 receptor antagonist approved for treatment of acute coronary syndrome. The objectives of these studies were to (1) evaluate the Ticagrelor 2-year rat carcinogenicity bioassay data; (2) investigate potential mode of action (MOA) and (3) interpret human relevance. METHODS: The following studies were done (1) rat two-year carcinogenicity study in male and female rats, (2) in vitro and in vivo genotoxicity assays, (3) quantitative whole body autoradiography (QWBA; male and female rats), (4) in vitro pharmacological profiling for more than 300 assays, and (5) in vivo ovariectomized rat assay. RESULTS: The carcinogenicity study indicated Ticagrelor increased uterine tumor incidence while decreasing mammary and pituitary tumors/hyperplasia incidences in only high dose female rats. However, this altered tumor incidences were not P2Y12 target related since marketed non-reversible P2Y12 receptor antagonists were not associated with alter tumor incidences. MOA studies determined Ticagrelor exposure in the anterior pituitary and Ticagrelor was (1) non-genotoxic, (2) peripherally-restricted, (3) a dopamine transport (DAT) inhibitor with an IC50 lower than systemic free exposure in the rat carcinogenic study and more than a log higher than the free systemic exposure seen in clinical trials and (4) an inhibitor of estradiol-induced prolactin secretion. DISCUSSION: Similar to Ticagrelor, centrally active dopamine agonists induce the same altered tumor incidence patterns that according to literature do not translate into the clinical setting, with a MOA involving decreased prolactin secretion. The Ticagrelor MOA data and literature suggest that altered dopamine levels in the hypophyseal part of the hypothalamus-hypophyseal axis (by Ticagrelor) will result in similar altered tumor incidences in rat that do not translate into the clinical setting, based on qualitative species differences. In conclusion Ticagrelor increased uterine tumors in the rat carcinogenesis study by a MOA consistent with reduced dopamine inhibition of prolactin, which is not a patient safety risk.