Yeshwant Singh, Mahendra Kumar Hidau,2△, Shio Kumar Singh*

1Pharmacokinetics & Metabolism Division, CSIR- Central Drug Research Institute, Lucknow, India-226031

2Academy of Scientific and Innovative Research, New Delhi, India

1. Introduction

Tuberculosis and malaria co-epidemics are very prevalent in most of the tropical and sub-tropical areas of the world that creates serious damage to socio-economic status besides public health concerns. More than six million people are killed by the co-infection of these two diseases with AIDS annually[1-3]. Patients are supposed to take co-medications for malaria and tuberculosis simultaneously,which may give rise to serious drug-drug interactions (DDIs). It has been reported that the greater the number of drugs the given to a patient, the more the risk of potential drug interactions. Incidences of such drug interactions raises up to 7% in those patients taking 6-10 drugs and 40% in those taking 16-20 drugs daily[4].

It is important to explore drug metabolism and pharmacokinetic(DMPK)properties of drug candidate in early stages of drug discovery and development. Besides DMPK, DDIs are routinely incorporated in the early phases of drug development pathway from a safety point of views. For this, preclinical studies are often carried out in experimental animals. From the preclinical data, the allometric scaling approaches that consider important anatomical and physiochemical variables in higher species as a power function of the body weight across species may be used to predict human pharmacokinetics (PK)aspects[5-8]. There are a number of genderdependent and gender-specific characteristics that are known to influence the PK of a drug. Physiological and anatomical differences between the genders of a species are the responsible factors influencing PK of a drug differently[9,10].

In the present study, in-vivo drug interactions were investigated between a novel trioxane anti-malarial candidate 97/78 (phaseⅠ clinical trials completed)and antitubercular drug rifabutin(interacting drug). Single oral dose drug interaction studies were performed in male and female Sprague Dawley (SD)rats to determine the changes in PK and intersexual interaction pattern of 97/78 upon rifabutin co-administration. The results of single dose interaction study were analyzed and in-vitro studies were performed to find out the mechanism behind the existence of these in-vivo interactions.

2. Materials and methods

2.1. Chemicals

Rifabutin was obtained as a gift sample from Lupin (Pune), India.Reference standards (purity ＞ 98%)of 97/78, 97/63 (Figure 1a)and internal standard (IS)arteether (Figure 1b)were obtained from the medicinal chemistry division, CDRI, Lucknow, India. Blank plasma was collected from drug free healthy male and female SD rats procured from the Laboratory Animal Services Division,CDRI. All other chemicals and reagents were of analytical liquid chromatographic (LC)grade. Dextran coated charcoal for plasma protein binding study was obtained from Sigma chemicals USA.Dulbecco’s Phosphate buffered saline (Ca2+and Mg2+free)was purchased from Hi media Lab. Pvt. Ltd., Mumbai. For metabolism stability studies, Tris (hydroxymethyl)aminomethane (tris base),KCl, MgCl2·6H2O, bovine serum albumin and a reduced form of β-nicotinamide adenine dinucleotide phosphate were purchased from Sisco Research Laboratory (Mumbai, Maharashtra, India).1-aminobenzotriazole, Bradford reagent and diethyl ether were procured from Qualigens (Mumbai), Loba Chemie (Mumbai)and TKM Pharma (Hyderabad, AP, India), respectively. Rat liver microsomes were prepared by using the methods adapted from literature[11].

2.2. Subjects and study design

Healthy male and female SD rats (250-270 g)free from any symptoms of pathophysiology were obtained from Laboratory Animal Services Division, CDRI (approval no. IAEC/2012/91Ns)were allowed to acclimatize for 3 d in ventilated polypropylene cases at standard laboratory conditions [12 h light-dark cycle, (22± 2)℃ temperature and (55 ± 5)% relative humidity]before the commencement of study. Subjects were fasted for about 10-12 h before and 2 h following drug administration with water ab libitum.Animals were cared in accordance with principles of the guide for care and use of laboratory animals [Department of health education and welfare, number (NIH)85-32].

