Targeted delivery of ursolic acid and oleanolic acid to lungs in the form of an inhaler for the management of tuberculosis: Pharmacokinetic and toxicity assessment.

INTRODUCTION: Ursolic acid (UA) and oleanolic acid (OA) are triterpenoids. They are used to treat numerous diseases, including tuberculosis. Combinations of these drugs provide new insight into the management of tuberculosis. The major obstacle is the effective delivery of these drugs to the lungs, which are mainly affected due to M. tuberculosis. A metered-dose inhaler (MDI) was developed to address this issue containing UA and OA, followed by in-vitro and in-vivo evaluation. METHODS: In the present study, MDI formulation was prepared by incorporating UA and OA at the dose level of 120 µg/ml in each actuation. In-vitro evaluation of this MDI formulation was performed to ensure its suitability to deliver UA and OA preciously. With prior approval of IAEC, a pharmacokinetic and acute inhalation toxicity study was conducted using MDI on Wistar rats. RESULTS: The pharmacokinetic study showed an increased biological half-life of UA (9.23±0.104 h) and OA (8.93±0.166 h) in combination therapy. In-vivo toxicity study demonstrated no adverse effects on body weight and vital organs in the treatment group compared with the control group. Histopathology examination of these essential organs showed no abnormalities. Mild alternation in the biochemical and hematological parameters was observed. However, these alterations did not affect the overall health of the animals. CONCLUSION: The present study documents a detailed study for the safety and pharmacokinetics of UA and OA in-vivo for their advanced application in tuberculosis disease.

Pharmacokinetics Comparison, Intestinal Absorption and Acute Toxicity Assessment of a Novel Water-Soluble Astragaloside IV Derivative (Astragalosidic Acid, LS-102).

BACKGROUND AND OBJECTIVES: Astragaloside IV (AGS IV) is the most important bioactive constituent of Radix Astragali. However, its disappointing clinical application is mainly caused by its very low solubility in biologic fluids, resulting in poor bioavailability after oral administration. We recently obtained a novel water-soluble derivative of AGS IV (astragalosidic acid, LS-102) that displayed significant cardioprotective potential against hypoxia-induced injury. The objective of this study was to investigate the intestinal absorption, main pharmacokinetic parameters and acute toxicity of LS-102 in rodents compared with AGS IV. METHODS: An oral dose of LS-102 and AGS IV (20 mg/kg) was administered to Sprague-Dawley (SD) rats, and blood samples were collected at predetermined time points. The plasma concentrations were detected by a validated UHPLC-MS/MS method, and pharmacokinetic parameters were calculated using a compartmental model. In the intestinal permeability study, the transport of LS-102 across Caco-2 cell monolayers was investigated at six concentrations from 6.25 to 250 µM. Moreover, the acute toxicity of LS-102 (40-5000 mg/kg) via a single oral administration was investigated in BALB/c mice. RESULTS: LS-102 was rapidly absorbed, attaining a maximum concentration of 248.7 ± 22.0 ng/ml at 1.0 ± 0.5 h after oral administration. The relative bioavailability of LS-102 was twice that of AGS IV. LS-102 had a Papp (mean) of 15.72-25.50 × 10-6 cm/s, which was almost 500-fold higher than that of AGS IV, showing that LS-102 had better transepithelial permeability and could be better absorbed in the intestinal tract. The acute toxicity study showed no abnormal changes or mortality in mice treated with LS-102 even at the single high dose of 5000 mg/kg body weight. CONCLUSIONS: Oral LS-102 produced a pharmacokinetic profile different from AGS IV with higher bioavailability, while the toxic tolerance was similar to previous estimates. Thus, we speculated that LS-102 might provide better clinical efficacy and be a potential candidate for the new drug development of Radix Astragali.

Assessment of Toxicity and Absorption of the Novel AA Derivative AA-Pme in SGC7901 Cancer Cells In Vitro and in Zebrafish In Vivo.

BACKGROUND Asiatic acid (AA; 2α,3ß,23-trihydroxyurs-12-ene-28-oic acid) is an active compound derived from Centella asiatica, a traditional medicinal plant used widely in many Asian countries, particularly for the treatment of cancer. However, the modified AA derivative N-(2α,3ß,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester (AA-PMe) has shown markedly better anti-tumor activity than AA. MATERIAL AND METHODS We evaluated the toxicity of AA and AA-PMe on zebrafish morphology, mortality, and hatching rate and determined the effect on SGC7901 cancer cells by acute toxicity assay. AA-PMe absorption in vitro in SGC7901 cells and in vivo in zebrafish was determined by establishing a highly accurate and reproducible HPLC protocol. RESULTS In zebrafish, the toxicity of AA-PMe was lower than AA, with an acute toxic dose of AA-PMe above 25 µM, compared to acute toxicity at doses above 10 µM for AA. However, chronic toxicity of AA-PMe began occurring at doses below 25 µM but became apparent for AA at doses below 10 µM. Although low doses of AA-PMe were tolerated acutely, it became chronically toxic during zebrafish development, resulting in morphological abnormalities, including peripheral and abdominal edema, hemorrhage, abnormal body shape, enlarged yolk sac, and reduced motility. At low concentrations, absorption of AA-PMe by cells and zebrafish embryos occurred in a dose-dependent manner, but this stabilized as the concentration increased. CONCLUSIONS This pharmacokinetic study outlines the cellular and organismal effects of AA-PMe and suggests a theoretical basis that may underlie its mechanism of action.

A 28-day Sub-acute Genotoxic and Behavioural Assessment of Garcinielliptone FC.

