Ashok D.B. Vaidya

Reverse Pharmacology and Systems Approaches for Drug Discovery and Development

While biotechnological advances, genomics and high throughput screenings or combinatorial and asymmetric syntheses have opened new vistas in drug discovery, the industry is facing a serious innovation deficit. Critics suggest that “we have become high throughput in technology, yet have remained low throughput in thinking”. Post marketing failures of blockbuster drugs have become major concerns of industries, leading to a significant shift in favor of single to multi targeted drugs and affording greater respect to traditional knowledge. Typical reductionist approach of modern science is being revisited over the background of systems biology and holistic approaches of traditional practices. Scientifically validated and technologically standardized botanical products may be explored on a fast track using innovative approaches like reverse pharmacology and systems biology, which are based on traditional medicine knowledge. Traditional medicine constitutes an evolutionary process as communities and individuals continue to discover practices transforming techniques. Many modern drugs have origin in ethnopharmacology and traditional medicine. Traditions are dynamic and not static entities of unchanging knowledge. Discovering reliable ‘living tradition’ remains a major challenge in traditional medicine. In many parts ‘little traditions’ of indigenous systems of medicine are disappearing, yet their role in bioprospecting medicines or poisons remains of pivotal importance. Indian Ayurvedic and traditional Chinese systems are living ‘great traditions’. Ayurvedic knowledge and experiential database can provide new functional leads to reduce time, money and toxicity the three main hurdles in the drug development. We begin the search based on Ayurvedic medicine research, clinical experiences, observations or available data on actual use in patients as a starting point. We use principles of systems biology where holistic yet rational analysis is done to address multiple therapeutic requirements. Since safety of the materials is already established from traditional use track record, we undertake pharmaceutical development, safety validation and pharmacodynamic studies in parallel to controlled clinical studies. Thus, drug discovery based on Ayurveda follows a ‘Reverse Pharmacology’ path from Clinics to Laboratories. Herein we describe such approaches with selected examples based on previous studies.

Inhibition of the growth of urinary calcium hydrogen phosphate dihydrate crystals with aqueous extracts of Tribulus terrestris and Bergenia ligulata

Urinary type calcium hydrogen phosphate dihydrate (CHPD) or Brushite crystals were grown by the single diffusion gel technique in silica hydro-gels. The gel framework acts as a three dimensional crucible in which the crystal nuclei are delicately held in the position of their formation and nutrients are supplied for their growth. This technique can be utilized as a simplified screening model to study the growth and dissolution of urinary stones in vitro. The action of the putatively litholytic medicinal plants Tribulus terrestris and Bergenia ligulata on the growth of CHPD crystals was studied . The effects of artificial reference urine (ARU) and human urine (HU), along with the plant extracts, are also reported. Attempts were made to understand the role of these inhibitors on urinary crystal formation. HU, ARU, extracts of B. ligulata and T. terrestris exhibit appreciable amounts of inhibition, but B.ligulata and T.terrestris with ARU and HU do not show inhibition at all.

Exploratory study to evaluate tolerability, safety, and activity of Ashwagandha (Withania somnifera) in healthy volunteers

Ashwagandha (Withania somnifera) (WS), a “rasayana” drug, is recommended for balavardhan and mamsavardhan. The study was intended to evaluate dose-related tolerability, safety, and activity of WS formulation in normal individuals. The design was prospective, open-labeled, variable doses in volunteers. Eighteen apparently healthy volunteers (12M:6F, age:18-30 years, and BMI: 19-30) were enrolled. After baseline investigations, they received WS capsules (Rx) (aqueous extract, 8:1) daily in two divided doses with increase in daily dosage every 10 days for 30 days (750 mg/day ×10 days, 1 000 mg/day × 10 days, 1 250 mg/day × 10 days). Volunteers were assessed for symptoms/signs, vital functions, hematological and biochemical organ function tests. Muscle activity was measured by hand grip strength, quadriceps strength, and back extensor force. Exercise tolerance was determined using cycle ergometry. Lean body weight and fat% were computed from skin fold thickness measurement. Adverse events were recorded, as volunteered by the subjects. Repeated measures ANOVA, McNemars test, and paired t test were employed. All but one volunteer tolerated WS without any adverse event. One volunteer showed increased appetite, libido, and hallucinogenic effects with vertigo at the lowest dose and was withdrawn from study. In six subjects, improvement in quality of sleep was found. Organ function tests were in normal range before and after the intervention. Reduction in total- and LDL- cholesterol and increase of strength in muscle activity was significant. Total body fat percentage showed a reduction trend. WS, in escalated dose, was tolerated well. The formulation appeared safe and strengthened muscle activity. In view of its traditional Rasayana use, further studies are planned to evaluate potential of this drug in patients of sarcopenia.

