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Please be advised that this information was generated on 2021-10-04 and may be subject to change. CLINICAL PHARMACOLOGICAL STUDIES IN TUBERCULOSIS PATIENTS:

‘Research from the Kilimanjaro Region, Tanzania’

Hadija Hamisi Semvua CLINICAL PHARMACOLOGICAL STUDIES IN TUBERCULOSIS PATIENTS: ‘Research from the Kilimanjaro Region, Tanzania’. Thesis, Radboud University Nijmegen Medical Centre, the Netherlands Design and Layout by: Offpage, Amsterdam, the Netherlands Printed by: Offpage, Amsterdam, the Netherlands ISBN/EAN: 978-94-6182-403-5 © 2014 Hadija Hamisi Semvua No part of this thesis may be reproduced or transmitted in any form or by any means, electronic or mechanical including photocopy, recording or otherwise without permission of the author. Printing and dissemination of this thesis was financially supported by the Radboud University Nijmegen Medical Centre. CLINICAL PHARMACOLOGICAL STUDIES IN TUBERCULOSIS PATIENTS:

‘Research from the Kilimanjaro Region, Tanzania’

Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het college van decanen in het openbaar te verdedigen op woensdag 26 maart 2014 om 13.00 uur precies

door

Hadija Hamisi Semvua geboren op 22 Augustus 1968 te Mwanga, Kilimanjaro, Tanzania Promotor Prof. dr. D.M. Burger

Copromotoren Dr. R.E. Aarnoutse Dr. M.J. Boeree Prof. dr. G.S. Kibiki, Kilimanjaro Christian Medical College, Moshi, Tanzania

Manuscriptcommissie Prof. dr. W. M. V. Dolmans Prof. dr. J. W. Mouton Dr. J.W.C. Alffenaar, Universitair Medisch Centrum Groningen, Nederland CLINICAL PHARMACOLOGICAL STUDIES IN TUBERCULOSIS PATIENTS:

‘Research from the Kilimanjaro Region, Tanzania’

Doctoral thesis to obtain the degree of doctor from Radboud University Nijmegen on the authority of the Rector Magnificus prof. dr. S.C.J.J. Kortmann, according to the decision of the Council of Deans to be defended on Wednesday, 26 March 2014 at 13:00 hours

by

Hadija Hamisi Semvua born in Mwanga, Kilimanjaro, Tanzania on 22 August 1968 Supervisor Prof. dr. D.M. Burger

Co-supervisors Dr. R.E. Aarnoutse Dr. M.J. Boeree Prof. dr. G.S. Kibiki, Kilimanjaro Christian Medical College, Moshi, Tanzania

Names of the members of the Doctoral Thesis Committee Prof. dr. W. M. V. Dolmans Prof. dr. J. W. Mouton Dr. J.W.C. Alffenaar, University Medical Center Groningen, the Nederlands Contents

Chapter 1 Introduction and outline of the thesis 11

Chapter 2a Pharmacological interactions between rifampicin and antiretroviral drugs: challenges and research priorities for resource-limited settings. Manuscript submitted 27

Chapter 2b AtriplaR/anti-TB combination in TB/HIV patients. Drug in focus. BMC Research Notes 2011;4:511 51

Chapter 3 Efavirenz, tenofovir and emtricitabine combined with first-line tuberculosis treatment in tuberculosis-HIV-co-infected Tanzania patients: a pharmacokinetic and safety study. Antiviral Therapy 2013;18(1):105-13 65

Chapter 4 Pharmacokinetics of first line tuberculosis drugs in Tanzanian patients Antimicrobial Agents and Chemotherapy 2013;57(7):3208 79

Chapter 5 Associations between salivary, protein-unbound and total plasma concentrations of rifampicin. Manuscript submitted 93

Chapter 6 The effect of diabetes mellitus on exposure to tuberculosis drugs in Tanzanian patients. Manuscript in final preparation 109

Chapter 7 Antituberculosis drug-induced hepatotoxicity is uncommon in Tanzanian hospitalized pulmonary tuberculosis patients. Tropical Medicine and International Health.2010;15:268-272 127

Chapter 8a Fluoroquinolone sale in northern Tanzania: a potential threat for fluoroquinolone use in tuberculosis treatment. J Antimicrob Chemother 2010;65:145–147 137

Chapter 8b Low rate of fluoroquinolone resistance in Mycobacterium tuberculosis isolates from northern Tanzania. Journal of Antimicrobial Chemotherapy 2011 Aug;66(8):1810-4 145

Chapter 9 General discussion 155

7 Chapter 10a Summary 171

Chapter 10b Samenvatting 177

Chapter 10c Muhtasari 183

List of publications 189

Acknowledgements 191

Curriculum Vitae 193

8

Introduction 1 and outline of the thesis

Introduction and outline of the thesis

Tuberculosis Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. It is easier 1 to catch M. tuberculosis in places where people are crowding together, such as in prisons, schools, mines or marketplaces. However, not everyone who acquires infection develops clinical symptoms, because our bodies fight M. tuberculosis. If the body does not win the fight, TB causes disease. The commonest form of TB is pulmonary tuberculosis (PTB), which affects the lungs [1]. People with lung TB may cough up phlegm that contains the germs, which is how TB is mostly spread. TB germs are very tough, and can live in dust for months. TB can also infect other parts of the body such as lymph nodes, bones and pleural cavities (commonly known as extra-pulmonary TB). Night sweats, fever and weight loss are the common symptoms of active TB. Identification of the tubercle bacilli by sputum smear microscopy is the first step in diagnosis. Sputum culture is a more sensitive and preferred method, however this method is slow and expensive [2]. TB remains a major global health problem. It is ranked as the second most mortality- causing infectious disease after human immunodeficiency virus (HIV) disease worldwide [3]. An estimated 1.4 million (range 1.2-1.5 million) people annually die from TB globally [4]. It is the leading cause of death from a curable infectious disease, despite the availability of efficacious treatment for decades. One-third of the world population is latently infected with M. tuberculosis and has a risk of developing active TB [5]. The World Health Organization (WHO) estimates 9 million new cases of TB (range 8.5-9.2 million) each year (Figure 1). TB incidence is highest in Africa; the number of incident cases in African countries was estimated at 2.3 million in 2010 (range, 2.1 million–2.5 million [4]. TB and HIV infection are overlapping epidemics. HIV infection is the strongest risk factor for the development of TB. It increases the progression from TB infection to active disease [6,7]. On the other hand, TB increases the risk of progression from HIV infection to AIDS [8,9]. Of the 9 million incident cases of TB worldwide, 13% were among people living with HIV and the majority of cases were adults aged 15-59 years [4]. Without combined treatment, mortality rates are high (up to 2.2 million deaths per year) [10]. The proportion of TB cases co-infected with

Figure 1. Estimated TB incidence rates 2010 (Source: WHO TB report 2011)

13 chapter 1

HIV is highest in the African countries (figure 2); overall, African countries accounted for 82% 1 of TB cases among people living with HIV [4]. Apart from HIV infection, there is another disease that tends to evolve as an epidemic and that overlaps with the TB epidemic, i.e. diabetes mellitus (DM). There is a rapid increase in the global prevalence of DM, especially in developing countries, where TB is endemic. Diabetes mellitus (DM) is a well-known risk factor for acquiring tuberculosis (TB) [11], with prevalence rates among TB patients ranging from 10 to 30% [11,12,13]. In addition, diabetes exerts a negative effect on the response to TB treatment. Tuberculosis continues to be among the major public health problems in Tanzania as well. The number of reported TB cases has steadily increased from 11,753 in 1983 to 64,269 in 2009 [14]. The WHO 2011 report showed that the majority of cases appear in young adult population groups aged 15-45 years [1].

Tuberculosis treatment The introduction of the sanatorium care provided the first real step in TB management. New advances then followed in rapid succession. In 1882, Robert Koch discovered a staining technique that enabled him to see M. tuberculosis. The world was excited by the certainty that now the fight against humanity’s deadliest enemy could really begin. TB drug development began in the 1940s when streptomycin was discovered [15], but after treating some patients, resistant mutants began to appear. The discovery of isoniazid in 1952 brought renewed hope. This potent agent resulted in rapid sterilization of sputum and, when used in conjunction with streptomycin, reduced the rates of drug-induced resistance. In 1954 pyrazinamide was discovered and later ethambutol (1962) and rifampicin (1963), which revolutionized TB treatment [16].

Figure 2. Estimated HIV prevalence in new TB cases (Source: WHO TB report 2011)

14 Introduction and outline of the thesis

The current treatment of tuberculosis consists of two phases, which are the so-called initial or intensive phase and the continuation phase. In the intensive phase, all actively dividing 1 bacteria should be killed as soon as possible. The continuation phase is meant to eradicate the persisters, i.e. presumably slower replicating or dormant bacteria. The recommended initial phase for the treatment of tuberculosis consists of rifampicin 10mg/kg (maximum 600mg), isoniazid 5mg/kg (maximum 300mg), pyrazinamide 15–30mg/kg (maximum 2g), and ethambutol 15–20mg/kg (maximum 1.6g) given daily for 8 weeks [17]. This is followed by the continuation phase of isoniazid 5mg/kg (maximum 300mg) and rifampicin 10mg/kg (maximum 600mg). Clearly, TB drugs are not used singly except in latent TB or chemoprophylaxis (giving medication to people who have no active disease and are currently well). This is because the regimens that use only a single drug result in the rapid development of resistance and treatment failure [18]. This risk is especially large among large vital bacilli populations such as present at the onset of therapy. This is why the intensive phase constitutes even four drugs. The different drugs in the regimen have different modes of action. Isoniazid is bactericidal against replicating bacteria. Ethambutol is bacteriostatic at low doses, but is used in TB treatment at higher, bactericidal doses. Rifampicin is bactericidal and has a sterilizing effect. Pyrazinamide is only weakly bactericidal, but is very effective against bacteria located in acidic environments, inside macrophages, or in areas of acute inflammation. In order to prevent non-adherence to TB drugs and treatment dropouts, the WHO has introduced the Directly Observed Therapy (DOT) strategy. One of the crucial components of this strategy is the direct observation by trained personnel or relatives of patients when they take their medicines, as a means to ensure adherence. There is a definite supply of drugs, and standardized reporting and recording of cases and treatment outcomes.

First-line and second-line anti-tuberculosis drugs Isoniazid. Isoniazid is highly bactericidal against replicating M. tuberculosis. It is normally taken orally but may be administered intramuscularly or intra­venously to critically ill patients. It is rapidly absorbed and diffuses readily into all fluids and tissues. The plasma half-life, which is genetically determined, varies from less than 1 hour in fast acetylators to more than 3 hours in slow acetylators [19]. Isoniazid is largely excreted in the urine within 24 hours, mostly as inactive metabolites. Isoniazid is used with caution and clinical monitoring (liver function tests,

Isoniazid chemical structure

15 chapter 1

if possible) should be performed dur­ing treatment of patients with pre-existing liver disease. 1 Patients at risk of peripheral neuropathy, because of malnutrition, chronic alcohol dependence, HIV infection, pregnancy, breastfeeding, renal failure or diabetes, should additionally receive pyri­doxine, 10 mg daily. It is contraindicated in case of known hypersensitivity and jaundice patients.

Rifampicin. Rifampicin is a semisynthetic derivative of rifamycin. It is a complex macrocyclic antibi­otic that inhibits ribonucleic acid synthesis in a broad range of microbial pathogens. It has bactericidal action and a potent sterilizing effect against M. tuberculosis in both cellular and extracellular locations [20]. Rifampicin should preferably be given at least 30 minutes before meals, since absorp­tion is reduced when it is taken with food [21]. Following oral administration, it is rapidly absorbed and distributed throughout the cellular tissues and body fluids. A daily dose of 600mg administered on an empty stomach produces an average peak serum concentration of about 10 mg/L in 2–4 hours, which subsequently decays, with a half-life of 2–3 hours [22]. It is extensively recycled in the enterohe­patic circulation, and metabolites formed by deacetylation in the liver are eventually excreted in the faeces [23,24]. Clinical monitoring (liver function tests, if possible) should be performed during treatment of all patients with pre-existing liver disease, who are at increased risk of further liver damage. Patients should be warned that treatment may cause reddish coloration of all body secretions (urine, tears, saliva, sweat, semen and sputum), and that contact lenses and clothing may be irreversibly stained.

Rifampicin chemical structure

Pyrazinamide. Pyrazinamide is a synthetic analogue of nicotinamide that is only weakly bactericidal against M. tuberculosis but has potent sterilizing activity, particularly in the relatively acidic intracellular environment of macrophages and in areas of acute inflammation. It is highly effective during the first 2 months of treatment while acute inflammatory changes persist [25]. Its use has enabled treatment regimens to be shortened and the risk of relapse to be reduced. Pyrazinamide is administered orally. It is readily absorbed from the gastrointestinal tract and is rapidly distributed throughout all tissues and fluids. Peak plasma concentrations are attained in 2 hours and the plasma half-life is about 10 hours. It is metabolized mainly in

16 Introduction and outline of the thesis the liver and excreted largely in the urine. It is contraindicated for known hypersensitivity and active jaundice. A common adverse effect of pyrazinamide is hepatotoxicity. 1

Pyrazinamide chemical structure

Ethambutol. Ethambutol is used in combination with other anti-TB drugs to prevent or delay the emergence of resistant strains. It is administered orally and is readily absorbed from the gastrointestinal tract. Plasma concentration peaks within 2–4 hours and decays with a half-life of 3–4 hours [19]. It is excreted in the urine both unchanged and as inactive hepatic metabolites. About 20% is excreted unchanged in the faeces [26]. Dosage must always be carefully calculated on a weight basis to avoid toxicity, and the dose or the dosing interval should be adjusted in patients with impaired renal function (creatinine clearance <70 ml/min) [27]. If creatinine clearance is less than 30 ml/minute, ethambutol should be administered 3 times per week. It is contraindicated for known hypersensitivity and pre-existing optic neuritis. Patients should be advised to discontinue treatment immediately and to report to a clinician if their sight or perception of colour deteriorates. Ocular examination is recommended before and during treatment.

Ethambutol chemical structure

Second-line TB drugs. The first-line TB drugs are also designated as TB drugs from group 1. The second-line TB drugs, used for multidrug resistant TB (MDR-TB) are then subdivided. Group 2 which are aminoglycoside injections, e.g. amikacine, kanamycin), group 3 (fluoroquinolones) e.g. ciprofloxacin, levofloxacin, moxifloxacin and ofloxacin, group 4 (e.g. cycloserine, ethionamide, p-aminosalicylic acid) and group 5 (many agents with unclear efficacy) [28]). Second-line drugs are generally less effective, more toxic and more expensive than the first- line TB drugs. The lower in the group hierarchy, the less evidence is available for their efficacy in TB treatment. However, the fluoroquinolones have shown promise to shorten the TB

17 chapter 1

treatment duration and are currently being tested as first-line drugs in a phase III clinical trial. 1 A concern is that fluoroquinolones are widely used for other infections including respiratory tract infections in patients with (undiagnosed) active TB, which could result in fluoroquinolone –resistant M. tuberculosis.

Pharmacological studies Pharmacology (from the Greek pharmakon, meaning “poison” in classic Greek and “drug” in modern Greek), is the branch of medicine concerned with the study of drug action while clinical pharmacology is the study of drugs in their clinical use. Traditionally there are two branches of pharmacology; pharmacokinetics and pharmacodynamics. Pharmacokinetics includes the study of the mechanisms of absorption, distribution, metabolism and excretion of an administered drug. It looks at the rate at which a drug action begins and the duration of the effect, the chemical changes of the substance in the body and the effects and routes of excretion of the metabolites of the drug. Important pharmacokinetic parameters are the maximum concentration, area under the concentration versus time curve, the time to reach the maximum concentration and the minimum concentration (Figure 3). Others are half-life, clearance and volume of distribution of the drug. All these pharmacokinetic parameters are normally derived from studies in which blood is sampled in patients after administration of TB drugs. Sampling and bio-analysis of saliva may be more patient-friendly, as it does not require the attendance of medical staff and may be less costly. However, there are limited data of TB Chapter 1 drugs in saliva. Pharmacodynamic studies examine the subsequent relationship between drug concentrations achieved and eventual desirable and undesirable effects of the drug. Stated otherwise, pharmacokinetic studies examine what the body does to the drug, and pharmacodynamic studies evaluate how the drug affects the body. Clinical pharmacology

C A max Product A

Cmax B Product B

AUC Drug concentration (mg/L)

Tmax B Time after dose (hours)

Tmax A

Cmax maximum plasma drug concentration Tmax time required to achieve a maximal concentration AUC total area under the plasma drug concentration-time curve Burkitt, Australian Prescriber, 2003 Figure 3: Pharmacokinetic curves after oral administration of different formulations Figure 3. Pharmacokinetic curves after oral administration of different formulations

Pharmacological concerns about anti-TB drugs 18 A. TB/HIV drug interactions The treatment of TB in individuals with HIV infection is the same as that for patients with TB who do not have HIV disease. Standard first-line therapy for TB is effective in patients with HIV infection–related TB. However, management of co-infected patients can be complex because of pharmacokinetic and pharmacodynamic interactions between TB drugs and antiretroviral drugs [32]. These interactions may cause decreased concentrations of some TB drugs, adverse events due to overlapping adverse effect profiles and immune reconstitution disease. More specifically, a rifampicin based TB regimen lowers the concentration of non-nucleoside reverse transcriptase inhibitors, protease inhibitors and some new antiretroviral drugs such as maraviroc, raltegravir and etravirine [33]. Therefore, continuous original studies and reviews on interactions between anti-tuberculosis and antiretroviral drugs are necessary.

B. Pharmacokinetic sampling Pharmacokinetic studies are very useful in determining pharmacological activity and toxicity of a drug. Therefore, large studies with frequent blood sampling are required. However, these studies necessitate the attendance of medical staff. Therefore, the use of saliva samples would facilitate studies to evaluate concentrations of drugs. The advantage of this technique is that sample collection is easy and inexpensive and can be carried out by the patient him or herself, even at home. Moreover, the risk of HIV transmission

18

Introduction and outline of the thesis currently comprises more than pharmacokinetic and pharmacodynamic studies, e.g. more epidemiological-type studies such as surveillance of drug use and adverse effects that are 1 performed in order to promote the safety of prescription, maximize drug effects and minimize adverse effects. Various types of clinical pharmacological studies are incorporated in this thesis. An interaction between drugs can be synergistic (when the drugs’ effects are increased) or antagonistic (when the drugs’ effect are decreased). The interactions may be alterations in the absorption, distribution, metabolism, and excretion of a drug (pharmacokinetic interaction). Alternatively, drug interactions may be the result of the alterations of pharmacodynamic properties of the drug. Obviously, it is important to find out these pharmacological interactions in real practice. Clinically significant interactions occurring during antituberculous chemotherapy principally involve rifampicin. Such interactions can be associated with consequences even amounting to therapeutic failure or toxicity. The cytochrome P450 iso- enzymes are responsible for many interactions during drug biotransformation (metabolism) in the liver and/or intestine [29]. Generally, rifampicin is known to be a very strong inducer of cytochrome P450 iso-enzymes, other metabolic enzymes and transporter proteins. Co- administration of rifampicin leads to significant reduction in the serum concentrations of many drugs [30,31].

Pharmacological concerns about anti-TB drugs A. TB/HIV drug interactions. The treatment of TB in individuals with HIV infection is the same as that for patients with TB who do not have HIV disease. Standard first-line therapy for TB is effective in patients with HIV infection–related TB. However, management of co-infected patients can be complex because of pharmacokinetic and pharmacodynamic interactions between TB drugs and antiretroviral drugs [32]. These interactions may cause decreased concentrations of some TB drugs, adverse events due to overlapping adverse effect profiles and immune reconstitution disease. More specifically, a rifampicin based TB regimen lowers the concentration of non-nucleoside reverse transcriptase inhibitors, protease inhibitors and some new antiretroviral drugs such as maraviroc, raltegravir and etravirine [33]. Therefore, continuous original studies and reviews on interactions between anti-tuberculosis and antiretroviral drugs are necessary.

B. Pharmacokinetic sampling. Pharmacokinetic studies are very useful in determining pharmacological activity and toxicity of a drug. Therefore, large studies with frequent blood sampling are required. However, these studies necessitate the attendance of medical staff. Therefore, the use of saliva samples would facilitate studies to evaluate concentrations of drugs. The advantage of this technique is that sample collection is easy and inexpensive and can be carried out by the patient him or herself, even at home. Moreover, the risk of HIV transmission to the medical staff and discomfort for the patients would be minimized and the technique may therefore be particularly useful in children. The feasibility of using saliva to monitor drug concentrations has been earlier demonstrated for a large variety of drugs, but the number of data on TB drugs is very limited [36]. It would be useful if saliva can be used as an alternative specimen in monitoring TB drugs concentrations.

19 chapter 1

C. Use of TB drugs in Diabetic mellitus patients. Diabetes mellitus (DM) is a metabolic 1 disorder of energy balance with many contributory causes and forms. As described previously, DM exerts a negative effect on TB treatment. One of the possible underlying mechanisms could be altered pharmacokinetics of anti-TB drugs. Lower concentrations of anti-TB drugs in plasma have been associated with clinical failure and acquired drug resistance [34,35]. It is important to study the impact of DM on the pharmacokinetics of TB drugs and on the outcome of TB treatment.

D. Toxicity of Anti-tubercular treatment. Anti-tubercular drugs show a great level of efficacy with an acceptable degree of toxicity; however, they may cause derangement of hepatic functions. Isoniazid, rifampicin and pyrazinamide are potentially hepatotoxic drugs (Table 1). They are metabolized in the liver, making this organ vulnerable for injury. Apart from hepatotoxicity, TB drug related adverse reactions such as peripheral neuropathy, gastro-intestinal intolerance and skin rashes commonly occur [37]. They can cause significant morbidity and therefore may compromise adherence to treatment, eventually contributing to treatment failure, relapse or emergence of resistant strains [38]. Malnutrition, pre-existing liver disease, genetic factors, use of concomitant drugs and alcohol are risk factors specifically related to liver toxicity [39-43]. Hepatotoxicity rates of 13–15% have been reported from India and Iran [44]. Other studies reported hepatotoxicity to be <2%, however, liver function was not closely monitored in these studies [45-47]. The data on toxicity of anti-tubercular drugs in sub Saharan Africa are scarce; the main reason may be absence of biochemistry analysis during TB treatment. Valid estimation of the incidence of toxicity due to TB treatment in the African or Tanzanian region is important considering the era of TB/HIV co-medication.

E. The use of fluoroquinolones: second-line drugs for TB treatment. As mentioned above, there are four classes of second-line drugs (group 2 to group 5) used for treatment of (multidrug-resistant) TB. A drug may be classed as second-line instead of first-line for one of three possible reasons: it may be less effective than the first-line drugs (e.g., p-aminosalicylic acid); or, it may have toxic side-effects (e.g., cycloserine); or it may be unavailable in many

Table 1. Shared adverse effects of TB and Antiretroviral Therapy

Side Effects ARVs Tuberculous Treatment Nausea, Vomiting ddl Pyrazinamide AZT Hepatitis Nevirapine Rifampin Efavirenz INH PZA Peripheral Neuropathy d4T INH ddl Rash Nevirapine Rifampin Efavirenz INH PZA Source: SA National TB control Programme Practical Guideline Abbreviations: ddI: didanosine, AZT: zidovudine, d4T: stavudine, INH: isoniazid, PZA: pyrazinamide

20 Introduction and outline of the thesis developing countries (e.g., fluoroquinolones). Although fluoroquinolones are readily available in Tanzania, they are prescribed for a variety of infectious diseases including respiratory 1 infection and urinary tract infection. As the extent of use increases, the chance of developing resistance to fluoroquinolones, when used to treat TB, increases. Therefore, it is important to evaluate the extent of fluoroquinolone use in resource-limited settings like Tanzania and to assess the extent of resistance that occurs.

Outline of the thesis The aim of the studies presented in this thesis is to address the pharmacological problems in the treatment of tuberculosis patients as outlined above. Therefore, this thesis incorporates pharmacokinetic studies focusing on TB/HIV co-infection, sampling of saliva in TB patients, diabetic TB patients, a survey on toxicity and a study on surveillance of TB drug use. All studies presented in this thesis were conducted in Kilimanjaro Region in Tanzania. The Tanzanian National Tuberculosis Hospital is located in the Kilimanjaro Region, Siha District. In addition, the Mawenzi hospital in Moshi is a large centre for TB treatment. Hence, these two sites were used to conduct different studies in this thesis. All studies were coordinated from the Kilimanjaro Clinical Research Institute (KCRI) located at Kilimanjaro Christian Medical Centre (KCMC Hospital) in Moshi, Kilimanjaro Region. The outline of this thesis is as follows:

Chapter 1: This is the introductory chapter where an epidemiological update, overlapping of TB, HIV disease and diabetes mellitus, the history of TB treatment and general information on the TB drugs are highlighted. Then follows an exploration of the pharmacological concerns of anti-TB drugs.

Chapter 2: A detailed review of the interaction between first-line TB drugs and antiretroviral drugs used in resource-limited setting and the challenges involved are discussed in chapter 2(a). Furthermore, the hypothesis that ‘AtriplaR as a fixed dose combination and first-line anti-TB drugs are having pharmacokinetic interactions according to the existing literature’ is discussed in chapter 2 (b).

Chapter 3: This chapter presents a clinical trial conducted in Tanzania on TB/HIV co-infected patients who were given emtricitabine/tenofovir/efavirenz as a fixed dose combination concurrently with first-line anti-tuberculosis drugs. The study focused on pharmacokinetics of the drugs involved, safety and tolerability.

Chapter 4: There is complete lack of pharmacokinetic data for TB drugs administered to Tanzanian TB patients. We conducted a study to describe pharmacokinetic properties of TB drugs in plasma of Tanzanian patients with pulmonary TB.

Chapter 5: The plasma and saliva concentrations of rifampicin were simultaneously measured in patients with pulmonary TB, as a means to explore the feasibility of saliva sampling instead of blood sampling.

21 chapter 1

Chapter 6: Changes in lifestyle and diet have contributed to an increased prevalence of diabetes 1 in many low-income and middle-income countries where the burden of TB is high. Diabetes triples the risk of developing TB consequently; rates of TB are higher in people with diabetes than in the general population. In addition, TB patients with DM show a worse response to TB treatment compared to non-diabetics. This may result from differences in pharmacokinetics of TB drugs between diabetics and non-diabetics. This chapter describes a pharmacokinetic study in patients with TB alone and TB with DM as concurrent disease.

Chapter 7: This chapter describes a survey conducted in Tanzanian TB patients to find out the incidence of TB drugs induced hepatotoxicity in Tanzania.

Chapter 8: This is divided in two sections. In chapter 8(a), the rate of fluoroquinolones sale for the treatment of other infections, which may cause their effectiveness against TB to be lost, is evaluated in an antimicrobial drug surveillance in the Moshi area. Then in chapter 8(b), the research question is whether fluoroquinolones resistance in M. tuberculosis is common in Tanzanian TB patients, considering the availability of fluoroquinolones in the general population.

Chapter 9: This chapter discusses the findings from the above studies and implications of the findings in solving pharmacological concerns about anti-TB drugs. Furthermore, recommendations for further research are highlighted.

References

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22 Introduction and outline of the thesis

11. Guptan, A., and A. Shah. Tuberculosis and diabetes: an appraisal. Indian J. Tuberc. 2000;47:3–8. 12. Singla, R., N. Khan, N. Al-Sharif, M. O. Al-Sayegh, M. A. Shaikh, and M. M. Osman. Influence of 1 diabetes on manifestations and treatment outcomeof pulmonary TB patients. Int. J. Tuberc. Lung Dis. 2006;10:74–79. 13. Stevenson, C. R., N. G. Forouhi, G. Roglic, B. G. Williams, J. A. Lauer, C. Dye, and N. Unwin. Diabetes and tuberculosis: the impact of diabetes epidemic on tuberculosis incidence. BMC Public Health 2007;7:234. 14. National Aids Control Programme (NACP); Fourth Edition, 2012. 15. Fox W, Ellard GA, Mitchison DA. Studies on the treatment of tuberculosis undertaken by the British Medical Research Council Tuberculosis Units 1946–1986 with relevant subsequent publications. Int J Tuberc Lung Dis 1999;3:s231–s279. 16. Www.sciencedirect.com. Development of new drugs for TB treatment. Accessed on 28.09.2012. 17. Guideline for treatment of tuberculosis-fourth edition. Geneva, World health organization 2010 (WHO/ HTM/TB/2009.420). 18. Wang JY, Hsueh PR, Jan IS, et al.”Empirical treatment with a fluoroquinolone delays the treatment for tuberculosis and is associated with a poor prognosis in endemic areas”. Thorax. 2006 Oct;61(10):903-8. 19. Holdiness MR. Clinical pharmacokinetic of the antituberculosis drugs. Clin Pharmacokinet. 1984 Nov- Dec;9(6):511-44. 20. Thornsberry C and Hill BC. Rifampin: Spectrum of antibacterial activity. Rev Infect Dis 1983;3:412-417. 21. Peloquin CA, Namdar R, Singleton M, Nix D. Pharmacokinetics of rifampin under fasting conditions, with food, and with antacids. Chest 1999;115:12-18. 22. Acocella G. Clinical Pharmacokinetic of Rifampicin 1978 Mar-Apr;3(2): 108-27. 23. Acocella G. Pharmacokinetics and metabolism of rifampin in humans. Rev Infect Dis 1983;3:S428-S432. 24. Kenny MT and Strates B. Metabolism and pharmacokinetics of the antibiotic rifampin. Drug Metab Rev 1981;12:159-218. 25. Peloquin CA, Jaresko GS, Yong CL, Keung ACF, Bulpitt AE, Jelliffe RW. Population pharmacokinetic modeling of isoniazid, rifampin, and pyrazinamide. Antimicrob Agents Chemother 1997;41:2670-2679. 26. Lee C. S., Brater D. C., Gambertoglio J. G., Benet L. Z. Disposition of ethambutol in man. J. Pharmacokinet. Biopharm. 1980;8:335–346. 27. Johnson Siv, Alistair Davidse, Justin Wilkins et al. Population pharmacokinetics of ethambutol in South Africa Tuberculosis Patients. AAC Sept 2011;55(9):4230-4237. 28. Guidelines for the surveillance of drug resistance in tuberculosis – 4th ed. Geneva, World Health Organization, 2010 (WHO/HTM/TB/2009.422). 29. Niemi M, Backman JT, Fromm MF, Neuvonen PJ, Kivisto KT. Pharmacokinetic interactions with rifampicin: clinical relevance. Clin. Pharmacokinet. 2003;42:819-850. 30. World Health Organization. Global Tuberculosis Programme. Treatment of tuberculosis: guidelines for national programmes, 9th ed. Geneva: World Health Organization, 2011. 31. Hopewell PC, Pai M, Maher D, Uplekar M, Raviglione MC. International standards for tuberculosis care. Lancet Infect Dis 2006;6(11):710–725. 32. McIllero H, Meintjes G, Burman WJ, Maartens G. Complications of antiretroviral therapy in patients with tuberculosis: drug interactions, toxicity, and immune re-constitution inflammatory syndrome. J Infect Dis 2007;196 (suppl 1):S63-75.

23 chapter 1

33. Dean GL, Edwards SG, Ives NJ, et al. Treatment of tuberculosis in HIV-infected persons in the era of 1 highly active antiretroviral therapy. AIDS 2002;16:75–83 34. Kimerling, M. E., P. Phillips, P. Patterson, M. Hall, C. A. Robinson, and N. E. Dunlap. Low serum antimycobacterial drug levels in non-HIVinfectedtuberculosis patients. Chest 1998;113:1178–1183. 35. Sahai, J., K. Gallicano, L. Swick, S. Tailor, G. Garber, I. Seguin, L. Oliveras, S. Walker, A. Rachlis, and D. W. Cameron. Reduced plasma concentrationsofantituberculosis drugs in patients with HIV infection. Ann. Intern. Med. 1997;127:289–293. 36. Rolinski B, Wintergerst U, Matuschke A, et al. Evaluation of saliva as a specimen for monitoring zidovudine therapy in HIV-infected patients. AIDS 1990;5:885-888. 37. Yee D, Valiquette C, Pelletier M, Parisien I, Rocher I, et al. Incidence of serious side effects from first- line antituberculosis drugs among patients treated for active tuberculosis. Am J RespirCrit Care Med 2003;167:1472–1477. 38. Kaona FA, Tuba M, Siziya S, Sikaona L. An assessment of factors contributing to treatment adherence and knowledge of TB transmission among patients on TB treatment. BMC Public Health 2004; 4:68. 39. Fernandez-Villar A, Sopena B, Fernandez-Villar J, Vazquez-Gallardo R, Ulloa F, et al. The influence of risk factors on the severity of antituberculosis drug-induced hepatotoxicity. Int J Tuberc Lung Dis 2004; 8:1499–1505. 40. Sharma SK, Balamurugan A, Saha PK, Pandey RM, Mehra NK. Evaluation of clinical and immunogenetic risk factors for the development of hepatotoxicity during antituberculosis treatment. Am J RespirCrit Care Med 2002; 166:916–919. 41. Tost JR, Vidal R, Cayla J, Diaz-Cabanela D, Jimenez A, et al. Severe hepatotoxicity due to anti-tuberculosis drugs in Spain. Int J Tuberc Lung Dis 2005; 9:534–540. 42. Ungo JR, Jones D, Ashkin D, Hollender ES, Bernstein D, et al. Antituberculosis drug-induced hepatotoxicity. The role of hepatitis C virus and the human immunodeficiency virus. Am J RespirCrit Care Med 1998; 157:1871–1876. 43. Tostmann A, Boeree MJ, Aarnoutse RE et al. Antituberculosis drug-induced hepatotoxicity: concise up- to-date review. Journal of Gastroenterology and Hepatology 2008; 23:192–202. 44. Baghaei P, Tabarsi P, Chitsaz E et al. Incidence, Clinical and Epidemiological Risk Factors, and Outcome of Drug-Induced Hepatitis Due to Antituberculous Agents in New Tuberculosis Cases. Am J Ther. 2010 Jan-Feb;17(1):17-22. 45. Perriens JH, St Louis ME, Mukadi YB et al. Pulmonary tuberculosis in HIV-infected patients in Zaire. A controlled trial of treatment for either 6 or 12 months. New England Journal of Medicine 1995;332;779– 784. 46. Johnson JL, Okwera A, Nsubuga P et al. Efficacy of an unsupervised 8-month rifampicin-containing regimen for the treatment of pulmonary tuberculosis in HIV-infected adults. Uganda-Case Western Reserve University Research Collaboration. International Journal of Tuberculosis and Lung Disease 2000; 4:1032–1040. 47. Tostmann A, Boeree MJ, Harries AD et al. Short communication: antituberculosis drug-induced hepatotoxicity is unexpectedly low in HIV-infected pulmonary tuberculosis patients in Malawi. Tropical Medicine and International Health 2007; 12:852–855.

24

Pharmacological interactions between rifampicin and antiretroviral drugs: challenges and research priorities for 2a resource-limited settings

Hadija H. Semvua, Gibson S. Kibiki, Elton R. Kisanga, Martin J. Boeree, David M. Burger, Rob Aarnoutse.

Manuscript submitted chapter 2a

Abstract Co-administration of antituberculosis and antiretroviral therapy is often inevitable in high- burden countries where tuberculosis is the most common opportunistic infection associated 2a with HIV/AIDS. Concurrent use of rifampicin and many antiretroviral drugs is complicated by pharmacokinetic drug-drug interactions. Rifampicin is a very potent enzyme inducer, which can result in sub-therapeutic antiretroviral drug concentrations. In addition, TB drugs and antiretroviral drugs have additive (pharmacodynamic) interactions as reflected in overlapping adverse effect profiles. This review provides an overview of the pharmacological interactions between rifampicin-based TB treatment and antiretroviral drugs in adults living in resource- limited settings. Major progress has been made to evaluate the interactions between TB drugs and antiretroviral therapy, however burning questions remain concerning nevirapine and efavirenz effectiveness during rifampicin-based TB treatment, treatment options for TB-HIV co-infected patients with NNRTI resistance or intolerance and exact treatment or dosing schedules for vulnerable patients including children and pregnant women. The current research priorities can be addressed by maximising the use of already existing data, creating new data by conducting clinical trials and prospective observational studies and to engage a lobby to make currently unavailable drugs available to those most in need.