Garcinielliptone FC (GFC) is a polyisoprenylated benzophenone isolated from Platonia insignis Mart (Clusiaceae) with promising anticonvulsant properties. However, its safe use and other effects on the central nervous system require assessment. This study assessed the toxicological effects of GFC using the comet assay and the micronucleus test in mice treated for 28 days. A behavioural model was employed to detect possible injuries on the central nervous system. Mice treated with GFC (2, 10 and 20 mg/kg; i.p.) daily for 28 days were submitted to rotarod test, open-field test and tail suspension test (TST). After the behaviour tasks, biological samples were assessed to evaluate genotoxic and mutagenic effects using the comet assay and the micronucleus test. Garcinielliptone FC did not impair the performance of the animals in the rotarod and open-field tests, with no antidepressant-like effect in TST. No genotoxic effects in blood and cerebral cortex were observable in the comet assay; however, there was a significant increase in index and frequency of damage in liver after treatment with GFC 20 mg/kg. Garcinielliptone FC did not increase micronucleus frequency in bone marrow. At the tested doses, GFC was not toxic to the CNS and did not induce genotoxic damage to blood or bone narrow cells. DNA damage to liver tissue was caused only by the highest dose, although no mutagenic potential was observed.

Comprehensive assessment of Cucurbitacin E related hepatotoxicity and drug-drug interactions involving CYP3A and P-glycoprotein.

BACKGROUND: Cucurbitacin E (CuE), a tetracyclic triterpenoid isolated from Cucurbitaceae, possesses many pharmacological activities especially anti-cancer. PURPOSE: The aim of this investigation was to comprehensively assess CuE related hepatotoxicity and potential drug-drug interactions involving CYP3A and P-glycoprotein (P-gp). STUDY DESIGN AND METHODS: Four common cytotoxicity assays (MTS, SRB, NRU and apoptosis assays) were used to evaluate the hepatotoxicity of CuE in human hepatocellular carcinoma HepG2 cells. Human and rat liver microsomes incubation system, Caco-2 transport model and 3D organoids model were used to investigate the effects of CuE on CYP3A and P-gp in vitro. The oral pharmacokinetics of indinavir was employed to evaluate the effects of CuE on CYP3A and P-gp in vivo. RESULTS: CuE induced the HepG2 apoptosis and exhibited acute cytotoxicity in MTS, SRB, and NRU assays with IC50 value at 15.98µM, 0.31µM, and 1.11µM, respectively. Moreover, CuE not only presented mechanism-based inhibition on human CYP3A4, but also decreased the efflux ratio of digoxin (P-gp substrate) across Caco-2 cell monolayers in vitro. Furthermore, CuE significantly inhibited the transport of Rh123 into 3D organoids, which was caused by the inhibition on P-gp. In Sprague-Dawley rat studies in vivo, acute administration of CuE significantly increased the maximum serum concentration (Cmax) and area under the concentration-time curve (AUC) of indinavir. In contrast, CuE treatment for three consecutive days significantly decreased indinavir Cmax and AUC in rats. CONCLUSION: These studies demonstrated that CuE has strong hepatotoxicity, and CuE presents potent inhibition on both CYP3A and P-gp activities in vitro. In animal in vivo studies, CuE induces CYP3A and P-gp after a long-term treatment but inhibits the activities of CYP3A and P-gp after an acute dosing. Therefore, CuE as a dual functional regulator of both CYP3A and P-gp may cause complex drug-drug interactions.

Trypanocidal activity and acute toxicity assessment of triterpene acids.

The present study evaluates the in vitro and in vivo trypanocidal activity of ursolic acid and oleanolic acid against the Bolivia strain of Trypanosoma cruzi. Their acute toxicity is also assessed on the basis of median lethal dose (DL50) determination and quantification of biochemical parameters. Ursolic acid is the most active compound in vitro, furnishing IC50 of 25.5 microM and displaying 77% of trypomastigote lysis at a concentration of 128 microM. In agreement with in vitro assays, the results obtained for the in vivo assay reveals that ursolic acid (at a dose of 20 mg/Kg/day) provides the most significant reduction in the number of parasites at the parasitemic peak. Results concerning the LD50 assay and the biochemical parameters evaluated in the present study demonstrate that these substances can be safely used on an experimental basis.

Assessment of embryo/fetotoxicity and teratogenicity of azadirachtin in rats.

To evaluate the potential effect of exposure to azadirachtin technical 12% throughout major organogenesis, rats were fed orally with 500, 1000 and 1500 mg/kg/day azadirachtin on gestation days 6-15 and examined for evidence of embryo/fetotoxicity and teratogenic effects. Technical azadirachtin at different doses did not produce any significant adverse effects in reproductive parameters. Significant embryo/fetotoxic effects were not observed at tested dose levels as evidenced by total number of implantations, post-implantation loss and fetal weight. There were no major malformations, while some minor variants found in high doses were not compound or dose related. The absence of anomalies in fetal gross, visceral morphology and skeleton suggests that technical azadirachtin is not teratogenic in rats at the doses tested.

Azadirachtin, a neem biopesticide: subchronic toxicity assessment in rats.

Azadirachtin, a biopesticide obtained from neem, was subjected to subchronic toxicological testing to document its safety for use as a pesticide. Azadirachtin technical 12% orally administered to male and female rats at doses of 500, 1000 and 1500 mg/kg/day for 90 days did not produce any signs of toxicity, mortality, changes in tissue weight, pathology and serum and blood parameters. It can be suggested that azadirachtin at the highest dose tested is well tolerated by rats of both sexes. The highest dose, 1500 mg/kg, can be used as a basal dose for the determination of the no-observed-effect level (NOEL) of azadirachtin to calculate its safety margin.