Single-dose Acute Toxicity Studies Research Articles

Acute toxicity “six-pack” studies supporting approved new drug applications in the U.S., 2015–2018

The acute toxicity “six-pack” is a battery of animal tests used to evaluate acute systemic toxicity by three routes of exposure, skin and eye irritation/corrosion, and skin sensitization. A perception exists that these tests are not required for pharmaceuticals. For the four years from 2015 through 2018, we tallied the number of corresponding tests submitted by sponsors in approved, original new drug applications, as reported by the U.S. Food and Drug Administration (FDA) in publicly available reviews. In 125 reviews, we identified 228 single dose acute toxicity studies, 62 in vivo local tolerance studies, and 32 in vivo skin sensitization studies, as well as 37 in vitro or ex vivo local tolerance studies. A total of 4798 animals were used in these studies; however, FDA's reporting was inconsistent, and we estimate the actual number of animals used to be 8998. For the evaluation of single dose acute toxicity, we accessed two guidance documents with conflicting recommendations regarding routes of administration and number of species to be used. For the evaluation of local tolerance and skin sensitization, most studies examined were conducted by routes other than that intended for human administration. Non-animal methods used to evaluate skin sensitization were not reported.

Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the brain

IntroductionSince the approval of three 18F labeled β-amyloid-targeting PET imaging agents, Amyvid (florbetapir f18, AV-45), Neuraceq (florbetaben f18, AV-1) and Vizamyl (flutemetamol f18, F-PIB), they have increasingly been employed to assist differential diagnosis of Alzheimer's disease in patients with dementia. Also, they are frequently used in selecting patients participating drug trials aiming to reduce β-amyloid (Aβ) plaques in the brain. The first approved tracer in this class was [18F]AV-45, which is metabolized rapidly in blood and some of its N-demethylated metabolites cross the blood brain barrier and resulted in lowering the image contrast. To improve metabolic stability of [18F]AV-45, we hypothesized that substituting N-CH3 with N-CD3 at the metabolically labile position, creating [18F]D3FSP, may reduce in vivo N-demethylation. We report the preclinical evaluation of [18F]D3FSP as an Aβ imaging agent. MethodsPreclinical pharmacology of [18F]D3FSP was evaluated using in vitro autoradiography and competitive binding assay. Biodistribution of [18F]D3FSP was evaluated in wild-type CD-1 mice. In vivo metabolism in mice and in vitro microsomal metabolism were analyzed by HPLC. Single dose acute toxicity of D3FSP was also performed in rats. Results[18F]D3FSP showed high binding affinity to β-amyloid plaques (Ki = 3.44 ± 1.22 nM, a value similar as AV-45 (Ki = 4.02 ± 0.22 nM)), and displayed excellent β-amyloid binding in AD brain sections consistent with known Aβ regional distribution. After an iv injection it exhibited good initial brain uptake and fast washout in wild-type CD-1 mice. In vitro microsomal metabolism and in vivo metabolism in mice did not result in any significant differences between [18F]D3FSP and [18F]AV-45. No treatment-related mortality or any adverse effects were observed in single dose acute toxicity studies administered at hundred-folds of maximum human dose. ConclusionA new small molecule, [18F]D3FSP, was prepared and tested as an alternative to [18F]AV-45 to reduce N-demethylation in vivo. This strategy did not lead to better in vivo stability. However, [18F]D3FSP displayed very similar Aβ targeting property comparable to [18F]AV-45. Preclinical studies suggest that [18F]D3FSP is useful as a β-amyloid-targeting PET imaging agent.

