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EVALUATION OF ORAL BIOAVAILABILITY ENHANCEMENT OF
RIFAMPICIN BY KAJJALI, AN AYURVEDIC PROPRIETARY
HERBOMINERAL PRODUCT
Shah Devanga, Seervi Madhava, Zala Vijayb, Damre Anaghac, Sathaye Sadhanaa
a
Dept. of Pharmaceutical Sciences, Institute of Chemical Technology, Matunga, Mumbai-400 019, India
b
Veeda Clinical Research, Plymouth, PL6 5HH, UK
c
Drug Metabolism and Pharmacokinetics, Piramal Life Sciences Limited, Goregaon (E), Mumbai-400 063,
India
Running Title: Effect of Kajjali on Rifampicin Pharmacokinetics
*Address of correspondence:
Dr. Sadhana Sathaye
Pharmacology Research Lab-II,
Department of Pharmaceutical Sciences and Technology,
Institute of Chemical Technology, Mumbai- 400 019, India.
E-mail: [email protected]
Contact no: 022 3361 2218
Fax:
022 3361 1020
ABSTRACT
Kajjali, an ayurvedic proprietory herbo-mineral product, is reported in ancient Ayurvedic literature
to have yogavahi property that is, it enhances the activity of drugs co-administered with it. This
study examined the effect of Kajjali on single dose oral pharmacokinetics of Rifampicin in healthy
Wistar rats. Two groups (n=6) were employed; of which one received single dose of Rifampicin
alone and other received Kajjali (15 mg/kg, p.o.) with Rifampicin (10 mg/kg, p.o.). Blood samples
were collected up to 10 h to estimate the plasma levels of Rifampicin. Further effect of Kajjali on
testosterone metabolism was investigated in vitro in rat liver microsomes. The formation of 6βhydroxy testosterone from testosterone was used as an index of CYP3A4 activity in rat liver
microsomes under controlled conditions and in the presence of varying concentrations of Kajjali.
Kajjali was found to be a weak inhibitor of CYP3A with a 50 % decrease in enzyme activity
occurring at a concentration of ~50 µg/ml (IC50) in rat liver microsomes. Ketoconazole was used as
a standard inhibitor of CYP3A4. In rats, co-administration with Kajjali increased the Cmax, AUC and
t1/2 of Rifampicin by ~1.75, 1.5 and 1.35-fold, respectively. Thus, bioavailability enhancement is the
possible mechanism of increased activity of drugs co-administered with Kajjali, which could be due
to inhibition of first-pass intestinal metabolism as in the case with Rifampicin.
1.0 INTRODUCTION
Pharmacokinetic plays a major role in drug discovery and development of new chemical
entities (NCE’s). Approximately, 50 % NCE’s fail to make in drug development process due to
inadequate pharmacokinetic data such as absorption, distribution, metabolism and excretion
(ADME)1. Most of the drugs available are administered via the oral route; hence oral bioavailability
is the key factor for a drug to possess a better therapeutic value. The factors affecting bioavailability
can be divided into physiological, physicochemical and biopharmaceutical, primarily first pass
metabolism (FPM). Many sites in the body like liver, gut wall, kidney, lungs, etc., are involved in
drug metabolism. The cytochrome P450 superfamily comprises of many isozymes that play a
crucial role in metabolism. Approximately, 80 % of xenobiotics are metabolized by CYP3A4, 2D6,
2C9, 2C19, and 1A2 isoforms.2
Role of various pharmaceutical excipients in inhibiting P450 enzymes has been checked by many
investigators.3 Grapefruit juice increases the plasma concentrations of drugs that are substrates for
CYP3A4 (e.g., Felodipine, Triazolam, Midazolam, Terfenadine, Cyclosporine, Nifedipine, and
Diazepam) by inhibiting the enzyme4,5,6,7 Although typically a drug-drug interaction is regarded as
an adverse event, if managed effectively, this interaction could be used to enhance plasma
concentrations of high-clearance compounds, leading to increased bioavailability and, presumably,
efficacy.8
It is estimated that one-third of the world’s population is infected with the tubercle bacillus9 (Dye et
al., 1999; WHO, 2004). A number of efficacious anti-tubercular agents were discovered in the late
1940s and 1950s with the last, Rifampicin, introduced in the 1960s.10 Rifampicin is readily
absorbed from the gastrointestinal tract (90%). Despite of the high absorption, approximately 85 %
of Rifampicin is metabolized by the liver microsomal enzymes, thus limiting its bioavailability.11
Hence, when combined with a CYP450 inhibitor, its bioavailability may increase thus reducing the
dose, mycobacterial resistances, and cost of treatment with better quality of life for patients.
Kajjali is one such ayurvedic proprietary herbomineral preparation which is known to exhibit the
yogavahi property i.e. it potentiates the action of a drug concomitantly administered with it. This
study was undertaken to investigate the effect of Kajjali on the oral bioavailability of Rifampicin in
Wistar rats. Further, its effect was also evaluated in vitro on the substrate of CYP3A4 i.e.
hydroxylation of testosterone in rat liver microsomes.