2.2.1. In-vivo studies

We conducted a parallel design, single dose/single dose pharmacokinetic interaction study that comprised of two groups of animals. One group of subjects was orally administered with 97/78 at 40 mg/kg, while other group received 97/78 followed by rifabutin at 40 mg/kg dose. Sparse sampling approach was used to collect blood samples through cardiac puncture (first sample)and inferior vena cava (terminal sample). Blood samples were collected before dosing(0 time point)and up to 96 h post dose. Blood samples were stored in ice until centrifugation. Plasma was separated by centrifugation at 2 200 g for 5 min and stored at –60 ℃ until analysis. Analysis was performed within a month of sample collection. All experimental procedures were approved and performed in accordance with the guidelines of the Institutional Animal Experimentation Ethics Committee (IAEC/CPCSEA/Regulatory Agencies).

The formulations used in the study were aqueous suspension of 97/78 and rifabutin in 0.25% carboxy methyl cellulose. Both the formulations were subjected to quality control checks to ensure strength and content uniformity. The strength of the formulations for 97/78 and rifabutin was 20 and 35 mg/mL respectively.

2.2.2. In-vitro studies

In-vitro studies were then conducted to find out the mechanism behind the in-vivo study observations. In-vitro plasma protein binding studies were conducted to find out the changes in the plasma protein bindings of 97/78, while in-vitro drug metabolism studies were conducted to find out the interactions at the level of metabolism.

Protein binding was estimated using modified charcoal adsorption method adopted from literature[12]. The study was conducted in three replicates each at 100 and 1 000 ng/mL spiked plasma concentrations. The reactions were carried out in two sets, one set involving spiked 97/63 only, while other set spiked with 97/63 and rifabutin. The spiked plasma was allowed to equilibrate for 20 min.before the commencement of study. Serial samples (200 µL)were withdrawn at 0, 5, 10 and 15 min. in polypropylene centrifuge tubes and centrifuged at 10 000 g for 4 min at 37 ℃. Supernatant was collected and stored at -70 ℃ till LC-MS/MS analysis.

97/63 alone, 97/63 with rifabutin and testosterone (as a control)were incubated separately in a shaking water bath in an incubation mixture of 0.1 M Tris buffer, 5 mM MgCl2·6H2O and 0.4 mg/mL of rat microsomes at [(37 ± 1)℃]. The concentrations used for incubation were 2 mM, for 97/63, 5 mM for rifampicin and 2 mM for testosterone. β-nicotinamide adenine dinucleotide phosphate(2 mM)was pre-incubation for 4 min. before the commencement of the study. Two sets of control were used in the study. In the first set, β-nicotinamide adenine dinucleotide phosphate was substituted with an equal volume of Tris buffer, while other set of controls was incubated consisting of all incubation components except microsomes. The reactions were quenched with ice cold ACN spiked with 4 µM of IS at different time points ranging from 0 to 60 min.Samples were processed and supernatant was subjected to UFLC analysis. Before starting these experiments, non-specific binding of drug 97/63 with microsomal proteins was determined by incubating 97/63 in an optimized incubation milieu (0.4 mg/mL of rat liver microsomes at [(37 ± 1)℃]in a shaking water bath at varying concentrations ranging from 0.2 to 30 µM for 5 min.

2.3. Pharmacokinetic sample processing and bioanalysis

All study samples were quantitatively estimated for the determination of 97/63 (active metabolite of 97/78). A partially validated method in Q-trap 5500 LC–MS/MS mass spectrometer(Applied Biosystems, MDS Sciex, USA)with Analyst 1.6 software was used to determine plasma concentration of 97/63. The assay was linear over the range 0.625-80 ng/mL. 10 µL of IS was spiked into each study sample followed by a single step protein precipitation method using acetonitrile. Mass spectrometry was performed in positive mode electro spray ionization with capillary voltage 5.5 KV.The MS/MS transitions of m/z 418.2→119.1 and 330.3→267.4 were used to quantify 97/63 and IS respectively. HPLC elution was carried out in isocratic mode with mobile phase comprising of 80:20 (v/v)ACN (0.1% formic acid)and ammonium acetate buffer (10 mM,pH 4.0)using a Phenomenex C 18 Columbus (50 mm × 2 mm, 5 µparticle size)equipped with a security guard cartridge. Separation was achieved at 450 µL/min flow rate with injection volume of 10µL[13,14]. Processed sample were maintained in auto sampler at 4 ℃.