EXTRACTION, ISOLATION AND STRUCTURAL ELUCIDATION OF FLAVONOID FROM CHROZOPHORA PLICATA LEAVES AND EVALUATION OF ITS ANTIOXIDATIVE POTENTIALS

Objective: This investigation involves the extraction, isolation, and characterization of flavonoid from a Euphorbiaceae family plant Chrozophoraplicata followed by evaluation of its antioxidant principles.Methods: The dried leaves were subjected to sequential soxhlation with polar and nonpolar solvents. Methanolic extract reveals the presence of largeamount of flavonoids. Methanolic extract was subjected to isolation using column chromatographic analysis with solvents such as petroleum ether,chloroform, hexane, ethyl acetate, methanol, and water. Further, the isolated compound was subjected to thin layer chromatography technique andspectral analysis such as infrared, 1HNMR, 13CNMR, mass spectroscopy, and high performance thin layer chromatography (HPTLC) finger printingtechniques. The compound was evaluated for in vitro antioxidant studies using 2,2-diphenyl-1-picrylhydrazyl (DPPH), NO assay, reducing power assay,H2O2 scavenging assay, superoxide anion scavenging assay and β-Carotene linoleate system and in vivo antioxidative studies using carbon tetrachloride(CCl4), and acetaminophen intoxicated rats.Results: The compound was isolated in methanol:water in the ratio of 80:20 using column chromatographic technique. On the basis of phytochemical,chromatographic, and spectral analysis, the isolated compound was identified as kaempferol and finally with HPTLC finger printing technique it wasfound that the Rf value of the isolated compound was found to be 0.58 which is nearly similar to the Rf value of standard kaempferol (0.55). Hence,the isolated compound was confirmed as kaempferol and is structurally elucidated as 3,5,7-trihydroxy-2-(4-hydroxyphenyl)chromen-4-one. Thiscompound was isolated for the first time from the C. plicata leaves. The in vitro antioxidant assay of isolated flavonoid has shown a dose-dependentincrease in free radical scavenging activity using DPPH, no assay, reducing power assay, H2O2 scavenging assay, superoxide anion scavenging assay, andβ-carotene linoleate system. Further, the methanolic extract of C. plicata (MECP) leaves was subjected to single dose acute toxicity study for 14 days infemale rats on the basis of OECD guidelines 423 and the therapeutically selected doses were 200 mg/kg and 400 mg/kg. In vivo antioxidant studies inCCl4 and acetaminophen intoxicated rats indicated that the MECP leaves have significantly decreased lipid peroxidation in a dose-dependent mannerand increased the levels of catalase, superoxide dismutase, and glutathione.Conclusions: By the above results, it was concluded that the isolated compound from C. plicata leaves was confirmed as kaempferol and it possessessignificant antioxidative potentials.Keywords: Chrozophora plicata leaves, Flavonoids, Extraction, Isolation, Characterization, Methanolic extract, Antioxidant activity, Carbontetrachloride, Acetaminophen.

Phase-0/phase-I study of dye-loaded lipid nanoparticles for near-infrared fluorescence imaging in healthy dogs

Near-infrared (NIR) fluorescence imaging using FDA-approved indocyanine green (ICG) has been the subject of numerous studies during the past few years. It could constitute a potentially exciting new paradigm shift in veterinary oncology, especially to develop in vivo fluorescence imaging diagnostics and surgery guidance methods. The objective of this study was to evaluate the pharmacologic and toxicological characteristics in healthy beagle dogs of LipImage™ 815, a formulation made of NIR-dye-loaded lipid nanoparticles. The initial dosage for the evaluation of biodistribution was extrapolated from data in mice and then adapted to define the more adapted dose (MAD) according to the fluorescence results obtained in 5 dogs using a Fluobeam® 800 imaging device (phase 0 study). A single dose acute toxicity study was then performed (3 dogs, phase I study). Before the systemic administration of LipImage™ 815, the dogs presented a very mild residual fluorescence, particularly in the liver and kidneys. After injection, the plasma fluorescence continuously decreased, and the signal was relatively homogeneously distributed throughout the different organs, though more pronounced in the liver and to a lesser extent in the steroid-rich organs (adrenal, ovaries), intestines, lymph nodes and kidneys. A MAD of 2.0μg/kg was found. No evidence of acute or delayed general, hepatic, renal or hematologic toxicity was observed at 1-fold, 5-fold or 10-fold MAD. The results of this phase-0/phase-I study showed that an optimal dosage of LipImage™ 815 of 2.0μg/kg allowed the achievement of a fluorescence signal suitable for surgery guidance application without any acute side effects.