A study of standardized extracts of Picrorhiza kurroa Royle ex Benth in experimental nonalcoholic fatty liver disease

As a major organ of intermediary metabolism, the liver is exposed to a variety of metabolic insults due to diseases and xenobiotics viz., insulin resistance (IR) drugs, toxins, microbial products, etc. One of the consequences of these metabolic insults including obesity and type 2 diabetes mellitus is the development of non-alcoholic fatty liver disease (NAFLD). The recent alarming increase in the prevalence of NAFLD compels the need to develop an appropriate animal model of the disease so as to evolve effective interventions. In this study, we have developed, in the rat, a new model of NAFLD showing several key features akin to the disease in humans. Male Wistar rats were challenged with 30% high fat diet (HFD) – butter, for 2 weeks to induce NAFLD. A hydroalcoholic extract of Picrorhiza kurroa was administered to study the possible reversal of fatty changes in the liver. The extract was given in two doses viz., 200mg/kg and 400 mg/kg b.i.d., p.o. for a period of 4 weeks. There were three control groups (n = 6/group) – vehicle with a regular diet, vehicle with HFD, and HFD with silymarin – a known hepatoprotective. Histopathology showed that the P. kurroa extract brought about a reversal of the fatty infiltration of the liver (mg/g) and a lowering of the quantity of hepatic lipids (mg/g) compared to that in the HFD control group (38.33 ± 5.35 for 200mg/kg; 29.44 ± 8.49 for 400mg/kg of P. kurroa vs.130.07 ± 6.36mg/g of liver tissue in the HFD control group; P<0.001). Compared to the standard dose of the known hepatoprotective silymarin, P. kurroa reduced the lipid content (mg/g) of the liver more significantly at the dose of 400mg/kg (57.71 ± 12.45mg/kg vs. 29.44 ± 8.49 for the silymarin group vs. 400mg/kg of P. kurroa, P<0.001). In view of the increasing prevalence of metabolic syndrome and NAFLD, P. kurroa should be investigated by the reverse pharmacology path as a potential drug for the treatment of NAFLD, and essential safety studies and preformulation research for concentration of the putative actives should be carried out.

Cassia auriculata: Aspects of Safety Pharmacology and Drug Interaction

Safety pharmacology studies help in identifying preclinical adverse drug reactions. We carried out routine safety pharmacology with focus on cardiovascular variables and pharmacokinetic herb-drug interaction studies on rats fed with standardized traditional hydro-alcoholic extract and technology-based supercritical extract of Cassia auriculata for 12 weeks. Our studies indicate that both these extracts are pharmacologically safe and did not show any significant adverse reactions at the tested doses. The traditional hydro-alcoholic extract did not show any significant effect on pharmacokinetics; however, the technology-based supercritical extract caused a significant reduction in absorption of metformin. Our results indicate the need to include pharmacokinetic herb-drug interaction studies as evidence for safety especially for technology-based extracts.

An advocacy for Vaidya-Scientists in Ayurvedic research

“Those who have handled sciences have been either men of experiment or men of dogmas. The men of experiment are like ants; they only collect and use: the reasoners resemble spiders, which make cobwebs out of their own substance. But the bee takes a middle course, it gathers its materials from the flowers of the garden and of the field, but transforms and digests it by a power of its own.” –Sir Francis Bacon[1]

Plasmodium DNA Fluoresces With Berberine A Novel Approach for Diagnosis of Malarial Parasites

The key to reducing mortality and morbidity associated with malaria is rapid diagnosis and early, effective therapy. Berberine, a plant alkaloid, has been used for fluorescent staining of the Y chromosome. We evaluated whether berberine can be used for staining of malarial parasites in 40 selected peripheral blood smears from patients with clinical symptoms of malaria; smears were evaluated with OptiMal (DiaMed, Miami, FL) and Giemsa stain. Twenty were positive with both OptiMal and Giemsa (Plasmodium vivax, 14; Plasmodium falciparum, 6); 10 were negative with both. The remainder were positive by OptiMal but negative by Giemsa and, therefore, were classified as equivocal. All slides were processed simultaneously, stained with berberine, and read under a fluorescent microscope. P vivax and P falciparum DNA fluoresced with berberine. The positives and negatives by berberine concurred with the Giemsa staining. Of the 10 equivocal smears, 5 were confirmed positive by berberine. Gametocytes were easily identifiable. This test has high sensitivity and high positive predictive value and, once standardized, can be used as a potential screening and diagnostic tool.