2.0 MATERIALS AND METHODS
2.1 Reagents
Kajjali was gifted by Dhootpapeshwar Pvt Ltd Mumbai. Rifampicin, testosterone, hydroxy
testosterone, ketoconazole, acetonitrile, trisodium citrate, chloroform, methanol, bovine serum
albumin, sodium dithionite, Nicotinamide adenine dinucleotide phosphate (NADPH) were of
analytical grade and procured from commercial suppliers.
2.2 Experiments design and pharmacokinetic studies (Effect of Kajjali on bioavailability of
Rifampicin in vivo)
The study was performed using healthy male Wistar rats (250-300 g) procured from Haffkines
institute, Mumbai. All the animal experiments were performed in accordance with
Guidelines of
Committee for the purpose of Control and Supervision on Experiments on Animals (CPCSEA),
Government of India and institutional animal ethics committee of Institute of Chemical Technology
(IAEC-ICT), Mumbai. Animals were acclimatized to standard conditions of temperature (23 + 1 oC)
and humidity. The acclimatization period of 8 days was provided. The animals were allowed free
access to food consisting of standard pellet diet (Amrut brand) and filtered water ad libitum. The
animals were exposed to 12 h day and night cycle. Two separate and independent pharmacokinetic
studies were planned to investigate the effects of kajjali on the kinetics of rifampicin. For the
pharmacokinetic experiments rats were divided into two groups, each consisting of sub-groups of
six animals. Group A received Rifampicin (10 mg/kg) only, while Group B received Kajjali (15
mg/kg) along with Rifampicin (10 mg/kg) as a suspension in 0.5 % sodium carboxy methyl
cellulose (NaCMC).
On the day after dosing, multiple serial blood samples (approximately 0.3mL) were collected
through the retro-orbital sinus puncture in 0.5 ml eppendorf tubes containing trisodium citrate (4 %)
as anti-coagulant at 0, 1, 2, 4, 6, 8 and 10 h. The blood samples were centrifuged at 10,000 rpm for
10 min at 50 C and plasma was separated and stored at -200 C till further analysis.
2.3 Determination of Rifampicin concentration in plasma
After thawing at room temperature, 0.5 ml of plasma was treated thrice with 0.5 ml of ethyl acetate
to extract Rifampicin. The recovery of Rifampicin was 97%. Supernatant was collected, pooled and
analyzed by a sensitive and reproducible HPTLC method. The TLC plates were kept in a twin
trough chamber pre-saturated with mobile phase system consisting of chloroform: methanol (9:1) at
25 + 1 oC and 55 + 5 % relative humidity. The plates were allowed to run up to 70 % of its length.
The developed plates were scanned at λmax 340 nm. HPTLC system (Camag, Switzerland) equipped
with Linomat 5, PC based TLC scanner with WINCATS software program were used for the study.
2.4 Preparation of rat liver microsomes
Livers from male Sprague Dawley rats (n=5) were isolated and stored at –70 oC till further use. The
excised livers were thawed and weighed. After fine chopping with a fine pair of scissors, the livers
were homogenized in four time volume of initial liver weight in 10 mM Tris-HCL buffer containing
0.25 M sucrose, pH 7.4 in a tissue homogenizer equipped with a Teflon pestle. The homogenate
was centrifuged at 9000 X g for 10 min at 4oC in a refrigerated centrifuge. The resultant supernatant
was separated and stored at –700 C as S9 fraction and the residue was discarded. The S9 fraction
was centrifuged at 1,00,000 X g for 60 min at 4o C in an ultracentrifuge.12 The firmly packed pellets
of microsomes in the residue were resuspended in 100 mM Tris-HCL containing 20 % glycerol and
10 mM EDTA, pH 7.4. The microsomal fraction isolated was characterized for protein content by
Bradeford Method13 and CYP450 content was measured by the method of Omura and Sato14
2.5 CYP3A4 inhibition assay
Rat liver microsomes were prepared as mentioned above and were used for CYP3A4 inhibition
studies. For these studies, the reaction mixture (final volume 200 µl) consisted of the testosterone as
a classical substrate (90 µM), no drug (control) or Kajjali at various concentrations (0.01, 0.1, 1.0,
3.0, 10.0, 50.0 and 100 µg/ml) or Ketoconazole (standard inhibitor 0.3 and 3.0 µM concentration)
and rat liver microsomes (protein concentration 0.25 mg/ml) in 0.1 M sodium phosphate buffer (pH
7.4). The reaction was initiated by the addition of NADPH (10 mM) and after the completion of the
incubation period (25 min), 100 µl of the reaction volume was terminated by 100 µl of acetonitrile.
The mixture was centrifuged and supernatant was injected directly into HPLC.