AmbiOnp: Solid Lipid Nanoparticles of Amphotericin B for Oral Administration

Amphotericin B is the most effective gold standard drug against various fungal infections, especially in second line treatment of leishmaniasis. However, its usefulness is limited due to severe nephrotoxicity, which may lead to kidney failure. Due to its poor oral bioavailability, it is often administered parenterally to patients suffering from systemic fungal infection or visceral leishmaniasis (kala azar). In this investigation, solid lipid nanoparticles were formulated for oral administration of Amphotericin B. For this purpose, novel microemulsion based nanoprecipitation technique was employed. The influence of process variables such as sonication and dialysis time was studied. The optimized formulation was characterized for parameters such as particle size, polydispersity index, zeta potential, drug content and entrapment efficiency. The pH stability of the developed Amphotericin B solid lipid nanoparticles (AmbiOnp) at pH 1.2, 4, 6.8 values demonstrated enhanced protection of entrapped Amphotericin B. Further, single dose acute toxicity study established the safety of AmbiOnp for oral administration. In vivo pharmacokinetic studies revealed increase in % relative bioavailability of AmbiOnp in comparison to the plain drug. Additionally, the t1/2 of encapsulated Amphotericin B was significantly greater than that of plain drug, indicating the controlled release of Amphotericin B from AmbiOnp. Overall, the developed formulation; AmbiOnp was found to be successful in oral delivery of Amphotericin B.

Abstract 3613: Dual inhibitors against receptor tyrosine kinases c-Met and VEGFR-2 as efficacious antitumor agents in mouse xenograft tumor models

Abstract Vascular endothelial growth factor (VEGF) and the receptor VEGFR-2 (KDR) are the major components of angiogenesis signaling pathway and proven targets for the treatment of several solid tumors. However, recent studies have revealed that agents targeting VEGF/VEGFR-2 can promote tumor invasiveness and distant metastasis. These findings suggest that any future antiangiogenic therapies should be combined with mechanisms targeting cancer invasiveness and metastasis. Hepatocyte growth factor (HGF) receptor c-Met (HGFR) is a receptor tyrosine kinase considered critically involved in the control of cancer development, invasion, and metastasis. Studies have suggested that Inhibition of c-Met can not only reduce c-Met dependent tumor growth, but also decrease tumor invasion and metastasis. Thus, it is conceivable that targeting both c-Met and VEGFR-2 simultaneously will inhibit tumor growth and, at the same time, prevent tumor from invasion and metastasis through the following mechanisms: 1) synergy between VEGFR-2 and c-Met inhibition in antiangiogenesis; 2) inhibition of c-Met driven tumor growth; 3) disruption of c-Met mediated tumor invasion and metastasis. Compounds including ASP-08001 and 08126 were identified as small molecule, ATP competitive inhibitors of both c-Met and VEGFR-2 kinases through biochemical assays. In cellular studies, these compounds inhibited potently the activating phosphorylation of cytoplasmic domains of both kinases as well as c-Met and VEGFR-2 driven phenotypes such as cell proliferation, invasion, and angiogenesis. In addition, pharmacodynamic studies revealed that a single oral dose of a compound was able to inhibit the phosphorylation of cytoplasmic domains of both targeted and downstream kinases in tumor xenografts for up to 16 hours. The level of kinase inhibition in tumor tissues was well correlated with the plasma concentration of the compound inversely indicative of a proof of mechanism of actions. The antitumor efficacies of the compounds were demonstrated in xenograft tumor models of several human cancers including NSCLC, breast, gastric, and prostate cancers. Complete tumor growth arrest or even regression induced by oral dosing (50 or 100 mg/kg, q.d.) of the compounds was observed. Furthermore, pharmacokinetic studies suggested a promising drug-like PK profile for these compounds. Finally, single-dose acute toxicity studies of the compounds could not find a lethal dose in mice even though dosing had reached 5000mg/kg, suggesting a therapeutic index of greater than 50 and a promising safety profile. In summary, we reported here the discovery of small molecule, ATP-competitive, c-Met and VEGFR-2 inhibitors such as ASP-08001 and −08126 that have demonstrated strong potency in target inhibition and desired efficacies against a variety of human tumor models suggesting strong therapeutic potentials against human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3613.