28 First-line TB drugs and antiretroviral pharmacological interactions review

Introduction The tuberculosis (TB) and human immunodeficiency virus (HIV) epidemics show overlapping epidemiology globally [1,2]. In 2011, about one million (13%) of the 8.7 million people who developed TB worldwide were HIV-positive; 79% of these HIV-positive TB cases were in the 2a African Region[3]. Since 2010, the World Health Organisation (WHO) has recommended antiretroviral treatment (ART) for TB patients regardless of their CD4 cell-count. TB treatment should be initiated first and ART should be started as soon as possible within eight weeks of starting TB treatment. Those with profound immunosuppression (e.g. less than 50 CD4 cells/mL) are recommended to start ART within the first two weeks of TB treatment [3]. This means that about one million people per year are eligible for using concurrent TB and HIV medication, and WHO estimated that in 2011 about half of them were started on ART during TB treatment [3]. Standard first line TB treatment takes six months and consists of a two-month intensive phase with isoniazid, rifampicin, pyrazinamide and ethambutol followed by a four months continuation phase with isoniazid and rifampicin[4]. The currently recommended antiretroviral drug regimen for TB-HIV coinfected patients is presented in Table 1 [5]. In summary, efavirenz should be used as the preferred non-nucleoside reverse transcriptase inhibitor in patients starting ART while on TB treatment and the choice of the protease inhibitor for second line treatment depends on the availability of rifabutin for TB treatment. Unfortunately, patients using concomitant TB and antiretroviral treatment face several problems. Firstly, multi-drug treatment is prescribed for both TB and HIV, resulting in a high daily pill counts, which may cause non-adherence to these therapies. Secondly, both TB

Table 1. Potential overlapping toxicity due to antiretroviral and tuberculosis drugs

Antiretroviral drugs First line tuberculosis drugs Adverse effect from HIV and/or TB treatment [5] [4] Drug eruptions (mild to severe, including Stevens- Nevirapine, efavirenz Johnson syndrome or toxic epidermal necrolysis) (less commonly) Anaemia and neutropaenia Zidovudine Isoniazid (rare) Peripheral neuropathy Stavudine Isoniazid (common), ethambutol Gastrointestinal intolerance All All Hepatotoxicity All ARVs Isoniazid, rifampicin, Particularly nevirapine pyrazinamide Central nervous system toxicity Efavirenz Isoniazid Drug fever All All Bone marrow suppression and blood dyscrasias Zidovudine, efavirenz Rifampicin Ocular effect Didanosine Ethambutol, rifabutin Renal toxicity (renal tubular dysfunction) TDF

Note: not all potential adverse effects are shown in this table, only toxicity that can be caused by both TB and HIV treatment. NRTIs = nucleoside reverse transcriptase inhibitors; PIs = protease inhibitors; AZT = zidovudine, FTC = emtricitabine

29 chapter 2a

and antiretroviral treatment regimens are associated with frequent and potentially severe adverse events. This poses a great challenge in managing each of these diseases [2,6]. Due to overlapping toxicity profiles of TB and HIV drugs, it is often difficult defining the cause 2a of an observed adverse reaction. Thirdly, so called immune reconstitution inflammatory syndrome could occur, a phenomenon that may lead to clinical deterioration upon starting antiretroviral treatment [7-9]. Finally, relevant pharmacokinetic drug-drug interactions exist, especially between rifampicin and two widely used classes of antiretroviral drugs: the non- nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) [10-12]. Such pharmacokinetic interactions can significantly decrease the plasma concentrations of the HIV drugs, which can affect treatment outcome of ART. This review addresses the pharmacological interactions between TB treatment and the antiretroviral drugs that are available to adults living in the resource-limited settings. The main focus will be on the effect of rifampicin on the NNRTIs and the PIs. We have described the main challenges for concurrent TB and HIV treatment for these settings, identified the most urgent research priorities and propose strategies to address these research questions.

Pharmacokinetic interactions between rifampicin and antiretroviral drugs When co-administering TB drugs and antiretroviral drugs, the TB drugs precipitate pharmacokinetic interactions, whereas the antiretroviral agents are object drugs. Most pharmacokinetic interactions are caused by rifampicin, the cornerstone drug of TB treatment, which induces the activity and/or expression of many liver enzymes and transporter proteins. Rifampicin is a very potent inducer of cytochrome P450 (CYP450) a super family of enzymes in the liver and in the intestinal wall [13,14]. It strongly induces the expression of CYP3A4 and thereby greatly reduces the plasma concentrations and effects of many CYP3A4 substrates, including several antiretroviral drugs. A large number of other CYP450 iso-enzymes, including CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19 and CYP3A5 are also induced by rifampicin [15]. Therefore, pharmacokinetic properties and treatment effect of antiretroviral drugs that are metabolized by one of these enzymes are likely to be effected by rifampicin. Pharmacokinetic studies suggest that CYP2D6 activity is only modestly or not increased by rifampicin [16,17]. Furthermore, rifampicin induces phase II drug-metabolizing enzymes such as UDP-glucuronyltransferases and sulfotransferases. Finally, rifampicin also induces the expression of transporter proteins, such as P-glycoprotein, multiple drug resistance protein 2 (MRP2), and organic anion-transporting polypeptide (OATP [18-20]. For the interaction potential of rifampicin, P-glycoprotein appears to be especially relevant. CYP3A4 and P-glycoprotein may act synergistically to limit oral drug bioavailability and to enhance drug metabolism and elimination, as suggested by their joint presence in enterocytes, hepatocytes and renal tubular cells and by the extensive overlap in their substrate specificities [21-23]. In fact, the function of P-glycoprotein may allow CYP3A4 to have repeated and prolonged access to its substrate molecules [24,25]. The antiretroviral NNRTIs and PIs are substrates of CYP450 iso-enzymes and /or P-glycoprotein. In contrast to inhibition of metabolic

30 First-line TB drugs and antiretroviral pharmacological interactions review enzymes and transport proteins, induction takes time to develop and disappear. Full induction after starting rifampicin is reached in roughly one week [26] and the activity of enzymes returns to baseline in about two weeks after discontinuing rifampicin treatment [27,28]. Apart from rifampicin, the TB drug isoniazid affects CYP450 enzymes as well. Isoniazid is 2a a relatively potent inhibitor of several CYP450 enzymes (CYP2C9, CYP2C19, CYP2E1) but it has less effect on CYP3A4 [29]. However, during TB treatment any inhibitory effect of isoniazid is generally outweighed by the strong inductive effect of concomitantly used rifampicin [30,31]. Currently, all HIV infected patients without active tuberculosis should be offered isoniazid preventive therapy for at least six months as prophylaxis for latent TB infection [5]. This includes HIV infected patients who are currently using ART, thus the inhibition of CYP3A and CYP2C19 by isoniazid and the consequent effect on plasma levels of antiretroviral drugs becomes relevant for a large group of HIV infected patients. The other first-line TB drugs, pyrazinamide and ethambutol do not have any inductive or inhibitory effect and are therefore not involved in pharmacokinetic interactions with antiretroviral drugs.

Interactions between rifampicin and NNRTIs Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are part of WHO recommended first- line antiretroviral regimens in resource-limited settings [5], hence the interaction between rifampicin and NNRTIs is particularly important. Efavirenz is the recommended NNRTI to be used in patients starting ART while on TB treatment [5]. It should be taken once daily and is generally well tolerated. Efavirenz is mainly metabolized by hepatic CYP2B6. As rifampicin is a potent inducer of CYP450 enzymes, concomitant use of rifampicin and efavirenz leads to unavoidable drug interactions. However, rifampicin has not been shown to consistently reduce efavirenz concentrations. The metabolism of efavirenz is extensively influenced by pharmacogenetic factors, particularly the CYP2B6 single nucleotide polymorphism 516G>T, which impairs metabolism resulting in high efavirenz concentrations. This polymorphism occurs commonly in South Africa [32] West Africa and India [33,34]. Factors influencing efavirenz concentrations include body weight and racial or ethnic background [35-37]. Thai and Afro-American patients have reduced efavirenz clearance and hence increased efavirenz concentrations [38]. Most pharmacokinetic studies have shown that exposure to efavirenz is decreased upon co- administration of rifampicin, but whether to increase the dose of efavirenz when concomitantly used with rifampicin is still the subject of controversy. A pharmacokinetic study in 12 European participants found a decrease in efavirenz area under the concentration curve (AUC) by 26% [10]. Also, a retrospective analysis of a therapeutic drug monitoring database found that efavirenz concentrations were significantly lowered (35%) with rifampicin [36]. The need for efavirenz dose increase when rifampicin is co-administered was recommended [39], however in recent studies it was concluded that there is no need to increase the dose of efavirenz while co administered with rifampicin [40,41]. A prospective pharmacokinetic study conducted in adults from Spain [37]showed a decrease in efavirenz concentration but studies in India and South Africa reported a non-significant reduction in efavirenz concentrations [34,42]. In these studies, patients showed similar efavirenz trough levels and adequate virological outcome in patients who took 600 mg efavirenz during

31 chapter 2a

and after co-administration of rifampicin. Previously, no significant difference in efavirenz concentrations was observed in a randomized study performed in Thailand where standard (600 mg once daily) and increased doses (800 mg once daily) of efavirenz were compared 2a [43,44]. Virological outcomes in both arms were similar as well. However, the interpretation of this study is limited by the inclusion of just one ethnic group of patients with a low mean body weight (<50kg) and by a small number of participants (84 patients). Most of the studies have shown that body weight is the important predictor of efavirenz concentration [45-47]. Many of the studies have shown that response to standard-dose efavirenz based ART is similar in patients with and without rifampicin -containing treatment [48,49]. Efavirenz, given at a standard dosage of 600 mg per day, was found adequate for suppression of HIV, despite variation in blood concentrations of the drug [50]. However, ethnicity plays an important role because ethnic Black Africans are frequently of the homologous CYP2B6 516-T/T genotype in comparison to other ethnic groups [51,52]. This fact is supported by the study conducted by Friedland et al [52], where the study results indicated that Black African patients suffered more frequently from central nervous system side effects although receiving a dose of 600 mg with rifampicin. Also, intersubject variability in pharmacokinetics of efavirenz due to CYP2B6 polymorphism was observed [53-57]. The suggestion that the effect of CYP2B6 genotype on efavirenz levels is greater than that of rifampicin was supported by a recent study from Manosuthi et al who showed that particular CYP2B6 genotypes (especially haplotype *6/*6) had the greatest impact on efavirenz concentrations in TB-HIV co-infected patients in Thailand [58]. Low body weight and concurrent receiving rifampicin were of less influence. Also, in vitro work supports the role of pharmacogenetics on efavirenz exposure [59]. Interestingly, CYP2B6 genotype was the explanation for an observed increase in efavirenz exposure during TB treatment in a group of black African patients [60]. The same was found in Ghana, where patients with CYP2B6 516TT genotype who were on concomitant TB and HIV treatment had higher efavirenz plasma concentrations than volunteers with the same genotype who did not receive TB treatment [55]. Because of the effect of CYP2B6 genotype on efavirenz levels when used with rifampicin, some argue the use of therapeutic drug monitoring and subsequent efavirenz dose adjustment [61]. In summary, there is still controversy about whether to increase the dose of efavirenz, in general or for certain subgroups of patients. Pharmacogenetic factors (CYP2B6) play a major role in efavirenz exposure and it is suggested that the effect of pharmacogenetics is larger than that of rifampicin. Therapeutic drug monitoring to assess whether a dose increase might be justified is an option offered by some researchers. However, more research is needed on the association between efavirenz plasma concentrations and virological and immunological treatment outcomes. Nevirapine, another NNRTI, is widely used in developing countries. It is cheaper than efavirenz and is recommended instead of efavirenz in women who might become childbearing [62], as efavirenz is teratogenic and therefore contraindicated during the first trimester of pregnancy. Nevirapine is mainly metabolized by CYP3A4 and to a lesser extent by CYP2B6 and is therefore vulnerable for interaction with rifampicin. The mean effect of rifampicin on nevirapine concentrations is stronger than its effect on efavirenz concentrations, as reductions up to 55% in nevirapine concentrations have been described [63,64]. Likewise, a relatively larger

32 First-line TB drugs and antiretroviral pharmacological interactions review proportion of co-treated patients can be expected to have trough levels of nevirapine below the target range [42,63, 65]. This is consistent with the finding that nevirapine associated with virological failure and death more frequently than efavirenz [66]. Despite the relatively strong effect of rifampicin on nevirapine concentrations, standard- 2a dose nevirapine combined with rifampicin was shown not to be associated with worse virological outcome compared to HIV patients on ART without TB treatment [67,68]. However, in another study virological outcome was worse in patients who received nevirapine based ART combined with TB treatment compared to those who had no concurrent TB. Increasing the dose of nevirapine upon co-administration of rifampicin can overcome the decreased exposure to nevirapine, but this approach raises concerns about hepatotoxicity, which may be dose- related [69]. This concern is especially relevant because isoniazid, rifampicin and pyrazinamide are also hepatotoxic drugs. Monitoring of hepatotoxicity has been recommended when it is used with rifampicin [70]. There is discussion about the lead-in dose of nevirapine when used with rifampicin-based TB treatment. Nevirapine initiation by dose escalation resulted in sub-therapeutic concentrations in an Ugandan study [71]. The authors suggest that this strategy should be avoided in rifampicin co-treated patients, because nevirapine concentrations were subtherapeutic during initiation with dose escalation and nevirapine initiation at the maintenance dose of 200 mg twice daily is preferred. Furthermore, the reduction of nevirapine levels by rifampicin is suggested to be reversible, as nevirapine exposure significantly increased after 10 weeks of combined TB and HIV treatment [72]. Although rifampicin decreases efavirenz and nevirapine concentrations, its effect on ART treatment outcome is not clear. Two recent studies showed that the subtherapeutic plasma concentrations of nevirapine or efavirenz due to rifampicin were not associated with virological failure. Patients had good clinical outcomes, and the combined treatment was tolerated well with a low incidence of hepatotoxicity [73]. The viral load and CD4 count of patients using nevirapine-based ART with versus without rifampicin were comparable [74]. The association between altered NNRTI pharmacokinetics and treatment efficacy warrants more research. Furthermore, there is insufficient data to make definitive recommendations about dose adjustment of the NNRTIs during rifampicin containing therapy [75].

Interactions between rifampicin and PIs Protease inhibitors (PIs) are part of second line antiretroviral treatment. PIs are used in case of resistance to NNRTIs and for individuals who cannot tolerate NNRTIs. Of the PIs recommended by the WHO saquinavir, lopinavir, indinavir, and atazanavir are all metabolized by CYP3A4 and also act as weak inhibitors of CYP3A4 [76]. Therefore, interaction with rifampicin is expected (i.e. decreased plasma concentrations). Nelfinavir is the only PI that is metabolized by both CYP2C19 and CYP3A4, which means that concomitant use of isoniazid will likely result in increased plasma levels due to CYP2C19 inhibition. Ritonavir is metabolized via CYP3A and CYP2D6 isoforms [77] and is an exceptionally potent inhibitor of CYP3A4. Less commonly, it can serve as an inducer of CYP3A4 and glucuronosyl S-transferase, or act as a mixed competitive/ noncompetitive inhibitor of CYP2D6 [78].

33 chapter 2a

Due to its potent inhibition of CYP3A4 as well as P-glycoprotein, ritonavir is frequently used with other PIs to pharmacologically ‘boost’ their concentrations. In such boosted PI regimens, ritonavir is used in a low dose (100 mg twice-daily, instead of the approved dose of 600 mg twice- 2a daily). A recommended option for combined use with rifampicin is so-called superboosting with an adjusted dose of ritonavir (200mg or 400 mg) [5]. Considering the metabolic profiles of the PIs and their transport by P-glycoprotein, it is not surprising that rifampicin can reduce PI plasma concentrations up to 80-95% [79]. The exposure to the therapeutic dose of ritonavir (600 mg twice daily) is less affected by rifampicin compared to the other PIs, because ritonavir is a potent inhibitor of CYP3A4 and P-glycoprotein, thereby compensating (part of) the inducing effect of rifampicin [80]. However, this PI is not well tolerated and its capsules need to be refrigerated, which is problematic in developing countries. Unfortunately, boosting the exposure to PIs with low dose ritonavir (100 mg BID) is not able to overcome the inducing effect of rifampicin. An increased dose of ritonavir (400 mg twice daily) to boost saquinavir or lopinavir may be adequate to compensate for the induction by rifampicin [8,81-85]. For example in a study with relatively small sample size, high-dose saquinavir/ritonavir (400/400 mg) coadministered with rifampicin was well tolerated, produced adequate concentrations, and was recommended for use [86]. Similarly adjusted doses of lopinavir/ritonavir (800/200 and 400/400 mg) resulted in acceptable mean trough levels, but a higher interpatient variability in exposure [82]. Unfortunately, combination of boosted PIs with rifampicin has been associated with unacceptable toxicity. In a study of 20 TB-HIV -coinfected patients who started this saquinavir/ ritonavir 400/400 mg after four weeks of rifampicin -containing TB treatment, 15 dropped out because of adverse events [8]. Likewise, in a study of 14 healthy volunteers who first were given 600 mg of rifampicin daily for 14 days and then also were given 1000/100 mg of Saquinavir/ritonavir twice daily, 9 had grade 3 or 4 hepatotoxicity; the trial was stopped early [83]. Similar results were obtained when adjusted doses of lopinavir/ritonavir were introduced in healthy volunteers on rifampicin [87]. Studies from South Africa showed that once patients were established on treatment with increased doses of lopinavir/ritonavir co-administered with rifampicin-based tuberculosis, treatment was tolerated and lopinavir predose concentrations were adequate [88]. Additionally, doubling the dose of the tablet formulation of lopinavir/ritonavir (to 800/200mg) overcomes the metabolism induction by rifampicin and was tolerated well in a cohort of HIV-infected participants [89]. Rifampicin reduces saquinavir concentrations up to 70% in healthy volunteers, however the reduction was only 46% in HIV infected patients [90]. These studies show that findings from healthy volunteers can be different from findings in HIV-infected patients. The effect of doubling the dose of lopinavir/ritonavir and superboosted lopinavir/ritonavir (1:1 ratio) should be evaluated further in HIV infected patients, with special emphasis on the virological and immunological outcomes as well as development of hepatotoxicity. Recent experience show that combined use of lopinavir/ritonavir and rifampicin is a problem as one need to balance between sub-optimal efficacy and toxicity [91]. As to actual TB patients, very few data are available about pharmacokinetics, efficacy and tolerability of (boosted) PIs and rifampicin-

34 First-line TB drugs and antiretroviral pharmacological interactions review based TB treatment. In a current study [92] rifabutin was found to be safe in combination with PIs and also cost saving when given with current recommended boosted PIs.

Interactions between rifampicin and NRTIs & NtRTIs Nucleoside reverse transcriptase inhibitors (NRTIs) serve as the backbone of antiretroviral 2a treatment regimens. Because CYP450 enzymes do not metabolize them, this class of drug is less prone to drug-interactions with rifampicin; NRTIs can therefore be combined with rifampicin [93]. However, rifampicin is known to reduce the exposure to zidovudine by almost 50% [46,47]. The effect of rifampicin on the intracellular concentrations of zidovudine-triphosphate is not known. Nevertheless, this interaction is thought to be of minor clinical relevance [94]. Tenofovir disoproxil fumarate, the only approved nucleotide reverse transcriptase inhibitor (NtRTI) is not a substrate, inhibitor, or inducer of the CYP450 system. A study in healthy volunteers [95] showed a clinically insignificant interaction between tenofovir and rifampicin, which reflects the possibility of combining this drug with rifampicin.

Interactions between rifampicin and other ARVs Antiretroviral drugs from other classes have been evaluated for interaction with rifampicin. Plasma concentrations of the fusion inhibitor enfuvirtide were not shown to be affected by ten days of concomitant use with rifampicin, showing that these drugs can be co-administered without dose-adjustments [96]. The integrase inhibitor raltegravir is metabolized by glucuronidation, an enzyme system that is induced by rifampicin, but raltegravir does not inhibit or induce CYP450 enzymes. Recent data show that the dose of raltegravir needs to be doubled when combined with rifampicin, but this increased dose (800 mg) tolerated well in a small study with two patients [97,98]. Raltegravir trough concentrations were at the lower limit of clinical experience when used with rifampicin so caution should be applied [99]. Raltegravir was used in increased dose in four Spanish patients to replace the PIs during rifampicin-based TB treatment [100]. Raltegravir maintained its virological and immunological efficacy and was tolerated well. These studies indicate that raltegravir may be a good substitute for the PIs when second line ART has to be combined with rifampicin-based TB treatment. A study from Thailand showed that using raltegravir at 400 mg once daily was associated with a low C(trough) and with a risk for HIV viremia. Raltegravir at 200 or 300mg twice daily should be studied. However, a higher dose raltegravir (800mg) might have a better virological response. Another investigational second-generation integrase inhibitor, dolutegravir, is a substrate of UGT1A1 and CYP3A. Dolutegravir were reduced when combined with rifampicin, however, when dolutegravir was given twice-daily mean pharmacokinetic parameters were 20%–33% higher compared to once daily use (both in combination with rifampicin) [101]. Maraviroc, a CCR5 antagonist, is a substrate of CYP3A4 and P-glycoproteine. As expected maraviroc exposure (Cmax and AUC12) was reduced by approximately 70% by rifampicin [102]. Etravirine is the first next-generation NNRTI that is approved for treatment of HIV infection in patients who have experienced virological failure while receiving an NNRTI-containing regimen. Because the drug is metabolized by CYP450 isoenzymes, it is not recommended for use with rifampicin, or high-dose ritonavir and atazanavir [103,104].

35 chapter 2a

The bioavailability of another NNRTI, rilpivirine, is also dramatically reduced during combined use with rifampicin and the study investigators concluded that concurrent administration of rilpivirine and rifampicin is not recommended [105]. 2a The pharmacokinetic properties of the fusion inhibitor enfuvirtide were not clinically affected by coadministration of rifampicin, or the protease inhibitors ritonavir and ritonavir- boosted saquinavir, indicating the potential for use with TB treatment [106]. Unfortunately, all these newer antiretroviral drugs are not yet available in resource-limited settings. However, if proven to be well tolerated and combinable with rifampicin, effort should be made to make these drugs available to those who would mostly benefit from these drugs: TB-HIV coinfected people who would need to use second line ART.

Interactions with isoniazid and antiretroviral drugs Currently, all HIV infected patients without active tuberculosis should be offered isoniazid preventive therapy for at least six months as prophylaxis for latent TB infection [5]. Isoniazid is an inhibitor of CYP2C19 and CYP3A [29]. Therefore increased efavirenz and lopinavir/ritonavir plasma concentrations are to be expected when isoniazid monotherapy for treatment of latent TB infection is given during ART. However, this has not yet been investigated.

Overlapping toxicity profiles Adverse effects to TB drugs appear to occur more commonly among HIV-infected versus non-HIV-infected patients, as suggested by most studies [107-109]. In case TB drugs and antiretroviral drugs are combined, the antiretroviral drugs as well as the TB drugs may cause major treatment-limiting adverse effects. In addition these drugs may have pharmacodynamic interactions, which generally involve additive, synergistic or antagonistic effects of drugs acting on the same receptors or physiological systems. In this case, TB drugs and antiretroviral drugs have an overlapping or additive adverse effect profile, related to (amongst others) hepatotoxicity, gastro-intestinal intolerance and peripheral neuropathy. Not surprisingly, drug toxicity has been implicated as a major cause of discontinuation or interruption of TB and/or antiretroviral therapy during treatment of TB-HIV co-infection. Adverse reactions that can occur due to both tuberculosis and antiretroviral treatment are summarized in Table 1, which clearly shows the overlap in toxicity profiles between these drugs. A recent study from Ethiopia indicated that 30% of TB-HIV co-treated patients developed hepatotoxicity during efavirenz based ART and rifampicin based anti-TB therapy. The predictors included slow acetylation status and a CYP2B6*6/*6 and ABCB13435TT genotype, which indicates that high efavirenz levels (caused by the latter two genotypes) might result in higher risk on hepatotoxicity [110]. However, actual data on adverse effects amongst patients who receive both TB drugs and antiretroviral drugs coadministered are relatively scarce and most studies are retrospective in nature. There is a need for prospective studies that evaluate toxicity to TB and antiretroviral drugs in patients with TB-HIV co-infection. Such studies may shed light on the effects of ethnicity, pharmacogenetic risk factors, immune suppression and malnutrition on adverse effects to TB and HIV drugs.

36 First-line TB drugs and antiretroviral pharmacological interactions review

In the mean time, clinicians should be aware of the overlapping adverse effect profiles, and it should be attempted to use agents with minimal overlapping or additive toxicities. In addition to patient education on signs and symptoms of toxicity, regulatory scheduled clinical and laboratory monitoring, and close communication between TB and HIV clinicians are critical 2a to minimizing treatment discontinuation due to adverse events.

Challenges for resource-limited settings From the above discussion of pharmacokinetic interactions between TB treatment, particularly rifampicin, and antiretroviral drugs it is clear that antiretroviral treatment with a backbone of NNRTIs and N(t)RTIs can be combined with rifampicin-based TB treatment. Overlapping adverse effect profiles may limit the acceptability of such combinations in individual patients. In case of such overlapping toxicity, unduly toxicity caused by NNRTIs, or treatment failure due to resistance to NNRTIs, a major problem arises: there is no obvious second-line protease inhibitor-based antiretroviral regimen that can be administered with rifampicin. The currently recommended second line ART schedule with the protease inhibitors lopinavir or saquinavir (boosted with ritonavir) has the problem that rifampicin significantly reduces the levels of the boosted PIs. The use of increased ritonavir doses (superboosting) can overcome this problem; however with increased risk of toxicity. The need for second line ART in resource limited settings will likely increase in the near future because ART is being rolled- out in developing countries. This means that the number of TB patients who will need second line ART will also increase: these patients need to overcome a period of (at least) six months of combined TB-HIV treatment one way or the other and these are the options:

The use of boosted PIs The currently recommended second line ART combined with rifampicin-based TB treatment is based on the protease inhibitors lopinavir or saquinavir, with boosting doses of ritonavir (lopinavir/ritonavir 400mg/400mg twice daily or lopinavir/ritonavir 800mg/200mg twice daily; or saquinavir/ritonavir 400mg/400mg twice daily). These drug combinations have resulted in severe toxicity in healthy volunteers. However, two recent studies from South Africa indicate that using increased lopinavir/ritonavir doses in HIV-infected TB patients were well tolerated [88,89]. This is a promising finding, which also indicates that drug-interactions should be studied in the relevant population and not only in healthy volunteers [111]. Data on lopinavir/ ritonavir used in combination with rifampicin is sparse and requires more investigation. Its safety and efficacy should be evaluated in larger cohorts of TB-HIV coinfected patients and whether the approach to double the lopinavir/ritonavir dose in two steps over a two-week period is the best approach to prevent severe hepatotoxicity [88].

Use of NRTI/NtRTI-only regimens Combination triple NRTI/NtRTIs regimens appear to be attractive for concurrent application with rifampicin-based TB therapy, as N(t)RTIs do not interact with rifampicin. However, triple NRTI/NtRTI combinations have been shown to be less potent than NNRTI or PI-based regimens in the treatment of HIV infection, as they have been associated with inferior virological

37 chapter 2a

responses in clinical trials [112,113]. Consequently such combinations are not recommended with rifampicin-based TB treatment and the role of triple NRTI/NtRI, or possibly quadruple NRTI/NtRTI, combined with rifampicin-based TB treatment has not been evaluated in clinical 2a trials so far. However, because these regimens would only be needed temporarily (for the duration of TB treatment) evaluating its efficacy would be worthwhile.

Use of new antiretroviral drugs Newer antiretroviral drugs such as the integrase inhibitor raltegravir could be good options for second line ART in combination with rifampicin-based TB treatment. Despite the effect of rifampicin on raltegravir metabolism, increasing the doses was shown to overcome the problem of decreased plasma concentrations and was tolerated well [98,100]. Unfortunately these drugs are currently not available in resource-limited settings; a well organized lobby should be triggered by the scientific community because of the high potential of these drugs in situations where NNRTIs are no longer useful and second line ART is needed. HIV treatment is for life. However, as combined HIV-TB treatment is only for a limited period of six months and the proportion of TB-HIV patients in need of second line ART is small, there should be options for currently unavailable drugs to become available in settings where they are mostly needed.

Use of other rifamycins instead of rifampicin The current guideline for concurrent TB treatment with second line ART suggests replacing rifampicin with rifabutin [3,5]. Rifabutin is a less potent inducer of the CYP450 system and is therefore preferred over rifampicin in patients on TB treatment that have to use second line ART regimens that include a protease inhibitor. However, unlike rifampicin, rifabutin is metabolized by CYP3A4. As most PIs are CYP3A4 inhibitors, this means that a reduction in the dose of rifabutin is required when co-administered with several PIs. Such two-way interactions are complex to manage, especially in developing countries where the high burden of TB and TB-HIV infection is preferably managed by standard treatment regimens. Apart from this, rifabutin is very expensive, making widespread use in developing countries challenging. In addition, even if the price of rifabutin were reduced it would be difficult to implement this drug in high-burden countries, because standard TB treatment is often available as fixed-dose combination tablets, making it complicated to replace rifampicin by rifabutin. Since 79% of patients with TB-HIV coinfections occur in the African Region [114], using rifabutin instead of rifampicin is probably only feasible for a minority of coinfected patients. Another rifamycin, rifapentine, is currently not recommended in HIV-infected patients, as it is associated with the emergence of rifamycin-monoresistant TB strains when used in a once- weekly dosing scheme among patients with TB-HIV co-infection [115,116].

Use of non rifamycin-based TB regimens A TB treatment regimen without rifampicin may be considered to circumvent the problem of pharmacokinetic interactions with PIs. This is not an attractive option, as use of rifampicin in TB-HIV co-infection has been associated with better responses to TB treatment, improved survival, and reduced recurrence rates of TB in HIV-co-infected persons [117,118]. In addition, TB treatment without rifampicin should be extended to 8 months instead of 6 months. In

38 First-line TB drugs and antiretroviral pharmacological interactions review instances where a rifampicin containing regimen is initially given during the initiation phase of TB therapy and then switched to a non-rifampicin regimen, to allow for PI-based ART to be initiated, rifampicin should be discontinued at least 2 weeks before the introduction of a PI-based regimen. This will allow for the induction effect of rifampicin on CYP3A4 to dissipate to 2a avoid sub-therapeutic concentrations of the PI. In summary, no clear second-line antiretroviral regimen is available for TB patients. The best option may arguably be the use of a triple-NRTI regimen for the duration of rifampicin-based TB treatment.

Conclusions and research priorities With about one million TB-HIV coinfected patients eligible for combined TB-HIV treatment each year, it is of utmost importance to optimise combined TB-HIV treatment in terms of safety and efficacy. Major progress has been made to evaluate the interactions between TB drugs and antiretroviral therapy, however burning questions remain concerning nevirapine and efavirenz effectiveness during rifampicin-based TB treatment, treatment options for TB-HIV coinfected patients with NNRTI resistance or intolerance and exact treatment or dosing schedules for vulnerable patients including children and pregnant women. The current research priorities can be addressed using different strategies that are equally important (table 2): to maximise the use of already existing programme and research data, to create new data by conducting clinical trials and prospective observational studies and to engage a lobby to make currently unavailable drugs available to those most in need. Evaluating program data and conducting meta-analyses or modeling exercises with trial data maximizes the use of already existing data. For example, more knowledge is needed on the effect of rifampicin on the effectiveness of ART, more specifically on the virological and immunological outcomes of HIV infected patients using efavirenz or nevirapine based ART with rifampicin. Conducting a meta-analysis or modeling exercise on all currently available clinical trial and observational study data could be an approach to solve currently unresolved issues, such as finding the optimal efavirenz dose during rifampicin-based TB treatment, elucidating the association between subtherapeutic efavirenz plasma concentrations and treatment outcome and whether nevirapine-based ART should be recommended. An example of modelling was the use of population pharmacokinetic analysis for dose optimization, which has been done by Zhang et al whose simulations predicted that almost all patients receiving doubled doses of lopinavir/ritonavir achieve satisfying morning trough concentrations of lopinavir during rifampicin co-administration [119]. Furthermore, actual data on adverse effects during TB-HIV combined treatment are scarce, and in addition to prospective studies on this subject, a thorough literature review or meta-analysis of existing data would be highly valuable. Retrospective research using already existing data would, however, not replace the need for prospective studies. Efavirenz is the preferred NNRTI to be used with rifampicin-based TB treatment. Rifampicin has an effect on plasma concentrations of the NNRTIs nevirapine and efavirenz. Despite the fact that nevirapine is the most widely used NNRTI in developing countries because it is cheaper than efavirenz and safe during pregnancy, there is much more clinical and pharmacokinetic data on rifampicin with efavirenz than with nevirapine. More research is needed on the

39 chapter 2a

Table 2. Pharmacological research priorities concerning combined TB and HIV treatment

NNRTIs Evaluation of 600mg and 800mg efavirenz and/or the need for individual efavirenz dose adjustment when 2a combined with rifampicin and the role of pharmacogenetic profile (CYP2B6) The effect of rifampicin on the efficacy of nevirapine-based ART (virological and immunological outcomes) Evaluate the pharmacokinetic and efficacy effects of omitting the lead-in dose of nevirapine with combined with rifampicin. Protease inhibitors Evaluate the efficacy (virological and immunological outcomes) and safety of bPI and sbPI-based regimens when combined with rifampicin in relevant populations (HIV-infected TB patients) Evaluation of the tolerability (hepatotoxicity) of lopinavir/ritonavir co-administered with rifampicin in African and Asian HIV-TB populations. NRTIs / NtRTIs Evaluating the efficacy of triple NRTI/NtRTIs regimens for temporal use during combined rifampicin-based TB treatment. New antiretroviral drugs Evaluation of the tolerability and efficacy of double-dose raltegravir in HIV-TB patients using concomitant rifampicin Evaluate the safety and efficacy of twice daily dolutegravir in combined use with rifampicin based TB treatment in HIV-TB coinfected patients. Other Effect of isoniazid monotherapy (treatment LTBI) on PK of efavirenz and lopinavir/ritonavir Effect of isoniazid monotherapy (treatment LTBI) on treatment outcome of efavirenz or lopinavir-based ART General Determine the incidence and type of adverse effects during TB-HIV combined treatment The development of simple and inexpensive analytical tools for TDM, e.g. for efavirenz. Development of safe and effective ART regimens for children Evaluate the efficacy of safe ART regimens (e.g. nevirapine based) in pregnant women

Legend: TDM, therapeutic drug monitoring; LTBI, latent tuberculosis infection; ART, antiretroviral treatment; PK, pharmacokinetic

effectiveness of nevirapine-based ART when combined with rifampicin and whether the lead-in dose could be omitted, as recent research indicated that nevirapine initiation at the maintenance dose of 200 mg twice daily is preferred [71]. There is no conclusive evidence concerning dose-adjustment of efavirenz during rifampicin-based TB treatment. In summary, more adequately powered observational cohort studies and randomized clinical trials should be conducted to assess the clinical relevance of the NNRTI-rifampicin interactions for both efavirenz and nevirapine. On the use of PIs and ritonavir-boosted PIs, these drugs only display suboptimal exposure when combined with rifampicin. Increasing the dose of protease inhibitors is recommended to compensate for the metabolism inducing effect of rifampicin, which can be achieved by increasing the ritonavir dose or by doubling the lopinavir/ritonavir dose. More research is

40 First-line TB drugs and antiretroviral pharmacological interactions review needed on the effect on virological and immunological treatment outcomes of such dose increases. Furthermore, toxicity remains an issue with boosted (or superboosted) PIs, although the severe adverse effects observed in volunteer subjects using rifampicin and boosted-PIs were not observed to the same extent in HIV-infected TB patients [88,89]. This underlines 2a the need of pharmacological interaction studies in the relevant populations (i.e. HIV-infected TB patients) because data from healthy volunteers cannot be readily translated to patient populations [111,120]. There are new antiretroviral drugs, for example raltegravir, that seem promising candidates for second line ART when combined with TB treatment in terms of safety and tolerability. Plasma concentrations of the integrase inhibitor raltegravir are reduced by rifampicin. However increasing the raltegravir dose overcomes this and a recent small study indicated a good tolerability of the increased dose [98]. However, more safety, pharmacokinetic and efficacy data of an increased raltegravir dose combined with rifampicin is definitely needed. Triple/quadruple NRTI/NtRTI treatment in combination with rifampicin-based TB treatment has not been evaluated in clinical trials. The efficacy and safety of triple/quadruple NRTI/ NtRTI treatment during rifampicin-based TB treatment for its use as a temporal ART during TB treatment should be evaluated. There is a need for simple and inexpensive modes of plasma concentration monitoring (Therapeutic Drug Monitoring) for antiretroviral drugs, such as a rapid test using a saliva sample. 107 This could potentially be useful for efavirenz, where there is a discussion around the need for individualized dosing based on pharmacogenetic factors [58]. A pragmatic approach with a simple TDM technique would be a way around the need to determine a patient’s CYP2B6 genotype prior to efavirenz use, which would not be feasible in resource-limited settings. The effect of isoniazid monotherapy for treatment of latent TB infections on efavirenz and lopinavir pharmacokinetics could be studied in prospective observational studied. However, the effect of isoniazid monotherapy on ART treatment outcome might be evaluated using already existent program data. Pharmacokinetic interaction studies with ARVs should be considered and planned early in the course of the TB drug development process, to ensure that HIV-infected TB patients can benefit from new treatment regimens for treatment of susceptible or multidrug resistant TB [120]. Such interaction studies must be conducted in the relevant populations, as opposed to healthy volunteers, to account for environmental and host factors such as HIV infection and pharmacogenetic factors. If pharmacological scientific data shows that new antiretroviral drugs would provide a way around the increased toxicity or decreased effectiveness of currently recommended second line ART regimens, a lobby to make currently unavailable drugs available to those most in need is eventually the ultimate way forward to achieve better treatment for TB-HIV coinfected patients. Emphasis should be put on the fact that it is only a relatively small proportion of all TB-HIV coinfected patients who would need different (i.e. new) antiretroviral drugs to overcome a limited period of time (six months) during which they have to combine second line ART with rifampicin-based TB treatment.