2.4. Statistical analysis

The study endpoints examined were area under the curve (AUC0-last), maximum plasma concentration (Cmax), time to attain Cmax(Tmax),elimination half life (T1/2)and mean residence time (MRT)and the relative bioavailability (RB). The PK parameters were calculated by non-compartmental analysis using Winnonlin software (Version 1.5,Pharsight Corporation). All results were expressed as mean ± SEM.RB was calculated as:

The results of the in-vitro metabolic study were expressed as the %drug remaining, which was calculated as:

The % plasma protein binding was calculated as,

It has been reported that 97/78 (prodrug)converts into 97/63(active metabolite)within a few minutes in biological matrices like plasma[15], thus samples were analyzed for the determination of 97/63, post 97/78 oral dose administration alone and with rifabutin in male and female rats. All the results are expressed as mean values± SEM, n=4. The values of various PK parameters for 97/78 alone and co-administration with rifabutin were statistically compared with‘2-tailed Students t-test’. The values were considered statistically significantly different for P＜0.05.

3. Results

3.1. Pharmacokinetic drug interaction studies

3.1.1. Male rats

Table 1 summarizes PK parameters of 97/63 post 97/78 administration alone and with rifabutin in male rats. The value of Cmaxwas (0.56 ± 0.18)µg/mL at corresponding Tmaxof (5.33 ± 0.76)h when 97/78 was given alone. While upon co-administration with rifabutin these values were found (0.13 ± 0.03)µg/mL and (1.83 ±1.25)h for Cmaxand Tmaxrespectively. Figure 2 represents plasma concentration-time profile of 97/63, when 97/78 was given alone and in combination with rifabutin in male rats.

3.1.2. Female rats

Table 1 summarizes PK parameters of 97/63 post 97/78 administration alone and with rifabutin in female rats. The value of Cmaxwas (0.79 ± 0.10)µg/mL at corresponding Tmaxof (6.00 ± 0.00)h when 97/78 was given alone. While upon co-administration with rifabutin these values were found (0.17 ± 0.03)µg/mL and (1.83 ±1.25)h for Cmaxand Tmaxrespectively. Figure 3 represents plasma concentration-time profile of 97/63, when 97/78 was given alone and in combination with rifabutin in female rats.

Table 1Comparative PK parameters of 97/63 after 97/78 alone and co-administration with rifabutin in male and female rats (mean ± SEM, n=3).

3.2. In-vitro drug interaction studies

3.2.1. In-vitro metabolism study

The experiment was carried out to determine the non-specific binding of 97/63 with microsomal proteins. Non-specific binding was predicted from the disappearance of 97/63, upon its incubation in an optimized incubation milieu of rat liver microsomes at various concentrations ranging from 0.1 to 20 µM for 5, 10, 15 min. It revealed that there were non-significant protein-ligand interactions(data not shown). Figure 4 represents the % drug remaining upon incubation of 97/63 alone and 97/63 with rifabutin in optimized microsomal mixture at various time intervals.

3.2.2. In-vitro plasma protein binding study

Table 2 represents % plasma protein binding of 97/63, when spiked alone and with rifabutin in SD rat plasma. It was found that there is slight increase in 97/63 metabolism when 97/63 was incubated with rifabutin, compared to metabolism of 97/63 upon alone incubation.

Table 2Percent plasma protein binding of 97/63, when spiked alone and with rifabutin in SD rat plasma.

Figure 5 represents plasma concentration-time profile of 97/63,when 97/78 was given alone in male and female rats.

On the other hand, when 97/78 was co-administered with rifabutin in male rats, statistically significant differences were reported in PK parameters of 97/63. It was observed that rifabutin co-administration reduced systemic exposure of 97/63 by a factor of 3-4. In terms of AUC0-last, the value was (4.03 ± 0.60)µg·h·mL-1when 97/78 was given alone, while it was decreased to (1.13 ± 0.10)µg·h·mL-1upon co-administration of rifabutin (Figure 6).alone in male and female rats (mean ± SEM, n=3).