2.6 Analytical procedure for estimation of 6-OH testosterone
After the termination of reaction, 50 µl of supernatant was injected into HPLC system for the
determination of 6-hydroxy testosterone formation. An analytical HPLC system of waters model
2998, equipped with Quaternary Pump, auto sampler, UV/Vis detection system was used for this
analysis. The stationary phase used was Agilent, Zorbax CN (4.6 x 250 mm), 5 µm. whereas the
mobile phase comprised of acetonitrile : water in ratio of 1:1 respectively was employed. The
analyte was eluted by isocratic method at a flow rate of 1.0 ml/min and detected at a wavelength of
240 nm at a retention time (RT) of 8.0 mins. The chromatograms were analyzed by Empower
software version 1.0
3.0 RESULTS AND DISCUSSION
3.1 Result
Pharmacokinetic indices of rifampicine were determined using non-compartmental analysis
(Kinetica 5 trial version). Doses were normalized based on body weight of the rat.
Pharmacokinetic parameters of Rifampicin in presence and absence of Kajjali have been
summarized in Table I and the plasma concentration-time profiles obtained are shown in Figure 1.
In the presence of Kajjali, the Cmax of Rifampicin increased by ~74% fold. Also the increase in
AUC0-10 h and AUC0-inf was 48 and ~65% respectively. Rifampicin achieved the maximum plasma
concentration within 2 h in presence of Kajjali as compared to 4 h when given alone. The plasma
half life (t1/2) increased by 1.35 times. Decrease in the elimination rate constant was also observed.
In the second part of experiment, effect of kajjali on CYP3A4 activity was determined in rat liver
microsomes. It was found that Kajjali inhibited the CYP3A activity in dose dependent manner with
IC50 being ~50 µg/ml (Figure 2).
3.2 Discussion
Pre-systemic intestinal and hepatic phase I metabolism plays a major role in oral drug
bioavailability. A drug, in possession of good intestinal absorption but high first pass metabolism
results in low bioavailability. CYP3A is estimated to metabolize about 50-70% of currently
administered drugs.15 Overall drug metabolizing enzymes limit the systemic bioavailability of
xenobiotics. It is very well known that high cellular expression of CYP3A in mature intestinal
enterocytes and liver creates hurdles in systemic availability of drugs. A Number of preclinical and
clinical studies suggest that the oral bioavailability of various CYP3A4 substrates can be increased
by concomitant administration of CYP3A4 inhibitors or inactivators.16 However; Cytochrome P450
inhibition has a significant role in clinical drug-drug interactions. Most commonly these type of
interactions lead to adverse effect and toxicity but they can be utilized to increase the therapeutic
value of those drugs with high first pass metabolism.
Rifampicin is one of the most commonly used drugs in the treatment of tuberculosis and leprosy. It
is a macrocyclic antibiotic which posses a major issue of oral bioavailability due to first pass
metabolism and autoinduction.17,18,19
This study was conducted to evaluate the effect of Kajjali, an ayurvedic herbomineral
bioavailability enhancer, on the oral pharmacokinetics of Rifampicin. The study showed that the
kajjali considerably increased the maximum plasma concentration (Cmax) of rifampicin by 74 % and
bioavailability by 48 %.
The increase in bioavailability can be due to the inhibition in CYP3A metabolism. To understand
the mechanism, we planned a study in which different concentration of kajjali was incubated with
testosterone (CYP3A4 substrate). The results showed that kajjali inhibited the CYP3A4 in dose
dependent manner and the IC50 was found to be around 50 µg/ml.
This pharmacological effect of Kajjali may be responsible for increase in bioavailability of
Rifampicin by causing inhibition of the CYP3A enzyme that hydrolyses Rifampicin. Thus
Rifampicin bioavailability can be greatly increased by administering along with kajjali an
Ayurvedic herbomineral preparation. This will be a breakthrough in Anti-tubercular therapy, where
the bioavailability of Rifampicin is compromised leading to a mycobacterial resistance and poor
prognosis of the disease.
4.0 CONCLUSION
Kajjali enhanced the bioavailability of Rifampicin by inhibiting its metabolism through CYP3A
enzymes. The experiment endorses the yogavahi property of kajjali as claimed in Ayurveda. The
yogavahi property of this ayurvedic herbomineral preparation can be exploited further to address the
cause of poor bioavailability of therapeutic drugs.
Conflict of interest
The authors declare that they have no conflict of interest.
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Table I: Effect of Kajjali on oral pharmacokinetics of Rifampicin
Rifampicin
Rifampicin (10 mg/kg) +
(10 mg/kg)
Kajjali (15 mg/kg)
Cmax (µg/ml)
7.86 + 0.10
13.66 + 0.45
AUC0-10 h (µg.h/ml)
49.8
73.5
AUC0-inf (µg.h/ml)
56.080
92.50
Tmax (h)
4.00
2.00
T1/2 (h)
2.64
3.58
Kel (hr-1)
0.263
0.193
Pharmacokinetic Parameter
Figure 1:
Plasma time profile of Rifampicin (10 mg/kg, p.o.) in presence and absence of
Kajjali (15 mg/kg, p.o.)
Figure 2: Inhibitory effect of Kajjali on 6-testosterone metabolism in rat liver microsomes