41 chapter 2a

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36. Stohr W, Back D, Dunn D, Sabin C, Winston A, Gilson R, et al. Factors influencing efavirenz and nevirapine plasma concentration: effect of ethnicity, weight and co-medication. Antivir. Ther. 2008; 13:675-685. 37. Lopez-Cortes LF, Ruiz-Valderas R, Viciana P, arcon-Gonzalez A, Gomez-Mateos J, Leon-Jimenez E, et al. Pharmacokinetic interactions between efavirenz and rifampicin in HIV-infected patients with 2a tuberculosis. Clin. Pharmacokinet. 2002;41:681-690. 38. Kappelhoff BS, van LF, Robinson PA, MacGregor TR, Baraldi E, Montella F, et al. Are adverse events of nevirapine and efavirenz related to plasma concentrations? Antivir. Ther. 2005;10:489-498. 39. CDC. CDC, Managing Drug interactions in the treatment of HIV-related tuberculosis, 2007. Available at: http://www.cdc.gov/tb/TB HIV Drugs/default.htm 2007; Accessed 16th August 2013 40. Orrell C, Cohen K, Conradie F, Zeinecker J, Ive P, Sanne I, et al. Efavirenz and rifampicin in the South African context: is there a need to dose-increase efavirenz with concurrent rifampicin therapy? Antivir Ther 2011;16:527-534. 41. Uttayamakul S, Likanonsakul S, Manosuthi W, Wichukchinda N, Kalambaheti T, Nakayama EE, et al. Effects of CYP2B6 G516T polymorphisms on plasma efavirenz and nevirapine levels when co-administered with rifampicin in HIV/TB co-infected Thai adults. AIDS Res Ther 2010;7:8. 42. Cohen K, Grant A, Dandara C, McIlleron H, Pemba L, Fielding K, et al. Effect of rifampicin-based antitubercular therapy and the cytochrome P450 2B6 516G>T polymorphism on efavirenz concentrations in adults in South Africa. Antivir. Ther. 2009;14:687-695. 43. Manosuthi W, Sungkanuparph S, Thakkinstian A, Vibhagool A, Kiertiburanakul S, Rattanasiri S, et al. Efavirenz levels and 24-week efficacy in HIV-infected patients with tuberculosis receiving highly active antiretroviral therapy and rifampicin. AIDS 2005;19:1481-1486. 44. Manosuthi W, Kiertiburanakul S, Sungkanuparph S, Ruxrungtham K, Vibhagool A, Rattanasiri S, et al. Efavirenz 600 mg/day versus efavirenz 800 mg/day in HIV-infected patients with tuberculosis receiving rifampicin: 48 weeks results. AIDS 2006;20:131-132. 45. Manosuthi W, Sungkanuparph S, Tantanathip P, Mankatitham W, Lueangniyomkul A, Thongyen S, et al. Body weight cutoff for daily dosage of efavirenz and 60-week efficacy of efavirenz-based regimen in human immunodeficiency virus and tuberculosis coinfected patients receiving rifampin. Antimicrob Agents Chemother 2009;53:4545-4548. 46. Manosuthi W, Sungkanuparph S, Tantanathip P, Lueangniyomkul A, Mankatitham W, Prasithsirskul W, et al. A randomized trial comparing plasma drug concentrations and efficacies between 2 nonnucleoside reverse-transcriptase inhibitor-based regimens in HIV-infected patients receiving rifampicin: the N2R Study. Clin Infect Dis 2009;48:1752-1759. 47. Villar J, Sanchez P, Gonzalez A, Sorli L, Montero MM, Guelar A, et al. Use of non-nucleoside analogues together with rifampin in HIV patients with tuberculosis. HIV Clin Trials 2011;12:171-174. 48. Boulle A, Van CG, Cohen K, Hilderbrand K, Mathee S, Abrahams M, et al. Outcomes of nevirapine- and efavirenz-based antiretroviral therapy when coadministered with rifampicin-based antitubercular therapy. JAMA 2008;300:530-539. 49. Nachega JB, Hislop M, Dowdy DW, Gallant JE, Chaisson RE, Regensberg L, et al. Efavirenz versus nevirapine-based initial treatment of HIV infection: clinical and virological outcomes in Southern African adults. AIDS 2008;22:2117-2125. 50. Haas DW, Ribaudo HJ, Kim RB, Tierney C, Wilkinson GR, Gulick RM, et al. Pharmacogenetics of efavirenz and central nervous system side effects: an Adult AIDS Clinical Trials Group study. AIDS 2004;18:2391- 2400.

44 First-line TB drugs and antiretroviral pharmacological interactions review

51. Kwara A, Tashima KT, Dumond JB, Poethke P, Kurpewski J, Kashuba AD, et al. Modest but variable effect of rifampin on steady-state plasma pharmacokinetics of efavirenz in healthy African-American and Caucasian volunteers. Antimicrob Agents Chemother 2011;55:3527-3533. 52. Friedland G, Khoo S, Jack C, Lalloo U. Administration of efavirenz (600 mg/day) with rifampicin results in highly variable levels but excellent clinical outcomes in patients treated for tuberculosis and HIV. 2a J. Antimicrob. Chemother. 2006;58:1299-1302. 53. Burger D, Van der Heiden I, laPorte C, Van der Ende M, Groeneveld P, Richter C, et al. Interpatient variability in the pharmacokinetics of the HIV non-nucleoside reverse transcriptase inhibitor efavirenz: the effect of gender, race, and CYP2B6 polymorphism. Br. J. Clin. Pharmacol. 2006;61:148-154. 54. Ngaimisi E, Mugusi S, Minzi O, Sasi P, Riedel KD, Suda A, et al. Effect of rifampicin and CYP2B6 genotype on long-term efavirenz autoinduction and plasma exposure in HIV patients with or without tuberculosis. Clin Pharmacol Ther 2011;90:406-413. 55. Kwara A, Lartey M, Sagoe KW, Court MH. Paradoxically elevated efavirenz concentrations in HIV/ tuberculosis-coinfected patients with CYP2B6 516TT genotype on rifampin-containing antituberculous therapy. AIDS 2011;25:388-390. 56. Kwara A, Lartey M, Sagoe KW, Rzek NL, Court MH. CYP2B6 (c.516G-->T) and CYP2A6 (*9B and/or *17) polymorphisms are independent predictors of efavirenz plasma concentrations in HIV-infected patients. Br J Clin Pharmacol 2009;67:427-436. 57. van Luin M, Brouwer AM, van der Ven A, de Lange W, van Schaik RH, Burger DM. Efavirenz dose reduction to 200 mg once daily in a patient treated with rifampicin. AIDS 2009;23:742-744. 58. Manosuthi W, Sukasem C, Lueangniyomkul A, Mankatitham W, Thongyen S, Nilkamhang S, et al. Impact of Pharmacogenetic Markers of CYP2B6, Clinical Factors, and Drug-Drug Interaction on Efavirenz Concentrations in HIV/Tuberculosis-Coinfected Patients. Antimicrob Agents Chemother 2013;57:1019- 1024. 59. Rekic D, Roshammar D, Mukonzo J, Ashton M. In silico prediction of efavirenz and rifampicin drug-drug interaction considering weight and CYP2B6 phenotype. Br J Clin Pharmacol 2011;71:536-543. 60. Gengiah TN, Holford NH, Botha JH, Gray AL, Naidoo K, Abdool Karim SS. The influence of tuberculosis treatment on efavirenz clearance in patients co-infected with HIV and tuberculosis. Eur J Clin Pharmacol 2012;68:689-695. 61. de la Calle C, Sanchez A, Codero M, Iglesias A, Luna G. Moderator effect of CYP2B6 genotype in HIV-1 patients with tuberculosis treated with rifampicin and efavirenz. Journal of the International AIDS Society 2012;15:18364. 62. von Hentig N, Carlebach A, Gute P, Knecht G, Klauke S, Rohrbacher M, et al. A comparison of the steady- state pharmacokinetics of nevirapine in men, nonpregnant women and women in late pregnancy. Br. J. Clin. Pharmacol. 2006;62:552-559. 63. Autar RS, Wit FW, Sankote J, Mahanontharit A, Anekthananon T, Mootsikapun P, et al. Nevirapine plasma concentrations and concomitant use of rifampin in patients coinfected with HIV-1 and tuberculosis. Antivir. Ther. 2005;10:937-943. 64. Ribera E, Pou L, Lopez RM, Crespo M, Falco V, Ocana I, et al. Pharmacokinetic interaction between nevirapine and rifampicin in HIV-infected patients with tuberculosis. J. Acquir. Immune. Defic. Syndr. 2001;28:450-453. 65. Cohen K, Van CG, Boulle A, McIlleron H, Goemaere E, Smith PJ, et al. Effect of rifampicin-based antitubercular therapy on nevirapine plasma concentrations in South African adults with HIV-associated tuberculosis. J. Antimicrob. Chemother. 2008;61:389-393.

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66. Swaminathan S, Padmapriyadarsini C, Venkatesan P, Narendran G, Ramesh Kumar S, Iliayas S, et al. Efficacy and safety of once-daily nevirapine- or efavirenz-based antiretroviral therapy in HIV-associated tuberculosis: a randomized clinical trial. Clin Infect Dis 2011;53:716-724. 67. Elsherbiny D, Cohen K, Jansson B, Smith P, McIlleron H, Simonsson US. Population pharmacokinetics of 2a nevirapine in combination with rifampicin-based short course chemotherapy in HIV- and tuberculosis- infected South African patients. Eur. J. Clin. Pharmacol. 2009;65:71-80. 68. Ramachandran G, Hemanthkumar AK, Rajasekaran S, Padmapriyadarsini C, Narendran G, Sukumar B, et al. Increasing nevirapine dose can overcome reduced bioavailability due to rifampicin coadministration. J. Acquir. Immune. Defic. Syndr. 2006;42:36-41. 69. Avihingsanon A, Manosuthi W, Kantipong P, Chuchotaworn C, Moolphate S, Sakornjun W, et al. Pharmacokinetics and 48-week efficacy of nevirapine: 400 mg versus 600 mg per day in HIV- tuberculosis coinfection receiving rifampicin. Antivir. Ther. 2008;13:529-536. 70. Mankhatitham W, Lueangniyomkul A, Manosuthi W. Hepatotoxicity in patients co-infected with tuberculosis and HIV-1 while receiving non-nucleoside reverse transcriptase inhibitor-based antiretroviral therapy and rifampicin-containing anti-tuberculosis regimen. Southeast Asian J Trop Med Public Health 2011;42:651-658. 71. Lamorde M, Byakika-Kibwika P, Okaba-Kayom V, Ryan M, Coakley P, Boffito M, et al. Nevirapine pharmacokinetics when initiated at 200 mg or 400 mg daily in HIV-1 and tuberculosis co-infected Ugandan adults on rifampicin. J Antimicrob Chemother 2011;66:180-183. 72. Matteelli A, Saleri N, Villani P, Bonkoungou V, Carvalho AC, Kouanda S, et al. Reversible reduction of nevirapine plasma concentrations during rifampicin treatment in patients coinfected with HIV-1 and tuberculosis. J Acquir Immune Defic Syndr 2009;52:64-69. 73. Sathia L, Obiorah I, Taylor G, Kon O, O’Donoghue M, Gibbins S, et al. Concomitant use of nonnucleoside analogue reverse transcriptase inhibitors and rifampicin in TB/HIV type 1-coinfected patients. AIDS Res Hum Retroviruses 2008;24:897-901. 74. Manosuthi W, Tantanathip P, Chimsuntorn S, Eampokarap B, Thongyen S, Nilkamhang S, et al. Treatment outcomes of patients co-infected with HIV and tuberculosis who received a nevirapine- based antiretroviral regimen: a four-year prospective study. Int J Infect Dis 2010;14:e1013-1017. 75. Kwara A, Ramachandran G, Swaminathan S. Dose adjustment of the non-nucleoside reverse transcriptase inhibitors during concurrent rifampicin-containing tuberculosis therapy: one size does not fit all. Expert Opin Drug Metab Toxicol 2010;6:55-68. 76. Piscitelli SC, Gallicano KD. Interactions among drugs for HIV and opportunistic infections. N. Engl. J. Med. 2001;344:984-996. 77. Barry M, Gibbons S, Back D, Mulcahy F. Protease inhibitors in patients with HIV disease. Clinically important pharmacokinetic considerations. Clin. Pharmacokinet. 1997;32:194-209. 78. von Moltke LL, Greenblatt DJ, Duan SX, Daily JP, Harmatz JS, Shader RI. Inhibition of desipramine hydroxylation (Cytochrome P450-2D6) in vitro by quinidine and by viral protease inhibitors: relation to drug interactions in vivo. J. Pharm. Sci. 1998;87:1184-1189. 79. Moreno S, Hernandez B, Dronda F. Antiretroviral therapy in AIDS patients with tuberculosis. AIDS Rev. 2006;8:115-124. 80. Braithwaite RS, Kozal MJ, Chang CC, Roberts MS, Fultz SL, Goetz MB, et al. Adherence, virological and immunological outcomes for HIV-infected veterans starting combination antiretroviral therapies. AIDS 2007;21:1579-1589.

46 First-line TB drugs and antiretroviral pharmacological interactions review

81. Burger DM, Agarwala S, Child M, Been-Tiktak A, Wang Y, Bertz R. Effect of rifampin on steady-state pharmacokinetics of atazanavir with ritonavir in healthy volunteers. Antimicrob. Agents Chemother. 2006;50:3336-3342. 82. la Porte CJ, Colbers EP, Bertz R, Voncken DS, Wikstrom K, Boeree MJ, et al. Pharmacokinetics of adjusted-dose lopinavir-ritonavir combined with rifampin in healthy volunteers. Antimicrob Agents 2a Chemother 2004;48:1553-1560. 83. Schmitt C, Riek M, Winters K, Schutz M, Grange S. Unexpected Hepatotoxicity of Rifampin and Saquinavir/Ritonavir in Healthy Male Volunteers. Arch. Drug Inf. 2009;2:8-16. 84. Ribera E, Azuaje C, Lopez RM, Domingo P, Curran A, Feijoo M, et al. Pharmacokinetic interaction between rifampicin and the once-daily combination of saquinavir and low-dose ritonavir in HIV- infected patients with tuberculosis. J. Antimicrob. Chemother. 2007;59:690-697. 85. Acosta EP, Kendall MA, Gerber JG, ston-Smith B, Koletar SL, Zolopa AR, et al. Effect of concomitantly administered rifampin on the pharmacokinetics and safety of atazanavir administered twice daily. Antimicrob. Agents Chemother. 2007;51:3104-3110. 86. Veldkamp AI, Hoetelmans RM, Beijnen JH, Mulder JW, Meenhorst PL. Ritonavir enables combined therapy with rifampin and saquinavir. Clin. Infect. Dis. 1999;29:1586. 87. Nijland HM, L’Homme R F, Rongen GA, van Uden P, van Crevel R, Boeree MJ, et al. High incidence of adverse events in healthy volunteers receiving rifampicin and adjusted doses of lopinavir/ritonavir tablets. AIDS 2008;22:931-935. 88. Decloedt EH, Maartens G, Smith P, Merry C, Bango F, McIlleron H. The safety, effectiveness and concentrations of adjusted lopinavir/ritonavir in HIV-infected adults on rifampicin-based antitubercular therapy. PLoS One 2012;7:e32173. 89. Decloedt EH, McIlleron H, Smith P, Merry C, Orrell C, Maartens G. Pharmacokinetics of lopinavir in HIV- infected adults receiving rifampin with adjusted doses of lopinavir-ritonavir tablets. Antimicrob Agents Chemother 2011;55:3195-3200. 90. Grub S, Bryson H, Goggin T, Ludin E, Jorga K. The interaction of saquinavir (soft gelatin capsule) with ketoconazole, erythromycin and rifampicin: comparison of the effect in healthy volunteers and in HIV- infected patients. Eur J Clin Pharmacol 2001;57:115-121. 91. L’Homme R F, Nijland HM, Gras L, Aarnoutse RE, van Crevel R, Boeree M, et al. Clinical experience with the combined use of lopinavir/ritonavir and rifampicin. AIDS 2009;23:863-865. 92. Loeliger A, Suthar AB, Ripin D, Glaziou P, O’Brien M, Renaud-Thery F, et al. Protease inhibitor- containing antiretroviral treatment and tuberculosis: can rifabutin fill the breach? Int J Tuberc Lung Dis 2012;16:6-15. 93. Yeni PG, Hammer SM, Carpenter CC, Cooper DA, Fischl MA, Gatell JM, et al. Antiretroviral treatment for adult HIV infection in 2002: updated recommendations of the International AIDS Society-USA Panel. JAMA 2002;288:222-235. 94. Burger DM, Meenhorst PL, Koks CH, Beijnen JH. Pharmacokinetic interaction between rifampin and zidovudine. Antimicrob. Agents Chemother. 1993;37:1426-1431. 95. Droste JA, Verweij-van Wissen CP, Kearney BP, Buffels R, Vanhorssen PJ, Hekster YA, et al. Pharmacokinetic study of tenofovir disoproxil fumarate combined with rifampin in healthy volunteers. Antimicrob. Agents Chemother. 2005;49:680-684. 96. Boyd MA, Zhang X, Dorr A, Ruxrungtham K, Kolis S, Nieforth K, et al. Lack of enzyme-inducing effect of rifampicin on the pharmacokinetics of enfuvirtide. J. Clin. Pharmacol. 2003;43:1382-1391.

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97. Siegert S, Schnierle P, Schnierle BS. Novel anti-viral therapy: drugs that block HIV entry at different target sites. Mini. Rev. Med. Chem. 2006;6:557-562. 98. Burger DM, Magis-Escurra C, van den Berk GE, Gelinck LB. Pharmacokinetics of double-dose raltegravir 2a in two patients with HIV infection and tuberculosis. AIDS 2010;24:328-330. 99. Wenning LA, Petry AS, Kost JT, Jin B, Breidinger SA, DeLepeleire I, et al. Pharmacokinetics of raltegravir in individuals with UGT1A1 polymorphisms. Clin Pharmacol Ther 2009;85:623-627. 100. Mena A, Vazquez P, Castro A, Lopez S, Bello L, Pedreira JD. Clinical experience of raltegravir-containing regimens in HIV-infected patients during rifampicin-containing treatment of tuberculosis. J Antimicrob Chemother 2011;66:951-952. 101. Dooley KE, Sayre P, Borland J, Purdy E, Chen S, Song I, et al. Safety, Tolerability, and Pharmacokinetics of the HIV Integrase Inhibitor Dolutegravir Given Twice Daily With Rifampin or Once Daily With Rifabutin: Results of a Phase 1 Study Among Healthy Subjects. J Acquir Immune Defic Syndr 2013;62:21-27. 102. Walker DK, Abel S, Comby P, Muirhead GJ, Nedderman AN, Smith DA. Species differences in the disposition of the CCR5 antagonist, UK-427,857, a new potential treatment for HIV. Drug Metab Dispos. 2005;33:587-595. 103. Johnson LB, Saravolatz LD. Etravirine, a next-generation nonnucleoside reverse-transcriptase inhibitor. Clin. Infect. Dis. 2009;48:1123-1128. 104. Kakuda TN, Scholler-Gyure M, Hoetelmans RM. Pharmacokinetic interactions between etravirine and non-antiretroviral drugs. Clin Pharmacokinet 2011;50:25-39. 105. Ford N, Lee J, Andrieux-Meyer I, Calmy A. Safety, efficacy, and pharmacokinetics of rilpivirine: systematic review with an emphasis on resource-limited settings. HIV AIDS (Auckl) 2011;3:35-44. 106. Patel IH, Zhang X, Nieforth K, Salgo M, Buss N. Pharmacokinetics, pharmacodynamics and drug interaction potential of enfuvirtide. Clin Pharmacokinet 2005;44:175-186. 107. Kwara A, Flanigan TP, Carter EJ. Highly active antiretroviral therapy (HAART) in adults with tuberculosis: current status. Int J. Tuberc. Lung Dis. 2005;9:248-257. 108. Kwara A, Carter EJ, Rich JD, Flanigan TP. Development of opportunistic infections after diagnosis of active tuberculosis in HIV-infected patients. AIDS Patient. Care STDS. 2004;18:341-347. 109. McIlleron H, Meintjes G, Burman WJ, Maartens G. Complications of antiretroviral therapy in patients with tuberculosis: drug interactions, toxicity, and immune reconstitution inflammatory syndrome. J. Infect. Dis. 2007;196 Suppl 1:S63-S75. 110. Yimer G, Ueda N, Habtewold A, Amogne W, Suda A, Riedel KD, et al. Pharmacogenetic & pharmacokinetic biomarker for efavirenz based ARV and rifampicin based anti-TB drug induced liver injury in TB-HIV infected patients. PLoS One 2011;6:e27810. 111. Toibaro JJ, Losso MH. Pharmacokinetics interaction studies between rifampicin and protease inhibitors: methodological problems. AIDS 2008;22:2046-2047. 112. Gulick RM, Ribaudo HJ, Shikuma CM, Lustgarten S, Squires KE, Meyer WA, III, et al. Triple-nucleoside regimens versus efavirenz-containing regimens for the initial treatment of HIV-1 infection. N. Engl. J. Med. 2004;350:1850-1861. 113. van Leeuwen R, Katlama C, Murphy RL, Squires K, Gatell J, Horban A, et al. A randomized trial to study first-line combination therapy with or without a protease inhibitor in HIV-1-infected patients. AIDS 2003;17:987-999. 114. WHO. WHO TB Report 2012 - Global tuberculosis report 2012 (WHO/HTM/TB/2012.6). Available at: http://www.who.int/tb/publications/global_report/gtbr12_main.pdf 2012; Accessed 20th Sept. 2013.

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115. Weiner M, Benator D, Burman W, Peloquin CA, Khan A, Vernon A, et al. Association between acquired rifamycin resistance and the pharmacokinetics of rifabutin and isoniazid among patients with HIV and tuberculosis. Clin. Infect. Dis. 2005;40:1481-1491. 116. Weiner M, Burman W, Vernon A, Benator D, Peloquin CA, Khan A, et al. Low isoniazid concentrations and outcome of tuberculosis treatment with once-weekly isoniazid and rifapentine. Am. J. Respir. Crit 2a Care Med. 2003;167:1341-1347. 117. Okwera A, Whalen C, Byekwaso F, Vjecha M, Johnson J, Huebner R, et al. Randomised trial of thiacetazone and rifampicin-containing regimens for pulmonary tuberculosis in HIV-infected Ugandans. The Makerere University-Case Western University Research Collaboration. Lancet 1994;344:1323-1328. 118. Elliot-Lopez C. Treatment news: new options for the future. Women Alive. 2000:7. 119. Zhang C, Denti P, Decloedt E, Maartens G, Karlsson MO, Simonsson US, et al. Model-based approach to dose optimization of lopinavir/ritonavir when co-administered with rifampicin. Br J Clin Pharmacol 2012;73:758-767. 120. Dooley KE, Kim PS, Williams SD, Hafner R. TB and HIV Therapeutics: Pharmacology Research Priorities. AIDS Res Treat 2012;2012:874083.

49

AtriplaR/anti-TB combination in TB/HIV 2b patients. Drug in focus

Hadija H Semvua and Gibson S Kibiki

BMC Research Notes 2011;4:511 chapter 2b

Abstract Background: Co-administration of anti-tuberculosis and antiretroviral therapy is often inevitable in high-burden countries where tuberculosis is the most common opportunistic 2b infection associated with HIV/AIDS. Concurrent use of rifampicin and several antiretroviral drugs is complicated by pharmacokinetic drug-drug interaction.

Method: Pubmed and Google search following the key words tuberculosis, HIV, emtricitabine, tenofovir efavirenz, interaction were used to find relevant information on each drug of the fixed dose combination AtriplaR.

Results: Information on generic name, trade name, pharmacokinetic parameter, metabolism and the pharmacokinetic interaction with Anti-TB drugs of emtricitabine, tenofovir, and efavirenz was obtained.

Conclusion: Fixed dose combination of emtricitabine/tenofovir/efavirenz (AtriplaR) which has been approved by Food and Drug Administration shows promising results as far as safety and efficacy is concerned in TB/HIV coinfection patients, hence can be considered effective and safe antiretroviral drug in TB/HIV management for adults and children above 3 years of age.

Keywords: Tuberculosis, HIV, Emtricitabine, Tenofovir, Efavirenz, Interaction

52 AtriplaR and First-line TB drugs review

Background Human immunodeficiency virus (HIV) and tuberculosis (TB) are overlapping epidemics that cause an immense burden of disease. Sub-Sahara Africa is a region most affected by both diseases including Tanzania [1]. Literatures show that more than 75% of TB patients have also 2b HIV, and possibly more than half of worldwide patients infected with HIV will also develop TB [2,3]. Furthermore TB contributed to 27% of all AIDS diagnoses [4]. For TB the currently used combination drug regimens produce cure rates that exceed 95%, given good patient adherence during the multiple months’ treatment period [5]. Also for HIV many regimens are available; howeveroptimal treatment regimens for TB/HIV co-infection are not yet clearly defined. As therapy for HIV disease becomes more available, physicians need to know how to treat these two diseases effectively while minimizing the risk of drug interactions and maintaining the shortest possible duration of treatment for TB. Current treatment of mycobacterium tuberculosis in most resource limited settings is comprised of a four-drug initial anti-tuberculosis regimen for 2 months (rifampicin, isoniazid, pyrazinamide andethambutol), followed by two-drugs continuation phase of anti-tuberculosis regimen for 4 months (rifampicin and isoniazid). For TB/HIV co-infected patients the guidelines which exist [6] have shown many challenges. Combining drug therapies for dual infection TB and HIV is made complex by alterations in the activity of the hepatic Cytochrome P450 (CYP) system, high pill Burdens, shared drug toxicities, drug-drug and drug-disease interactions, immune reconstitution inflammatory syndrome, co-morbid diseases and drug resistance in both bacillus and virus [7,8]. The CYP isoform enzymes are responsible for many interactions [9] (especially those involving rifampicin and isoniazid) during drug biotransformation (metabolism) in the liver and/or intestine, due to it is enzyme induction effect.

Presentation of the hypothesis Adherence to a complex regimen is often a significant barrier to treatment success. Following the current guideline where a patient has to take 4-fixed dose combination for TB and the complex triple combination therapy selected for HIV treatment. The consequence of many pills reduce compliance and hence adherence to the treatment regimen leading to suboptimal TB and HIV treatment, hence increasing the possibility of drug resistance and shared drug toxicities [10]. These toxicities may necessitate therapy discontinuation, which exacerbates immune suppression and predisposes to other opportunistic infections.

TB/HIV co-infection simultaneous treatment have shown pharmacokinetic interactions produced with mainly rifampicin [(a cornerstone in TB treatment) and the non nucleoside reverse transcriptase inhibitors (NNRTIs)]. Enzyme inducer decreases elimination halve life of nevirapine [11]. When rifampicin and nevirapine are given together there is an observed 31% to 58% decrease in plasma levels of nevirapine due to rifampicin induction effect [12-15]. Available literatures explain that rifampicin combination with efavirenz there is reduction of serum levels of 13-33% [16], however no virological failure reported to be significant [17]. HIVinfected patients achieve somewhat lower concentrations of the orally administered first-

53 chapter 2b

line anti-tubercular drugs [18,19]. The effect of rifampicin on the concentrations of Protease Inhibitors (PI) is well elaborated in a study of Moreno et al. [20]. Immune reconstitution inflammatory syndrome (IRIS), which is due to dysregulated immune recovery [21], occurs in 2b severely immune-suppressed HIV patients normally 1 to 4 weeks after ART initiation [22,23]. In tuberculosis-related IRIS which is an inflammatory reaction is directed towards mycobacterial antigens [24], resulting in worsening pulmonary infiltrates. Risk factors for TB-IRIS include low baseline CD4 count, high baseline viral load, short duration between TB and ART initiation and disseminated tuberculosis [22,25], making difficult to determine the optimal time to initiate ART in severely immune-suppressed TB patients [26,27]. Immune reconstitution is based on the patient’s ability to respond to treatment. For the most part, in patients with CD4 counts of 200-350 cells/mm3 who start treatment, their immune systems still have the ability to be activated to produce more CD4 cells [28]. In contrast, in patients with low CD4 counts (< 100 cells/mm3), it may be more difficult to activate their immune systems to produce many CD4 cells because of existing damage from the virus. In the management of TB/HIV co-infection directly observed therapy and other adherence promoting strategies should be used in all patients with HIV-related TB [29]. Whenever possible, the care for HIV-related TB should be provided by or in consultation with experts in management of both TB and HIV. The care for persons with HIV-related TB should include close attention to the possibility of TB treatment failure, antiretroviral treatment failure, paradoxical reactions, side effects for all drugs used, and drug toxicities associated with increased serum concentrations of rifampicin. Due to these accompanied complications, selecting an appropriate antiretroviral therapeutic regimen is warranted. This involves addressing multiple interdependent issues, including patient adherence, pharmacokinetic properties of the drugs (including food effects and drugdrug interactions), drug resistance, and overlapping adverse effects.

On 12 July 2006 Bristol-Myers Squibb and Gilead Sciences announced that the US Food and Drug Administration (FDA) had cleared AtriplaR, their fixed-dose combination tablet. AtriplaR is a complete regimen in a single, fixed-dose combination tablet that contains: efavirenz 600 mg, emtricitabine 200 mg and tenofovir disoproxil fumarate 300 mg. It is a novel co formulation drugs from two different classes, simplifying administration and increasing adherence too [30].

Testing the hypothesis A new formulation combining fixed doses of the nucleoside reverse transcriptase inhibitors emtricitabine (200 mg) and tenofovir disoproxil fumarate (tenofovir DF; 300 mg) with the non-nucleoside reverse transcriptase inhibitor efavirenz (600 mg) represents the first once- daily, one-tablet antiretroviral regimen. Co-formulated efavirenz/emtricitabine/tenofovir DF demonstrated excellent potency, tolerability and favorable safety profile [31]. Co administration of co-formulated efavirenz/emtricitabine/tenofovir DF with rifampicin based TB regimen clinical trials is going on in TB/HIV patients of Tanzania, therefore is important to have knowledge of each individual drug. Also a once-daily regimen of efavirenz, emtricitabine and tenofovir DF (administered as individual agents) was superior to once- daily efavirenz plus twice-daily co-formulated lamivudine/zidovudine in terms of virological

54 AtriplaR and First-line TB drugs review suppression, immunological recovery and adverse events [32]. Individually, these agents have long half-life that allow for once-daily dosing. Emtricitabine emtrivaR a cytosine analogue is the newest of the nucleoside reverse transcriptase inhibitor (NRTI) drugs. It was well tolerated in clinical trials where most adverse 2b events were consistent with the NRTI class. Moreover, emtricitabine based regimens were as well tolerated as those with lamivudine [33]. Emtricitabine has no currently known phase I (glucuronidation) or phase II (cytochrome P450 and others) interactions [34]. It may be taken with or without food [35]. Emtricitabine displays dose-proportional pharmacokinetics. Bioavailability is not affected by food intake. Mean steady-state Cmax, Tmax and AUC values were 1.7 mg/L, 2 h, and 10 mg/L.h respectively in 6 patients with HIV infection. Plasma elimination half-life is 8-10 h, but the intracellular half-life of the active compound, emtricitabine- triphosphate is much longer: 39 h which support the once daily dosing. Tenofovir disoproxil fumarate VireadR is an analogue of adenosine five monophosphate. It is a nucleotide analogue and consequently its mechanism of action differs from that of nucleoside analogues. It is a prodrug, therefore after absorption the drug is hydrolyzed and the active compound tenofovir-diphosphate is released. It has a longer serum half-life than most other NRTIs allowing a once daily dosing. The plasma half-life is 14.4h, but the intracellular elimination half-life of the active compound, tenofovir-diphosphate is much longer > 49h. This drug has broad tissue distribution, aided by its small molecular size and very low protein binding, and is eliminated as unchanged drug in the urine through glomerular filtration and active tubular secretion. Because of this latter characteristic, dosage adjustments are required in patients with renal insufficiency [36]. 70-80% of a dose is recovered unchanged in the urine, suggesting minimal influence of hepatic metabolism. Tenofovir mean steady-state Cmax, Tmax and AUC were 0.33mg/L, 2.3 h, and 3.0 mg/L.h respectively in patients infected with HIV. Adverse reactions are relatively uncommon, and nausea is the most often reported symptom [37]. The bioavailability of tenofovir disoproxil fumarate is increased in the presence of a high fat diet (40%-50%), and the drug should be taken with food [38,39]. Tenofovir disoproxil fumarate is not a substrate, inhibitor, or inducer of the P450 system, within the class of antiretroviral agents, an increase in the bioavailability of didanosine has been described, leading to the recommendation that the dose of didanosine be reduced when used in combination with tenofovir. Tenofovir can be used without adjustments with other nucleoside and nonnucleoside reverse transcriptase inhibitors. Equally, tenofovir seems to have no effect on the pharmacokinetics of protease inhibitors although these latter agents may produce a slight increase in the bioavailability of tenofovir, which seems to be of little clinical relevance. Efavirenz SustivaR is a NNRTI which is principally metabolized by cytochrome P450, more precisely isoenzymes CYP2B6 and CYP3A4. No active metabolites are formed. Efavirenz has a long terminal half-life of 40-55h after multiple dosing. Neuropsychiatric symptoms are the common side effects observed. Fumaz et al. [40], in a randomized, prospective, two-arm controlled study compared quality of life and neuropsychiatric side effects in patients receiving an efavirenz-containing regimen versus a group whose treatment did not include efavirenz. They found that the group receiving an efavirenz-containing regimen reported a better quality of life, particularly because their regimen was easier. They found, however, that 13% of their

55 chapter 2b

patients reported “character change”. Meals of normal composition have no appreciable effect on the pharmacokinetics of efavirenz. The bioavailability is increased following a high fat meal. Time to peak plasma concentration is 3-5 h. Steady state concentrations are achieved 2b after 6-7 days. Mean steady state Cmax, Cmin and AUC(0-24 h) were 4.1mg/L, 1.8 mg/L and 58.1 h*mg/L respectively after multiple dosing of 600 mg efavirenz once daily. Several pharmacokinetic studies have been conducted showing the interaction of rifampicin with efavirenz. Lopez-Cortez et al. [16] in Spain did on 24 patients with concomitant infections of HIV and tuberculosis. It was nonblinded study where group A (n = 16) received antituberculosis drugs without rifampicin but with added efavirenz 600 mg/day ARV regimen. The patients were then switched to a standard antituberculosis regimen containing rifampicin with efavirenz 600 mg/day (group A1; n = 8) or efavirenz 800 mg/day (group A2; n = 8). The result showed that levels of AUC, Cmax, and Cmin of efavirenz decreased respectively by 24%, 25%, and 22%, after addition of rifampicin to the efavirenz of 600mg/day group. Significant association of body weight, dose of efavirenz and its pharmacokinetic parameters was observed and the dose increase to 800 mg/day when efavirenz is used with rifampicin was recommended. Manosuthi et al. [17,41] conducted a non-blinded randomized clinical trial to Thai patients and they were given rifampicin for more than 1 month with regimen containing efavirenz 600 mg/day (n = 42), or efavirenz 800 mg/day (n = 42). The plasma concentrations of efavirenz were determined for both groups 12 h after drug administration and on day 14. Both groups had 12-h efavirenz concentrations (C12) of same value (median3.0 mg/L vs 3.3 mg/L); but the upper C12 levels werehigher in the group receiving efavirenz at the dosage of 800 mg/day (21.3 mg/L) in comparison with the group on 600 mg/day (12.2 mg/L). The study concluded that efavirenz with a dose of 600 mg/day is adequate for the majority of HIV-infected patients in Thailand (as their body weight is around 50 kg) but more studied should be performed to other ethnic groups. This is in line with a recent study by Orrell [42] which showed that 600 mg dose of efavirenz is adequate. Another study was conducted aiming at determining the effects of higher body weights and differing ethnicities on exposure to efavirenz. 9 patients with body weights of over 50 kg were enrolled in the study [43]. The patients received an antiretroviral regimen containing efavirenz 800 mg/day and antituberculosis therapy containing rifampicin. Plasma concentrations of efavirenz were monitored for 2 years. All patients had sub-therapeutic trough concentrations of efavirenz (median 11.7 mg/L; range 5.37-19.6 mg/L). The expected therapeutic range of trough concentrations of efavirenz were between 1.2 and 4.0 mg/L. Seven out of the 9 patients with HIV/TB co-infection had toxicity (6 of them black and 1 caucasian). Around 20% of ethnic Black Africans possess homologous G516T alleles versus 3% of Caucasians [44]. This CYP2B6 polymorphism is associated with exposure to higher concentrations of efavirenz. Existence of a wide variability in efavirenz concentrations among black Africans was observed in the study of Friedland et al. [45]. This study indicated that inter-subject variability in efavirenz concentrations was greater when the drug was administered with rifampicin (coefficient of variation [CV] 157%) compared with the intersubject variability after discontinuation of rifampicin (58%), with much of consistent intra-subject variation over time (CV 24%). However, Cmin was not significantly different during versus post TB therapy.

56 AtriplaR and First-line TB drugs review

Regarding metabolism studies have shown that CYP2B6 genetic polymorphisms influence very much efavirenz elimination [46-48]. CYP2B6 polymorphisms have been associated with altered PK of efavirenz in HIV-infected adults and children [49-51] resulting in a higher efavirenz exposure and an increase in central nervous system side effect [44,46]. CYP2B6 patients are 2b poor metabolizer of the drug and dose reduction has been proposed to people with this genotype [52,53]. Of note: is the high prevalence of efavirenz related CYP2B6 polymorphisms in patients of African region which represent the majority of the HIV-infected population in the world [54-58]. This indicates the need for prospective clinical studies to evaluate the utility of dose adjustments in these populations.

Use of efavirenz in children The use of efavirenz as a component of first-line antiretroviral therapy in adult and children above 3 years has been accepted worldwide [59], however safety and efficacyof efavirenz in children less than 3 years of age has not been performed [60-63] and the data on the efavirenz exposure in mothers and their breastfed infants is also limited [64] Despite the report that efavirenz is highly effective and well tolerated in the majority of pediatric patients [65], efavirenz plasma concentrations have been reported to be suboptimal in a large proportion of children and adolescents dosed according to current pediatric guidelines [60,61] Also interindividual variability has been observed in paediatric population [66]. Recently data on population pharmacokinetics (PK) of efavirenz in children predicts sub- therapeutic efavirenz exposure in a significant proportion on children with the currently recommended efavirenz dose [61,67]. Authors in ARROW study which was done recently in Uganda and Zimbabwe [68] suggested the use of higher paediatric efavirenz doses, as per WHO 2010 recommendations, however they concluded that children with toxic level of efavirenz concentration may be increased. Also efavirenz pharmacokinetics was shown to demonstrate significant age effects with apparent clearance much greater in young infants than older children. High efavirenz dose requirements and pharmacokinetic variability in infants will require additional pharmacokinetic studies to determine appropriate efavirenz dosing strategies. Therefore priorities should be made to access the developmental changes of efavirenz exposure in children and adolescents and application of therapeutic drug monitoring to the settings where the facility is available.