4. Discussion

It is important to assess drug’s safety and effectiveness during the drug development phase in terms of drug interactions between investigational new drug and other drugs, so as to define existence of any potential DDIs that in turn indicates the need for additional therapeutics monitoring, dose adjustments and /or contraindication to concurrent use[8]. Generally, the more the number of drugs is given simultaneously the greater is the potential of existence of DDIs. Keeping this in view, the study was designed to determine the influence of rifabutin co-administration on PK of 97/63 in male and female SD rats to assess co-administered and intersexual differences.

It was found that 97/63 exhibited irregular plasma concentrationtime profile in both the genders when 97/78 was either given alone or with rifabutin. However, when 97/78 was given alone, there were no intersexual differences in PK parameters of 97/63 ie. both the genders exhibited similar PK profile as evident from the data in our study. Though, the value of Cmaxin female rats was found to be greater than male rats, but the difference was statistically non-significant in terms of P＞0.05. All other PK parameters were comparable for both the genders.

On the other hand, when 97/78 was co-administered with rifabutin in male rats, statistically significant differences were reported in PK parameters of 97/63. It was observed that rifabutin co-administration reduced systemic exposure of 97/63 by a factor of 3-4. In the similar manner, 3-4 fold decrease was observed in Cmaxwhich is explicitly indicated by the reduced maximum plasma concentration of 97/63 from (0.56 ± 0.18)to (0.13 ± 0.03)µg/mL upon rifabutin coadministration. Statistically significant difference was also observed for Tmaxvalues. However, T1/2and MRT values were comparable in both the cases i.e. no significant differences were observed.

Similarly, in female rats statistically significant differences were reported in PK parameters of 97/63 when 97/78 was co-administered with rifabutin. It was observed that rifabutin co-administration reduced systemic exposure of 97/63 by a factor of 3-4 like male rats. In terms of AUC0-last, the value was (5.44 ± 1.15)µg·h·mL-1when 97/78 was given alone, while it was decreased to (1.23 ± 1.13)µg·h·mL-1upon co-administration of rifabutin. Similar to male rats,maximum plasma concentration of 97/63 was reduced from (0.79± 0.10)to (0.17 ± 0.03)µg/mL-1upon rifabutin co-administration in females too, indicating 3-4 fold decrease in Cmax. Statistically significant difference was observed for Tmaxvalues, while T1/2and MRT values were comparable in both the cases i.e. no significant differences were observed.

As discussed earlier, no significant differences (in terms of P-value)were observed in PK parameters of 97/63 when 97/78 was given alone in male and female rats ie. no intersexual influences were reported. In the similar way, when rifabutin was co-administered with 97/78, no intersex differences were observed in the interaction pattern. In both the genders, 3-4 fold decrease was reported in the systemic exposure of 97/63 upon rifabutin co-administration with 97/78.

Overall, in-vivo studies have shown that there was a significant decrease in the systemic exposure of 97/63 upon rifabutin coadministration in both the genders. However, we hypothesized that there could be muti-fold decrease in the systemic exposure of 97/63,when 97/78 would have been co-administered with rifabutin for long duration, since rifabutin is a moderate inducer of hepatic CYP 3A.Thus, a multiple dose study may be required to estimate and explore these aspects of interactions. The results of our study thus provide abasic information about the existence of drug interactions between rifabutin and 97/78, which can be used to conduct a multiple dose drug interaction study.

Further, we exercised to find out the mechanism behind the existence of interaction between 97/78 and rifabutin in-vitro. Invitro metabolism studies in rat liver microsome revealed that upon rifabutin co-incubation with 97/63, there was 10%-12% increase in the metabolism of 97/63 compared to 97/63 incubation alone.It was found that about 48% of 97/63 get metabolized upon alone incubation, compared to about 60% metabolism of 97/63 upon rifabutin co-incubation. Thus, it could be reasoned that the decreased systemic exposure of 97/63 upon rifabutin co-administration can be in-part attributed to the increased metabolism of 97/63 in presence of rifabutin. However, others factors like interference in the absorption may be one among the reasons responsible for decreased systemic exposure of 97/63.

In-vitro plasma protein binding studies have shown that there was a very strong displacement of 97/63 from plasma proteins when 97/63 was added with rifabutin into rat plasma. The extent of plasma protein binding of 97/63 was found to be decreased from 54%-55%to a very low value of 6%-8% upon rifabutin addition. It could be speculated from the results of plasma protein bindings that upon displacement of 97/63 from plasma proteins, the increased free drug concentration of 97/63 is more susceptible to extraction by hepatocytes and hence more liable to get metabolized by hepatic CYPs, thus contributing to the decrease in the systemic exposure of 97/63.

