National Center for Natural Products Research
The University of Mississippi

Herb-drug / Herb-herb Interaction Studies

Herb-drug (and more recently herb-herb) interaction is a major safety concern for the consumers as well as health care professionals especially with the rising use of herbal products / botanicals for general well being as well as their concomitant use with the prescription drugs. Assessment of the potential of herb-drug interaction can be made in terms of the ability of the herb to interfere with the activity and the expression of drug metabolizing enzymes and transporters which may lead to loss of efficacy or enhanced toxicity of the concomitantly used drug or another herb. Following bioassays have been established at NCNPR to assess the herb-drug interaction potential :

  • CYP inhibition
  • P-gp inhibition
  • PXR activation
  • mRNA expression of CYPs and P-gp

Skin Sensitization (cosmetic products)

Skin sensitization is an important toxicological end-point to be characterized for the safety evaluation of chemicals such as ingredients used in the formulations of drugs, cosmetics, fragrances, traditional herbal remedies and other household products, which can come into contact with the skin.

There is a growing trend among regulatory bodies worldwide to embrace the 3R approach (Reduce, Replace and Refine) in order to progressively minimize the need of animal methods for risk assessment.

At the present, characterization of skin sensitizers using non-animal methods requires integrated approaches, where each key event (haptenation, elicitation of inflammatory response in keratinocytes and activation of dendritic cell responses) is assessed independently in targeted assays.

The facilities at the NCNPR offer a complete collection of non-animal methods for skin sensitization risk assessment. Methods available include validated methods accepted by regulatory agencies as well as in house techniques developed by the NCNPR team to overcome some of the common hurdles encountered with other methods. The two method (NMR-DCYA and HTS-DCYA) have been developed as orthogonal approaches which could potentially be used for the testing of complex (un)characterized mixtures, such as botanicals and cosmetic ingredients.

DCYA methods. Electrophilicity is a key feature of chemical sensitizers. The ability of chemicals to react covalently with nucleophilic amino acids (especially cysteine and lysine) is considered the driving force behind the haptenation phase, which is the Molecular Initiating Event (MIE). Electrophilicity of test articles can be easily assessed by evaluating the chemical reactivity toward model nucleophiles designed to mimic the reactivity of skin proteins. A team at the NCNPR developed two chemical assays based on a small nucleophilic dansylated molecule (DCYA) as model nucleophile the detection of reactive compounds.

  • The first method is a fluorescence-based high throughput method (HTS-DCYA) where reactivity is estimated by spectrophotometric quantification of fluorescent covalent adducts. The workflow enables a fast, sensitive and rapid estimation of reactivity which can be used to characterize the MIE (also called Key Event 1). The method enables the testing of 30 chemicals simultaneously, has the advantage of being suitable for testing of complex test articles (including cosmetic formulations) and it can be used for untargeted investigation of uncharacterized mixtures.
  • The second method is based on quantification of reactivity by nuclear magnetic resonance (NMR-DCYA). The method exploits the advantages of NMR techniques to directly quantify the depletion of the chemical and can provide essential insight on the mechanism and site of reaction. 

Both methods can be used to complement validated methods (listed below) and can provide useful complementary information for a more comprehensive risk assessment decision without the need of animal testing.

DPRA. The Direct Peptide Reactivity Assay (DPRA) has been the first (and so far the only) chemical method to have completed full inter-laboratory validation scrutiny. The method has been incorporated by the Organization for Economic Co-Operation and Development as guideline OECD 442C. The method relies on the use of Cys- or Lys-heptapeptides as model nucleophiles. The chemical reactivity is estimated by quantification of unreacted peptide after 24 h reaction with the test article (pure compounds only) and can be used for characterization of the MIE.

KeratinoSensTM. The activation of inflammatory pathways in keratinocytes is considered the second key event (KE2). Once the compound is internalized (through haptenation), further KEAP1-ARE-Nrf-mediated signaling pathways are activated in the keratinocytes. This KE can be assessed by in vitro methods such as the KeratinoSensTM (OECD 442D). An immortalized adherent cell line derived from HaCaT human keratinocytes is used. The cell line is stably transfected with a selectable plasmid containing a luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitizer. This method enables the characterization of early inflammation responses through fluorescence-based spectrophotometric assays on whole cell models, and can be applied to non-cytotoxic compounds and mixtures.

hCLAT. The third KE can be assessed through the human Cell Line Activation Test (hCLAT), described in regulatory guideline OECD 442E. The hCLAT method can be applied to characterize the activation of cellular responses in dendritic cells (DC) following exposure to the test article. The test is performed using human monocytic leukemia cell line (THP-1). Changes in the expression of cell surface markers associated with the process of activation of monocytes and DC (CD86 and CD54) are quantified by cytofluorimetric assays by measuring changes in relative fluorescence intensity (RFI). Integrated approaches with the KeratinoSens and DPRA are available at the NCNPR.

Examples of studies conducted at the NCNPR include:

  • Investigation of the stability of allergenic fragrance ingredients and correlation of stability with skin sensitization adverse effects
  • Investigation the applicability of non-animal methods to the safety investigation of botanicals
  • Identification of potential compounds of concern from chamomile and tea tree oils using non-animal methods

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.