Implication of the hypothesis HAART regimens have contained very high pill burdens, complex dosing schedules, and specific food requirements. The management of HIV-related tuberculosis disease seems to be complex although the treatment of TB in persons with HIV is essentially the same as for patients without HIV. A major concern in treating TB in HIV-infected persons is the interaction of rifampin with certain antiretroviral agents, PIs and NNRTIs [69,70]. Rifabutin, which has fewer drug interactions, may be used as an alternative to rifampicin but is expensive and not available in resource limited area. Pharmacokinetic data which are present indicate that rifampicin can be combined with efavirenz [16,17].or nevirapine,[14] although for TB/HIV co-infected patients

57 chapter 2b

who got 600 mg rifampicin, adverse events were reported to be higher in nevirapine based regimen compared to efavirenz based regimen [71]. Non-boosted PIs cannot be administered with rifampicin because of considerable 2b decreases in plasma concentrations of PIs, which may cause sub-therapeutic concentrations exceptritonavir [72,73]. Ritonavir is a strong inhibitor of CYP3A4 and P-glycoprotein, and when it is low dose is combined with other PIs an important increase in their plasma concentrations is achieved, however this is not a solution as substantial decreases in plasma concentrations of indinavir,[74] lopinavir [75] and atazanavir [76] have been observed when co-administered with rifampicin, even when small doses of ritonavir are given. Another promising PI is saquinavir although in a study of Ribera et al. [77] a decrease of 40%, 35% and 49% in the median saquinavir AUC0-24, Cmax and Ctrough, respectively, were observed. In vitro study reveals the benefit from the use of combination therapy achieved with Atripla in terms of the emtricitabine and tenofovir intracellular drug concentration [78]. However in this study patients were HIV positive only hence more data are needed from TB/HIV co-infected patient. HIV fusion inhibitors represent a novel class of antiretroviral drugs and enfuvirtide is the first drug within this class to be approved [79]. When combined with active ARV drugs, different studies reported findings of substantial improvements in virological, immunological and clinical outcomes [80-83]. It has little potential for drug-drug interactions as far as coadministration with TB drugs is concerned [79,84,85]. The major limitation of this drug is the twice daily dosing which may interfere with the treatment adherence. Injection-site reactions occurred almost universally in its recipients, although did not necessitate stopping treatment. Co-formulated atriplaR (emtricitabine/tenofovir/efavirenz) have demonstrated excellent potency, tolerability and favorable safety profile. The drug is now available in most of the limited-resources countries. The drug is given once per day simplifying administration and increasing adherence too, hence may be recommended as a way to jointly treat these two diseases.

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83. Youle M, Staszweski S, Clotet B, Arribas JR, Blaxhult A, Carosi G, et al: Concomitant use of an active boosted protease inhibitor withenfuvirtide in treatment-experienced HIV-1 infected individuals: recentdata and consensus recommendations. HIV lin trials 2006;7:86-96. 84. Ruxrungtham K, Boyd M, Bellibas SE, et al: Lack of interaction between enfuvirtide and ritonavir or ritonavir-boosted saquinavir in HIV-1- infected patients. J Clin Pharmacol 2004;44:793-803. 2b 85. Boyd MA, Zhang X, Dorr A, Ruxrungtham K, et al: Lack of enzyme-inducing effect of rifampicin on the pharmacokinetics of enfuvirtide. J ClinPharmacol 2003;43:1382-1391.

63

Efavirenz, tenofovir and emtricitabine combined with first-line tuberculosis treatment in tuberculosis-HIV-co-infected Tanzania patients: a pharmacokinetic 3 and safety study

Hadija H Semvua, Charles M Mtabho, Quirine Fillekes, Jossy van den Boogaard, Riziki M Kisonga, Liberate Mleoh, Arnold Ndaro, Elton R Kisanga, Andre van der Ven, Rob E Aarnoutse, Gibson S Kibiki, Martin J Boeree, David M Burger

Antiviral Therapy 2013;18(1):105-13 chapter 3

Abstract Background: To evaluate the effect of rifampicin-based tuberculosis (TB) treatment on the pharmacokinetics of efavirenz/tenofovir/emtricitabine in a fixed-dose combination tablet, and vice versa, in Tanzanian TB-HIV-coinfected patients.

Method: This was a Phase II open-label multiple dose pharmacokinetic and safety study. This 3 study was conducted in TB-HIV-coinfected Tanzanian patients who started TB treatment (rifampicin/isoniazid/pyrazinamide/ethambutol) at week 1 to week 8 and continued with rifampicin and isoniazid for another 16 weeks. Antiretroviral treatment (ART) of efavirenz/ tenofovir/emtricitabine in a fixed-dose combination tablet was started at week 4 after initiation of TB treatment. A 24-h pharmacokinetic sampling curve was recorded at week 8 (with TB treatment) and week 28 (ART alone). For TB drugs, blood samples at 2 and 6 h post-dose were taken at week 3 (TB treatment alone) and week 8 (with ART).

Results: A total of 25 patients (56% male) completed the study; 21 had evaluable pharmacokinetic profiles. The area under the concentration-time curve 0-24 h post-dose of efavirenz, tenofovir and emtricitabine were slightly higher when these drugs were coadministered with TB drugs; geometric mean ratios (90% CI) were 1.08 (0.90, 1.30), 1.13 (0.93, 1.38) and 1.05 (0.85, 1.29), respectively. For TB drugs, equivalence was suggested for peak plasma concentrations when administered with and without efavirenz/tenofovir/ emtricitabine. Adverse events were mostly mild and no serious adverse events or drug discontinuations were reported.

Conclusion: Coadministration of efavirenz, tenofovir and emtricitabine with a standard first- line TB treatment regimen did not significantly alter the pharmacokinetic parameters of these drugs and was tolerated well by Tanzanian TB patients who are coinfected with HIV.

66 Efavirenz/tenofovir/emtricitabine with first line TB drugs

Introduction Co-infection with tuberculosis (TB) and HIV is an important public health problem. Nearly 40 million people are living with HIV worldwide and one-third is co-infected with TB [1-3]. The majority of TB cases in people living with HIV/AIDS occur in Africa, where 39% of people who develop TB are HIV-positive. The continent accounts for 82% of new TB cases living with HIV worldwide [1]. Management of the dual infection requires combining TB treatment and 3 antiretroviral treatment (ART) which markedly decrease the risk of morbidity and mortality to those patients [4]. Co-administering TB drugs with ART precipitates pharmacokinetic interactions, mostly caused by rifampicin. Rifampicin is a well-known inducer of many iso-enzymes of the hepatic cytochrome P450 (CYP) enzyme system, which metabolizes a large number of drugs [5]. In addition, it stimulates the expression of the multi-drug resistance transporter, P-glycoprotein [6,7]. Current guidelines for ART in patients also using rifampicin recommend the use of efavirenz plus two nucleoside reverse transcriptase inhibitors (NRTIs) [8,9]. Although combined use of rifampicin and efavirenz leads to an average decrease of about 18 – 26% in efavirenz exposure due to induction of the CYP2B6 enzyme [10,11], several studies have demonstrated adequate virological response in patients using both drugs [4,12]. A fixed-dose combination (FDC) tablet of efavirenz, tenofovir and emtricitabine has been approved by the Food and Drug Administration in the USA in 2006. This combination tablet is widely used as it is highly effective and can be administered as a single tablet once-daily [13]. There are no data available on the combined use of a rifampicin-based TB regimen and this FDC in TB/HIV co-infected patients. The aim of this study was to evaluate the effect of rifampicin- based TB treatment on the pharmacokinetics (PK) and safety of the FDC of efavirenz, tenofovir and emtricitabine. The secondary objective was to evaluate the effect of this FDC on the PK of the TB medication.

Methods Study design and population. This was a multiple-dose, open-label, three-period, phase II PK and safety study. The enrollment started on November 2008 and ended in February 2010. Patients included were 18-65 years and TB/HIV co-infected with CD4 counts of 50-350 cells/mm3 (later amended to 0-350 cells/mm3). Patients were excluded if they were already using ART, they were pregnant or breastfeeding, they had liver dysfunction (objectified by biochemistry results, and hepatitis B and C antigen tests) and if they were hypersensitive to either of the regimens. Another exclusion criterion was a Karnofsky score <40 based on the activities a patient could perform. The study protocol was approved by the Institutional Review Board of the Kilimanjaro Christian Medical University Centre, Moshi, Tanzania, and the National Institute for Medical Research, Dar-es-salaam, Tanzania. Written informed consent was obtained from each patient. Eligible patients were admitted at Kibong’oto National Tuberculosis Hospital, Kilimanjaro, Tanzania for the first eight weeks of TB treatment (intensive phase). Patients <50kg were given TB treatment consisting of rifampicin 450mg, isoniazid 225mg, pyrazinamide 1200mg and ethambutol 825mg, all administered once-daily for eight weeks. If

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3

Figure 1. Trial study design

patients were >50kg, they received rifampicin 600mg, isoniazid 300mg, pyrazinamide 1600mg and ethambutol 1100mg once-daily for eight weeks. After a negative sputum culture at week 8, TB treatment was continued with rifampicin and isoniazid for another 16 weeks. TB drugs were administered 30 minutes after a light breakfast. TB drugs were provided by the Tanzanian National TB program, and they were manufactured by Sandoz, Mumbai India and donated by Novartis. The ART regimen of once-daily one FDC tablet of efavirenz 600mg, tenofovir 300mg and emtricitabine 200mg (Atripla®, Merck & Co. Inc. USA) was initiated at week 4 after starting TB treatment and was administered together with the TB drugs. The study was divided in three phases (Figure 1): From week 1 to the end of week 4 patients were exposed to TB drugs alone (phase I). From week 5 to the end of week 24 they were exposed to concomitant use of TB drugs and a FDC tablet containing efavirenz, tenofovir, and emtricitabine (phase II). After 24 weeks, when TB treatment was ended, patients were exposed to ART alone (phase III). Both TB and HIV treatment were administered under supervised care including directly observed treatment for 8 weeks. After 8 weeks, patients were discharged. A one month dose of efavirenz, tenofovir and emtricitabine and standard TB treatment was dispensed and patients were asked to come for follow-up visits at the clinic every month.

Blood sampling and bio-analysis procedures. Eight weeks after starting TB treatment, a 24-hour PK sampling session was done. Samples were taken 5-20 minutes before directly observed intake of efavirenz, tenofovir and emtricitabine (t=0) and 1, 2, 3, 4, 6, 8, 10, 12, 16 and 24 hours later. At 28 weeks, four weeks after cessation of TB treatment, intensive plasma PK sampling was repeated. In addition, for the determination of rifampicin, isoniazid, pyrazinamide and ethambutol plasma concentrations, blood samples were taken after initiation of TB treatment at three weeks (without ART) and eight weeks (with ART). These blood samples were drawn at two and six hours after observed medication intake to be able to assess (‘catch’) peak plasma concentrations of TB drugs, which was taken as highest of the two. Breakfast (non- milky tea and/or porridge) was provided 5-20 minutes after pre-dose sampling, together with TB and/or HIV medication. Six mL of blood was collected per time point and centrifuged at 2800rpm for 10min. Within one hour plasma was stored at -20°C for one day and then shifted to a -80°C freezer until transportation on dry-ice to Radboud University, The Netherlands,

68 Efavirenz/tenofovir/emtricitabine with first line TB drugs for bio-analysis. Plasma concentrations of all drugs except isoniazid were assayed by validated high-performance liquid chromatography methods [14-17]. Isoniazid was measured with liquid/liquid extraction followed by UPLC with ultraviolet (UV) detection. Accuracy was between 98% and 107%, dependent on the concentration level. The intra- and interassay coefficients of variation were less than 13% over the ranges of 0.051 to 15.1 mg/L. The lower limit of quantitation was 0.051 mg/l. Isoniazid containing samples were stable (<5% loss) for at least 12 months at -80oC. 3

Safety and tolerability monitoring. Baseline clinical, hematology and biochemistry parameters were taken and, when applicable, a pregnancy test was done. Safety and tolerability of the trial treatment were assessed by a medical doctor on the basis of the occurrence of adverse events (AEs) about which patients were interviewed every two weeks during the first eight weeks, and every one month after hospital discharge. The onset, severity, and potential relationship of any AE to the study medication were recorded. Severity was rated according to the Common Terminology Criteria for AEs (v4, 2009) and the Division of AIDS table for grading the severity of Adult and Paediatrics AEs (DAIDS, 2004). In addition, the use of concomitant medication was reported, as were the results of a physical examination including measurement of vital signs. Clinical laboratory testing (hematology and biochemistry parameters) was performed at baseline, week 2, 4, 6, 8, 12, 16, 24 and 28. Follow-up sputum collection, CD4 lymphocyte count and plasma HIV-1 RNA quantification (using Abbott Real Time HIV-1 assay) was done at baseline, week 4, 8, 16, and 28.

PK and statistical analyses. PK parameters (area under the concentration-time curve

0-24 hours post-dose [AUC0-24h], Cmin, Cmax of efavirenz, tenofovir and emtricitabine were calculated by non-compartmental analysis using WinNonlin (version 5.2; Pharsight, CA).

AUC0-24h was calculated using the linear-log trapezoidal rule. PK parameters, geometric means

(GM) with standard deviation (SD), geometric mean ratios (GMRs) for AUC0-24h, Cmax and Cmin for the antiretroviral drugs with and without TB treatment and the corresponding 90% confidence intervals (CIs) were calculated. PK parameters of the antiretroviral drugs with vs. without TB drugs were considered bioequivalent if the GMR and corresponding 90%CI fell completely within the 0.80-1.25 range. Equivalence was ‘suggested’ in case the GMR is within the 0.80-1.25 range, but with one confidence interval limit outside this range. Inequivalence was to be concluded if the GMR and corresponding 90%CI fell completely outside the 0.80-1.25 range. Inequivalence was ‘suggested’ if the GMR is outside the 0.80-1.25 range, with one confidence interval inside this range [18]. PK analysis included only those patients with two evaluable PK curves (at week 8 and week 28). All statistical analyses were done in SPSS, version 18.0. To ensure an evaluable PK dataset of at least 20 patients, a total of 30 patients were included (based on an expected maximum drop-out of 33% during the trial after ART initiation).

Results Study population. We screened 66 HIV-infected, patients with confirmed smear-positive pulmonary TB. Thirty-eight (58%) patients were not eligible: 19 had a CD4 count> 350cells/mm3,

69 chapter 3

eight had a CD4 count <50 cells/mm3 (before protocol amendment), five were already on ART, two had a positive hepatitis antigen test, three had a Karnofsky score <40 and one had relapse TB. Of the 28 patients (42%) enrolled in the trial three withdrew consent; the remaining 25 patients completed follow-up and were evaluable for analysis. For PK evaluation four patients were excluded due to non-adherence or incorrect dosing, as indicated by undetectable plasma concentrations for efavirenz, tenofovir and emtricitabine prior to observed intake during 3 the intensive PK sampling day. Hence, 21 subjects were qualified for the PK evaluation. In the patients who completed follow-up, 14 patients (56%) were male, the median (interquartile range, IQR) baseline age and body weight were 32 (27.5-42.5) years and 48.4 (43.5-52.8) kg, respectively. In our study population body weight (mean+SD, kg) increased significantly from 53.4 (8.0) at week 8 to 56.8 (6.5) at week 28. The median (IQR) CD4 count at baseline was 155 (71-208) cells/mm3, the median (IQR) viral load (VL) was 129,779 (71,214-310,141) copies/mL. Baseline characteristics are in Table 1.

Table 1. Enrolment characteristics of the study participants (N=25)

Characteristics Total Number of patients followed up 25 Male, n (%) 14 (56%) Age, years 32 (27.5-42.5) Weight, kg 48.4 (43.5-52.8) CD4 count, cells/mm3 155 (71-208) HIV viral load, copies/ml 129,779 (71,214 -310,141)

Note: Values are N (%) for categorical variables and median ( IQR) for continuous variables.

PK of ART. Figure 2 illustrates the effect of the TB treatment on the geometric mean

concentration-time profiles of the ART. The average efavirenz AUC0-24h, Cmax and Cmin were slightly higher when efavirenz was co-administered with rifampicin-based TB treatment. The

GMRs lie within the range of 0.80-1.25 with the upper limit of the 90% CIs for AUC0-24h and C24h just above 1.25 (Table 2). GMRs (90%CI) were 1.08 (0.90-1.30), 1.02 (0.8-1.23) and 1.11 (0.90-1.37)

for AUC0-24h, Cmax and C24h, respectively, which means that equivalence was suggested (i.e. no effect of rifampicin on the efavirenz PK) Three of the 21 (14%) patients had subtherapeutic efavirenz plasma concentrations (<1.0 mg/L [19]) after observed intake at one or both PK sessions: one patient at week 8 only, one patient at week 28 only and one at both PK days. Potentially toxic plasma levels (>4.0 mg/L [19]) after observed intake were found in ten (48%) of the patients. Eight of these patients had potentially toxic levels at both PK sessions. Two patients had a Cmin and Cmax both >10mg/ml. The time to reach the maximum concentration

(Tmax) was observed to be higher during co-administration with TB drugs. Similarly, for tenofovir and emtricitabine the GMRs and 90% CIs of the PK parameters

suggest a slightly higher exposure in combination with TB drugs. GMRs (90%CIs) of AUC0-24h,

Cmax and C24h for tenofovir were 1.1 (0.93-1.4), 0.98 (0.78-1.4) and 1.09 (0.86-1.4), respectively,

70 Efavirenz/tenofovir/emtricitabine with first line TB drugs

6.0

5.0

4.0

3.0 without TB treatment

EFV (mg/L) EFV 2.0 with TB treatment

1.0 3

Geometric mean concentration of of concentration mean Geometric 0.0 0 4 8 12 16 20 24 Figure 2a Time after observed intake (hours)

2.0

1.6

1.2

without TB treatment 0.8

FTC (mg/L) FTC with TB treatment

0.4

Geometric mean concentration of of concentration mean Geometric 0.0 0 4 8 12 16 20 24 Time after observed intake (hours) Figure 2b

0.30

0.25

0.20

0.15 without TB treatment

TDF (mg/L) TDF 0.10 with TB treatment

0.05

Geometric mean concentration of 0.00 0 4 8 12 16 20 24 Time after observed intake (hours) Figure 2c

Figure 2. Curves of mean plasma concentration versus time for efavirenz (2A), emtricitabine (2B) Figureand tenofovir 2: (2C) Curves (order of from mean above plasma downwards) concentration after observed intakeversus of FDC time tablet for efavirenz/ efavirenz (2A), emtricitabine (2B) tenofovir/emtricitabine and tenofovir alone (grey (2C) curve) (order and in combinationfrom above with downwards) TB treatment (black after curve) observed intake of FDC tablet efavirenz/tenofovir/emtricitabine alone (grey curve) and in combination with TB while for emtricitabine treatment they (black were 1.05 curve) (0.85-1.3), 0.97 (0.75-1.3) and 1.3 (1.04-1.5), respectively. The PK parameters (AUC , C , C , T , elimination half-life (t ), volume of distribution 0-24h max 24h max 1/2 (V/F) and clearance (CL/F)) for efavirenz, tenofovir and emtricitabine are summarized in Table 2.

PK of TB drugs. The plasma concentrations of all TB drugs tended to be slightly lower during

co-administration in combination with ART. The bio-equivalence approach applied to the Cmax values for TB drugs suggested bioequivalence of the TB drugs when given alone versus in combination with ART (Table 3).

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Table 2. Pharmacokinetic parameters of efavirenz, tenofovir and emtricitabine alone and in combination with TB treatment

Week 28 (ART alone; Week 8 (ART with TB geometric mean treatment; geometric PK parameter (95% CI); mg/L) mean (95% CI); mg/L) GMR (90% CI) Efavirenz AUC , h.mg/L 81 (58-114) 88 (60-129) 1.08 (0.90-1.30) 3 0-24h Cmax, mg/L 5.7 (4.2-7.5) 5.7 (4.3-7.7) 1.02 (0.80-1.23)

Cmin, mg/L 2.4 (1.6-3.5) 2.6 (1.6-4.2) 1.11 (0.90-1.37)

Tmax, h 3.1 (1.0-10.1) 4.0 (1.1-8.0) -

t1/2, h 27 (25-35) 29 (20-47) 1.08 (0.77-1.41) Cl/F, L/h 7.4 (5.3-10.4) 6.8 (4.6-10.1) 0.92 (0.77-1.11) V/F, L 313 (232-421) 301 (236-383) 0.96 (0.72-1.29)

Tenofovir AUC0-24h, h.mg/L 2.6 (2.1-3.2) 2.9 (2.4-3.5) 1.13 (0.93-1.38)

Cmax, mg/L 0.33 (0.26-0.41) 0.32 (0.26-0.40) 0.98 (0.78-1.38)

Cmin, mg/L 0.05 (0.04-0.06) 0.05 (0.04-0.07) 1.09 (0.86-1.38)

Tmax, h 1.0 (0.9-8.0) 2.1 (0.0-6.2) -

t1/2, h 14 (12-15) 11 (10-12) 0.81 (0.75-0.86) Cl/F, L/h 94 (76-117) 83 (69-100) 0.88 (0.73-1.08) V/F, L 1852 (1438-2385) 1319 (1099-1584) 0.71 (0.59-0.86)

Emtricitabine AUC0-24h, h.mg/L 14 (12-16) 15 (11-18) 1.05 (0.85-1.29)

Cmax, mg/L 2.1 (1.8-2.3) 2.0 (1.5-2.7) 0.97 (0.75-1.25)

Cmin, mg/L 0.10 (0.09-0.12) 0.12 (0.10-0.16) 1.26 (1.04-1.53)

Tmax, h 2.0 (1.0-8.0) 3.0 (0.0-6.0) -

t1/2, h 6.6 (5.8-7.5) 7.3 (6.1-8.6) 1.10 (0.93-1.31) Cl/F, L/h 18 (15-21) 17 (13-22) 0.96 (0.77-1.18) V/F, L 171 (139-210) 180 (124-261) 1.05 (0.75-1.48)

Note: Tmax is presented as median (IQR)

Efficacy. Twenty-five patients completed follow-up to the end of week 28. At week 4 of the study (prior to ART) they had a median (IQR) VL of 134,204 (71,414-401,004) copies/mL. At week 28 (after starting ART), 17 (68%) patients had an undetectable VL (<40 copies/ml). The remaining eight patients (32%) had a median (IQR) VL of 51(40-532) copies/mL. From 23 of the patients (92%), CD4 counts were available both at baseline and at the end of the study. Their median (IQR) CD4 count increased from 119 (69-203) cells/mm3 to 238 (147-424) cells/mm3 (p<0.01; Signed Rank). By the end of eight weeks of TB treatment, 19 of 25 patients (76%) had negative sputum smears (confirmed by negative sputum cultures) and continued with rifampicin and isoniazid for 16 weeks. Six patients (24%) had positive sputum smear results after 8 weeks of treatment, of which two had negative and four had a positive cultures for Mycobacterium tuberculosis (MTB). Drug susceptibility testing of the isolates obtained from these four patients showed normal susceptibility patterns and therefore, those four patients continued with intensive phase TB

72 Efavirenz/tenofovir/emtricitabine with first line TB drugs

Table 3. Peak plasma concentrations (Cmax) of rifampicin, isoniazid, pyrazinamide and ethambutol alone and in combination with ART, based on sampling at 2 h and 6 h post dose

GM Week 2 GM Week 8 Drug (without HIVdrug; mg/L) (with HIV drug; mg/L) GMR (90% CI) Rifampicin 4.60 3.97 1.16(0.88-1.53) Isoniazid 1.24 1.18 1.06(0.85-1.31) 3 Pyrazinamide 31.64 35.95 0.88(0.78-0.99) Ethambutol 2.09 2.11 0.99(0.78-1.26)

treatment for another four weeks. At week 12, sputum smear results of all four patients were negative. Of the four, two had an undetectable VL at week 16, the remaining two had had 659 copies/mL and 8,032 copies/mL each. All 25 patients had negative sputum culture results at the end of TB treatment.

Safety. Twelve patients (48%) developed elevated biochemistry parameters. These included elevated alkaline phosphatase in 11 patients (all grade 1 except for two patients who had grade 2), elevated creatinine in three patients (two of which were grade 2 and one was grade 3), and elevated pancreatic amylase and ALT in one patient each (both grade 1). The patient with grade 3 elevation of creatinine recovered spontaneously. Nineteen patients (76%) developed decreased hematological parameters. These were mostly leucopenia and neutropenia (12 subjects each), and were all grade 1. Four patients had anemia, of which two were grade 1, one was grade 2 and the remaining one was grade 3. Two patients had thrombocytopenia (one grade 1 and the other grade 2). The laboratory abnormalities had all resolved at completion of the study.

Tolerability. Before administration of ART (Phase 1), 24 of 25 (96%) patients had experienced one or more clinical AEs; with a total of 99 events (65% grade 1, 34% grade 2, 1% grade 3). After starting ART (Phase 2), 23 of the 25 (92%) subjects experienced one or more new or aggravated AEs at some time during follow-up. These were in total 95 AEs, mostly mild disturbances (64% grade 1, 33% grade 2, 3% grade 3). There were no serious AEs reported and no modifications or discontinuations of treatment were done. Thirteen percent of the clinical events were judged to have no relationship to the drugs, 83% had either a doubtful or possible relationship and 4% had a possible relationship. At the end of follow-up, all events were recovered except for four patients who still had either loss of appetite, body rash, general body weakness or constipation. Fifty percent of the 10 patients who had toxic levels of efavirenz experienced a clinical AE compared to 40% among patients without toxic levels (p=0.70) and 30% of the events were grade 2 for the group with toxic levels versus 38.5% for the sub therapeutic group (p=0.23). Lab events were reported in 90% of the patients with toxic levels of efavirenz and 93% of patients without toxic levels. Two patients with efavirenz concentration above 10 mg/L throughout showed only mild (grade 1) AEs.

73 chapter 3

Discussion This study is the first to report on the interactions of efavirenz, tenofovir emtricitabine with first line rifampicin-based TB treatment. No interactions were observed in the pharmacokinetic

analysis. The observed slightly higher AUC0-24h, Cmax and C24h of efavirenz when co-administered with the rifampicin-based TB treatment in our study is, consistent with a recent study that showed 3 the absence of an effect of rifampicin on efavirenz PK when patients are poor metabolizers of CYP2B6 [20]. Our data are also in agreement with recent studies in South Africa, Tanzania and India showing that efavirenz concentrations do not decrease when it is co-administered with a rifampicin-based TB treatment regimen [21-23]. The metabolism of efavirenz is extensively influenced by pharmacogenetic factors [21,24-26]. A single nucleotide polymorphism at position 516 on the CYP2B6 gene has been widely reported to play an important role in the metabolism of efavirenz and nevirapine [27-29]. Genetic polymorphisms with CYP2B6 occur in all populations, and this may affect the exposure to efavirenz and its susceptibility to rifampicin- based enzyme induction [30-32]. The slightly higher efavirenz levels during combined TB and HIV treatment in our study could be explained by genetic differences [20,23,25] A once-daily dose of efavirenz 600mg during TB therapy is reported to be adequate [22,33,34]. However, in our study potentially toxic plasma levels (>4.0 mg/L) were observed in 48% of the patients. Also sub-therapeutic levels of efavirenz (<1.0mg/L) were found in 14% of the patients. The observed high intervariability of drug levels suggests inter-patient differences in the expression of the drug metabolizing enzymes [11]. For observed potentially toxic levels, no further action was taken as the patients were already discharged in a good condition. The levels occurred both before and during co-administration with TB treatment. Nevertheless the frequent presence of high levels in our African patient population may warrant the use of therapeutic drug monitoring of efavirenz plasma concentrations to improve the safe use of ART during TB treatment, and the need for individual dose adjustment [10]. Comparison of exposure to tenofovir with and without rifampicin-based TB treatment did not suggest any effect of rifampicin, although PK equivalence (lack of interaction) could not be concluded. Studies elsewhere have also shown no difference in tenofovir concentration while observing bioequivalence [15]. However, participants in these studies were healthy volunteer subjects who received tenofovir only shortly, which implicate that less potential factor may have affected the PK of tenofovir in those subjects. PK parameters may be different in patients as compared to healthy volunteers due to changes brought by both HIV and TB infection [35]. It is known that tenofovir is eliminated unchanged by glomerular filtration and active tubular secretion [36], while rifampicin is extensively metabolized in intestinal and hepatic metabolism [7] which minimizes the interactions. Emtricitabine has also been reported to have no PK drug-drug interactions with other antiretroviral drugs and rifampicin.

The time to reach maximum concentrations (Tmax) of emtricitabine, tenofovir and efavirenz was longer when co-administered with rifampicin than without rifampicin, although the median difference was only one hour for all three drugs. The reason for one of these drugs may be delayed absorption upon co-administration of rifampicin. Clearance of efavirenz could be affected in patients with genotype CYP2B6. We did not analyze genetic aspects of these

74 Efavirenz/tenofovir/emtricitabine with first line TB drugs patients, hence further studies are warranted. No large differences were found in clearance and volume of distribution. This study showed a small decrease in levels of TB drugs with versus without co- administration of efavirenz, tenofovir and emtricitabine. We do not consider this to be clinically significant. Studies elsewhere have also found no statistically significant effect of efavirenz on rifampicin [5,7,22,37]. In our study, rifampicin and isoniazid peak plasma concentrations were lower than the reference peak values (8-24 mg/L rifampicin; 3-5 mg/L isoniazid) [38]. One 3 possible explanation could be that the values in this study, obtained from either two or six hours after intake, were not the actual peak values. Secondly, our patients took medications just after food intake which is known to lower concentrations of rifampicin and isoniazid [39]. All patients had a positive response to treatment in terms of sputum culture and decrease of VL. An important finding of our study is that we detected non-adherence in four patients included in the trial when pre-dose samples were analyzed. This can be explained by the fact that the patients were discharged home after eight weeks, and subjects were asked to come for follow-up visits at the clinic every month. We recommend strong coordination between the national TB; community DOT system and the AIDS control programs. The main shortcoming of this study is that we did not look into genetic factors of the patients, which could have provided an explanation of high efavirenz drug levels and for the extent of the interaction between efavirenz and rifampicin-based TB treatment. Also, the study did not examine the effect of anti-TB treatment on intracellular PK of tenofovir and emtricitabine, as intracellular diphosphate or triphosphate levels are associated with clinical effects. It is possible that rifampin-containing TB treatment may not influence plasma PK but could affect intracellular PK through induction or inhibition of drug transporters. In conclusion, our findings suggest that co-administration of the standard first line TB treatment regimen with efavirenz, tenofovir, and emtricitabine does not alter pharmacokinetic parameters. The combination is tolerated well by Tanzanian TB/HIV co-infected patients. Hence, a combination of efavirenz, tenofovir and emtricitabine may be considered in managing HIV infection in African patients who are co-infected with TB.

References

1. World Health Organization. Global tuberculosis control: WHO report 2011. In. 2011 ed. Geneva: World Health Organization; 2011. 2. Corbett EL, Marston B, Churchyard GJ, De Cock KM. Tuberculosis in sub-Saharan Africa: opportunities, challenges, and change in the era of antiretroviral treatment. Lancet 2006,367:926-937. 3. Lawn SD, Churchyard G. Epidemiology of HIV-associated tuberculosis. Curr Opin HIV AIDS 2009,4:325-333. 4. Manosuthi W, Kiertiburanakul S, Sungkanuparph S, Ruxrungtham K, Vibhagool A, Rattanasiri S, et al. Efavirenz 600 mg/day versus efavirenz 800 mg/day in HIV-infected patients with tuberculosis receiving rifampicin: 48 weeks results. AIDS 2006,20:131-132. 5. Niemi M, Backman JT, Fromm MF, Neuvonen PJ, Kivisto KT. Pharmacokinetic interactions with rifampicin : clinical relevance. Clin. Pharmacokinet. 2003,42:819-850. 6. Finch CK, Chrisman CR, Baciewicz AM, Self TH. Rifampin and rifabutin drug interactions: an update. Arch. Intern. Med. 2002,162:985-992.

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7. Burman WJ, Gallicano K, Peloquin C. Comparative pharmacokinetics and pharmacodynamics of the rifamycin antibacterials. Clin. Pharmacokinet. 2001,40:327-341. 8. World Health Organisation. Treatment of Tuberculosis: Guidelines. In. Fourth Edition ed. Geneva: World Health Organisation; 2010. 9. World Health Organisation. Antiretroviral Therapy for HIV Infection in Adults and Adolescents: Recommendations for a Public Health Approach. In. 2010 Revision ed. Geneva: World Health 3 Organisation; 2010. 10. Kwara A, Tashima KT, Dumond JB, Poethke P, Kurpewski J, Kashuba AD, et al. Modest but variable effect of rifampin on steady-state plasma pharmacokinetics of efavirenz in healthy African-American and Caucasian volunteers. Antimicrob Agents Chemother 2011,55:3527-3533. 11. Lopez-Cortes LF, Ruiz-Valderas R, Viciana P, Alarcon-Gonzalez A, Gomez-Mateos J, Leon-Jimenez E, et al. Pharmacokinetic interactions between efavirenz and rifampicin in HIV-infected patients with tuberculosis. Clin. Pharmacokinet. 2002,41:681-690. 12. Friedland G, Khoo S, Jack C, Lalloo U. Administration of efavirenz (600 mg/day) with rifampicin results in highly variable levels but excellent clinical outcomes in patients treated for tuberculosis and HIV. J. Antimicrob. Chemother. 2006,58:1299-1302. 13. Semvua HH, Kibiki GS. AtriplaR/anti-TB combination in TB/HIV patients. Drug in focus. BMC Res Notes 2011,4:511. 14. Aarnoutse RE, Grintjes KJ, Telgt DS, Stek M, Jr., Hugen PW, Reiss P, et al. The influence of efavirenz on the pharmacokinetics of a twice-daily combination of indinavir and low-dose ritonavir in healthy volunteers. Clin Pharmacol Ther. 2002,71:57-67. 15. Droste JAH, Verweij-van Wissen CPWGM, Kearney BP, Buffels R, vanHorssen P, Hekster YA, et al. Pharmacokinetic Study of Tenofovir Disoproxil Fumarate Combined with Rifampin in Healthy Volunteers. Antimicrob. Agents Chemother. 2005,49:680-684. 16. la Porte CJ, Colbers EP, Bertz R, Voncken DS, Wikstrom K, Boeree MJ, et al. Pharmacokinetics of Adjusted-Dose Lopinavir-Ritonavir Combined with Rifampin in Healthy Volunteers. Antimicrob. Agents Chemother. 2004,48:1553-1560. 17. Ruslami R, Nijland HM, Alisjahbana B, Parwati I, van Crevel R, Aarnoutse RE. Pharmacokinetics and tolerability of a higher rifampicin dose versus the standard dose in pulmonary tuberculois patients. Antimicrob. Agents Chemother. 2007. 18. Williams RL, Chen ML, Hauck WW. Equivalence approaches. Clin. Pharmacol. Ther. 2002,72:229-237. 19. Marzolini C, Telenti A, Decosterd LA, Greub G, Biollaz J, Buclin T. Efavirenz plasma levels can predict treatment failure and central nervous system side effects in HIV-1-infected patients. AIDS 2001,15:71-75. 20. Cohen K, Grant A, Dandara C, McIlleron H, Pemba L, Fielding K, et al. Effect of rifampicin-based antitubercular therapy and the cytochrome P450 2B6 516G>T polymorphism on efavirenz concentrations in adults in South Africa. Antivir. Ther. 2009,14:687-695. 21. Ngaimisi E, Mugusi S, Minzi O, Sasi P, Riedel KD, Suda A, et al. Effect of rifampicin and CYP2B6 genotype on long-term efavirenz autoinduction and plasma exposure in HIV patients with or without tuberculosis. Clin Pharmacol Ther 2011,90:406-413. 22. Orrell C, Cohen K, Conradie F, Zeinecker J, Ive P, Sanne I, et al. Efavirenz and rifampicin in the South African context: is there a need to dose-increase efavirenz with concurrent rifampicin therapy? Antivir Ther 2011,16:527-534. 23. Ramachandran G, Hemanth Kumar AK, Rajasekaran S, Kumar P, Ramesh K, Anitha S, et al. CYP2B6 G516T polymorphism but not rifampin coadministration influences steady-state pharmacokinetics

76 Efavirenz/tenofovir/emtricitabine with first line TB drugs

of efavirenz in human immunodeficiency virus-infected patients in South India. Antimicrob. Agents Chemother. 2009,53:863-868. 24. Kwara A, Lartey M, Sagoe KW, Xexemeku F, Kenu E, Oliver-Commey J, et al. Pharmacokinetics of efavirenz when co-administered with rifampin in TB/HIV co-infected patients: pharmacogenetic effect of CYP2B6 variation. J. Clin. Pharmacol. 2008,48:1032-1040. 25. Uttayamakul S, Likanonsakul S, Manosuthi W, Wichukchinda N, Kalambaheti T, Nakayama EE, et al. Effects of CYP2B6 G516T polymorphisms on plasma efavirenz and nevirapine levels when co-administered with 3 rifampicin in HIV/TB co-infected Thai adults. AIDS Res Ther 2010,7:8. 26. di Iulio J, Fayet A, Arab-Alameddine M, Rotger M, Lubomirov R, Cavassini M, et al. In vivo analysis of efavirenz metabolism in individuals with impaired CYP2A6 function. Pharmacogenet Genomics 2009,19:300-309. 27. Haas DW, Gebretsadik T, Mayo G, Menon UN, Acosta EP, Shintani A, et al. Associations between CYP2B6 polymorphisms and pharmacokinetics after a single dose of nevirapine or efavirenz in African americans. J. Infect. Dis. 2009,199:872-880. 28. Powers V, Ward J, Gompels M. CYP2B6 G516T genotyping in a UK cohort of HIV-positive patients: polymorphism frequency and influence on efavirenz discontinuation. HIV Med 2009,10:520-523. 29. King J, Aberg JA. Clinical impact of patient population differences and genomic variation in efavirenz therapy. AIDS 2008,22:1709-1717. 30. Nyakutira C, Roshammar D, Chigutsa E, Chonzi P, Ashton M, Nhachi C, et al. High prevalence of the CYP2B6 516G-->T(*6) variant and effect on the population pharmacokinetics of efavirenz in HIV/AIDS outpatients in Zimbabwe. Eur J Clin Pharmacol 2008,64:357-365. 31. Blumberg HM, Burman WJ, Chaisson RE, Daley CL, Etkind SC, Friedman LN, et al. American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: treatment of tuberculosis. Am. J. Respir. Crit Care Med. 2003,167:603-662. 32. Klein K, Lang T, Saussele T, Barbosa-Sicard E, Schunck WH, Eichelbaum M, et al. Genetic variability of CYP2B6 in populations of African and Asian origin: allele frequencies, novel functional variants, and possible implications for anti-HIV therapy with efavirenz. Pharmacogenet. Genomics 2005,15:861-873. 33. Friedland G, Churchyard GJ, Nardell E. Tuberculosis and HIV coinfection: current state of knowledge and research priorities. J Infect Dis 2007,196 Suppl 1:S1-3. 34. Abdool Karim SS, Naidoo K, Grobler A, Padayatchi N, Baxter C, Gray A, et al. Timing of initiation of antiretroviral drugs during tuberculosis therapy. N Engl J Med 2010,362:697-706. 35. Lee BL, Wong D, Benowitz NL, Sullam PM. Altered patterns of drug metabolism in patients with acquired immunodeficiency syndrome. Clin. Pharmacol. Ther. 1993,53:529-535. 36. Barditch-Crovo P, Deeks SG, Collier A, Safrin S, Coakley DF, Miller M, et al. Phase i/ii trial of the pharmacokinetics, safety, and antiretroviral activity of tenofovir disoproxil fumarate in human immunodeficiency virus-infected adults. Antimicrob. Agents Chemother. 2001,45:2733-2739. 37. Kwara A, Lartey M, Sagoe KW, Court MH. Paradoxically elevated efavirenz concentrations in HIV/ tuberculosis-coinfected patients with CYP2B6 516TT genotype on rifampin-containing antituberculous therapy. AIDS 2011,25:388-390. 38. Peloquin CA. Therapeutic drug monitoring in the treatment of tuberculosis. DRUGS 2002,62:2169-2183. 39. Lin MY, Lin SJ, Chan LC, Lu YC. Impact of food and antacids on the pharmacokinetics of anti-tuberculosis drugs: systematic review and meta-analysis. Int J Tuberc Lung Dis 2010,14:806-818.