Preferential inhibition of xanthine oxidase by 2-amino-6-hydroxy-8-mercaptopurine and 2-amino-6-purine thiol

BackgroundThe anticancer drug, 6-mercaptopurine (6MP) is subjected to metabolic clearance through xanthine oxidase (XOD) mediated hydroxylation, producing 6-thiouric acid (6TUA), which is excreted in urine. This reduces the effective amount of drug available for therapeutic efficacy. Co-administration of allopurinol, a suicide inhibitor of XOD, which blocks the hydroxylation of 6MP inadvertently enhances the 6MP blood level, counters this reduction. However, allopurinol also blocks the hydroxylation of hypoxanthine, xanthine (released from dead cancer cells) leading to their accumulation in the body causing biochemical complications such as xanthine nephropathy. This necessitates the use of a preferential XOD inhibitor that selectively inhibits 6MP transformation, but leaves xanthine metabolism unaffected.ResultsHere, we have characterized two such unique inhibitors namely, 2-amino-6-hydroxy-8-mercaptopurine (AHMP) and 2-amino-6-purinethiol (APT) on the basis of IC50 values, residual activity in bi-substrate simulative reaction and the kinetic parameters like Km, Ki, kcat. The IC50 values of AHMP for xanthine and 6MP as substrate are 17.71 ± 0.29 μM and 0.54 ± 0.01 μM, respectively and the IC50 values of APT for xanthine and 6MP as substrates are 16.38 ± 0.21 μM and 2.57 ± 0.08 μM, respectively. The Ki values of XOD using AHMP as inhibitor with xanthine and 6MP as substrate are 5.78 ± 0.48 μM and 0.96 ± 0.01 μM, respectively. The Ki values of XOD using APT as inhibitor with xanthine and 6MP as substrate are 6.61 ± 0.28 μM and 1.30 ± 0.09 μM. The corresponding Km values of XOD using xanthine and 6MP as substrate are 2.65 ± 0.02 μM and 6.01 ± 0.03 μM, respectively. The results suggest that the efficiency of substrate binding to XOD and its subsequent catalytic hydroxylation is much superior for xanthine in comparison to 6MP. In addition, the efficiency of the inhibitor binding to XOD is much more superior when 6MP is the substrate instead of xanthine. We further undertook the toxicological evaluation of these inhibitors in a single dose acute toxicity study in mice and our preliminary experimental results suggested that the inhibitors were equally non-toxic in the tested doses.ConclusionWe conclude that administration of either APT or AHMP along with the major anti-leukemic drug 6MP might serve as a good combination cancer chemotherapy regimen.

Acute and subacute toxicity and efficacy of S-nitrosylated captopril, an ACE inhibitor possessing nitric oxide activities

