Scientific data obtained from preclinical research is valuable in evaluating the pharmacology and safety profile of a botanical dietary supplement and potential pharmaceutical product. The NCNPR has expertise is ADME studies as well as in vitro and in vivo toxicology testing.

ADME Studies

Characterization of ADME (Absorption, Distribution, Metabolism, Excretion ) properties is an integral part of drug development process and preclinical studies.

A series of in vitro bioassays have been established at NCNPR to characterize ADME properties of potential drug candidates / phytochemical / botanicals under investigation:

• Intestinal absorption / permeability using Caco-2 cell monolayer model
• BBB absorption / permeability using MDR-MDCKII cell monolayer model
• Metabolic stability (PhaseI and Phase II drug metabolizing enzymes)
• Stability in biological fluids (Plasma, gastric, intestinal fluid)
• Plasma protein binding (equilibrium dialysis method)
• Pharmacokinetics and tissue distribution

In vitro and in vivo toxicology

In vitro toxicity

Assessment of general in vitro cytotoxicity of bioactive molecules / natural product extracts is done through a cell viability assay in a panel of 3 cell lines (HepG2, LLC-PK1, and VERO). In this assay a dose dependent response of the test material on cell viability is determined.

Cell viability assays can also be combined with apoptosis assays to provide more information about mechanisms of cell death. The procedures include AnnexinV staining, caspase activity, DAPI staining, and cell cycle analysis by propidium iodide staining using flow cytometer.

In vivo toxicity

Maximum Tolerated Dose

Prior to initiation of in vivo efficacy testing, the maximum tolerated dose (MTD) of the product is determined to guide the choice of dose. The product is administered by a selected route (oral or IP) at an arbitrary dose (eg: 5 mg/kg) in mice and observed for any observable discomfort for one hour. If the mice survive with no untoward signs, an increased dose is given to fresh mice and observed for another one hour. The maximum dose rarely exceeds 1.0 G/kg. The MTD is the one at which the mice do not show signs of toxicity or death within one hour.

Hepatotoxicty in vivo

Mice (ND4) are used for these studies. Heathy and health compromised mice (mice pre-treated with a single i.p dose of LPS) are administered the product via the selected route (oral or ip) for a period of up to 28 days. Appropriate controls are also taken into consideration. At the end of the study, half of animals from each group are sacrificed. Remaining half of animals in each group serve as satellite groups to be observed for additional 2 weeks without any treatment. Blood samples are collected through cardiac puncture for hematological and biochemical studies. A complete blood count (CBC) (RBC, WBC, Platelets), hemoglobin, hematocrit will be determined using a hematology analyzer. For biochemical analysis, heparinized blood is subjected to comprehensive liver profile (alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), alkaline phosphatase, blood urea nitrogen (BUN), total bilirubin, and creatinine is determined using a Biochemical Analyzer. Histopathology with special stains (if required) are done on representative liver samples from each group to visualize any lesion.