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Pharmacokinetics of first line tuberculosis drugs 4 in Tanzanian patients

Alma Tostmann, Charles M. Mtabho, Hadija H. Semvua, Jossy van den Boogaard, Gibson S. Kibiki, Martin J. Boeree, Rob E. Aarnoutse

Antimicrobial Agents and Chemotherapy 2013;57(7):3208 chapter 4

Abstract Introduction: East Africa has a high tuberculosis (TB) incidence and mortality, yet there is very limited data on exposure to TB drugin patients from this region.We therefore determined the pharmacokinetic characteristics of firstline TB drugs in Tanzanian patients using intensive pharmacokinetic sampling.

Methods: In twenty adult TB patients, plasma concentrations were determined just before 4 and at 1, 2, 3, 4, 6, 8, 10 and 24 hours after observed drug intake with food to estimate the area under the curve (AUC0-24h) and peak plasma concentrations (Cmax) of isoniazid, rifampicin, pyrazinamide and ethambutol. Acetylator status for isoniazid was assessed phenotypically using the isoniazid elimination half-life and the acetylisoniazid/isoniazid metabolic ratio at 3 hours post dose.

Results: The geometric mean AUC0-24h of isoniazid was 11.0 h*mg/L, rifampicin 39.9h*mg/L,

pyrazinamide 344 h*mg/L and ethambutol 20.2 h*mg/L.Cmax was below the reference range for

isoniazid in 10/19 patients and for rifampicin in 7/20 patients.In none of the patients, the Cmax values for pyrazinamide and ethambutol were below the reference range. Elimination half-life and metabolic ratio of isoniazid gave discordant phenotyping results in only 2/19 patients.

Discussion: A substantial proportion of patients had an isoniazid and/or rifampicin Cmax below the reference range. Intake of TB drugs with food may partly explain these low drug levels, but such drug intake reflects common practice. The finding of low TB drug concentrations is concerning because low concentrations have been associated with worse treatment outcome in several other studies.

80 Descriptive pharmacokinetic of TB drugs

Introduction The pharmacokinetic properties of tuberculosis (TB) drugs are well described especially in Caucasian populations [1,2]. Pharmacokinetic data from EastAfricaare limited, especially data that are based on intensive pharmacokinetic sampling during the full dosing interval. Such intensive pharmacokinetic sampling enables an accurate assessment of the total exposure to TB drugs (area under the time versus plasma concentration curve; AUC0-24h) and the peak plasma concentration (Cmax) in individual patients. Large inter-patient variability generally exists in thesekey pharmacokinetic parameters of TB drugs [1]. As a result, a proportion of 4 patients achieves drug concentrations that are below the reference range for TB drugs. Several studies have shown that such lower exposures to TB drugs are associated with suboptimal response [3-8]. A recent study showed thatpharmacokinetic variability to just a single drug in the multidrug TB drug regimen is associated with treatment failure and acquired drug resistance [9,10]. On the other hand, unduly high exposures may cause toxicity and interruption of TB treatment.It is therefore important to have more knowledge on pharmacokinetic properties of TB treatment in populations from East Africa, as a high TB incidence and TB mortalityis found in this region [11]. HIV infection [12-14] and malnutrition [15] have been associated with decreased plasma concentrations ofTBdrugs.Both are highly prevalent amongst East African TB patients, therefore low TB drug levels are to be expected in this population [11]. The objective of this study was to describe the pharmacokinetic parameters of isoniazid, rifampicin, pyrazinamide and ethambutol using intensive sampling during the full dosing interval in Tanzanian TB patients.

Methods Study design. We conducted an observational pharmacokinetic study at the Kilimanjaro Clinical Research Institute at the Kilimanjaro Christian Medical Centre (KCMC) in Moshi, Tanzania, using the standard two-stage approach. With this approach individual pharmacokinetic parameters are estimated in the first stage. In the second stage, population characteristics of each parameter are derived by obtaining measures of central tendency and spread of all the subjects’ individual parameters.

Study participants. Study participants were recruited from an outpatient tuberculosis treatment clinic at Mawenzi Hospital in Moshi. Adult TB patients who were in the intensive phase of treatment and who were not using medication that could interfere with TB drug plasma concentrations were eligible for participation. All participants gave written informed consent. The study was approved by the local Institutional Research Board at KCMC and by the Tanzanian National Institute of Medical Research. Tuberculosis was treated with fixed-dose combination tablets (FDC tablets) manufactured by Sandoz, Mumbai India and are on the WHO List of Prequalified Medicinal Products. The drugs were donated by Novartis through the WHO Global Drug Facility (GDF), which only provides high quality drugs that meet stringent WHO standards.Patients with a body weight

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less than 50 kg usethree FDC tablets per day (i.e. 225 mg isoniazid, 450 mg rifampicin, 1200 mg pyrazinamide and 875 mg ethambutol) and patients with a body weight>50 kg use four FDC tablets per day (i.e. 300 mg isoniazid, 600 mg rifampicin, 1600 mg pyrazinamide and 1100 mg ethambutol) [16].

Data collection. Basic demographic and clinical information was collected from all participants, including age, sex, body weight and height (to calculate body mass index; BMI), co-morbidities (including HIV infection and hepatitis B and C) and concomitant drug use. Malnutrition was 4 defined as BMI < 18.5 kg/m2 [17]. Sample collection. Pharmacokinetic sampling took place at KCMC hospital. Patients had to be on TB treatment for at least ten days because of the expected steady state in the pharmacokinetics of the TB drugs at that point. Patients had refrained from food at least eight hours before drug intake. On the sampling day, patients took their drugs under our supervision at 8am in the morning. Then they had a standardized breakfast within 30 minutes after drug intake, which reflected the usual drug intake procedures in this population. The standardized breakfast consisted of a cup (125 ml) of tea with whole milk and sugar and either a small bowl of porridge or mandaazi, a typical East African fried pasty (analogous to doughnut) that is rather fat. Serial venous blood samples were collected just before, and at 1, 2, 3, 4, 6, 8, 10 and 24 hours after observed TB drug intake. Plasma was separated and stored at -80oC immediately until transport on dry ice to The Netherlands for bio-analysis.

Bio-analysis. The plasma concentrations of isoniazid, rifampicin, pyrazinamide and ethambutol were assessed by validated high-performance liquid chromatography (HPLC) as described before [18]. Isoniazid and acetylisoniazid were measured with liquid-liquid extraction followed by Ultra Performance Liquid Chromatography (UPLC) with ultraviolet (UV) detection. Accuracy was between 97.8% and 106.7% for isoniazid and between 98.0% and 108.9% for acetylisoniazid, dependent on the concentration level. The intra- and inter-assay coefficients of variation were less than 13.4 % and less than 3.2% (dependent on the concentration) over the range of 0.05-15.1 mg/L for isoniazid; and less than 4.2% and less than 5.7% over the range of 0.16-16.2 mg/L for acetylisoniazid. Lower limits of quantification were 0.05 mg/L for isoniazid and 0.16 mg/L for acetylisoniazid. Isoniazid and acetylisoniazid containing samples were stable (<5% loss) for at least 12 months at -80oC.

Pharmacokinetic analysis. Pharmacokinetic evaluations were performed using non- compartmental methods in WinNonLin Version 5.3 (Pharsight Corp., Mountain View, California

US). The highest observed plasma concentration was defined as Cmax with the corresponding

sampling time as Tmax. The log-linear period (log C versus t) was based on the last data points (at least three). The absolute value of the slope (β/2.303, in which β is the first order elimination rate constant) was calculated using linear regression analysis. The elimination half-life (T½)

was calculated as 0.693/β. If C24h was below the limit of quantification of the assay (often the case for isoniazid and rifampicin), it was estimated based on the last measurable concentration -(β*(24-Tlast) (Clast) and β using the formula C24h = Clast*e . The area under the plasma concentration-

82 Descriptive pharmacokinetic of TB drugs

time curve (AUC0-24h) was calculated using the linear/log-trapezoidal rule from zero up to the last concentration at 24h. The apparent clearance of the drug (Cl/F; in which F is bioavailability) was calculated as dose/AUC0-24h and the apparent volume of distribution (Vd/F) was calculated as (Cl/F)/β. The reference ranges for Cmax were 3-5 mg/L for isoniazid, 8-24 mg/L for rifampicin, 20-50 mg/L for pyrazinamide and 2-6 mg/L for ethambutol [19].

These reference ranges represent the normal Cmax concentrations that can be expected in adults after the standard doses of anti-TB drugs. They are based on data that were compiled from all available sources (both healthy volunteers and TB patients) by, amongst others, Holdiness [1] and Peloquin [2]. Subsequently, the ranges were validated in a range of phase I 4 studies in healthy volunteers [20].

Determination of acetylator status. We determined the acetylator status phenotypically by assessing the T½ of isoniazid. Participants with aT½ greater than 130 minutes were classified as slow metabolisers and those with a t½ less than 130 minutes were fast/intermediate metabolisers [21-23]. As another means to assess acetylator phenotype, the metabolic ratio of acetylisoniazid concentration over isoniazid concentration at 3 hours post dose was calculated. Patients with a ratio above 1.5 were considered fast/intermediate metabolisers, patients with a ratio below 1.5 were slow metabolisers [21]. In addition, we also explored a metabolic ratio of 0.55 at 3 hours post dose as a cut-off to distinguish fast/intermediate from slow metabolisers, as this evolved from a study in African patients [23].

Statistical analysis. Most pharmacokinetic parameters were presented as geometric means. Sample size was considered too small to test for associations between patient characteristics and pharmacokinetic parameters. We only tested the effect of acetylator status on the pharmacokinetics of isoniazid. The correlations between Cmax and AUC0-24hof each of the drugs were explored using Spearman’s Rhocorrelation.

To explore the potential of limited sampling for estimating the AUC0-24hbased on sampling earlyin the pharmacokinetic curve, univariate linear regression was used to determine the association between the plasma concentration at 2, 3, 4 and 6 hours post dose and the AUC0-24h for each of the drugs. The R-squared was presented as a measure of variance in the AUC0-24hthat is explained by the variance in the concentration at that time point. Statistical analyses were performed in STATA version 10.1 (Stata Corp LP, College Station, Texas, USA).

Results Twenty tuberculosis patients were enrolled for this study.All were under community-based directly observed treatment.Theirmedian age was 38 years (inter-quartile range [IQR] 30-42), fifteen patients (75%) were male, seven (35%) were HIV positive and seven (35%) were considered malnourished based on their BMI, see Table 1.

Descriptive pharmacokinetics. Intensive pharmacokinetic sampling took place after a median of 19 days after start of TB treatment (range 11-49 days).The pharmacokinetic parameters for isoniazid, rifampicin, pyrazinamide and ethambutol are presented in Table 2. In ten patients (53%), the isoniazid peak plasma concentration was below the reference range (3-5 mg/L). In

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Table 1. Characteristics of 20 tuberculosis patients in northern Tanzania

Characteristic Male sex, n (%) 15 (75%) Age, median years (IQR) 38 (30-42) Body weight, mean kg (SD) 55.7 (6.4) BMI, mean (SD) 19.5 (2.4) Malnutrition, n (%)a 7 (35%) 4 HIV positive, n (%) 7 (35%) Type of tuberculosis, n (%) Pulmonary 19 (95%) Extra pulmonary 1 (5%) Concomitant drugs, n (%) Amoxycillin 1 (5%) ARV b 2 (10%) Co-trimoxazole 3 (15%) Diclofenac 3 (15%) Vitamin B complex 2 (10%) Dose (mg) per kg body weight, mean (SD) Isoniazid 5.2 (0.46) Rifampicin 10.4 (0.91) Pyrazinamide 27.8 (2.4) Ethambutol 19.1 (1.7)

Acetylator status based on INH t1/2, n (%) Slow 12 (63%) Fast/ intermediate 7 (37%) Acetylator status based on ac-INH/INH ratio (cut-off 1.5), n (%) Slow 10 (53%) Fast/ intermediate 9 (47%) Acetylator status based on ac-INH/INH ratio (cut-off 0.55), n (%) Slow 8 (42%) Fast/intermediate 11 (58%)

IQR = interquartile range; BMI = body mass index; a Malnutrition defined as BMI < 18.5 kg/m2;b ARV = antiretroviral treatment.

seven patients (35%), the rifampicin Cmax was below the reference range (8-24 mg/L); in five

of them isoniazid Cmax was also below the reference range. In none of the patients the Cmax of pyrazinamide or ethambutol was below the reference range.

There was a positive correlation between the AUC0-24h and Cmax of isoniazid (correlation coefficient 0.68; P=0.001), pyrazinamide (correlation coefficient 0.69; P=0.001) and ethambutol (correlation coefficient 0.72; P=0.003), but not of rifampicin (correlation coefficient 0.35 (P=0.14).

84 Descriptive pharmacokinetic of TB drugs

4

Figure 1. The relation between the acetylator status (slow vs. fast/intermediate) based on two methods: the t1/2 of isoniazid and the acetyl-INH/INH metabolic ratio in the 3h post-dose plasma sample. Patients with a t1/2 below 130 minutes (=below the horizontal dashed line) were considered fast/intermediate metabolisers.[17-19] Patients with anacetyl-INH/INH ratio greater than 0.55 [19] or greater than 1.5 17(=right of the first or second vertical dashed line, respectively) were considered fast/ intermediate metabolisers based on metabolic ratio. Note that phenotyping by t1/2 and metabolic ratio resulted in discordant results (in the upper right quadrant) in 4 patients when a metabolic ratio of 0.55 was used as cut-off compared to 2 patients when a metabolic ratio of 1.5 was used as cut-off.

Table 2. Pharmacokinetic parameters of tuberculosis drugs in 20 Tanzanian tuberculosis patients

PK parameters, geometric mean (min-max) Isoniazid Rifampicin Pyrazinamide Ethambutol

AUC0-24, h*mg/L 11 (3.7-22.7) 39.9 (27.4-68.3) 344 (209-610) 20.2 (13.4-32.0)

Cmax, mg/L 2.8 (1.0-4.6) 8.9 (5.9-14.8) 38.2 (29.0-50.8) 3.3 (2.2-5.8) tmax, h 1.2 (0.7-2.9) 1.3 (0.9-3.0) 1.2 (0.7-3.0) 1.5 (0.9-2.2) CL/F, L/h 25.8 (13.0-60.5) 14.4 (8.8-21.9) 4.5 (2.6-6.5) 52 (34.3-71.3) Vd/F, L 99 (69.6-173.4) 37.3 (22.7-56.5) 40.3 (29.5-98.3) 719 (491-965) t1/2, h 2.9 (1.3-4.2) 1.8 (1.1-3.8) 5.5 (4.1-15.7) 9.5 (6.9-13.5) 2 Reference range Cmax 3-5 mg/L 8-24 mg/L 20-50 mg/L 2-6 mg/L

Proportion with Cmax below reference range 10 (52%) 7 (35%) 0 (0%) 0 (0%) Isoniazid plasma concentrations were only determined in 19 patients.

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Assessment of acetylator status. Based on the elimination half-life of isoniazid, 7 (37%) patients were fast or intermediate metabolisers and 12 (63%) were slow metabolisers. Based on the 3-houracetyl-INH/INH ratio with a cut-off of 1.5, nine (47%) patients were fast/intermediate metabolisers and ten patients (53%) were slow metabolisers (See Figure 1). Two patients had a discrepancy in acetylator phenotype determined by the two methods. With a cut-off of 0.55, 11 (58%) were fast/intermediate metabolisers and eight patients (42%) were slow metabolisers.

Acetylator status based on the half-life method was associated with the AUC0-24h and Cmax of isoniazid. In slow metabolisers geometric mean AUC was 17.2 h*mg/L (range 9.5-22.7) 4 0-24h and Cmax 3.4 mg/L (range 2.0-4.6) and in intermediate/fast metabolisers geometric mean

AUC0-24h was 5.2 h*mg/L (range 3.7-8.1) and Cmax2.0 mg/L (range 1.0-3.3); (P<0.001 and P=0.01, respectively).

Association between the 2, 3, 4 and 6h concentration and AUC0-24h. The isoniazid

concentrations at 2, 3, 4 and 6 hours post-dose were associated with AUC0-24h and the R-squared

ranged from 0.91 to 0.96, meaning that 91-96% of the variance in the AUC0-24h is explained by the variance in the concentration at a particular sampling time point. For rifampicin the R-squared was highest for the concentration 4 hours post-dose (0.82). For pyrazinamide the R-squared was highest for the concentrations 4 and 6 hours post dose (0.84 and 0.86, respectively). For the different ethambutol concentrations the R-squared was low, varying from 0.40 to 0.71. This

indicates that limited sampling to estimate the AUC0-24h may be feasible for isoniazid, rifampicin and pyrazinamide but less for ethambutol.

Discussion This report provides important data on pharmacokinetic characteristics of isoniazid, rifampicin, pyrazinamide and ethambutol based on intensive 24-hour pharmacokinetic sampling in East African TB patients.

Half of the patients from our study had anisoniazid peak plasma concentration (Cmax) below

the reference range and a third of patients had a rifampicin Cmax below the reference range. This may partly be explained by the breakfast that patients took within half an hour after the intake of TB drugs. This approach was chosen to mimic the real-life situation for TB patients, as most TB patients take their pills just before or with their breakfast in order to prevent or alleviate gastro- intestinal adverse effects. Previous studies have shown that intake of a meal with a high fat or carbohydrate content just before drug intake decreases the rate of absorption and significantly

reduces the isoniazid Cmax with 20% to 51% [24-27]. Rifampicin Cmax can be reduced up to −36%

when it is taken directly after a meal [24,28]. There is no significant effect of food on the Cmax of

pyrazinamide and ethambutol [24,29,30]. Of note, in the current study the average Tmax values for the TB drugs were short (about 1 h for isoniazid, rifampicin and pyrazinamide, see table 2),

indicating that the delay in absorption and a possible related decrease in Cmax were probably limited. Furthermore, it should be noted that pharmacokinetic studies from other African

countries found even higher proportions of patients with Cmax below the reference range for isoniazid (30% in Botswana [8,31], 89% in Kenya [32]) and rifampicin (69% in South Africa [12], 78-84% in Botswana [27,28] and 90% in Kenya [32]), despite administration of drugs on an empty

86 Descriptive pharmacokinetic of TB drugs

stomach in the studies from Botswana and Kenya. The relatively high Cmax values of the TB drugs in our study among African patients may also relate to the average body weight of 55.7 kg (table 1), which is just above 50 kg above which 4 FDC tablets (rather than 3) are administered.

Values for total exposure (AUC0-24h) may be even more relevant to the efficacy of first-line TB drugs than the Cmax [33]. AUC0-24h values can best be compared to those recorded in Indonesian TB patients [18] and in a racially mixed population of patients in The Netherlands who used similar doses on a mg/kg basis (Magis et al, submitted), as the pharmacokinetics in these studies were assessed with the same analytical methodology. Average exposures to rifampicin were 4 39.9, 48.5 and 41.1 h*mg/L in Tanzanian patients, Indonesian patients and a mixed population, pyrazinamide AUC0-24h values were 344, 473 and 380 h*mg/L respectively, and exposures to ethambutol were 20.2, 14.4 and 23.5 h*mg/L. Isoniazid was not measured in Indonesian TB patients and we used another analytical method in the mixed population (average AUC0-24h was 15.2 h*mg/L compared to 11,0 in the current study). These comparisons show no drastically lower total exposure to TB drugs between Tanzanian patients and the Indonesian patients and the mixed population from the Netherlands. Even though the exposure to TB drugs in Tanzanian patients may be high compared to exposurefrom other African studies and similar to those in other populations, a large number of patients had low plasma concentrations of isoniazid and rifampicin., Several clinical studies have pointed towards a possible association between low TB drug concentrations and poor treatment outcome [3-5]. A preclinical model showed that interindividual variability in pharmacokinetics is relevant to the emergence of resistance [7] and a recent meta-analysis revealed that variability in exposure to isoniazid is associated with failure of therapy and acquired drug resistance [10]. On the other hand, other studies have found no association between plasma concentrations and effect to first-line TB drugs [34, 35].

We could not readily explain the observed low isoniazid and rifampicin AUC0-24h and Cmax values by patient characteristics, as the sample size was considered too small for analysis of association between patient characteristics and pharmacokinetic parameters. However, we evaluated the effect of genetic polymorphisms in N-acetyl transferase 2 (NAT2), a phase II metabolic enzyme, on the pharmacokinetics of isoniazid.Slow acetylation of isoniazid into acetylisoniazid is a homozygous recessive trait. Genotypically, homozygous fast, heterozygous fast (or intermediate) and slow acetylators are distinguished [22,36]. In our study acetylator status was assessed phenotypically using the isoniazid elimination half-life and the acetylisoniazid/isoniazid ratio 3 hours post-dose with a cut-off of 1.5. Based on these two methods, 63% or 53% of patientsin our study were slow metabolisers. This is consistent with available data showing that 50-60% percent of European (Caucasian), African and Indian populations are slow metabolisers [37]. Clearly, assessment of the acetylator status by the metabolic ratio of acetylisoniazid/isoniazid at a single time point post dose is more convenient. There was good concordance between the two methods when 1.5 was used as the cut-off for the metabolic ratio (2 discordant patients). In the future, limited sampling may allow for an assessment of acetylator status as well as estimation of AUC values of TB drugs, considering the high correlation between concentrations measured in the early part of the pharmacokinetic

87 chapter 4

curve and exposures to isoniazid, rifampicin and pyrazinamide. AUC0-24h and Cmax values of isoniazid, pyrazinamide and ethambutol were also correlated, but for rifampicin no such association was found. We have considered whether this may be explained by the nonlinear, saturable pharmacokinetics of rifampicin, but disproportional changes in exposure above

certain concentrations are expected to affect both Cmax as well as AUC0-24h. In summary, this study provides pharmacokinetic data of first-line TB drugs based on intensive 24h sampling in East African TB patients.Half of the patients had isoniazid peak plasma levels and a third had rifampicin peak plasma concentrations below the reference ranges. 4 Although the pharmacodynamics of the currently used first line TB drugs is not entirely clear, low isoniazid and rifampicin concentrations have been associated with suboptimal treatment response. Exploring the effect, tolerabilityand pharmacokinetics of higher dose of isoniazid and rifampicin in African tuberculosis patients is therefore recommended.

Acknowledgements We wish to thankthe following people: all patients for participating in this study; Dr C. Irongo (Regional TB and Leprosy coordinator in the National TB and Leprosy Programme Tanzania), staff of the TB clinic at Mawenzi Hospital in Moshi and the research nurses at KCMC for their cooperation and effort; the laboratory technicians at KCMC and at the Department of Pharmacy of the RUNMC, The Netherlands for their technical support.

Financial support A. Tostmann received the UNESCO/L’Oreal For Young Women in Science Fellowship 2008 to coordinate this study. C. Mtabho, H. Semvua and J. van den Boogaard and bio-analysis of drugs were sponsored by the African Poverty Related Infection Oriented Research Initiative (APRIORI), a research network granted by the Netherlands-African partnership for Capacity development and Clinical interventions Against Poverty-related diseases (NACCAP).

References

1. Holdiness, M. R. 1984. Clinical pharmacokinetics of the antituberculosis drugs. Clin.Pharmacokinet. 9:511-544. 2. Peloquin, C. A. 1991. Antituberculosis drugs: pharmacokinetics., p. 59-88. In L. Heifets (ed.), Drug susceptibility in the chemotherapy of mycobacterial infections. CRC Press, Boca Raton. 3. Kimerling, M. E., P. Phillips, P. Patterson, M. Hall, C. A. Robinson, and N. E. Dunlap. 1998. Low serum antimycobacterial drug levels in non-HIV-infected tuberculosis patients. Chest 113:1178-1183. 4. Mehta, J. B., H. Shantaveerapa, R. P. Byrd, Jr., S. E. Morton, F. Fountain, and T. M. Roy. 2001. Utility of rifampin blood levels in the treatment and follow-up of active pulmonary tuberculosis in patients who were slow to respond to routine directly observed therapy. Chest 120:1520-1524. 5. Weiner, M., W. Burman, A. Vernon, D. Benator, C. A. Peloquin, A. Khan, S. Weis, B. King, N. Shah, and T. Hodge. 2003. Low isoniazid concentrations and outcome of tuberculosis treatment with once- weekly isoniazid and rifapentine. Am.J.Respir.Crit Care Med. 167:1341-1347.

88 Descriptive pharmacokinetic of TB drugs

6. Weiner, M., D. Benator, W. Burman, C. A. Peloquin, A. Khan, A. Vernon, B. Jones, C. Silva-Trigo, Z. Zhao, and T. Hodge. 2005. Association between acquired rifamycin resistance and the pharmacokinetics of rifabutin and isoniazid among patients with HIV and tuberculosis. Clin.Infect.Dis. 40:1481-1491. 7. Srivastava, S., J. G. Pasipanodya, C. Meek, R. Leff, and T. Gumbo. 2011. Multidrug-resistant tuberculosis not due to noncompliance but to between-patient pharmacokinetic variability. J.Infect.Dis. 204:1951- 1959. 8. Chideya, S., C. A. Winston, C. A. Peloquin, W. Z. Bradford, P. C. Hopewell, C. D. Wells, A. L. Reingold, T. A. Kenyon, T. L. Moeti, and J. W. Tappero. 2009. Isoniazid, rifampin, ethambutol, and pyrazinamide pharmacokinetics and treatment outcomes among a predominantly HIV-infected cohort of adults with 4 tuberculosis from Botswana. Clin.Infect.Dis. 48:1685-1694. 9. Egelund, E. F., and C. A. Peloquin. 2012. Editorial commentary: pharmacokinetic variability and tuberculosis treatment outcomes, including acquired drug resistance. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 55:178-179. 10. Pasipanodya, J. G., S. Srivastava, and T. Gumbo. 2012. Meta-analysis of clinical studies supports the pharmacokinetic variability hypothesis for acquired drug resistance and failure of antituberculosis therapy. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 55:169-177. 11. WHO. 2012. Global tuberculosis control: WHO report 2012. World Health Organization. 12. McIlleron, H., P. Wash, A. Burger, J. Norman, P. I. Folb, and P. Smith. 2006. Determinants of rifampin, isoniazid, pyrazinamide, and ethambutol pharmacokinetics in a cohort of tuberculosis patients. Antimicrob.Agents Chemother. 50:1170-1177. 13. Gurumurthy, P., G. Ramachandran, A. K. Hemanth Kumar, S. Rajasekaran, C. Padmapriyadarsini, S. Swaminathan, P. Venkatesan, L. Sekar, S. Kumar, O. R. Krishnarajasekhar, and P. Paramesh. 2004. Malabsorption of rifampin and isoniazid in HIV-infected patients with and without tuberculosis. Clin. Infect.Dis. 38:280-283. 14. Sahai, J., K. Gallicano, L. Swick, S. Tailor, G. Garber, I. Seguin, L. Oliveras, S. Walker, A. Rachlis, and D. W. Cameron. 1997. Reduced plasma concentrations of antituberculosis drugs in patients with HIV infection. Ann.Intern.Med. 127:289-293. 15. Polasa, K., K. J. Murthy, and K. Krishnaswamy. 1984. Rifampicin kinetics in undernutrition. Br.J.Clin. Pharmacol. 17:481-484. 16. The United Republic of Tanzania, M. o. H. a. S. W. 2006. Manual of the National Tuberculosis and Leprosy Programme in Tanzania. Fifth edition. 17. Organization, W. H. 2012. BMI classification. In: WHO nutrition. 18. Ruslami, R., H. M. Nijland, B. Alisjahbana, I. Parwati, R. van Crevel, and R. E. Aarnoutse. 2007. Pharmacokinetics and tolerability of a higher rifampin dose versus the standard dose in pulmonary tuberculosis patients. Antimicrob Agents Chemother 51:2546-2551. 19. Peloquin, C. A. 2002. Therapeutic drug monitoring in the treatment of tuberculosis. Drugs 62:2169-2183. 20. Peloquin, C. A. 2011. 9/17/2011. 21. Hutchings, A., and P. A. Routledge. 1986. A simple method for determining acetylator phenotype using isoniazid. Br.J.Clin.Pharmacol. 22:343-345. 22. Parkin, D. P., S. Vandenplas, F. J. Botha, M. L. Vandenplas, H. I. Seifart, P. D. van Helden, B. J. van der Walt, P. R. Donald, and P. P. van Jaarsveld.1997. Trimodality of isoniazid elimination: phenotype and genotype in patients with tuberculosis. Am.J.Respir.Crit Care Med. 155:1717-1722.

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23. Seifart, H. I., D. P. Parkin, F. J. Botha, P. R. Donald, and B. J. van der Walt. 2001. Population screening for isoniazid acetylator phenotype. Pharmacoepidemiol.Drug Saf 10:127-134. 24. Lin, M. Y., S. J. Lin, L. C. Chan, and Y. C. Lu. 2010. Impact of food and antacids on the pharmacokinetics of anti-tuberculosis drugs: systematic review and meta-analysis. Int.J.Tuberc.Lung Dis. 14:806-818. 25. Peloquin, C. A., R. Namdar, A. A. Dodge, and D. E. Nix. 1999. Pharmacokinetics of isoniazid under fasting conditions, with food, and with antacids. Int.J.Tuberc.Lung Dis. 3:703-710. 26. Zent, C., and P. Smith. 1995. Study of the effect of concomitant food on the bioavailability of rifampicin, isoniazid and pyrazinamide. Tuber.Lung Dis. 76:109-113. 4 27. Joshi, M. V., Y. S. Saraf, N. A. Kshirsagar, and V. N. Acharya. 1991. Food reduces isoniazid bioavailability in normal volunteers. J.Assoc.Physicians India 39:470-471. 28. Peloquin, C. A., R. Namdar, M. D. Singleton, and D. E. Nix. 1999. Pharmacokinetics of rifampin under fasting conditions, with food, and with antacids. Chest 115:12-18. 29. Peloquin, C. A., A. E. Bulpitt, G. S. Jaresko, R. W. Jelliffe, J. M. Childs, and D. E. Nix. 1999. Pharmacokinetics of ethambutol under fasting conditions, with food, and with antacids. Antimicrob.Agents Chemother. 43:568-572. 30. Peloquin, C. A., A. E. Bulpitt, G. S. Jaresko, R. W. Jelliffe, G. T. James, and D. E. Nix. 1998. Pharmacokinetics of pyrazinamide under fasting conditions, with food, and with antacids. Pharmacotherapy 18:1205-1211. 31. Tappero, J. W., W. Z. Bradford, T. B. Agerton, P. Hopewell, A. L. Reingold, S. Lockman, A. Oyewo, E. A. Talbot, T. A. Kenyon, T. L. Moeti, H. J. Moffat, and C. A. Peloquin. 2005. Serum concentrations of antimycobacterial drugs in patients with pulmonary tuberculosis in Botswana. Clin.Infect.Dis. 41:461- 469. 32. Choudhri, S. H., M. Hawken, S. Gathua, G. O. Minyiri, W. Watkins, J. Sahai, D. S. Sitar, F. Y. Aoki, and R. Long. 1997. Pharmacokinetics of antimycobacterial drugs in patients with tuberculosis, AIDS, and diarrhea. Clin.Infect.Dis. 25:104-111. 33. Hall, R. G., R. D. Leff, and T. Gumbo. 2009. Treatment of active pulmonary tuberculosis in adults: current standards and recent advances. Insights from the Society of Infectious Diseases Pharmacists. Pharmacotherapy 29:1468-1481. 34. Narita, M., M. Hisada, B. Thimmappa, J. Stambaugh, E. Ibrahim, E. Hollender, and D. Ashkin. 2001. Tuberculosis recurrence: multivariate analysis of serum levels of tuberculosis drugs, human immunodeficiency virus status, and other risk factors. Clin.Infect.Dis. 32:515-517. 35. Chang, K. C., C. C. Leung, W. W. Yew, K. M. Kam, C. W. Yip, C. H. Ma, C. M. Tam, E. C. Leung, W. S. Law, and W. M. Leung. 2008. Peak plasma rifampicin level in tuberculosis patients with slow culture conversion. Eur.J.Clin.Microbiol.Infect.Dis. 27:467-472. 36. Aarnoutse, R. E. 2011. Pharmacogenetics of antituberculosis drugs, p. 176-190. In P. R. Donald and P. D. van Helden (ed.), Antituberculosis Chemotherapy. Progress in Resporatory Research, Basel, Switzerland. 37. Ellard, G. A. 1984. The potential clinical significance of the isoniazid acetylator phenotype in the treatment of pulmonary tuberculosis. Tubercle. 65:211-227.

90

Associations between salivary, protein-unbound and total plasma 5 concentrations of rifampicin

Hadija H. Semvua‡, Charles M. Mtabho‡, Marga Teulen, Angela Colbers, Rogathe Machange, Arnold Ndaro, André van der Ven, David Burger, Gibson S. Kibiki, Rob E. Aarnoutse

‡ These authors contributed equally to this work

Manuscript submitted chapter 5

Abstract Introduction: Plasma is the traditional biological sample for PK studies and Therapeutic Drug Monitoring (TDM) of the pivotal anti-tuberculosis (TB) drug rifampicin. Saliva may be an attractive alternative matrix, also considering that it may reflect protein-unbound, active plasma concentrations. The objectives of this study were (1) to compare the PK of rifampicin in saliva and plasma and (2) to assess whether saliva could be an alternative matrix for PK studies and TDM with this drug.

Methods: A descriptive PK study was performed among 15 adult Tanzanian TB patients who were in the intensive phase of TB treatment. Time-matched samples of stimulated saliva 5 (obtained with a Salivette® device containing citric acid) and plasma were collected at predose and at 1, 2, 3, 4, 6, 8, 10 and 24 hours after intake of rifampicin. Salivary, total (protein-unbound plus bound) and unbound plasma concentrations of rifampicin were measured with validated HPLC methods. Salivary and plasma PK parameters were assessed, ratios of salivary to (total and unbound) plasma concentrations were calculated and the performance of salivary concentrations to predict plasma concentrations was evaluated using the jackknife method.

Results: The geometric mean AUC0-24h of rifampicin in saliva (3.1 h*mg/L) was slightly but

significantly lower than the protein-unbound AUC0-24h in plasma (5.3 h*mg/L) and these

were much lower than the plasma AUC0-24h based on total concentrations (32.7 h*mg/L).

Corresponding geometric mean Cmax values were 0.64, 1.0 and 6.8 mg/L. Average (geometric mean) ratios of loose salivary versus total or unbound plasma concentrations were 0.099 and 0.614 and these ratios were not dependent on time post dose or associated rifampicin plasma concentrations (repeated measures ANOVA). The prediction of total and unbound plasma concentrations based on salivary concentrations of rifampicin resulted in median percentage prediction errors (MPPE) of 13.4% and 6.0% and median absolute percentage prediction errors (MAPE) of 35.7% and 23.0%, respectively, which means that these predictions were sufficiently accurate (<15%) yet imprecise (>15%).

Conclusions: AUC0-24h and Cmax of rifampicin in saliva were much lower than those in plasma

based on total plasma concentrations. The AUC0-24h of rifampicin in saliva was significantly

lower than the protein-unbound plasma AUC0-24h. It is not possible to predict total or protein- unbound plasma concentrations from salivary concentrations, due to inadequate precision associated with this prediction.

94 Saliva study

Introduction The global burden of tuberculosis (TB) remains immense, with an estimated 8.7 million new cases of TB and 1.4 million people dying from this infectious disease in 2011 [1]. Still too many patients do not respond adequately to treatment, experience a relapse of TB after treatment completion, or are confronted with the emergence of drug resistant strains of M. tuberculosis [1]. Inadequate exposure to TB drugs constitutes one of the factors underlying suboptimal treatment response and development of resistance, as evidenced by results from the in vitro hollow fibre model [2], clinical studies on relationships between drug concentrations and response [3-7], and findings of a recent meta-analysis [8]. In view of the relevance of exposure to TB drugs, drug dosing may be individualized based on measurement of drug concentrations (Therapeutic Drug Monitoring, TDM), a practice applied in a few centres around the 5 world [9-13]. Yet in the long term it seems preferable to develop new TB regimens that are pharmacokinetically optimized, enabling fixed dosing for all patients. This requires multiple pharmacokinetic studies during development of such regimens. Plasma is the traditional biological sample for pharmacokinetic studies and TDM, but saliva may be an attractive alternative matrix. Advantages of saliva include the easy sample collection, which can be standardized with specific saliva collection devices; and the painless collection of saliva, which seems particularly advantageous for children [14,15]. Furthermore, salivary concentrations may reflect the protein-unbound (free) fraction of drug in plasma, which is active and available to be transported to the sites of action, rather than the total (protein- unbound plus bound) plasma concentration [14]. A clear prerequisite for the use of saliva in pharmacokinetic studies and TDM is a strong relationship between the salivary concentrations and the (unbound or total) plasma concentrations of a drug. The plasma pharmacokinetics of the TB drug rifampicin are well-characterized [16-18]. This pivotal TB drug is now being repurposed as a component of future TB drug regimens for adults, but at higher doses [19-20]. In addition, higher doses of rifampicin are currently advised for treatment of TB in children, requiring pharmacokinetic evaluations as well [21]. Therefore, the objectives of this study were (1) to describe the pharmacokinetics of rifampicin in saliva and plasma (total and protein-unbound concentrations) and (2) to assess whether saliva could be an alternative matrix for pharmacokinetic studies and TDM with this essential TB drug.