The toxicity and efficacy of S-nitrosocaptopril (CapNO), a novel vasodilator possessing the capacities of both a nitric oxide donor and an angiotensin converting enzyme (ACE) inhibitor, were examined in rodents. In single-dose acute toxicity studies in ICR mice, the median lethal dose (LD 50) for CapNO was 674±94 mg/kg (iv) and 2078±100 mg/kg (po), whereas for oral captopril was 4286±173 mg/kg. S-nitrosoglutathione, containing the same S-nitroso moiety as CapNO, showed an LD 50 equal to CapNO when the values were expressed by the mol/kg. The cause of acute death by the high doses of CapNO was lethal hypotension. In the subacute toxicity studies, oral CapNO was well tolerated in normotensive and hypertensive rats at doses up to 500 mg/kg/day for 3 months, except for considerable reductions in food consumption and growth rate observed in the 500 mg/kg/day group. Serum chemistry and hematology tests performed in the subacute toxicity studies revealed no adverse effects of oral CapNO except for a significant decrease in cholesterol levels in hypertensive SHR rat. At autopsy, no histopathological changes in major organs were observed over the subacute period. Administration of a therapeutic dose of CapNO (iv, 250 μg/kg which produced 25% decreases in blood pressure) revealed no changes in the hematological parameters. Subchronic treatment of SHR and SS/Jr rats with oral CapNO (50 mg/kg/day) significantly reduced mean arterial pressure to the normotensive level. Considering the absence of adverse effects of CapNO in the subchronic toxicity study, CapNO appears to be a safe drug for further clinical trials, but particular caution must be taken because it can cause hypotension when overdosed.

Formulation development and antitumor activity of a filter-sterilizable emulsion of paclitaxel.

Paclitaxel is currently administered i.v. as a slow infusion of a solution of the drug in an ethanol:surfactant:saline admixture. However, poor solubilization and toxicity are associated with this drug therapy. Alternative drug delivery systems, including parenteral emulsions, are under development in recent years to reduce drug toxicity, improve efficacy and eliminate premedication. Paclitaxel emulsions were prepared by high-shear homogenization. The particle size of the emulsions was measured by dynamic light scattering. Drug concentration was quantified by HPLC and in vitro drug release was monitored by membrane dialysis. The physical stability of emulsions was monitored by particle size changes in both the mean droplet diameter and 99% cumulative distribution. Paclitaxel potency and changes in the concentration of known degradants were used as chemical stability indicators. Single dose acute toxicity studies were conducted in healthy mice and efficacy studies in B 16 melanoma tumor-bearing mice. QW8184, a physically and chemically stable sub-micron oil-in-water (o/w) emulsion of paclitaxel, can be prepared at high drug loading (8-10 mg/mL) having a mean droplet diameter of <100 nm and 99% cumulative particle size distribution of <200 nm. In vitro release studies demonstrated low and sustained drug release both in the presence and absence of human serum albumin. Based on single dose acute toxicity studies, QW8184 is well tolerated both in mice and rats with about a 3-fold increase in the maximum-tolerated-dose (MTD) over the current marketed drug formulation. Using the B16 mouse melanoma model, a significant improvement in drug efficacy was observed with QW8184 over Taxol. QW8184, a stable sub-micron o/w emulsion of paclitaxel has been developed that can be filter-sterilized and administered i.v. as a bolus dose. When compared to Taxol, this emulsion exhibited reduced toxicity and improved efficacy most likely due to the composition and dependent physicochemical characteristics of the emulsion.

Acute and subacute toxicity study of water-soluble polyalkylsulfonated C60 in rats.