From the above discussion, it could be concluded that rifabutin co-administration altered PK parameters of 97/63 in SD rats. A significant decrease was reported in the systemic exposure of 97/63, which indicates that the combination of these two drugs may require some dose adjustments. However, no intersexual influences were reported in the interaction pattern. The results of in-vivo interaction studies were well correlated with in-vitro studies and attempts were made to explain the mechanism behind the existence of drug interaction. Though this was a single dose interaction study in SD rats, multiple dose drug interaction study can be conducted on the basis of the informations obtained in this study. Further,clinical studies are required to determine the potential of these drug interactions in humans. The results obtained in this study can be extrapolated to humans using proper simulation models keeping in view all the gender and species differences in relation to physiochemical variations.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

The authors are sincerely thankful to the Council for Scientific and Industrial Research (CSIR), India, for providing research fellowships and to the Director, CDRI, for providing facilities and infrastructure for the study. DRI communication number is 8972.

[1]Li XX, Zhou XN. Co-infection of tuberculosis and parasitic diseases in humans: a systematic review. Parasit Vectors 2013; 6: 79.

[2]UNDP, World Bank: WHO Special Programme for Research and Training in Tropical Diseases (TDR): Tropical Disease Research:Progress 1999–2000. Geneva, Switzerland: World Health Organization Press; 2001. WHO/TDR/GEN/01.5

[3]Shapiro HM, Perlmutter NG. Killer applications: Toward affordable rapid cell-based diagnostics for malaria and tuberculosis. Cytometry Part B:Clinical Cytometry 2008; 74: S152-S64.

[4]Puckett WH, Visconti J. An epidemiological study of the clinical significance of drug-drug interactions in a private community hospital.American J Health-System Pharm 1971; 28: 247-253.

[5]Damanhonri Z, Gumnbleton M, Nicholls P, Shaw M. In-vivo effects of itraconazole on hepatic mixed-function oxidase. J Antimicrob Chemother 1988; 21: 187-194.

[6]Ishigami M, Kawabata K, Takasaki W, Ikeda T, Komai T, Ito K, et al.Drug interaction between simvastatin and itraconazole in male and female rats. Drug Metabolism Disposition 2001; 29: 1068-1072.

[7]Poggesi I. Predicting human pharmacokinetics from preclinical data.Curr Opin Drug Discovery Dev 2004; 7: 100-111.

[8]Drug interaction studies-Study design, data analysis, implications for dosing, and labeling recommendations, U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER).

[9]Ciccone G, Holdcroft A. Drugs and sex differences: a review of drugs relating to anaesthesia. Survey Anesthesiol 1999; 43: 293-294.

[10]Martignoni M, Groothuis GM, de Kanter R. Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism,inhibition and induction. Expert Opin Drug Metab Toxicol 2006.

[11]Nelson AC, Huang W, Moody DE. Variables in human liver microsome preparation: impact on the kinetics of l- -acetylmethadol (LAAM)N-demethylation and dextromethorphano-demethylation. Drug Metab Dispos 2001; 29: 319-325.

[12]Khurana M, Paliwal JK, Kamboj VP, Gupta RC. Binding of centchroman with human serum as determined by charcoal adsorption method. Int J Pharm 1999; 192: 109-114.

[13]Kushwaha HN. Drug drug interaction and pharmacokinetic studies of novel lucknow: antimalarial molecules with antiepileptic drugs.Lucknow: Integral University； 2013.

[14]Singh RP, Sabarinath S, Gautam N, Gupta RC, Singh SK.Pharmacokinetic study of the novel, synthetic trioxane antimalarial compound 97-78 in rats using an LC-MS/MS method for quantification.Arzneim Forsch 2011; 61: 120-125.

[15]Shafiq N, Rajagopalan S, Kushwaha HN, Mittal N, Chandurkar N,Bhalla A, et al. Single ascending dose safety and pharmacokinetics of CDRI-97/78: First-in-human study of a novel antimalarial drug. Malaria Res Treatment 2014; 2014: 372521.