Methods Study design and population. We performed a descriptive pharmacokinetic study in Moshi, Tanzania. Adult TB patients who were in the intensive phase of TB treatment were eligible for participation. They had to be on TB treatment for at least two weeks because of the expected steady state of rifampicin at that time. Patients with a body weight less than 50 kg used three Fixed Dose Combination (FDC) tablets per day (i.e. 225 mg isoniazid, 450 mg rifampicin, 1200 mg pyrazinamide and 825 mg ethambutol) and patients with a body weight ≥50 kg used four FDC tablets per day (i.e. 300 mg isoniazid, 600 mg rifampicin, 1600 mg pyrazinamide and 1100 mg ethambutol). All participants gave written informed consent. Intensive pharmacokinetic sampling for both plasma and saliva took place during 24 h.

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This was an explorative study and no sample size calculation was performed. The study aimed to recruit 15 patients, providing for an expected number of circa 80 time-matched detectable salivary, protein-unbound and total (protein-unbound plus bound) concentrations of rifampicin. Study participants were recruited from the outpatient TB clinic at Mawenzi Hospital in Moshi and pharmacokinetic sampling was done at the patient unit of the Kilimanjaro Clinical Research Institute. The study was approved by the Research Ethics Committee at the Kilimanjaro Christian Medical Centre (KCMC) and by the Tanzanian National Institute for Medical Research.

Pharmacokinetic sampling. Patients were asked not to eat for at least six hours (i.e. overnight) 5 before start of intensive pharmacokinetic sampling. On the sampling day, patients took their drugs under supervision of a nurse in the morning, and then had a standardized breakfast within 30 minutes after drug intake, which is the usual procedure in this population. The standardized breakfast consisted of a cup (250 ml) of tea with milk and sugar and two pancakes. Serial venous blood samples were collected just before, and at 1, 2, 3, 4, 6, 8, 10 and 24 hours after TB drug intake. Plasma was centrifuged at 2800 rpm for 10 minutes and stored at -80oC immediately thereafter. Stimulated saliva was collected immediately (i.e within 2 minutes) after every blood sample. The patients were asked to rinse their mouth with water. Then a Salivette® was provided, which a plastic device is containing a dental cotton roll impregnated with citric acid that stimulates the salivary flow (Sarstedt, Etten-Leur, The Netherlands). The patients were asked to chew on the cotton roll inside the Salivette®. After one minute, the patient was asked to put the cotton roll back in the insert of the Salivette® centrifuge vessel. The centrifuge vessel was centrifuged at 3000g for 5 minutes, which allows saliva to be collected in the extended tip of the Salivette® tube. The saliva was put in polypropylene tubes and stored at -80°C. The total process until storage in the freezer took a maximum of 45 minutes after sampling. Both the plasma and saliva samples weretransported on dry ice to The Netherlands, where bio-analysis took place.

Bio-analysis. Total (protein-bound plus unbound) plasma concentrations of rifampicin were measured with a validated HPLC method as described before [22]. For the analysis of rifampicin in saliva, liquid-liquid extraction followed by HPLC was used. Briefly, 250 µL of saliva was mixed with 25 µL ascorbic acid (20 g/L), 25 µL 0.2 M acetate buffer pH5, 25 µL of internal standard (sulindac) solution and 1 ml of iso-octane / dichloromethane (3:2 %v/v) and the mixture was centrifuged at 1910 g for 5 minutes. The water layer was frozen in a cryobath (-40°C), the

organic layer was decanted and evaporated at 37°C under a N2 flow until the tube was dry.

The residue was reconstituted in 200 µL H2O / CH3CN (70/30 %v/v) at 4°C by shaking it for 10 minutes. Immediately after centrifugation at 1910 g for 10 minutes at 4°C, 50 µL was injected into the HPLC system. To measure protein-unbound rifampin, ultrafiltration followed by HPLC was used. Briefly, 0.5 ml of plasma was added into a Centrifree YM-30 tube (Millipore, Amsterdam, the Netherlands). The tube was centrifuged for 15 min at 1650 g at 25oC and 50 µL of the clear ultrafiltrate was injected into the HPLC system. One HPLC system was developed for measurement of salivary

96 Saliva study and protein-unbound rifampicin concentrations. The analytical column was an OmniSpher 5 C18 column (250 × 4.6 mm ID; particle size 5 µm) protected by a Chromguard RP ss 10 x 3 mm column (Varian, Middelburg, The Netherlands). The mobile phase components were 30% acetonitrile and 70% 10 mM phosphate buffer with pH 5, run during a HPLC gradient with different flow rates. Total run time was 17 min. UV detection was set at 334 nm. The autosampler liquid system was set at 4°C. For measurement of rifampicin in saliva, accuracy and intra-day precision ranged from 98 to 105% and from 3.7 to 7.3 %, respectively, dependent on the concentration which ranged from 0.1 (limit of quantitation) to 10.4 mg/L. For measurement of unbound plasma concentrations of rifampicin, the average accuracy of ultrafiltrate spiked with rifampin was 107% and intraday precision in measurement of rifampicin in ultrafiltrate varied from 1.5 to 1.9% dependent 5 on the concentration measured. The range of the method for unbound rifampicin plasma concentrations was from 0.06 mg/L (limit of quantitation) to 13 mg/L.

Pharmacokinetic analysis. The area under the plasma concentration-time curve (AUC0-24h), the highest observed concentration (Cmax) with the corresponding sampling time Tmax, apparent clearance (Cl/F; in which F is bioavailability), apparent volume of distribution (Vd/F) and elimination half-life (T½) were assessed by noncompartmental pharmacokinetic methods using WinNonLin Version 5.3 (Pharsight Corp., Mountain View, California US), as described before [22]. C24h was estimated based on the last measurable concentration (Clast) taken at time - *(24-Tlast) Tlast and the elimination rate constant β, using the formula C24h = Clast*e β .

Statistical analysis. Differences in pharmacokinetic parameters of rifampicin based on salivary, protein-unbound and total plasma concentrations were assessed with repeated- measures (within-subject) analysis of variance (ANOVA) performed on log-transformed pharmacokinetic parameters, followed by three separate paired-samples T-tests performed at a Bonferroni-corrected significance level of 0.05/3. maxT values of rifampicin were tested with the related-samples (within-subject) Friedman’s analysis of variance by ranks. Correlation between pharmacokinetic parameters was performed on untransformed data using Spearman’s rho (rank correlation). To assess whether saliva could be an alternative matrix for pharmacokinetic studies and TDM, the time-matched salivary concentrations, protein-unbound and total plasma concentrations of rifampicin were first correlated to eachother using Spearman’s rho. Subsequently ratios of salivary concentrations versus total and protein-unbound plasma concentrations of rifampicin were calculated for all time-matched samples. The effect of sampling time and the effect of the associated rifampicin plasma concentrations on the concentration ratios was assessed with repeated-measures (within-subject) ANOVA on log-transformed concentration ratios. Geometric means were calculated for the saliva to plasma (total and unbound) concentration ratios; the reciprocal average concentration ratios were used as conversion factors to estimate total and unbound rifampicin plasma concentrations based on the measured salivary concentrations [23]. The predictive performance of these estimations was assessed by calculation of conversion factors based on datasets in which one out of the 15 patients was omitted, followed by use of these

97 chapter 5

conversion factors to estimate the (total and unbound) plasma concentrations of the 15th patient (jackknife method [24]). Such predictions were performed for plasma concentrations of all 15 patients. Potential bias in the predictions was assessed using the median percentage prediction error (MPPE). This is the median of all percentage prediction errors, which were defined as: 100% * (predicted concentration – measured concentration) / measured concentration. Imprecision was assessed using the median absolute percentage prediction error (MAPE). This is the median of all absolute percentage prediction errors. The absolute percentage prediction error was defined as: 100% * absolute ((predicted concentration – measured concentration) / measured concentration). For an acceptable predictive performance, both MPPE and MAPE had to be <15% [24,25]. 5 As an alternative means to estimate plasma concentrations and assess predictive performance, a linear regression analysis was performed on log-transformed salivary concentrations versus log-transformed plasma (total or protein-unbound) concentrations of rifampicin, yielding a least squares regression line with equation: log (plasma concentration) = a + b* log (salivary concentration), in which a is the intercept and b is the slope of the regression line [26]. This equation was derived in a randomly selected set of eight patients (index set) and was used to estimate plasma concentrations in the remaining seven patients (validation set) [24,26]. This procedure was repeated three times. Again, MPPE and MAPE were used as measures of bias and imprecision. All statistical analyses were performed with SPSS for Windows version 20.0 (SPSS Inc, Chicago, IL). P values of below 0.05 were considered significant.

Results Patients. Fifteen TB patients were enrolled in the study. Twelve patients were male (80%), their median age was 37 years (range 19-50 years), their median body weight was 49.5 kg (range 41.5-73.6 kg) and they received a median rifampicin dose of 10.2 mg/kg (range 8.2 -11.3 mg/kg).

Pharmacokinetics of rifampicin based on salivary, protein-unbound and total plasma

concentrations. The geometric mean AUC0-24h of rifampicin in saliva (3.1 h*mg/L) was lower

than the geometric mean protein-unbound AUC0-24h in plasma (5.3 h*mg/L) and these were

much lower than the geometric mean plasma AUC0-24h based on total concentrations

(32.7 h*mg/L, table 1, figure 1). Repeated-measures ANOVA on log-transformed AUC0-24h values showed a significant main effect based on type of concentration measured (<0.001) and paired

t-tests with Bonferroni correction revealed significant differences between all three AUC0-24h

values (p<0.001). For Cmax values of rifampicin, the difference in rifampicin Cmax in saliva and

Cmax based on protein-unbound plasma concentrations was not significant, but both these Cmax

values were significantly lower than the Cmax based on total plasma concentrations (p<0.001).

Rifampicin AUC0-24h values based on salivary concentrations, protein-unbound plasma concentrations and total plasma concentrations were all strongly (Spearman’s rho at least 0.71)

and significantly (p≤ 0.003) correlated to eachother. Rifampicin Cmax values based on protein- unbound and total concentrations were also correlated (rho = 0.90, p<0.001) but these were

not correlated with rifampicin Cmax values in saliva (rho = 0.41, p=0.13 and rho = 0.43, p =0.11).

98 Saliva study

Table 1. Pharmacokinetics of rifampicin in saliva and plasma (n=15)1

Plasma, Ratio Ratio protein- saliva / plasma Ratio plasma protein- PK unbound Plasma, total protein- saliva / plasma unbound / parameter saliva2 conc. 2 conc.2,3 unbound4 total3,4 plasma total3,4

AUC0-24h 3.1 5.3 32.7 0.59 0.096 0.161 (h*mg/L) (0.70-8.6) (1.8-10.3) (11.7-73.6) [0.50-0.71] [0.077-0.119] [0.146-0.178]

Cmax (mg/L) 0.64 1.0 6.8 0.62 0.094 0.151 (0.12-1.7) (0.50-2.2) (3.2-17.4) [0.42-0.91] [0.063-0.140] [0.136-0.167]

Tmax (h) 3.0 2.1 2.1 - - - (1.0-6.1) (1.0-6.0) (1.0-6.0) Cl/F (L/h) 164.1 97.6 15.7 1.68 10.4 6.21 5 (69.9-646.3) (58.1-256.5) (8.2-38.6) [1.42-2.00] [8.4-12.9] [5.61-6.87] Vd/F (L) 469.3 318.8 47.7 1.47 9.8 6.69 (223.3-1673.6) (167.9-1022.4) (24.4-139.5) [1.26-1.72] [8.1-11.9] [5.69-7.85]

T1/2(h) 2.0 2.3 2.1 0.88 0.94 1.08 (1.3-2.7) (1.6-4.9) (1.4-3.9) [0.76-1.01] [0.84-1.06] [0.96-1.21]

1. Abbreviations: AUC0-24h: area under the concentration versus time curve, Cmax: peak plasma concentration, Tmax: time

to peak plasma concentration, Cl/F: apparent clearance, Vd/F: apparent volume of distribution, T1/2: elimination half-life, conc.: concentration Chapter2. Geometric 5 means and ranges, apart from Tmax (median and range) 3. Total plasma concentrations refer to protein-unbound plus bound concentrations 4. Geometric means and 95% confidence intervals

1 FigureFigure 1. 1:Geometric Geometric mean steady-state mean concentrationssteady-state ofconcentrations rifampicin versus of time rifampicin (N=15)1 versus time (N=15) Concentrations of rifampicin1. Concentrations (Y-axis) are of displayed rifampicin in (Y-axis)mg/L. Time are displayedpost dose in (X-axis) mg/L. isTime in hours. post dose (X-axis) is in hours.

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Table 2. Effect of sampling time and associated concentrations on concentration ratios

Time Total plasma Concentration ratio Concentration ratio Concentration ratio post concentration of saliva to protein- of saliva to total of protein-unbound dose (mg/L, unbound plasma plasma to total plasma (h)1 geometric mean)2 concentrations 3 concentrations 2,3 concentrations2,3 1 2.0 0.773(0.301-5.511) N=9 0.111(0.048-0.539) N=9 0.145(0.098-0.197) N=12 2 4.2 0.575(0.338-1.067) N=14 0.091(0.047-0.142) N=14 0.154(0.106-0.229) N=11 3 5.6 0.528(0.087-1.593) N=15 0.082(0.014-0.214) N=15 0.155(0.110-0.218) N=15 4 4.2 0.658(0.289-1.011) N=15 0.104(0.045-0.168) N=15 0.157(0.113-0.229) N=15 6 3.0 0.656(0.313-1.072) N=15 0.107(0.047-0.171) N=15 0.163(0.105-0.238) N=15 5 8 1.6 0.581(0.210-1.015) N=13 0.098(0.035-0.210) N=13 0.169(0.100-0.232) N=14 10 0.79 0.593(0.376-1.086) N=7 0.114(0.061-0.182) N=7 0.184(0.116-0.263) N=14

1. Both salivary and protein-unbound plasma concentrations were below the limit of quantitation in all samples at T=0 h and T=24 h post dose. 2. Total plasma concentrations refer to protein-unbound plus bound concentrations 3. Geometric means and ranges

Tmax of rifampicin in saliva appeared to be larger in saliva compared to plasma (3.0 h versus 2.1 h and 2.1 h, table 1), but this difference did not reach statistical significance (p=0.08). Elimination half-lives of rifampicin based on salivary, protein-unbound and total plasma concentrations were similar (see table 1, p=0.10).

Evaluation of saliva as alternative sampling matrix: correlation analysis and time and concentration dependency of concentration ratios. As expected based on the short elimination half-life of rifampicin, loose concentrations of rifampicin were below the limits of quantitation at predose (T=0h) and at 24 h post dose for all salivary, protein-unbound and total plasma concentration measurements. Salivary and unbound plasma concentrations were still below the limit of quantitation at T=1 (6 patients) and T=2h (1 patient) and they returned below the limit of quantitation at T=8h (2 patients) and T=10h post dose (T=8 patients). A total of 88 time-matched salivary and protein-unbound and total plasma concentrations were available based on concentrations measured above the limits of quantitation of the assays. In 100 time-matched samples, protein-unbound and total plasma concentrations were measurable. Salivary concentrations were significantly correlated with total plasma concentrations (rho = 0.78, p<0.001, n=88) and with protein-unbound plasma concentrations (rho = 0.79, p<0.001, n=88, figure 2). Protein-unbound and total plasma concentrations showed a stronger correlation (rho = 0.96, p<0.001, n=100). The geometric mean concentration ratio for salivary versus total plasma concentrations was 0.099 (95% CI: 0.089-0.110, range 0.014-0.539) and that for salivary versus protein-unbound plasma concentrations was 0.614 (95% CI: 0.553-0.682, range 0.086-5.51). The concentration ratio of salivary versus total plasma concentrations of rifampicin did not seem to be dependent of time of sampling (table 2) and this was confirmed by repeated-measures ANOVA performed over the sampling times 1h to 10h (p=0.59, n=4), 2h to 10h (p=0.91, n=6) and 2h to 8h (p=0.43,

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n=12) post dose; no trends over the sampling times were shown either. Concentration ratios for salivary versus unbound plasma concentrations were not dependent of sampling time either (1h to 10h post dose: p=0.23, n=5; 1h to 8 h: p=0.31, n=9). However, concentrations of unbound versus total plasma concentrations showed an increase with sampling time according to a significant linear trend (sampling times 1h to 10h: p=0.01, n=12, table 2).

Evaluation of saliva as alternative sampling matrix: predictive performance of salivary concentrations. The conversion factor to predict total (protein-bound plus unbound) rifampicin plasma concentrations from salivary concentrations was 10.14 (n=88, all 15 patients) and this factor varied slightly when derived from different sets of 14 patients (range: 9.64 to 10.53). The jackknife method showed that the median percentage prediction error (MPPE) for 5 estimation of total rifampicin plasma concentrations based on salivary concentrations was 13.4% and the median absolute percentage prediction error (MAPE) was 35.7%. The conversion factor to predict protein-unbound rifampicin plasma concentrations from salivary concentrations was 1.63 (n=88, all 15 patients, varying from 1.56 to 1.70 in the sets of 14 patients). The MPPE and MAPE for estimation of protein-unbound rifampicin plasma Chapter 5 concentrations based on salivary concentrations were 6.0 and 23.0%.

FigureFigure 2: 2. ScatterScatter plot plot and and regression regression line line of log-transformedof log-transformed salivary salivary and and total total (protein-bound (protein-bound plus plus unbound) plasma unbound) concentrations plasma concentrations of rifampicin of (R rifampicin2 =0.608, (R P<0.001,2 =0.608, n=88) P<0.001, n=88)

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Use of a linear regression equation to predict total plasma concentrations in three index and validation sets resulted in MPPE values of 43.1, 3.8 and -22.4%, and MAPE values were 43.3, 37.5 and 34.6%. For prediction of protein-unbound plasma concentrations, MPPE and MAPE values were 23.5 and 28.1% for one index and validation set, data for the other two sets are pending.

Discussion The first objective of this study was to describe the pharmacokinetics of rifampicin in saliva and compare these to plasma pharmacokinetics based on protein-unbound and total concentrations. It appeared that the salivary AUC and C of rifampicin were much lower 5 0-24h max than those in plasma as based on total (protein-bound plus unbound) concentrations (table 2). This was anticipated, as only the protein-unbound (free) fraction of a drug in plasma can pass into the salivary compartment [14,15,27,28]. In fact, saliva concentrations of most drugs are thought to reflect unbound drug concentrations in plasma [14,15,27,28]. In this study, we actually measured the protein-unbound rifampicin plasma concentrations by using a validated analytical method based on ultrafiltration.

The AUC0-24h of rifampicin in saliva was of the same order of magnitude as the protein-unbound

plasma AUC0-24h, yet it was significantly lower (table 1). This may be explained by one or several other factors that (apart from protein binding) determine the extent of diffusion of drugs into saliva, including lipid solubility, the degree of ionisation in blood and in saliva, and salivary flow [14,15]. The rifampicin molecule exhibits high lipid solubility [16]; its degree of ionisation is dependent on the pH of the solution in which it resides. In solution, rifampin behaves as a zwitterion. Its acidic function (pKa = 1.7) is associated mainly to its hydroxyl groups, while its basic function (pKa = 7.9) is related to a piperazine-nitrogen functional group. In acidified saliva obtained with the Salivette® device (pH=3 [29]), the hydroxylgroup (pKa 1.7) will be largely unprotonated and charged negative, whereas the piperazine-nitrogen will be charged positive, resulting in neutral molecule. The neutral molecule of rifampicin is not expected to be ‘trapped’ in saliva (causing higher salivary than plasma concentrations) as is the case for many basic drugs that are charged positive in acidified saliva.

The appearance of rifampicin into saliva was rapid as reflected in a Tmax of 3.0h (table 1) and salivary to plasma concentration ratios were not dependent on sampling time or associated rifampicin concentrations. The rapid appearance of rifampicin in saliva and the absence of saturation of passage of rifampicin from blood into saliva suggest that this TB drug enters saliva by passive diffusion instead of active transport. Relatively few comparable studies are available on diffusion of rifampicin into saliva. Kenny and Strates have summarized the literature on rifampicin up to 1981 and stated that highest rifampicin levels in saliva and sputum are obtained up to 4 hr after peak serum levels and that these are only 17-25% of peak serum levels [16]. Gurumuthy et al showed that peak concentrations of rifampicin in saliva were 10.6% of those in plasma [30], very similar to data from our study. Contrary to the current study, Gurumuthy et al found a clear increase in the

102 Saliva study saliva to serum concentration ratio with the sampling time post dose. Orisakwe et al found a much higher saliva to plasma ratio for AUC0-24h (i.e. a ratio of 0.67) in their study with chewing gum stimulated saliva flow and intensive pharmacokinetic sampling in healthy volunteers [31]. This is one of very few studies that assessed protein-unbound concentrations of rifampicin in all samples of the recorded pharmacokinetic curves. The average protein binding of 84% as assessed in this study based on unbound/total plasma AUC0-24h ratios (table 1) is very close to the 80% protein binding which is often stated for rifampicin [17,18]. Of note, the ratio of protein-unbound versus total concentrations of rifampicin steadily increased with higher (total) rifampicin concentrations, which is relevant to rifampicin being repurposed at higher doses. 5 The second objective of this study was to assess whether saliva could be used as an alternative matrix for pharmacokinetic studies and TDM with rifampicin. Based on experience in the past [29,32] we deliberately chose to sample ‘stimulated’ saliva using a standardized, reproducible and convenient sampling technique with the Salivette® device. It was considered that stimulated collection results in less variation of salivary pH and possibly less variation in saliva to plasma ratios compared to simple expectoration of saliva [14]. The patients were asked to rinse their mouth with water before sampling of saliva to avoid contamination of the oral cavity with drugs due to prior drug administration, which could lead to spurious findings [14]. As required for a good prediction, saliva to plasma concentration ratios were not dependent of sampling time and rifampicin plasma concentrations. Prediction of total or unbound rifampicin plasma concentration based on salivary concentrations resulted in an acceptable median percentage prediction error (MPPE), which means that systematic bias was limited when many individual predictions (each with positive or negative bias) were considered. Unfortunately, the average absolute error as measured by the median absolute percentage prediction error (MAPE) was above the predefined criterium, which means that the overall predictive performance of salivary concentrations was insufficient. Use of a regression equation rather than a straightforward conversion factor resulted in a similarly imprecise prediction of (total or unbound) plasma concentrations based on salivary concentrations. Of note, the current study also showed that monitoring of saliva concentrations of rifampicin has limited value in evaluating adherence to this drug, as salivary concentrations of rifampicin were below the limits of quantitation at predose (T=0h), at T=1 and starting from T=10h post dose in many patients who took rifampicin under observation. This means that early or late sampling of saliva may incorrectly suggest nonadherence to rifampicin.

In summary, this study showed that total exposure (AUC0-24h) and Cmax of rifampicin in saliva were much lower than those in plasma based on total (protein-unbound plus bound) plasma concentrations. The AUC0-24h of rifampicin in saliva was of the same order of magnitude as the protein-unbound plasma AUC0-24h, but was significantly lower. Rifampicin appeared rapidly in saliva and probably enters saliva through passive diffusion. It appeared not to be possible to predict total or protein-unbound plasma concentrations from salivary concentrations, due to inadequate precision associated with this prediction.

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Acknowledgements We wish to thank all people who in one way or another facilitated the conduct of this study, particularly all patients for participating in this study; dr Chelangwa (Kilimanjaro Regional TB and Leprosy coordinator in the National TB and Leprosy Programme Tanzania), the staff of the TB clinic at Mawenzi Hospital in Moshi and the research nurses Rose Malya, Mono Batuli and Taji Mnzava for their cooperation and effort during pharmacokinetic sampling; the laboratory technicians at KCMC-Biotechnology and at the Department of Pharmacy of Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands for analysing samples; and the Kilimanjaro Clinical Research Institute (KCRI) staff for their support. 5 Financial support The African Poverty Related Infection Oriented Research Initiative (APRIORI), a research network granted by the Netherlands-African partnership for Capacity development and Clinical interventions Against Poverty-related diseases (NACCAP), sponsored the activities of PhD students involved in this project. The Kilimanjaro Clinical Research Institute (KCRI) sponsored the transport, participant’s incentives and food during pharmacokinetic sampling. Bio-analysis of drugs was sponsored by the Department of Pharmacy of Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

References

1. Global Tuberculosis Report 2012, WHO, 2012. Available at http://www.who.int/tb/publications/global_ report/en/ 2. Srivastava S, Pasipanodya JG, Meek C, Leff R, Gumbo T. Multidrug-resistant tuberculosis not due to noncompliance but to between-patient pharmacokinetic variability. J Infect Dis 2011;204:1951-9. 3. Kimerling ME, Philips P, Patterson P, Hall M, Robinson CA, Dunlap DA. Low serum antimycobacterial drug levels in non-HIV infected tuberculosis patients. Chest 1998;113:1178-83. 4. Mehta JB, Shantaveerapa H, Byrd RP Jr, Morton SE, Fountain F, Roy TM. Utility of rifampin blood levels in the treatment and follow-up of active pulmonary tuberculosis in patients who were slow to respond to routine directly observed therapy. Chest 2001;120:1520-4. 5. Weiner M, Burman W, Vernon A, Benator D, Peloquin CA, et al. Low isoniazid concentrations and outcome of tuberculosis treatment with once-weekly isoniazid and rifapentine. Am J Resp Crit Care Med 2003;167:1341-7. 6. Weiner M, Benator D, Burman W, Peloquin CA, Khan A, et al. Association between acquired rifamycin resistance and the pharmacokinetics of rifabutin and isoniazid among patients with HIV and tuberculosis. Clin Infect Dis 2005;40:1481-91. 7. Chideya S, Winston CA, Peloquin CA, Bradford WZ, Hopewell PC, et al. Isoniazid, rifampin, ethambutol, and pyrazinamide pharmacokinetics and treatment outcomes among a predominantly HIV-infected cohort of adults with tuberculosis from Botswana. Clin Infect Dis 2009;48:1685-94. 8. Pasipanodya JG, Srivastava S, Gumbo T. Meta-analysis of clinical studies supports the pharmacokinetic variability hypothesis for acquired drug resistance and failure of antituberculosis therapy. Clin Infect Dis:2012;55:169-77.

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9. Peloquin CA. Therapeutic drug monitoring in the treatment of tuberculosis. Drugs 2002;62:2169-83. 10. Ray J, Gardiner I, Marriott D. Managing antituberculosis drug therapy by therapeutic drug monitoring of rifampicin and isoniazid. Internal Medicine Journal 2003;33:229-34. 11. Holland DP, Hamilton CD, Weintrob AC, Engemann JJ, Fortenberry ER, et al. Therapeutic drug monitoring of antimycobacterial drugs in patients with both tuberculosis and advanced human immunodeficiency virus infection. Pharmacotherapy 2009;29:503-10. 12. Heysell SK, Moore JL, Keller SJ, Houpt ER. 2010. Therapeutic drug monitoring for slow response to tuberculosis treatment in a state control program, Virginia, USA. Emerging infectious diseases 2010;16:1546-53. 13. Magis-Escurra C, van den Boogaard J, Ijdema D, Boeree M, Aarnoutse R. Therapeutic drug monitoring in the treatment of tuberculosis patients. Pulmonary pharmacology & therapeutics 2012;25:83-6. 5 14. Liu H, Delgado MR. Therapeutic drug concentration monitoring using saliva samples. Focus on anticonvulsants. Clin Pharmacokinet. 1999;36:453-70. 15. Mullangi R, Agrawal S, Srinivas NR. Measurement of xenobiotics in saliva: is saliva an attractive alternative matrix? Case studies and analytical perspectives. Biomed Chromatogr. 2009;23:3-25. 16. Kenny MT, Strates B. Metabolism and pharmacokinetics of the antibiotic rifampin. Drug Metab Rev. 1981;12:159-218. 17. Acocella G. Clinical pharmacokinetics of rifampicin. Clin Pharmacokinet. 1978;3:108-27. 18. Holdiness MR. Clinical pharmacokinetics of the antituberculosis drugs. Clin Pharmacokinet 1984;9: 511-44. 19. Ginsberg AM. Drugs in development for tuberculosis. Drugs 2010;70:2201-14. 20. van Ingen J, Aarnoutse RE, Donald PR, Diacon AH, Dawson R, Plemper van Balen G, Gillespie SH, Boeree MJ. Why Do We Use 600 mg of Rifampicin in Tuberculosis Treatment?Clin Infect Dis 2011;52:e194-9. 21. Rapid Advice. Treatment of tuberculosis in children. WHO, 2010. Available at: http://whqlibdoc.who. int/publications/2010/9789241500449_eng.pdf 22. Ruslami R, Nijland HM, Alisjahbana B, Parwati I, van Crevel R, Aarnoutse RE. Pharmacokinetics and tolerability of a higher rifampin dose versus the standard dose in pulmonary tuberculosis patients. Antimicrob Agents Chemother 2007;51:2546-51 23. Van Heeswijk RPG, Veldkamp AI, Mulder JW, Meenhorst PL, Beijnen JH, Lange JMA, Hoetelmans RMW. Saliva as an alternative body fluid for therapeutic drug monitoring of the nonnucleoside reverse transcription inhibitor nevirapine. Ther Drug Monit. 2001;23:255-8. 24. Ting LS, Villeneuve E, Ensom MH. Beyond cyclosporine: A systematic review of limited sampling strategies for other immunosuppressants. Ther. Drug Monit.2006; 28:419–30. 25. Barraclough KA, Isbel NM, Johnson DW, Hawley CM, Lee KJ, McWhinney BC, Ungerer JP, Campbell SB, Leary DR, Staatz CE. A limited sampling strategy for the simultaneous estimation of tacrolimus, mycophenolic acid and unbound prednisolone exposure in adult kidney transplant recipients. Nephrology. 2012;17:294-9. 26. Koks CHW, Crommentuyn KML, Hoetelmans RMW, Mathot RAA, Beijnen JH. Can fluconazole concentrations in saliva be used for therapeutic drug monitoring ? Ther Drug Monit 2001;23:449-53. 27. Jusko WJ, Milsap RL. Pharmacokinetic principles of drug distribution in saliva. Ann N Y Acad Sci. 1993;694:36-47. 28. Haeckel R. Factors influencing the saliva/plasma ratio of drugs. Ann N Y Acad Sci. 1993;694:128-42.

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29. Hugen PW, Burger DM, de Graaff M, ter Hofstede HJ, Hoetelmans RM, Brinkman K, Meenhorst PL, Mulder JW, Koopmans PP, Hekster YA. Saliva as a specimen for monitoring compliance but not for predicting plasma concentrations in patients with HIV treated with indinavir. Ther Drug Monit. 2000;22:437-45. 30. Gurumurthy P, Rahman F, Narayana ASL, Sarma GR. Salivary levels of isoniazid and rifampicin in tuberculous patients. Tubercle 1990;71: 29-33. 31. Orisakwe OE, Akunyili DN, Agbasi PU, Ezejiofor NA. Some plasma and saliva pharmacokinetics parameters of rifampicin in the presence of pefloxacin. Am J Ther.2004;11:283-7. 32. L’homme RF, Muro EP, Droste JA, Wolters LR, van Ewijk-Beneken Kolmer NW, Schimana W, Burger DM. 5 Therapeutic drug monitoring of nevirapine in resource-limited settings. Clin Infect Dis. 2008;47:1339-4.

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The effect of diabetes mellitus on exposure to tuberculosis drugs in 6 Tanzanian patients

Charles M. Mtabho, Hadija H. Semvua, Jossy van den Boogaard, Constantine F. Irongo, Martin J. Boeree, Angela Colbers, David M Burger, Reinout van Crevel, Andre J.A.M. van der Ven, Gibson S. Kibiki, Alma Tostmann, Rob E. Aarnoutse

Manuscript in final preparation chapter 6

Abstract Introduction: Diabetes mellitus (DM) is a well-known risk factor for tuberculosis (TB) and is associated with poor TB treatment outcome and an increased risk of TB relapse. With the current global increase in cases of type 2 DM, attention needs to be increased on optimum treatment of TB in patients with diabetes. Previous studies examining the possible role of altered pharmacokinetic (PK) profiles of TB drugs in DM patients have met with different results, and no studies have been conducted in Africa. We performed a study to describe the PK of TB drugs in Tanzanian TB patients with and without diabetes mellitus.

Methods: We collected blood samples from 20 diabetic TB patients and a control group of 20 6 non-diabetic TB patients during the intensive phase of TB treatment at a Tanzanian outpatient TB clinic. A full pharmacokinetic profile (0-24 hours) was determined for isoniazid, rifampicin, pyrazinamide and ethambutol, and plasma concentrations were measured using validated HPLC methods. PK parameters were calculated using non-compartmental methods and were compared using the independent samples T-test on log-transformed data. Multiple linear regression analysis was performed to assess the effect of other exploratory variables on the PK of TB drugs.

Results: The geometric mean exposures (AUC0-24) to rifampicin and isoniazid were lower in diabetic than non-diabetic TB patients (39.9 versus 29.9 h*mg/L for rifampicin, p=0.052;

5.4 versus 10.6 h*mg/L for isoniazid, p=0.015). Cmax of isoniazid was also lower in diabetic TB

patients (1.7 versus 2.8 mg/L, p=0.010). Forty-seven percent versus 35% of patients had Cmax of rifampicin below the reference range in the diabetic TB group as compared to the non-diabetic TB group, and for isoniazid, the proportions were 74% against 55%. Fast and slow acetylators for isoniazid were not statistically different in proportion between diabetic and non-diabetic TB patients. In a multiple linear regression analysis with age, sex, dose per kg, HIV status and acetylator status as additional explanatory variables, DM remained an independent predictor of the PK of isoniazid and rifampicin, next to acetylator status for isoniazid. Fasting blood glucose also predicted exposure to isoniazid and rifampicin. No PK parameters for pyrazinamide and ethambutol were significantly different between diabetic and non-diabetic TB patients.

Conclusion: Exposure to isoniazid is reduced in Tanzanian diabetic patients with TB and a clear trend to a reduction in exposure to rifampicin was observed. These effects are most likely explained by the diabetes disease. Increasing the doses of TB drugs in treating diabetic TB patientsmay be considered in view of accumulating evidence that exposure to TB drugs is related to response.

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Introduction Diabetes mellitus (DM) was a well-known risk factor for tuberculosis (TB) in the past, but this was largely forgotten during the second half of the 20th century, with the advent of widely available treatment for both diseases [1]. With the current global increase in cases of type 2 DM, the association between TB and DM is re-emerging [2]. The greatest increase in cases of type 2 DM will occur in developing countries, where TB is highly endemic [3]. Patients with DM have a higher risk on developing TB [4,5]. This is probably because of impaired immunity [6]. On top of that, TB is more difficult to treat in diabetic patients. Recent studies have shown that TB patients with DM have a less favourable response to TB treatment as shown by higher failure, relapse, and death rates [7]. It has been shown that patients with DM have lower plasma concentrations of certain drugs 6 [8,9]. Apparently, absorption, distribution, metabolism and/or excretion of drugs are changed in patients with DM. If this also applies to anti-TB drugs, this may at least partly explain the slower response to TB treatment in patients with DM, as lower plasma concentrations of anti-TB drugs have been associated with clinical failure and acquired drug resistance [10-13]. Recently simulations also showed that interindividual variability in pharmacokinetics of TB drugs, rather than non-adherence, causes multidrug resistant TB [14]. In addition, a meta-analysis showed that variability in exposure to a single drug is associated with failure of TB therapy and acquired drug resistance [15]. In one study in Indonesia, TB patients with DM had lower rifampicin concentrations than TB patients without DM [16]. In that study, TB patients with diabetes and age- and sex-matched TB patients without DM were studied during the continuation phase of TB treatment. The exposure to rifampicin up to six hours post dose (area under the curve from 0-6 hours; AUC0–6 h) was 53% lower in patients with TB and DM, compared with patients with TB only; this was attributed to DM per se and to the higher weight of patients with DM. In a follow up study, Indonesian TB patients with and without DM in the intensive phase of treatment were matched for weight and all four TB drugs (isoniazid, rifampicin, pyrazinamide and ethambutol) were analysed; in contrast to previous observations, no differences in drug exposure were found between the two groups of patients [17]. Another study in Peruvian patients also did not find a difference in pharmacokinetic parameters between DM and non DM patients taking TB medication [18]. Clearly, these findings need more research. It needs to be explored whether DM affects TB drug concentrations in populations other than Asian and South-American. Pharmacokinetics can differ between different races and ethnic groups, because of genetic variation in the metabolic and transporter enzymes involved [19]. It is important to study this subject also in African TB patients because the African continent has the highest TB incidence and mortality rates [3]. Therefore, we designed a pharmacokinetic study in northern Tanzania where we studied the effect of type 2 diabetes on the steady state pharmacokinetics of isoniazid, rifampicin, pyrazinamide and ethambutol. Tanzania is a high burden country for tuberculosis. In 2011, the incidence rate was approximately 169 per 100,000 population per year and there were about 82,000 tuberculosis cases [3]. The diabetes prevalence in the Kilimanjaro Region is estimated 1-1.5%, and the prevalence is estimated to be higher in urban Tanzania, namely around 5.5%, which can at least partly be explained by differences in overweight [20].