Shirodhara : A psycho-physiological profile in healthy volunteers

Background: Shirodhara is a classical and a well-established ayurvedic procedure of slowly and steadily dripping medicated oil or other liquids on the forehead. This procedure induces a relaxed state of awareness that results in a dynamic psycho-somatic balance. Objectives: The objective of the study is to evaluate the psychological and physiological effects of Shirodhara in healthy volunteers by monitoring the rating of mood and levels of stress, electrocardiogram (ECG), electroencephalogram (EEG), and selected biochemical markers of stress. Materials and Methods: The study was conducted in the human pharmacology laboratory. The study design was open labeled, comparing the baseline variables with values after Shirodhara. The subjects (n = 16) chosen were healthy human volunteers who gave an informed consent. Shirodhara was preceded by Abhyanga – whole body massage. The Shirodhara method was standardized for rate of dripping with peristaltic pump and temperature was controlled with a thermostat. Mood and stress levels were assessed by validated rating scales. The pre- and post-Shirodhara ECG and EEG records were evaluated. Results: Students paired “t” test was applied to the means + SE of the variables to calculate statistical significance at P <0.05. There was a significant improvement in mood scores and the level of stress (P <0.001). These changes were accompanied by significant decrease in rate of breathing and reduction in diastolic blood pressure along with reduction in heart rate. The relaxed alert state, after Shirodhara, was co-related with an increase in alfa rhythm in EEG. Conclusion: A standardized Shirodhara leads to a state of alert calmness similar to the relaxation response observed in meditation. The clinical benefits observed with Shirodhara in anxiety neurosis, hypertension, and stress aggravation due to chronic degenerative diseases could be mediated through these adaptive physiological effects.

Single dose metformin kinetics after co-administration of nisha-amalaki powder or mamejwa ghanavati, ayurvedic anti-diabetic formulations: A randomized crossover study in healthy volunteers

Objective: The aim was to study herb-drug interaction of two ayurvedic formulations - DMFN01 (Nisha-Amalaki) powder and DMFN02 (Mamejava) ghanavati with metformin at a single dose in healthy volunteers. Materials and Methods: This was an open-labelled, single dose, crossover, and randomized volunteer study. Healthy volunteers were studied in two groups (6/group). Volunteers were randomized to oral metformin (500 mg single dose) alone or with concurrent DMFN01 (10 g), or DMFN02 (750 mg). Venous blood samples were collected at different time points from 0 to 24 h. Plasma metformin concentrations were measured by high performance liquid chromatography coupled with an ultraviolet detector. Results: Simultaneous administration of DMFN01 with metformin showed a reduction in the mean area under the curve (AUC [0-24 h]) of metformin by 51% (P < 0.002) when compared with metformin alone. However, co-administration of DMFN02 did not show any significant difference in the mean AUC of metformin (P = 0.645). One volunteer had a reduction of 41% in AUC of metformin with DMFN 02. Conclusions: These data raise relevant questions on therapeutic control of hyperglycemia when DMFN01 choorna is given concurrently with metformin. Based on known absorption pattern of metformin an interval of 2 h between the oral doses of metformin and ayurvedic formulations would be advisable to avoid interactions. In reverse pharmacological studies, at an early stage, such interaction studies are desirable.

Comparative study of amrutbhallataka and glucosamine sulphate in osteoarthritis: Six months open label randomized controlled clinical trial

Background: AmrutBhallatak (ABFN02), a ‘rasayana’ drug from Ayurveda is indicated in degenerative diseases and arthritis. Objective: To evaluate safety and efficacy of ABFN02 in osteoarthritis (OA) and compare it with Glucosamine sulphate (GS) Materials and Methods: This was a randomized open comparative study. Ambulant OPD patients of OA knees (n = 112) were enrolled for 24 weeks. Tablets (750mg each) of GS and ABFN02 were matched. Three groups of patients: (A) GS, one tablet × twice/day × 24 weeks. (B) ABFN02, incremental pulse dosage (one tablet x twice/day × two weeks, two tablets × twice/day × two weeks, three tablets × twice/day × two weeks), two such cycles of drug and non-drug phases alternately for six weeks each (C) ABFN02 continuous dosage akin to GS. Pain visual analogue score (Pain-VAS) and Western Ontario and Mc-Master University Osteoarthritis Index (WOMAC) were the primary outcome measures. Secondary outcome measures were Health assessment questionnaire (HAQ), paracetamol consumption, 50 feet walking, physician and patient global assessment, knee stiffness, knee status, urinary CTX II, serum TNFa-SRI, SRII and MRI knee in randomly selected patients. Results: ABFNO2 and GS demonstrated, adherence to treatment 87.75% and 74.3%, reduction in Pain-VAS at rest 61.05% and 57.1%, reduction in pain-VAS on activity 57.4% and 59.8%, WOMAC score drop 62.8% and 59.1% respectively. Secondary outcome measures were comparable in all groups. Safety measures were also comparable. No serious adverse events reported. However, asymptomatic reversible rise in liver enzymes was noted in the ABFNO2 group. Conclusions: ABFN02 has significant activity in OA; the formulation needs further investigation.