Polyalkylsulfonated C60, or FC4S, a highly water-soluble caged fullerene derivative, is believed to be a free radical remover or an antioxidant in biological systems. A 50 mg/ml aqueous solution was prepared as a master solution and administered to female Sprague-Dawley CD(Crl:CD(SD)BR) rats in a single-dose acute toxicity study or a 12-day subacute toxicity study where rats were given the solution daily. In a study of the median lethal dose (LD50), no rats died after oral administration, and thus FC4S was considered to be nontoxic if administered orally. In an LD50 intraperitoneal injection study, rats died within 30 hr after injection; the LD50 was determined to be approximately 600 mg per kilogram of body weight. Rats injected with the compound intraperitoneally or intravenously immediately eliminated the compound through the kidney; the kidney appeared to be the primary target organ. The compound induced a distinct lysosome-overload nephrosis, a phagolysosomal nephropathy characterized by a tinctorial difference between the outer cortex and the inner cortex and the medulla. The affected outer cortex showed a diffuse degeneration, with the presence of numerous large vacuoles and cytoplasmic aggregates in the tubular epithelium. The phagolysosomal nephropathy was detected in rats after acute exposure as well as in the surviving rats following 1 intraperitoneal injection of 500 mg/kg or intravenous injection of 100 mg/kg. Ultrastructural investigation revealed numerous membranous conglomerates characteristic of phagolysosomal and/or lysosomal inclusions in the cytoplasm of the renal tubular epithelium. These conglomerates were confined to the vacuole, electron-dense, and unevenly stained. They varied in size and shape and were fused or aggregated. Occasional phagolysosomes were also observed in the endothelial cells of the peritubular plexus. A preliminary study of microsomal enzyme activity analysis revealed a suppression effect of liver cytochrome P-450-dependent monooxygenase activities, including cytochrome P-450, cytochrome b5, and benzo(a)pyrene hydroxylase, but an increased level of kidney cytochrome P-450-dependent monooxygenase activities, including NADPH-cytochrome P-450 reductase. The significance of these enzyme alterations was not well determined. Further study is needed to clarify the correlation between the alterations of microsomal enzyme activity and the nephropathy of lysosomal overload-induced changes. These changes may serve as a biological marker in toxicity screening tests for this class of compound.

Nonclinical Toxicology Studies with Zidovudine: Acute, Subacute, and Chronic Toxicity in Rodents, Dogs, and Monkeys

In single dose acute toxicity studies in CD-1 mice and CD rats, the median lethal dose (MLD) for zidovudine (ZDV) was >750 mg/kg after iv dosing and >3000 mg/kg after po administration (recommended human dose is 100 mg every 4 hr while awake). Because of the short half-life in rats (0.8 hr), dogs (1.0 hr), and monkeys (0.8 hr), the daily dose of ZDV in most studies was given in two equal portions approximately 6 hr apart. Intravenous administration of ZDV was well tolerated in beagle dogs at dose levels up to 42.5 mg/kg bid for 2 weeks and in CD rats at dose levels up to 75 mg/kg bid for 4 weeks. In a 2-week dose range-finding study in beagle dogs, cytostatic effects were noted at po dose levels of 62.5 to 250 mg/kg bid in certain tissues with rapid cell replication rates. In contrast, in 3- to 12-month oral toxicity studies in CD rats and cynomolgus monkeys, the principal toxicologic finding was reversible macrocytic normochromic anemia which occurred at 225–250 mg/kg bid in rats and 17.5–150 mg/kg bid in monkeys. In the 12-month rat study, RBC was decreased at 25 and 75 mg/kg bid. In the 12-month monkey study WBC was slightly decreased at 150 mg/kg bid.

Nonclinical Toxicology Studies with Zidovudine: Acute, Subacute, and Chronic Toxicity in Rodents, Dogs, and Monkeys

In single dose acute toxicity studies in CD-1 mice and CD rats, the median lethal dose (MLD) for zidovudine (ZDV) was > 750 mg/kg after iv dosing and > 3000 mg/kg after po administration (recommended human dose is 100 mg every 4 hr while awake). Because of the short half-life in rats (0.8 hr), dogs (1.0 hr), and monkeys (0.8 hr), the daily dose of ZDV in most studies was given in two equal portions approximately 6 hr apart. Intravenous administration of ZDV was well tolerated in beagle dogs at dose levels up to 42.5 mg/kg bid for 2 weeks and in CD rats at dose levels up to 75 mg/kg bid for 4 weeks. In a 2-week dose range-finding study in beagle dogs, cytostatic effects were noted at po dose levels of 62.5 to 250 mg/kg bid in certain tissues with rapid cell replication rates. In contrast, in 3- to 12-month oral toxicity studies in CD rats and cynomolgus monkeys, the principal toxicologic finding was reversible macrocytic normochromic anemia which occurred at 225-250 mg/kg bid in rats and 17.5-150 mg/kg bid in monkeys. In the 12-month rat study, RBC was decreased at 25 and 75 mg/kg bid. In the 12-month monkey study WBC was slightly decreased at 150 mg/kg bid.