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Methods Study design. We conducted a prospective observational pharmacokinetic study in which the plasma concentrations of the anti-TB drugs isoniazid, rifampicin, pyrazinamide and ethambutol were compared between Tanzanian TB patients with and without diabetes. The plasma concentrations were measured during the intensive phase of TB treatment. The patients were using TB treatment for drug-sensitive Mycobacterium tuberculosis, as prescribed by the Tanzanian National Tuberculosis Guidelines.

Study subjects. Forty adult TB patients were included in this study; 20 TB patients without diabetes were recruited at Mawenzi Hospital, an outpatient TB treatment clinic in Moshi in 6 northern Tanzania, and 20 TB patients with diabetes were recruited from Mawenzi hospital as well as other hospitals around the region that treat TB. TB was diagnosed by these hospitals according to the Tanzanian guideline and practice. The diagnosis was based on clinical symptoms and signs, chest x-ray examination, and sputum smear microscopy. Diabetic patients were included if they had a previously established diagnosis of DM and were attending a diabetic clinic. In addition, DM was confirmed at the time of pharmacokinetic sampling using WHO criteria [21] where a fasting blood glucose concentration greater than 7 mmol/L (126 mg/dL) was considered to indicate diabetes. Only patients above 18 years of age were eligible for participation. Participants gave written informed consent and the study was approved by the Tanzanian National Institute of Medical Research.

Drug treatment. Tuberculosis was treated with fixed-dose combination tablets (FDC tablets) manufactured by Sandoz, Mumbai India and donated by Novartis through the WHO Global Drug Facility (GDF) that monitors the quality of TB drugs according to stringent WHO standards. Patients with a body weight above 50 kg daily received four FDC tablets daily (i.e. 300 mg isoniazid, 600 mg rifampicin, 1600 mg pyrazinamide and 900 mg ethambutol), those below 50 kg received three FDC tablets daily (i.e. 225 mg isoniazid, 450 mg rifampicin, 1200 mg pyrazinamide and 675 mg ethambutol). Patients were all under community-based directly observed treatment. Diabetic patients either were on dietary management alone, or were treated with oral hypoglycaemic agents and/or injectable insulin.

Sample collection. The pharmacokinetic sampling took place when patients were on TB treatment for at least two weeks, given the expected steady state of the pharmacokinetic parameters at that point. On the sampling day, serial venous blood samples were collected just before, and at 1, 2, 3, 4, 6, 8, 10 and 24 hours after observed tuberculosis drug intake. Patients fasted at least eight hours (from the preceding dinner to the next morning dose) before drug intake and took a standardised breakfast within 30 minutes after drug intake, which reflected the usual drug intake procedures in this population. The standardized breakfast consisted of a cup (125 ml) of tea with milk and sugar and either a small bowl of porridge or maandazi, a typical east African doughnut-like pasty that is rather fat. Plasma was separated immediately and kept frozen at -80oC until transport on dry ice to the Netherlands for bio-analysis.

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Bio-analysis. The total (protein bound plus unbound) plasma concentrations of isoniazid, acetylisoniazid, rifampicin, desacetylrifampicin, pyrazinamide and ethambutol were assessed by validated high-performance liquid chromatography (HPLC) as described before [22]. Isoniazid and acetylisoniazid were measured with liquid-liquid extraction followed by Ultra Performance Liquid Chromatography (UPLC) with ultraviolet (UV) detection. Accuracy was between 97.8% and 106.7% for isoniazid and between 98.0% and 108.9% for acetylisoniazid, dependent on the concentration level. The intra- and inter-assay coefficients of variation were less than 13.4 % and less than 3.2% (dependent on the concentration) over the range of 0.05-15.1 mg/L for isoniazid; and less than 4.2% and less than 5.7% over the range of 0.16-16.2 mg/L for acetylisoniazid. Lower limits of quantification were 0.05 mg/L for isoniazid and 0.16 mg/L for acetylisoniazid. Isoniazid and acetylisoniazid containing samples were stable 6 (<5% loss) for at least 12 months at -80oC.

Pharmacokinetic analysis. Pharmacokinetic evaluations were performed using non- compartimental methods in WinNonLin Version 4.1 (Pharsight Corp., Mountain View, California

US). The highest observed plasma concentration was defined as Cmax with the corresponding sampling time as Tmax. The area under the plasma concentration-time curve (AUC0-24h) was calculated using the linear/log-trapezoidal rule from zero up to the last concentration at 24h

(Clast). The terminal log-linear period (log C versus t) was based on the last data points (at least three). The absolute value of the slope (β / 2.303, in which β is the first order elimination rate constant) was calculated using linear regression analysis. The elimination half-life (t1/2) was calculated as 0.693/β. The apparent clearance of the drug (CL/F; in which F is bioavailability) was calculated as dose/AUC0-24h and the apparent volume of distribution (Vd/F) was calculated as (CL/F)/β. Reference ranges for Cmax values were 3-5 mg/L for isoniazid, 8-24 mg/L for rifampicin, 20-50 mg/L for pyrazinamide and 2-6 mg/L for ethambutol [23]. The acetylator status for isoniazid was determined phenotypically by calculating the metabolic ratio of acetylisoniazid concentration over isoniazid concentration at 3 hours post dose. Patients with a ratio above 1.5 were considered fast or intermediate metabolisers; patients with a ratio below 1.5 were slow metabolisers [24].

Statistical analysis. Demographic data were presented as mean plus standard deviation when normally distributed and as median plus interquartile range when data were not normally distributed. Continuous variables were compared between diabetic and non-diabetic TB patients using a two-sample t-test or Mann-Whitney U test. Associations between numerical variables were determined using parametric correlation (Pearson’s correlation) when data were normally distributed, and non-parametric correlation (Spearman’s Rho correlation) when not normally distributed.

For pharmacokinetic parameters AUC0-24, Cmax, volume of distribution, clearance, and elimination half-life, analysis was performed on logarithm transformed data and geometric means were presented. The differences in these parameters between diabetic and non- diabetic TB patients were calculated with an independent sample T-test on the transformed data. Tmax values were not transformed and were compared with the Mann-Whitney U test.

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Pearson Chi-square test was used to determine the difference in proportions of patients who reached reference peak plasma concentrations of the TB drugs. Univariate analyses were performed in the whole group to assess the effects of age, sex, body weight, dose in mg/kg, HIV status, acetylator status, fasting plasma glucose and HbA1con

the AUC0-24h and Cmax of the four first-line TB drugs. A multivariate analysis with multiple linear regression was used to assess the effects of the explanatory variables DM, age, sex, body weight, dose in mg/kg, HIV status and acetylator status

on the log-transformed AUC0-24h and Cmax of the TB drugs by using the stepwise method with Probability-of-F-to-enter ≤ 0.05 and Probability-of-F-to-remove ≥ 0.10. In addition, a second regression analysis was performed in which fasting blood glucose was assessed as independent 6 variable for the log-transformed AUC and C values. The presence of DM and fasting blood 0-24h max glucose cannot be assessed in one model simultaneously as these variables are highly correlated. All statistical analyses were performed using SPSS version 20 (spss inc, Chicago II).

Results Demographics/ baseline characteristics. We enrolled 40 subjects, 20 diabetic and 20 non- diabetic TB patients. For pharmacokinetic (PK) analysis, the data of one subject (with DM) were excluded because the patient had high concentrations for all TB drugs at time zero hours, indicating that the patient had incorrectly taken the drugs at home prior to pharmacokinetic sampling. Patient characteristics of the remaining 39 patients are summarized in table 1. Of these, 78.5% were male, 35% were HIV infected and the median age was 41 yrs. One patient had extrapulmonary TB (TB of the spine), all others had pulmonary TB. There was no difference in body weight, body mass index (BMI), sex, and dose per kg of the TB drugs between diabetic and non-diabetic TB patients. Diabetic TB patients were older than non-diabetic TB patients (median age 49 versus 38 years). As expected, the median fasting blood glucose (FBG) was higher for diabetic TB patients than non-diabetic TB patients (15.9 versus 6.95 mmol/L). Common presenting symptoms are also shown in table. All diabetic

patients had glycosylated haemoglobin (HbA1c; range, 65 to 147 mmol/mol) above the target limit of 53 mmol/mol for good control. All but one of the diabetic patients knew they had DM, and were enrolled to a diabetic clinic. Three of the diabetic patients were not currently on antidiabetic medications; two were on insulin, 11 on metformin, 10 on chlorpropamide, and six on glibenclamide. Other co- administered drugs were not known to interact with anti-TB drugs (data not shown).

Pharmacokinetic parameters. Table 2 shows the average of PK parameters in diabetic and non-diabetic TB patients and figure 1 compares the plasma concentration-time curves of TB drugs between diabetic and non-diabetic TB patients.

The average AUC0-24h for rifampicin was 25% (p=0.052) lower in diabetic TB patients. Rifampicin clearance showed a trend for an increase (p=0.06) and its elimination half-life was significantly shorter in diabetic versus non-diabetic TB patients. In addition, time to maximum

concentration (Tmax) of rifampicin was longer in diabetic (2.1h; IQR 1-3) than non-diabetic TB patients (1.08h; IQR 0.98-2.05, p=0.027, table 2).

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Table 1. Characteristics of 20 tuberculosis (TB) and 20 TB-diabetes (TB-DM) patients in northern Tanzania

Measure Parameter TBDM TB p value Sex (female, n[%]) 5[25%] 4[20%] 0.705** Age (years; median[IQR]) 49(40-56) 38(30-42) 0.001* Body weight (kg; mean[SD]) 58.7(12.3) 55.7(6.4) 0.346 BMI(kg/m2 mean[SD]) 20.6(4.0) 19.5(2.4) 0.293 TB drugs Number of FDC tablets, n[%] 6 3FDCs 3[15%] 5[25%] 4FDCs 17[85%] 15[75%] 0.695** Dose per kg (mg/kg, mean[SD]) Rifampicin 9.8(1.4) 10.4(0.9) 0.118 Isoniazid 4.9(0.7) 5.2(0.5) 0.118 Pyrazinamide 25.5(3.8) 27.8(2.4) 0.029 Ethambutol 18(2.5) 19.1(1.7) 0.118 FBG(mmol/L, median(IQR)) 15.9(13.1-18) 6.95(5.7-7.4) <0.001* HIV positive, n(%) 7(35%) 7(35%) - HBA1c(mmol/mol; mean[SD]) 106.4[22.7] - - Diabetes treatment Dietary only 3(15%) Metformin 11(55%) Chlorpropamide 10(50%) Glibenclamide 6(30%) Presenting symptoms, n(%) Cough 14(70%) 15(75%) Night sweats 14(70%) 17(85%) Weight loss 17(85%) 20(100%) Chest pain 2(10%) 2(10%) Anorexia 2(10%) 4(20%) Shortness of breath 2(10%) - Fever 7(35%) 4(20%) Hemoptysis 3(15%) 1(5%) *test performed is Mann-Whitney U **test performed is chi square

The average AUC0-24h for isoniazid was 49% (p=0.015) lower in diabetic TB patients. Compared to non-diabetics, diabetic TB patients also had significantly lower values of the isoniazid maximum concentration (Cmax, p = 0.01). Both clearance and volume of distribution were increased in diabetics compared to non-diabetics (table 2).

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AUC0-24h and Cmax of pyrazinamide showed a trend to lower exposure in diabetic versus non- diabetic TB patients, yet all PK parameters for pyrazinamide as well as for ethambutol were not significantly different between diabetic and non-diabetic TB patients.

Table 2. Pharmacokinetic parameters of tuberculosis drugs in Tanzanian diabetic and non-diabetic TB patients in northern Tanzania (continued)

Value for group Ratio of TB-DM to Drug/PK parameter TB-DM TB TB value[95%CI] P 6 Rifampicina

AUC0-24, h*mg/L 29.91[1.77] 39.91[1.26] 0.75[0.56-1.03] 0.052 Cmax, mg/L 7.92[1.71] 8.92[1.28] 0.89[0.67-1.17] 0.384 Proportion with Cmax below reference 9[47.4] 7[35.0] - 0.433 range, n[%]c Tmax, h[IQR]b 2.12[1.03-3.30] 1.08[0.98-2.05] - 0.027 T1/2, h 1.44[1.30] 1.80[1.38] 0.80[0.66-0.97] 0.026 Vz, L 38.74[1.79] 37.30[1.31] 1.04[0.77-1.40] 0.798 CL, L/h 18.60[1.74] 14.40[1.24] 1.29[0.98-1.71] 0.072 Desacetylrifampina

AUC0-24, h*mg/L 3.97[1.81] 4.90[1.45] 0.81[0.57-1.16] 0.240 Cmax, mg/L 0.73[1.97] 0.91[1.32] 0.80[0.56-1.14] 0.206 T1/2, h 1.61[1.44] 2.00[1.30] 0.81[0.64-1.01] 0.066 Desacetylrifampin/rifampin ratiod AUC 0.125[0.057] 0.133[0.035] - 0.672 Cmax 0.100 [0.039] 0.107[0.029] - 0.576 Isoniazida

AUC0-24, h*mg/L 5.41[2.61] 10.61[1.94] 0.51[0.30-0.87] 0.015 Cmax, mg/L 1.65[2.15] 2.77[1.45] 0.60[0.40-0.89] 0.013 Proportion with Cmax below reference 14 [73.7] 11 [55.0] - 0.224 range, n[%]c Tmax, h[IQR] b 1.03[0.97-2.08] 1.07[0.98-1.19] - 0.855 T1/2, h 2.55[1.51] 2.54[1.61] 1.00[0.75-1.34] 0.985 Vz, L 188.78[2.26] 99.19[1.26] 1.90[1.27-2.85] 0.003 CL, L/h 51.31[2.78] 26.89[1.84] 1.91[1.09-3.32] 0.024 Acetylisoniazida

AUC0-24, h*mg/L 21.70[2.00] 22.56[1.50] 0.96[0.67-1.39] 0.831 Cmax, mg/L 2.29[2.29] 2.18[1.80] 1.05[0.66-1.67] 0.831 T1/2, h 5.50 [1.76] 5.03[1.49] 1.10[0.80-1.50] 0.562 Acetylisoniazid/isoniazid ratio AUC 4.01 [3.6] 2.125[2.75] 0.53 [0.25-1.12] 0.095 Cmax 1.40[3.36] 0.788[2.3] 0.57 [0.29-1.11] 0.095

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Table 2. Pharmacokinetic parameters of tuberculosis drugs in Tanzanian diabetic and non-diabetic TB patients in northern Tanzania (continued)

Value for group Ratio of TB-DM to Drug/PK parameter TB-DM TB TB value[95%CI] P Pyrazinamidea

AUC0-24, h*mg/L 289.63[1.41] 344.16[1.29] 0.84[0.69-1.02] 0.083 Cmax, mg/L 34.47[1.23] 38.19[1.17] 0.90[0.80-1.02] 0.090 Proportion with Cmax below reference 0 0 - range, n[%]c Tmax, h[IQR] b 1.12[1.00-3.00] 1.07[0.97-1.76] - 0.252 6 T1/2, h 5.35[1.36] 6.28[1.45] 0.85[0.68-1.07] 0.154 Vz, L 39.54[1.37] 40.35[1.33] 0.98[0.81-1.19] 0.832 CL, L/h 5.12[1.45] 4.45[1.27] 1.15[.094-1.41] 0.170 Ethambutola

AUC0-24, h*mg/L 19.64[1.57] 20.24[1.22] 0.97[0.77-1.22] 0.789 Cmax, mg/L 3.15[1.56] 3.31[1.31] 0.95[0.75-1.21] 0.672 Proportion with Cmax below reference 0 0 - range, n[%]c Tmax, h[IQR] b 2.00[1.00-3.00] 1.98[0.99-2.09] - 0.317 T1/2, h 8.59[1.65] 9.58[1.20] 0.90[0.70-1.16] 0.384 Vz, L 643.74[1.67] 719.03[1.24] 0.90[0.69-1.16] 0.394 CL, L/h 51.92[1.62] 52.04[1.17] 1.00[0.78-1.27] 0.985

aData are presented as geometric mean(standard deviation) unless stated otherwise bBy Mann-Whitney U test cBy Pearson’s chi-square test dData are presented as mean(standard deviation). An independent-samples T-test was used for testing.

With respect to metabolite desacetylrifampicin, there appeared to be no statistically significant difference in average AUC0-24h and Cmax values or corresponding desacetylrifampicin / rifampicin ratios between diabetics and nondiabetics. Despite the lower exposure to isoniazid in diabetics, the AUC0-24h and Cmax values for metabolite acetylisoniazid were similar in diabetics and nondiabetics, resulting in nonsignificantly higher acetylisoniazid/isoniazid ratios in diabetics versus nondiabetics. The proportion of patients with maximum concentrations (Cmax) of rifampicin and isoniazid below reference ranges was higher in diabetic than in non-diabetic TB patients (table 2), yet the distribution of these proportions was not statistically different between diabetic and non-diabetic TB patients. For pyrazinamide and ethambutol all patients had Cmax within reference ranges. Based on the metabolic ratio of acetylisoniazid concentration over isoniazid concentration at 3 hours post dose, acetylator status of 37 patients was determined. Nineteen (51.4%) patients were found to be fast acetylators. Breaking them down by diabetes group, 55.6% of the diabetic versus 47.4% of the non-diabetic TB patients were fast acetylators. The difference was not statistically significant (chi square = 0.248, p value 0.618).

117 chapter 6 Chapter 6

UChapternivariate 6 analysis. Analysis of the association between PK parameters AUC0-24 and Cmax of the TBA drugs and other possible explanatory variables (age, sex, body weight, dose per kilogram, HIV status, acetylator status, fasting blood glucose, HbA1c) showed that age significantly A correlated only with Cmax of isoniazid (Spearman’s rho = -0.392, p = 0.014). Between men and

6

B B

Figure 1. Geometric mean steady-state plasma concentration-time profiles of rifampicin (A), isoniazid (B), pyrazinamide (C), and ethambutol (D) in TB-DM patients (solid line graph, n=19) and TB only patients (dotted graph, n=20)

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Diatub study Page 132 Diatub study Chapter 6 women, rifampicin Cmax (geometric means 7.8 versus 10.7 mg/L, p = 0.048) and pyrazinamide Cmax (35.1 versus 40.7 mg/L, p =0.036) were different. Body weight and dose per kilogram showed no correlations with AUC and C of the TB drugs. HIV positive and negative patients only differed 0-24 max with respect to the Cmax of pyrazinamide (geometric means 39.7 versus 34.7 mg/L, p = 0.033). Fast C

6

D

Figure 1. (Continued) Figure 1: Geometric mean steady-state plasma concentration-time profiles of rifampicin (A), isoniazid (B), pyrazinamide (C), and ethambutol (D) in TB-DM patients (solid line graph, n=19) and TB only patients (dotted graph, n=20 119

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acetylators had significantly lower AUC0-24 (geometric means 4.1 versus 16.2 h*mg/L, p = <0.001)

and Cmax (1.6 versus 3.1 mg/L, p = 0.001) valuesof isoniazid than slow acetylators.

Fasting blood glucose showed a significant correlation with AUC0-24 of rifampicin

(Spearman’s rho = -0.406, p =0.013) and with AUC0-24 (Spearman’s rho = -0.376, p =0.022) and

Cmax (Spearman’s rho = -0.421, p =0.009) of isoniazid. For the diabetic group, HbA1c did not show a significant correlation with any of the PK parameters of rifampicin, isoniazid, pyrazinamide, and ethambutol.

Multivariate analysis. Multiple regression with the stepwise method and diabetes mellitus, age, sex, dose in mg/kg, HIV status and acetylator status as explanatory variables yielded 6 significant models with regression equations that only contained diabetes mellitus as explanatory variable for log AUC0-24 and log Cmax of rifampicin, as well as for log AUC0-24 and

log Cmax of pyrazinamide. Diabetes mellitus and acetylator status were the only variables that

remained in regression equations for log AUC0-24 and log Cmax of isoniazid. Similarly, multiple regression analyses with fasting blood glucose instead of DM yielded

fasting blood glucose as a significant predictor for log AUC0-24 and log Cmax of rifampicin,

pyrazinamide and isoniazid as well as acetylator status as a predictor for log AUC0-24 and log

Cmax of isoniazid. These results show that the effect of diabetes mellitus and the effect of fasting blood glucose on the pharmacokinetic parameters of TB drugs remained (or even became) significant when other explanatory variables were taken into consideration.

Discussion This is the first report describing the pharmacokinetic (PK) parameters of tuberculosis (TB) drugs in diabetic and non-diabetic TB patients using intensive PK sampling in an African population.

We have found a clear, nearly significant trend for a decrease in AUC0-24 of rifampicin in diabetic

versus nondiabetic TB patients. Similarly, AUC0-24 and Cmax of isoniazid were significantly decreased in diabetic TB patients. The effect of DM correlated with a similar effect of fasting blood glucose on the exposure to these TB drugs. In multivariate analyses, the effects of DM and fasting blood glucose remained significant and acetylator status was another predictor of

the AUC0-24 and Cmax of isoniazid Similar studies in Indonesia and Peru have shown contradicting results [16-18]. The first Indonesian study [16] concluded that DM was associated with a strong decrease in plasma concentrations of rifampicin whereas a follow up study in the same population [17], and a Peruvian study [18] revealed no significant difference in exposure to TB drugs between diabetic and non-diabetic TB patients. Our study was carried out in a distinctively different ethnic population (Africans) and involved intensive (9 sampling points over 24 hour interval) PK sampling. We analysed all four standard TB drugs in 40 patients with validated methods. In the second Indonesian study, the authors implicate the low levels of TB drugs seen in their first study to weight differences between diabetic and non-diabetic TB patients [17]. Although this is still a clinically relevant finding, they matched their patients for body weight in their follow-up study to disentangle

120 Diatub study the effects of weight and DM per se. Although we did not match our patients for body weight, the distribution of body weight (and therefore drug dose per kilogram weight) were the same between diabetic and non-diabetic TB patients (table 1); and besides that body weight was no predictor of exposure to TB drugs in our multiple linear regression analyses. There is no evidence that antidiabetic drugs lower the concentration of TB drugs [25]. Therefore, we believe the observed differences in PK parameters are due to diabetes mellitus. This is further substantiated by similar associations that we found between fasting blood glucose levels and exposure to rifampicin and isoniazid in our population of diabetic and non-diabetic patients. Unlike previous studies, for the first time, we have performed the analysis of isoniazid in diabetic and non-diabetic TB patients and it is actually isoniazid that was found to be most adversely affected in its values for AUC and C . Next to diabetes mellitus, acetylator status of 6 0-24 max isoniazid appeared to be a predictor of the AUC0-24 and Cmax of isoniazid. It should be noted that we realize that we evaluated the effect of DM on the pharmacokinetics of isoniazid and at the same time used a phenotyping method (which is based on relative exposure to acetylisoniazid and isoniazid) to assess the acetylator status. Theoretically, an incidental overrepresentation of (genotypic) fast acetylators among the diabetic TB patients might explain the lower exposure to isoniazid in this group, and this might by concealed by an effect of DM on the phenotypic assessment of acetylator status. Therefore, a genotypic assessment of the acetylator status of isoniazid is currently planned. Although we cannot readily translate suggested diabetes-mediated changes in pharmacokinetics of particular drugs to TB drugs, documented mechanisms suggest that diabetes influences the pharmacokinetics of various drugs by affecting (i) absorption, due to changes in subcutaneous and muscle blood flow and delayed gastric emptying; (ii) distribution, due to non-enzymatic glycation of albumin; (iii) biotransformation, due to differential regulation of enzymes involved in drug biotransformation and drug transporters; and (iv) excretion, due to nephropathy [8,9,26,27]. As to rifampicin, the current study suggests that the clearance of rifampicin may be increased by DM, yet this appears not to be mediated by an effect on the metabolism of rifampicin into desacetylrifampicin (table 2). For isoniazid, both clearance and volume of distribution were increased in diabetic TB-patients, ultimately resulting in a relative increase in the formation of metabolite acetylisoniazd. Evidence has shown that diabetic patients who get TB have poor treatment response to TB treatment and poor treatment outcome [28,29]. Moreover accumulating evidence suggests that total exposure (AUC) and maximum concentration (Cmax) of TB drugs are very important for efficacy of the drugs [30]. Since increasing the dose of TB drugs may result in increased plasma concentrations of the drugs and improved treatment outcome [31], individualization of the dosages and therapeutic drug monitoring in diabetic TB patients are necessary. However because therapeutic drug monitoring is not feasible in developing countries, increasing the doses of TB drugs (especially rifampicin and isoniazid) in the whole population of diabetic patients seems to be a better strategy so as attain average plasma concentrations that are associated with good treatment outcome in the majority of patients. To this end, higher doses of TB drugs have been shown to be safe and tolerable [22,31-33]. Besides our findings and conclusions, further studies in African and other ethnic groups are required to confirm our hypothesis.

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Our study findings may be limited by unequal age distribution between the groups, the diabetic TB group having on average older patients than the TB group. Age has been found not to be associated with pharmacokinetics of TB drugs in many studies [34,35]. Similarly we did not match the groups for sex and weight; however the distribution of these parameters was the same in the two groups. In addition, age, gender and dose/kg were no significant predictors in multiple regression analyses. The number of patients (n=39) may be high for a PK study with intensive PK sampling, yet this number is relatively low to perform regression analyses with several possible explanatory variables. In summary, we have shown a reduction in isoniazid plasma concentrations and a clear trend towards a reduction in exposure to rifampicin in Tanzanian diabetic patients with TB. We 6 conclude that diabetes disease is likely to be responsible for the observed effects. More African and other studies are needed to confirm our findings, and higher doses of the drugs need to be considered.

Acknowledgements We wish to thank all the patients for participating in this study; the staff of the TB clinic at Mawenzi Hospital in Moshi and the research nurses at KCMC for their cooperation and effort; the laboratory technicians at KCMC and at the Department of Pharmacy of the Radboud University Medical Center, The Netherlands for their technical support.

Financial support This study was co-supported by the African Poverty Related Infection Oriented Research Initiative (APRIORI), a research network granted by the Netherlands-African partnership for Capacity development and Clinical interventions Against Poverty-related diseases (NACCAP) and by a funding from the UNESCO/L’Oreal For Young Women in Science Fellowship 2008, which was awarded to A. Tostmann.

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7. Harries AD, Lin Y, Satyanarayana S, Lonnroth K, Li L, Wilson N, et al. The looming epidemic of diabetes- associated tuberculosis: learning lessons from HIV-associated tuberculosis. Int J Tuberc Lung Dis 2011 Nov;15(11):1436-44, i. 8. Gwilt PR, Nahhas RR, Tracewell WG. The effects of diabetes mellitus on pharmacokinetics and pharmacodynamics in humans. Clin Pharmacokinet 1991 Jun;20(6):477-90. 9. Dostalek M, Akhlaghi F, Puzanovova M. Effect of diabetes mellitus on pharmacokinetic and pharmacodynamic properties of drugs. Clin Pharmacokinet 2012 Aug 1;51(8):481-99. 10. Kimerling ME, Phillips P, Patterson P, Hall M, Robinson CA, Dunlap NE. Low serum antimycobacterial drug levels in non-HIV-infected tuberculosis patients. Chest 1998 May;113(5):1178-83. 11. Mehta JB, Shantaveerapa H, Byrd RP, Jr., Morton SE, Fountain F, Roy TM. Utility of rifampin blood levels in the treatment and follow-up of active pulmonary tuberculosis in patients who were slow to respond 6 to routine directly observed therapy. Chest 2001 Nov;120(5):1520-4. 12. Weiner M, Burman W, Vernon A, Benator D, Peloquin CA, Khan A, et al. Low isoniazid concentrations and outcome of tuberculosis treatment with once-weekly isoniazid and rifapentine. Am J Respir Crit Care Med 2003 May 15;167(10):1341-7. 13. Weiner M, Benator D, Burman W, Peloquin CA, Khan A, Vernon A, et al. Association between acquired rifamycin resistance and the pharmacokinetics of rifabutin and isoniazid among patients with HIV and tuberculosis. Clin Infect Dis 2005 May 15;40(10):1481-91. 14. Srivastava S, Pasipanodya JG, Meek C, Leff R, Gumbo T. Multidrug-resistant tuberculosis not due to noncompliance but to between-patient pharmacokinetic variability. J Infect Dis 2011 Dec 15;204(12):1951-9. 15. Pasipanodya JG, Srivastava S, Gumbo T. Meta-analysis of clinical studies supports the pharmacokinetic variability hypothesis for acquired drug resistance and failure of antituberculosis therapy. Clin Infect Dis 2012 Jul;55(2):169-77. 16. Nijland HM, Ruslami R, Stalenhoef JE, Nelwan EJ, Alisjahbana B, Nelwan RH, et al. Exposure to rifampicin is strongly reduced in patients with tuberculosis and type 2 diabetes. Clin Infect Dis 2006 Oct 1;43(7):848-54. 17. Ruslami R, Nijland HM, Adhiarta IG, Kariadi SH, Alisjahbana B, Aarnoutse RE, et al. Pharmacokinetics of antituberculosis drugs in pulmonary tuberculosis patients with type 2 diabetes. Antimicrob Agents Chemother 2010 Mar;54(3):1068-74. 18. Requena-Mendez A, Davies G, Ardrey A, Jave O, Lopez-Romero SL, Ward SA, et al. Pharmacokinetics of rifampin in Peruvian tuberculosis patients with and without comorbid diabetes or HIV. Antimicrob Agents Chemother 2012 May;56(5):2357-63. 19. Wilkinson GR. Drug metabolism and variability among patients in drug response. N Engl J Med 2005 May 26;352(21):2211-21. 20. Aspray TJ, Mugusi F, Rashid S, Whiting D, Edwards R, Alberti KG, et al. Rural and urban differences in diabetes prevalence in Tanzania: the role of obesity, physical inactivity and urban living. Trans R Soc Trop Med Hyg 2000 Nov;94(6):637-44. 21. World Health Organization. Definition, diagnosis, and classification of diabetes mellitus and its complications. World Health Organization, Geneva, Switzerland. 1999. 22. Ruslami R, Nijland HM, Alisjahbana B, Parwati I, van CR, Aarnoutse RE. Pharmacokinetics and tolerability of a higher rifampin dose versus the standard dose in pulmonary tuberculosis patients. Antimicrob Agents Chemother 2007 Jul;51(7):2546-51.

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23. Peloquin CA. Therapeutic drug monitoring in the treatment of tuberculosis. Drugs 2002;62(15):2169-83. 24. Hutchings A, Routledge PA. A simple method for determining acetylator phenotype using isoniazid. Br J Clin Pharmacol 1986 Sep;22(3):343-5. 25. Niemi M, Backman JT, Fromm MF, Neuvonen PJ, Kivisto KT. Pharmacokinetic interactions with rifampicin: clinical relevance. Clin Pharmacokinet 2003;42(9):819-50. 26. Okabe N, Hashizume N. Drug binding properties of glycosylated human serum albumin as measured by fluorescence and circular dichroism. Biol Pharm Bull 1994 Jan;17(1):16-21. 27. Cashion AK, Holmes SL, Hathaway DK, Gaber AO. Gastroparesis following kidney/pancreas transplant. Clin Transplant 2004 Jun;18(3):306-11. 28. Baker MA, Harries AD, Jeon CY, Hart JE, Kapur A, Lonnroth K, et al. The impact of diabetes on tuberculosis 6 treatment outcomes: a systematic review. BMC Med 2011;9:81. 29. Syed Suleiman SA, Ishaq Aweis DM, Mohamed AJ, Razakmuttalif A, Moussa MA. Role of diabetes in the prognosis and therapeutic outcome of tuberculosis. Int J Endocrinol 2012;April 18 (0) 30. Hall RG, Leff RD, Gumbo T. Treatment of active pulmonary tuberculosis in adults: current standards and recent advances. Insights from the Society of Infectious Diseases Pharmacists. Pharmacotherapy 2009 Dec;29(12):1468-81. 31. Magis-Escurra C, van den BJ, Ijdema D, Boeree M, Aarnoutse R. Therapeutic drug monitoring in the treatment of tuberculosis patients. Pulm Pharmacol Ther 2012 Feb;25(1):83-6. 32. Steingart KR, Jotblad S, Robsky K, Deck D, Hopewell PC, Huang D, et al. Higher-dose rifampin for the treatment of pulmonary tuberculosis: a systematic review. Int J Tuberc Lung Dis 2011 Mar;15(3):305-16. 33. Katiyar SK, Bihari S, Prakash S, Mamtani M, Kulkarni H. A randomised controlled trial of high- dose isoniazid adjuvant therapy for multidrug-resistant tuberculosis. Int J Tuberc Lung Dis 2008 Feb;12(2):139-45. 34. McIlleron H, Rustomjee R, Vahedi M, Mthiyane T, Denti P, Connolly C, et al. Reduced antituberculosis drug concentrations in HIV-infected patients who are men or have low weight: implications for international dosing guidelines. Antimicrob Agents Chemother 2012 Jun;56(6):3232-8. 35. Jonsson S, Davidse A, Wilkins J, Van der Walt JS, Simonsson US, Karlsson MO, et al. Population pharmacokinetics of ethambutol in South African tuberculosis patients. Antimicrob Agents Chemother 2011 Sep;55(9):4230-7.

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Antituberculosis drug-induced hepatotoxicity is uncommon in Tanzanian hospitalized pulmonary 7 tuberculosis patients

Alma Tostmann, Jossy van den Boogaard, Hadija Semvua, RizikiKisonga, Gibson S. Kibiki, Rob E. Aarnoutse, Martin J. Boeree

Tropical Medicine and International Health.2010;15:268-272 chapter 7

SUMMARY Data on antituberculosis drug-induced hepatotoxicity in sub Saharan Africa are limited, probably because liver function tests are not carried out routinely during tuberculosis treatment in most African countries. We monitored the liver function of 112 Tanzanian hospitalized pulmonary tuberculosis patients during the first two months (i.e. the intensive phase) of tuberculosis treatment. The rate of hepatotoxicity in our study was 0.9% (95% CI 0.04 - 4.3%). It is encouraging to find a lower rate of antituberculosis drug-induced hepatotoxicity than one would expect based on the high prevalence of risk factors such as HIV and hepatitis B.

Keywords: antitubercular treatment, adverse reactions, liver toxicity, Africa 7

128 TB drugs hepatotoxicity

Introduction With nine million new cases and almost two million deaths in 2007, tuberculosis (TB) remains a major cause of illness and death worldwide. The highest incidence rate is found in Africa, due to the HIV epidemic. TB is the leading cause of death among HIV patients [1]. Standard TB treatment consists of a six-month course with isoniazid, rifampicin, pyrazinamide and ethambutol. In a clinical setting, this regimen is able to cure more than 95% of the patients with active TB caused by normally sensitive Mycobacterium tuberculosis. However, in practice cure rates are lower. Adverse effects have a negative impact on therapy adherence, which may decrease treatment success rates and could eventually enhance treatment failure, relapse or the emergence of drug-resistance [2-4]. Isoniazid, rifampicin and pyrazinamide are potentially hepatotoxic drugs. They are metabolised in the liver, making this organ vulnerable for injury. Antituberculosis drug-induced hepatotoxicity (ATDH) is a serious adverse effect that can be fatal if therapy is not interrupted 7 in time [5,6]. ATDH occurs in 2 to 28% of the tuberculosis patients; the variation is large due to different definitions of hepatotoxicity and differences between the populations studied [6]. Data on ATDH in sub Saharan Africa are limited, probably because liver function tests are not carried out during tuberculosis treatment in most African countries. ATDH is reported to be less than 2%, but liver function was not closely monitored in these studies [7-9]. It is important to have a valid estimation of the incidence of ATDH in this region. It may help to explain low cure rates and is particularly relevant considering the continued introduction of antiretroviral treatment, which enhances hepatotoxicity when combined with tuberculosis drugs [10]. In addition, new tuberculosis treatment regimens containing moxifloxacin and a high dose of rifampicin are currently being tested in sub-Saharan Africa. To estimate the rate of ATDH in an African population, we closely monitored the liver function in Tanzanian hospitalized patients during the intensive phase of TB treatment.

Methods This study was conducted between April 2007 and July 2008 at the Kibong’oto National Tuberculosis Hospital in SanyaJuu, Northern Tanzania. All adults who were not on antiretroviral therapy before starting tuberculosis treatment, were eligible for participation. The study was approved by the Tanzanian National Institute for Medical Research and all patients gave written informed consent. Tuberculosis was treated according to guidelines of the Tanzanian National Tuberculosis and Leprosy Programme. During the intensive phase of treatment, patients with a body weight <50 kg received 225 mg isoniazid, 450 mg rifampicin, 1200 mg pyrazinamide and 675 mg ethambutol daily (three fixed-dose combination [FDC] tablets, Novartis®) and patients >50 kg received 300 mg isoniazid, 600 mg rifampicin, 1600 mg pyrazinamide and 900 mg ethambutol daily (four FDC tablets). All patients received full facility-based directly observed therapy (DOT), as they were admitted in the hospital during the intensive phase of treatment. Demographic data was collected prior to treatment. Patients were tested for HIV (SD BioLineTM [HIV rapid test]; if positive then confirmed by DetermineTM; Abbott). Hepatitis B

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surface antigen (HbsAg) and hepatitis C virus antibodies were detected with a qualitative rapid chromatographic immunoassay (ACON Laboratories Inc, San Diego, USA). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin were determined at baseline and after 2, 4, 6 and 8 weeks of TB treatment, using the Roche Reflotron® (Kerkhof Medical Service, the Netherlands). Hepatotoxicity was defined as serum ALT > three times the upper limit of normal (ULN) in the presence of hepatotoxicity symptoms or ALT > 5x ULN without symptoms of hepatotoxicity.6 The reference values were 41 U/l for ALT, 40 U/l for AST and 17 µmol/L for bilirubin. Every two weeks, a research nurse screened the patients for symptoms of hepatotoxicity (jaundice, abdominal pain, nausea and vomiting). Parameters that were presented as mean (standard deviation; SD) when normally distributed or median (inter-quartile range; IQR) if not normally distributed. Statistical analysis 7 were performed using SPSS for Windows (version 14.0; SPSS). Results We included 117 patients in this study. Five patients were excluded from analysis: three patients died in the first week of treatment, for reasons other than possible drug-induced hepatotoxicity and two patients withdrew from the study immediately after baseline. Of the 112 patients included in the analysis, 98 (87.5%) completed the two month study period. Eleven patients (9.8%) were HIV positive and six of them were started antiretroviral treatment during the intensive phase of TB treatment. Table 1 shows the patients’ characteristics. None of the patients developed hepatotoxicity during the first two months of TB treatment. Seven patients (6.3%) developed slight ALT increases, all less than 3 times the ULN. The highest ALT observed was 87 U/L; the median of peak ALT values during the study period was 12 U/L (IQR 9-21 U/L). The highest AST was 64 U/L, with a median peak value of 17 U/l (IQR 12-22 U/L). Table 2 shows the liver biochemistry at baseline and during the intensive phase of treatment. One patient developed hepatotoxicity symptoms in the sixth week of treatment: she experienced serious abdominal pain, nausea and vomiting and after intake of the TB drugs and got discoloration of the conjunctives after drug-intake which disappeared again after a few hours. Because serum ALT and bilirubin were completely normal, this patient did not have hepatotoxicity according to our definition. She had lost a lot of weight during treatment (from 54 kg to 34 kg) and was using 4 FDC tablets a day (for > 50 kg). Treatment was interrupted and relieved the symptoms, after which she continued with 3 FDC tablets. Three patients developed hyperbilirubinemia (bilirubin levels > 2.5 times ULN). ALT and AST levels were within the normal range and jaundice was not reported in these patients. Fourteen patients (12.5%) dropped out during the study. Nine patients withdrew because they refused to give any more blood samples, due to cultural believes concerning blood; three after week 2, four after week 4 and two after week 6 of treatment.

Discussion This is the first study to date where the liver function is closely monitored during TB treatment in a sub-Saharan African population. None of our 112 Tanzanian pulmonary TB patients developed

130 TB drugs hepatotoxicity

Table 1. Patient characteristics

N = 112 Age, median years (IQR) 32 (27-40) Male sex, n (%) 88 (78.6%) Weight, mean kg (SD) 52.4 (7.6) HIV, n (%) Positive 11 (9.8%) Negative 90 (80.4%) Unknown 11 (9.8%) Hepatitis B, n (%) Positive 8 (7.1%) Negative 88 (78.6%) 7 Unknown 16 (14.3%) Hepatitis C, n (%) Positive 3 (2.7%) Negative 93 (83.0%) Unknown 16 (14.3%) Study outcome, n (%) Study completed 98 (87.5%) LTF between week 2 and 4 3 (2.7%) LTF between week 4 and 6 5 (4.5%) LTF between week 6 and 8 6 (5.4%) Concomitant drugs, n (%) Antiretroviral drugs * 6 (5.4%) Cotrimoxazole 8 (7.1%)

Legend. LTF = Lost to follow-up; IQR = inter quartile range; SD = standard deviation; * Antiretroviral treatment in Tanzania: Combivir® (zidovudine + lamivudine) + efavirenz. hepatotoxicity during the intensive phase of TB treatment. One patient developed serious symptoms of hepatotoxicity. Although her liver biochemistry remained normal, the symptoms could have been caused by the TB drugs. Therefore, the rate of hepatotoxicity in our study was 0.9% (95% CI 0.04 - 4.3%) at the most. Clinical trials with moxifloxacin and a high dose of rifampicin are in preparation in the sub Saharan African region. Rifampicin-induced hepatotoxicity may be dose-related [11]. It is encouraging to observe that the rate of TB drug-induced hepatotoxicity is lower than expected in an area with HIV and hepatitis B. Although case reports of moxifloxacin-induced hepatotoxicity have been published, moxifloxacin is considered safe and is not expected to increase the risk of hepatotoxicity [12,13]. Our findings are in line with previous reports that indicate a low rate of ATDH in sub Saharan African TB patients. Recently, a study from South Africa reported that only two out of 400 TB patients (0.5%) developed hepatotoxicity during treatment [14]. In a trial on cotrimoxazole

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Table 2. Liver biochemistry during the intensive phase of treatment

Liver function parameter ALT AST Bilirubin Baseline Median (IQR) 10 U/l (7-15) 13 U/l (10-18) 11 µmol/l (9-16) Maximum 53 U/l 91 U/l 43 µmol/l Intensive phase * Median (IQR) 12 U/l (9-21) 17 U/l (12-22) 13 µmol/l (10-19) Maximum 87 U/l 64 U/l 47 µmol/l

Legend: ALT = alanine aminotransferase; AST = aspartate aminotransferase; Bilirubin = total bilirubin; * based on highest value measured during the intensive phase of TB treatment; IQR = inter quartile range 7 prophylaxis in Malawian HIV-positive pulmonary TB patients, about 2% of the patients developed hepatotoxicity during TB treatment [7]. Since cotrimoxazole can be hepatotoxic in patients with AIDS, the ATDH rate may even be slightly overestimated [15]. Two studies from Congo and Uganda reported no hepatotoxicity and 1% hepatotoxicity, respectively, during standard TB treatment [8,9]. Even though liver function was not routinely monitored and treatment was not directly observed, this indicates that hepatotoxicity during TB treatment is not a big problem. It is difficult to explain why the hepatotoxicity rate is low in sub-Saharan Africa despite the prevalence of risk factors such as HIV and hepatitis B. Hepatotoxicity rates of 13-15% have been reported from such as India and Iran [16]. Our study population was characterised by a low proportion of women, a relatively young age, 10% HIV and 7% hepatitis B carriers. Old age, female sex, malnutrition, HIV, alcohol usage, underlying liver disease such as hepatitis and several genetic polymorphisms are risk factors for ATDH [5,6]. Several genetic polymorphisms in drug metabolising enzymes have been associated with TB drug-induced hepatotoxicity, such as slow acetylator status (N-acetyltransferase 2), cytochrome P450 2E1 c1/c1 genotype and a glutathione S-transferaseM1 homozygote null genotype [17]. Slow and intermediate acetylators are highly prevalent in African populations [18,19]. Almost 95% of the black South Africans had a CYP2E1 c1/c1 genotype[20] and 33% of the Tanzanians have a GSTM1 null genotype [21]. These studies suggest that ‘high risk genotypes’ are prevalent amongst Africans, which would contradict the low rate of hepatotoxicity found in our study. We monitored the liver function for two months only. Even though ATDH most often occurs in the first two months of treatment [6], this may have slightly underestimated the hepatotoxicity rate. Our study was limited to hospitalised patients only, which could have overestimated the rate of hepatotoxicity, since generally higher rates of hepatotoxicity are seen in hospitalised patients. To estimate the rate of hepatotoxicity in sub Saharan African patients more accurately, a similar study could be performed in a larger group of TB patients that are not hospitalised. Ten percent of the patients from our study were HIV positive; six of them had started antiretroviral therapy during TB treatment (zidovudine, lamivudine and efavirenz). None of

132 TB drugs hepatotoxicity them developed any increase in ALT or AST. Concurrent TB-HIV therapy is often complicated by overlapping toxicities and drug-drug interactions.10 Drug toxicity, including hepatotoxicity, is a major cause of TB or HIV treatment interruption during combined TB-HIV treatment [22]. Nevirapine is the most hepatotoxic non-nucleoside reverse transcriptase inhibitor [23]. The majority of the nucleoside reverse transcriptase inhibitors (e.g. didanosine and stavudine) are potentially hepatotoxic as and hepatotoxicity has been described for some protease inhibitors (e.g. ritonavir, indinavir and saquinavir). The worldwide incidence of hepatotoxicity during antiretroviral treatment ranges from 2 to 18% [24]. In sub Saharan Africa, data are conflicting. In the study of Marks et al (that wasmentioned before) the rate of hepatotoxicity during TB treatment in HIV positive (n=141) and HIV negative (n=240) TB patients was low (0.5%), while 16.3% of the HIV positive patients received antiretroviral treatment [14]. These findings are in contrast with another South African study by Hoffmann et al, where 4.6% of the 868 patients that used antiretroviral drugs 7 (zidovudine, lamivudine and efavirenz) developed hepatotoxicity (grade 3 or 4; i.e. ALT > 5 times the ULN) and where the use of TB therapy increased the risk of hepatotoxicity eightfold [25]. More knowledge on hepatotoxicity during combined HIV-TB treatment is warranted, since a significant proportion of the TB patient population in sub-Saharan Africa is HIV positive and would need to use combined TB-HIV treatment [26]. A suggestion for future research would be to get more insight in country specific rates of hepatotoxicity during TB treatment and the effect of HIV and antiretroviral therapy by using data of the national TB programs. International guidelines recommend alanine aminotransferase (with or without bilirubin) for the monitoring of hepatotoxicity [5,6]. Regarding the low rate of TB drug-induced hepatotoxicity found in our study as well as other studies, routine liver function monitoring will not be necessary in sub-Saharan Africa. Nevertheless, it remains important that TB patients receive clear instructions on possible signs and symptoms of hepatotoxicity and they should be instructed to visit their doctor in case such symptoms occur.

References

1. World Health Organisation. Global Tuberculosis Control 2009: surveillance, planning, financing: WHO Report 2009. WHO/HTM/TB/2009.411. http://www.who.int/tb/publications/global_report/2009/pdf/ full_report.pdf 2. Kaona FA, Tuba M, Siziya S, Sikaona L. An assessment of factors contributing to treatment adherence and knowledge of TB transmission among patients on TB treatment. BMC Public Health 2004;4:68. 3. Wares DF, Singh S, Acharya AK, Dangi R. Non-adherence to tuberculosis treatment in the eastern Tarai of Nepal. Int J Tuberc Lung Dis 2003;7(4):327-335. 4. World Health Organisation/IUATLD Global project on anti-tuberculous drug Resistance Surveillance. Anti-tuberculosis drug resistance in the world. Fourth global report. WHO/HTM/TB/2008.394. 2008. http://whqlibdoc.who.int/hq/2008/WHO_HTM_TB_2008.394_eng.pdf 5. Saukkonen JJ, Cohn DL, Jasmer RM et al. An Official ATS Statement: Hepatotoxicity of Antituberculosis Therapy. Am J RespirCrit Care Med 2006;174(8):935-952. 6. Tostmann A, Boeree MJ, Aarnoutse RE, de Lange WC, van d, V, Dekhuijzen R. Antituberculosis drug- induced hepatotoxicity: concise up-to-date review. J Gastroenterol Hepatol 2008;23(2):192-202.

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7. Tostmann A, Boeree MJ, Harries AD, Sauvageot D, Banda HT, Zijlstra EE. Short communication: antituberculosis drug-induced hepatotoxicity is unexpectedly low in HIV-infected pulmonary tuberculosis patients in Malawi. Trop Med Int Health 2007;12(7):852-855. 8. Johnson JL, Okwera A, Nsubuga P et al. Efficacy of an unsupervised 8-month rifampicin-containing regimen for the treatment of pulmonary tuberculosis in HIV-infected adults. Uganda-Case Western Reserve University Research Collaboration. Int J Tuberc Lung Dis 2000;4(11):1032-1040. 9. Perriens JH, St Louis ME, Mukadi YB et al. Pulmonary tuberculosis in HIV-infected patients in Zaire. A controlled trial of treatment for either 6 or 12 months. N Engl J Med 1995;332(12):779-784. 10. Kwara A, Flanigan TP, Carter EJ. Highly active antiretroviral therapy (HAART) in adults with tuberculosis: current status. Int J Tuberc Lung Dis 2005;9(3):248-257. 11. Burman WJ, Gallicano K, Peloquin C. Comparative pharmacokinetics and pharmacodynamics of the rifamycinantibacterials. Clin Pharmacokinet 2001;40(5):327-341. 7 12. Ho CC, Chen YC, Hu FC, Yu CJ, Yang PC, Luh KT. Safety of fluoroquinolone use in patients with hepatotoxicity induced by anti-tuberculosis regimens. Clin Infect Dis 2009;48(11):1526-1533. 13. Bertino J, Jr., Fish D. The safety profile of the fluoroquinolones. ClinTher 2000; 22(7):798-817. 14. Marks DJ, Dheda K, Dawson R, Ainslie G, Miller RF. Adverse events to antituberculosis therapy: influence of HIV and antiretroviral drugs. Int J STD AIDS 2009;20(5):339-345. 15. Kovacs JA, Hiemenz JW, Macher AM et al. Pneumocystis carinii pneumonia: a comparison between patients with the acquired immunodeficiency syndrome and patients with other immunodeficiencies. Ann Intern Med 1984;100(5):663-671. 16. Baghaei P, Tabarsi P, Chitsaz E et al. Incidence, Clinical and Epidemiological Risk Factors, and Outcome of Drug-Induced Hepatitis Due to Antituberculous Agents in New Tuberculosis Cases. Am J Ther 2009 [epub ahead of print]. 17. Sun F, Chen Y, Xiang Y, Zhan S. Drug-metabolising enzyme polymorphisms and predisposition to anti- tuberculosis drug-induced liver injury: a meta-analysis. Int J Tuberc Lung Dis 2008;12(9):994-1002. 18. Matimba A, Del-Favero J, Van BC, Masimirembwa C. Novel variants of major drug-metabolising enzyme genes in diverse African populations and their predicted functional effects. Hum Genomics 2009;3(2):169-190. 19. Sabbagh A, Langaney A, Darlu P, Gerard N, Krishnamoorthy R, Poloni ES. Worldwide distribution of NAT2 diversity: implications for NAT2 evolutionary history. BMC Genet 2008;9:21. 20. Chelule PK, Pegoraro RJ, Gqaleni N, Dutton MF. The frequency of cytochrome P450 2E1 polymorphisms in Black South Africans. Dis Markers 2006;22(5-6):351-354. 21. Dandara C, Sayi J, Masimirembwa CM et al. Genetic polymorphism of cytochrome P450 1A1 (Cyp1A1) and glutathione transferases (M1, T1 and P1) among Africans. ClinChem Lab Med 2002;40(9):952-957. 22. Dean GL, Edwards SG, Ives NJ et al. Treatment of tuberculosis in HIV-infected persons in the era of highly active antiretroviral therapy. AIDS 2002;16(1):75-83. 23. Sanne I, Mommeja-Marin H, Hinkle J et al. Severe hepatotoxicity associated with nevirapine use in HIV- infected subjects. J Infect Dis 2005;191(6):825-829. 24. Nunez M. Hepatotoxicity of antiretrovirals: Incidence, mechanisms and management. J Hepatol 2005. 25. Hoffmann CJ, Charalambous S, Thio CL et al. Hepatotoxicity in an African antiretroviral therapy cohort: the effect of tuberculosis and hepatitis B. AIDS 2007;21(10):1301-1308. 26. Corbett EL, Watt CJ, Walker N et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med 2003;163(9):1009-1021.

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Sale of fluoroquinolones in northern Tanzania: a potential threat for fluoroquinolone use in 8a tuberculosis treatment

Jossy van den Boogaard, Hadija H. Semvua, Martin J. Boeree, Rob E. Aarnoutse and Gibson S. Kibiki

J Antimicrob Chemother 2010;65:145–147 chapter 8a

Abstract Objectives: Fluoroquinolones have a potential role in shortening tuberculosis (TB) treatment duration. They are currently used in the treatment of other infections. This has raised concerns about mycobacterial resistance development. The current study evaluates the sale of fluoroquinolones (among other antibacterials) in Moshi, Tanzania, one of the world’s countries with the highest burden of TB.

Methods: Trained pharmacy assistants registered the sale of fluoroquinolones during February and March 2009 to outpatients in Moshi in all 14 pharmacies that are authorized to sell antibacterials for systemic use. The sale of all antibacterials of the Anatomic Therapeutic Chemical (ATC) J01 class was expressed in Defined Daily Doses (DDD) per 1000 inhabitants per day (DID). The availability of fluoroquinolones in drug outlets that are not authorized to sell antibacterials for systemic use, was assessed in 15 randomly selected outlets in Moshi.

Results: The sale of antibacterials to outpatients in Moshi by authorized pharmacies was 8a 4.99 DID. The sale of fluoroquinolones was 0.63 DID (12% of total antibacterial sale). Ciprofloxacin was available in all 15 unauthorized drug outlets.

Conclusions: The substantial sale of the fluoroquinolones by authorized pharmacies and the wide availability of fluoroquinolones in unauthorized drug outlets in Moshi constitute a challenge to the use of fluoroquinolones in TB treatment in Tanzania. Control of antibacterial use in Tanzania requires the implementation of surveillance systems for antibacterial use and resistance, and adequate restriction of antibacterial sale by authorized pharmacies only.

138 Fluoroquinolone use

Introduction Fluoroquinolones are used to treat a variety of conditions, including genitourinary, respiratory tract and gastrointestinal infections, sexually transmitted diseases and a number of skin, bone and soft tissue infections [1]. In addition, the fluoroquinolones form a promising class of drugs in the treatment of tuberculosis (TB) [2]. Second and third generation fluoroquinolones are used in the treatment of multi-drug resistant (MDR) and extensively drug resistant (XDR) TB, and in case of intolerance to first-line TB drugs [3]. Moreover, moxifloxacin and gatifloxacin are being evaluated for their potential to shorten TB treatment duration, one of the major strategies for TB control [2]. The use of fluoroquinolones against various infections has raised concerns about the risk of mycobacterial resistance development in patients who are co-infected with actively replicating Mycobacterium tuberculosis.[1]Importantly, reduced mycobacterial susceptibility to one fluoroquinolone results in reduced susceptibility to all fluoroquinolones [4]. Whereas the European Surveillance of Antimicrobial Consumption (ESAC) project [5] provides data on fluoroquinolone consumption in Europe and the IMS Health has collected 8a similar data for the United States [6], to our knowledge no systematic data regarding fluoroquinolone consumption is available for sub-Saharan Africa. Evaluating the extent of fluoroquinolone use in this part of the world with the highest burden of TB [7] is highly relevant given the role of the fluoroquinolones in current and future TB treatment regimens. The aim of this study therefore was to systematically evaluate fluoroquinolone sale among other antibacterials by pharmacies in northern Tanzania.

Methods Data were collected from February 1st 2009 until March 31st 2009 (the dry, hot season) in Moshi, the capital of the Kilimanjaro Region in northern Tanzania. Drug sale in Moshi is covered by fourteen part I pharmacies, i.e. run by a registered pharmacist and allowed to sell both prescription-only and over-the-counter drugs, and 105 part II pharmacies, i.e. authorized to sell drugs for minor conditions, but no prescription-only drugs such as antibacterials for systemic use [8]. All fourteen part I pharmacies, serving a total population of 170500 (Moshi Urban Municipality, 2009), participated in data collection. Pharmacy assistants were trained to register the generic name, amount and strength per unit dose of each antibacterial sold to outpatients, on pre-designed forms that had been tested during a one-week pilot phase. Antibacterials were classified in accordance with the international Anatomic Therapeutic Chemical (ATC) system. Only antibacterials of the ATC J01 class (antibacterials for systemic use, excluding antifungals, antibacterials for tuberculosis and topical antibiotics) [9] were included. Data collection was closely monitored by the investigators who visited the pharmacies on a daily basis in the first week and at least once weekly throughout the remaining period of data collection, and who were fulltime available by phone in case of queries. Data were expressed in Defined Daily Dose (DDD) per 1000 inhabitants per day (DID) [9]. To calculate the DID per antibacterial, the total amount (in grams) of the antibacterials sold during the study period was calculated first. This amount was divided by the DDD conversion factor

139 chapter 8a

(ATC/DDD index 2009) [9]. The DID for each antibacterial was calculated by dividing the DDDs by 59 days (the study period) and dividing it by 170,5 (1000-inhabitants). To verify whether antibacterials for systemic use were indeed not available from part II pharmacies, fifteen randomly selected part II pharmacies (15% of all part II pharmacies) were visited by the investigators who requested a dose of ciprofloxacin, without referring to the study, but also without showing a prescription, in each pharmacy. The study was approved by the Institutional Review Board (IRB) of the Kilimanjaro Christian Medical Centre (KCMC) in Moshi.

Results In Table 1, the sale of antibacterials for systemic use (ATC J01 class) is summarized. The total sale of antibacterials to outpatients in Moshi was 4.99 DID. The penicillins accounted for the majority (2.18 DID; 44%) of antibacterials sold, followed by the quinolones (0.63 DID; 13%). A wide spectrum of fluoroquinolones was available, but ciprofloxacin was sold most frequently 8a (74% of fluoroquinolones). Contrary to drug sale regulations, ciprofloxacin was available without prescription in all fifteen part II pharmacies visited.

Discussion To our knowledge, this is the first study in sub-Saharan Africa in which the sale of antibacterials (in particular of the fluoroquinolones) was evaluated thoroughly. The total sale of antibacterials to outpatients by authorized drug outlets was 4.99 DID in Moshi. In comparison, outpatient antibacterial consumption in Europe ranged from 9.8 DID in the Netherlands to 31.4 DID in Greece [10], and was 24.92 DID in the Unites States [6]. The dominant class of antibacterials sold to outpatients in Moshi was the penicillins (44%). In Europe and the United States, the penicillins also represent the most frequently used antibacterials (46% and 39%, respectively) [6]. The total sale of quinolones to outpatients in Moshi by part I pharmacies was 0.63 DID (fluoroquinolones: 0.62 DID). Outpatient quinolone consumption ranged from 0.25 DID in Denmark to 3.10 DID in Portugal5 and was 2.47 DID in the United States [6]. Given the high burden of infectious diseases in Tanzania and the limited diagnostic facilities resulting in empirical use of antibacterials, higher DIDs had been expected from our study. However, we also revealed the unregulated availability of antibacterials in part II pharmacies. This finding suggests that the sale of antibacterials in northern Tanzania by part I pharmacies is an incomplete representation of the availability of antibacterials in reality, and the actual antibacterial consumption in Moshi is likely to be higher than found in the current study. Considering the high incidence of active TB infection in Tanzania [7], the widespread availability of the fluoroquinolones raises concerns about mycobacterial resistance development. Resistance to the fluoroquinolones reduces second-line treatment options for drug resistant TB and will limit future use of moxifloxacin and gatifloxacin in shorter, first-line treatment regimens.

140 Fluoroquinolone use

Table 1. Antibacterials sold in Moshi, presented in accordance with the ATC* classification (includingsubstance specification for fluoroquinolones) and expressed in Defined Daily Doses per 1000 inhabitants per day (DID)

ATC* code Corresponding antibacterial (sub)class or substance name DID# (% of total) J01C Penicillins 2.18 (44) J01CA Penicillins with extended spectrum 1.39 J01CE Beta-lactamase sensitive penicillins 0.14 J01CF Beta-lactamase resistant penicillins 0.29 J01CR Combinations of penicillins 0.36 J01M Quinolones 0.63 (13) J01MA Fluoroquinolones 0.62 J01MA02 Ciprofloxacin 0.46 J01MA06 Norfloxacin 0.04 J01MA07 Lomefloxacin 0.04 J01MA12 Levofloxacin 0.04 8a J01MA01 Ofloxacin 0.03 J01MA09 Sparfloxacin <0.01 J01MA03 Pefloxacin <0.01 J01MB Other quinolones 0.01 J01F Macrolides, lincosamides and streptogramins 0.61 (12) J01A Tetracyclines 0.57 (11) J01E Sulphonamides and trimethoprim 0.33 (7) J01X Other antibacterials 0.31 (6) J01XD Imidazole derivatives 0.30 J01XE Nitrofuran derivatives 0.01 J01D Cephalosporins 0.20 (4) J01B Amphenicols 0.11 (2) J01G Aminoglycoside antibacterials 0.05 (1) Total 4.99 (100)

Note: *ATC, Anatomic Therapeutic Chemical classification; #DID, Defined Daily Dose (DDD) per 1

This study has some limitations. It is unknown whether the antibacterials that were sold were actually used by patients. The data represent a single measurement period of two months in a limited area only, whereas antibacterial consumption generally shows seasonal and regional variation [5,10]. No differentiation was made between antibacterial sale with and without prescription. Alarmingly, antibacterials are available in either way according to pharmacy assistants (personal communication). In conclusion, the substantial and inadequately controlled sales of the fluoroquinolones in northern Tanzania constitute a challenge to the incorporation of the fluoroquinolones in TB

141 chapter 8a

treatment. Restricting their use to TB treatment only will pose a major practical challenge. To improve control on antibacterial use in Tanzania, the implementation of surveillance systems for antibacterial consumption and resistance is warranted, and the sale of antibacterials should be restricted to part I pharmacies only.

Acknowledgements The authors gratefully acknowledge the participation of the owners and assistants of the fourteen part I pharmacies in Moshi.

Funding This work was supported by the African Poverty Related Infection Oriented Research Initiative (APRIORI), a research network sponsored by the Netherlands-African partnership for capacity development and clinical interventions against poverty-related diseases (NACCAP) (WOTRO/ 8a NACCAP 07.05.203).

References

1. Oliphant CM, Green GM. Quinolones: a comprehensive review. Am Fam Physician 2002;65: 455-64. 2. Van den Boogaard J, Kibiki GS, Kisanga ER et al. New drugs against tuberculosis: problems, progress, and evaluation of agents in clinical development. Antimicrob Agents Chemother 2009;53:849-62. 3. World Health Organization. Guidelines for the programmatic management of drug-resistant tuberculosis. WHO/HTM/TB/2006.361. Geneva: WHO, 2006. 4. Ginsburg AS, Grosset JH, Bishai WR. Fluoroquinolones, tuberculosis, and resistance. Lancet Infect Dis; 2003;3:432-42. 5. Ferech M, Coenen S, Malhotra-Kumar S et al. European Surveillance of Antimicrobial Consumption (ESAC): outpatient quinolone use in Europe. J Antimicrob Chemother 2006;58:423-7. 6. Goossens H, Ferech M, Coenen S et al. Comparison of outpatient systemic antibacterial use in 2004 in the United States and 27 European countries. Clin Infect Dis 2007;44:1091-5. 7. World Health Organization. Global tuberculosis control 2009: epidemiology, strategy, financing. WHO/ HTM/TB/2009.411. Geneva: WHO, 2009. 8. WHO Collaborating Centre for Drug Statistics Methodology. The ATC/DDD system.http://www.whocc. no/atcddd (3 August 2009, date last accessed). 9. Strategies for Enhancing Access to Medicines Program. Access to essential medicines: Tanzania, 2001. Arlington, VA: Management Sciences for Health, 2003. 10. Ferech M, Coenen S, Malhotra-Kumar S et al. European Surveillance of AntimicrobialConsumption (ESAC): outpatient antibiotic use in Europe. J Antimicrob Chemother 2006;58:401-7.

142

Low rate of fluoroquinolone resistance in Mycobacterium tuberculosis isolates 8bfrom northern Tanzania

Jossy van den Boogaard, Hadija H. Semvua, Jakko van Ingen, Solomon Mwaigwisya, Tridia van der Laan, Dick van Soolingen, Gibson S. Kibiki, Martin J. Boeree, Rob E. Aarnoutse

Journal of Antimicrobial Chemotherapy 2011 Aug;66(8):1810-4 chapter 8b

Abstract Objectives: Fluoroquinolones are used in second-line treatment of tuberculosis (TB) and have a potential role in shortening TB treatment duration. The wide use of fluoroquinolones in the treatment of other infections including respiratory tract infections in patients with (undiagnosed) active TB, could result in fluoroquinolone resistant Mycobacterium tuberculosis (M. tuberculosis). We determined the rate of fluoroquinolone resistance in M. tuberculosis isolates obtained from Tanzanian patients and linked this to previous fluoroquinolone exposure and mycobacterial resistance to rifampicin and isoniazid.

Methods: A total of 291 M. tuberculosis isolates were obtained between April 2009 and June 2010 from patients with smear-positive pulmonary TB and tested for susceptibility to ciprofloxacin, moxifloxacin, rifampicin and isoniazid. Information on previous fluoroquinolone use was obtained by interviewing patients and checking their medical files.

Results: Only two (0.7%) of the 291 M. tuberculosis isolates were resistant to ciprofloxacin; 8b one of which was intermediately resistant to moxifloxacin as well. These two isolates were susceptible to rifampicin and isoniazid. Twenty-two (8%) of the 291 patients had a history of fluoroquinolone use (median: 7 days, interquartile range: 5-10). The patients from whom the fluoroquinolone resistant M. tuberculosis isolates were obtained had no known history of previous fluoroquinolone use.

Conclusions: Our findings indicate that the rate of fluoroquinolone resistant M. tuberculosis in Tanzanian patients with TB is low and not related to previous, brief episodes of exposure to fluoroquinolones. The findings favour future application of fluoroquinolones in TB treatment regimens of shorter duration.

146 Fluoroquinolone resistance

Introduction Fluoroquinolones, a class of broad spectrum antibacterial drugs, are pivotal second-line drugs in the treatment of multidrug-resistant (MDR) tuberculosis (TB) and they are used as substitute agents in patients who are intolerant to the first-line TB drugs [1,2]. Moreover, in vitro and in vivo experiments have shown that the newer fluoroquinolones moxifloxacin and gatifloxacin have the potential to shorten TB treatment duration; an important strategy in the fight against TB [3-6]. The agents are currently being tested in phase III clinical trials for their role in TB treatment regimens of shorter duration [7]. A potential problem of using fluoroquinolones in first-line TB treatment is that they are prescribed for treatment of a wide variety of bacterial infections, including empirical treatment of community-acquired pneumonia [8,9]. Exposure to fluoroquinolone monotherapy in patients with yet undiagnosed active TB can result in the emergence of fluoroquinolone resistant Mycobacterium tuberculosis (M. tuberculosis) and this could jeopardize the application of the fluoroquinolones in TB treatment regimens, particularly in regimens of shorter duration [10,11]. Fluoroquinolone resistance in M. tuberculosis has been observed 8b in a number of studies around the world and the resistance rates ranged from <1% to >35% [12-15]. In several studies, M. tuberculosis resistance to fluoroquinolones was associated with MDR TB (resistance to at least rifampicin and isoniazid) [16-20], probably as a consequence of adding a fluoroquinolone to a failing multidrug TB treatment regimen [11]. In a number of studies, the association between previous fluoroquinolone use for any kind of infection and the development of fluoroquinolone resistance in patients with active TB was assessed. While the association was confirmed in some studies [14,21], other studies yielded more ambiguous results with respect to the existence of such a correlation [16,19,22] or showed a risk increment of fluoroquinolone resistance development only after substantial (i.e. >10 days) exposure to these drugs [23]. The occurrence of fluoroquinolone resistance inM. tuberculosis and its possible association with previous fluoroquinolone exposure has not yet been studied in Tanzania, one of the 22 countries with the highest burden of TB [24]. In a previous study, we assessed the extent of fluoroquinolone use for all kinds of infections in outpatients from the Kilimanjaro Region in northern Tanzania. We confirmed that the fluoroquinolones are widely used in Tanzania; they account for 12% of the total sale of antibacterial drugs in authorized pharmacies and they are readily available in unauthorized drug outlets as well [25]. In the present study, we determined the occurrence of fluoroquinolone resistance in M. tuberculosis isolates from TB patients in the Kilimanjaro Region and we linked this to previous fluoroquinolone exposure and resistance to isoniazid and rifampicin.

Materials & methods Setting. The study was conducted in the Tanzanian Kilimanjaro Region where the annual TB case notification rate (new and retreatment cases) is around 178 per 100,000 population [25]. Forty-one percent of TB patients in the region are diagnosed with smear-positive pulmonary TB and 59% with smear-negative pulmonary or extrapulmonary TB. Thirty-one percent of TB

147 chapter 8b

patients are co-infected with HIV [26,27]. TB diagnosis is primarily based on clinical symptoms and Ziehl-Neelsen (ZN) smear microscopy for the detection of acid-fast bacilli (AFB), complemented by radiology in case of AFB-negative smears. At the time of the conduct of this study, no TB culture facilities were available for routine practice in the Kilimanjaro Region, and there was no second-line treatment for drug resistant TB [28].

Study population and procedures. We conducted a cross-sectional study between April 2009 and June 2010 in patients who were diagnosed with smear-positive pulmonary TB and had not yet started TB treatment. Smear-positivity was defined as at least one AFB-positive smear out of two sputum samples: one spot and one morning sample [28]. District TB and Leprosy Coordinators from all seven districts of the Kilimanjaro Region were responsible for enrolling patients from their district’s clinics. Because of logistical constraints (in particular, long travelling time from the most distant districts to the culture laboratory), convenience sampling was applied, meaning that as many patients as logistically possible were enrolled. Demographic and clinical data such as TB classification (new or retreatment case) and 8b HIV-status were collected from medical files. A new TB case was defined as a patient who had not received more than 1 month of TB treatment in the past. Retreatment cases included patients classified as treatment failures, returning after default and relapse cases. Trained clinicians interviewed patients on the use of antibacterial drugs with or without prescription in the previous 6 months (a longer period was expected to result in substantial recall bias) and explicitly asked about ciprofloxacin use (the fluoroquinolone that is most frequently used in Tanzania) [25]. Furthermore, medical files were checked for fluoroquinolone prescription in the previous 6 months. One spot sputum sample for culture and drug susceptibility testing to isoniazid, rifampicin, ciprofloxacin and moxifloxacin was collected from every patient. Ciprofloxacin was selected because this fluoroquinolone is sold most extensively in Tanzania [25], and moxifloxacin was chosen because this is one of the newer fluoroquinolones with the potential to shorten TB treatment duration [6]. The study protocol was approved by the Institutional Review Board of the Kilimanjaro Christian Medical Centre (Moshi, Tanzania) and the National Institute for Medical Research (Dar es Salaam, Tanzania). Written informed consent was obtained from all participants.

Laboratory analysis. After collection, sputum samples were refrigerated for a maximum of 1 week and transported to the TB culture laboratory of the Kilimanjaro Christian Medical Centre (KCMC; Moshi, Tanzania). After decontamination, sputum samples were incubated in an automated liquid culture system (BACTEC MGIT960, BD Biosciences, Erembodegem, Belgium). The content of positive MGIT tubes was inoculated on blood agar plates for 24 h to assess possible contamination, and ZN microscopy was done to confirm the presence of AFB. In case of AFB positivity and growth on the blood agar plates, MGIT tube sediments underwent renewed decontamination and incubation. In case of AFB positivity and no growth on the blood agar plates, concentrated MGIT tube sediments were subcultured on Löwenstein-Jensen (LJ) slopes. Growth on LJ slopes was assessed after 8 weeks of incubation and the isolates obtained in this way were stored in glycerol at -80°C until transport to the

148 Fluoroquinolone resistance

National Tuberculosis Reference Laboratory of the National Institute for Public Health and the Environment (RIVM) in The Netherlands. At the RIVM, the isolates were first identified as M. tuberculosis complex by spoligotyping [29]. Drug susceptibility testing was performed using BACTEC MGIT960; the drugs included in the panel and their critical concentrations were isoniazid 0.1 mg/L, rifampicin 1 mg/L, ciprofloxacin 1 mg/L and moxifloxacin 0.5 mg/L [30]. The process was controlled using EpiCenter software; standard MGIT960 conditions applied. Cultures were incubated an additional 3 days after full appearance of the growth control. If growth was detected in a fluoroquinolone- containing tube after the additional 3 days incubation but not before, the isolate was labelled ‘intermediately resistant [31]’. All fluoroquinolone-resistant isolates were subjected to gyrA gene mutation analysis using the GenoType MTBDRsl reverse line blot assay (Hain Lifescience, Nehren, Germany) [30]. The results were discussed with the regional TB coordinators and laboratory technicians, to provide them with feedback on the quality of ZN microscopy in the region. 8b Results Patient characteristics. A total of 379 sputum samples were collected. M. tuberculosis isolates were obtained from 291 of these 379 sputum samples. Thirty samples were culture-negative, 35 showed growth but no M. tuberculosis, and 23 were contaminated. The characteristics of the 291 patients with culture-confirmed TB are shown in Table 1. Twenty-two (8%) of the 291 patients had a history of fluoroquinolone use in the previous 6 months: 11 (50%) had used the drug for respiratory symptoms and the others for gastrointestinal problems. Seven patients had used fluoroquinolones in the past month and 15 patients between

Table 1. Patient characteristics of the total study population (n=291) and by drug resistance pattern

Patients Patients Patients Patients Patients with pan- with INH with RIF with INH + with FQ Total group susceptible resistant resistant RIF resistant resistant Characteristic (n=291) MTB (n=262) MTB (n=23) MTB (n=1) MTB (n=3) MTB (n=2) Age, median (IQR) 35 (27-44) 35 (27-45) 36 (27-42) 25 39 (34-50) 30 (20-40) Male sex, n (%) 208 (72) 183 (70) 20 (87) 1 (100) 3 (100) 1 (50) Retreatment cases, 29 (10) 27 (10) 0 (0) 0 (0) 1 (33) 0 (0) n (%)* HIV-positive, n (%) 63 (22) 55 (21) 6 (26) 0 (0) 1 (33) 1 (50) Positive history of 22 (8) 21 (8) 1 (4) 0 (0) 0 (0) 0 (0) FQ use, n (%)

* Of the 29 retreatment cases, two were defaulters who returned and 27 were classified as relapse cases. The two patients who returned after default both had pan-susceptible Mycobacterium tuberculosis isolates. MTB = Mycobacterium tuberculosis IQR = interquartile range INH = isoniazid RIF = rifampicin FQ = fluoroquinolone

149 chapter 8b

1 and 3 months ago. The median duration of fluoroquinolone use was 7 days (interquartile range (IQR): 5-10), the maximum duration was 10 days. None of the patients had used the fluoroquinolones more than once in the previous 6 months.

Drug susceptibility test results. Isoniazid resistance was found in 26 M. tuberculosis isolates (8.9%), three of which were resistant to rifampicin as well, giving an MDR rate of 1.0%. Mono- resistance to rifampicin occurred in one isolate (0.4%). Ciprofloxacin resistance was found in two isolates (0.7%): one was intermediately resistant to ciprofloxacin and susceptible to moxifloxacin, the other was resistant to ciprofloxacin and intermediately resistant to moxifloxacin. Both were susceptible to isoniazid and rifampicin. The isolate resistant to ciprofloxacin and intermediately resistant to moxifloxacin harboured an A90V mutation in the gyrA gene, but wild type sequences were detected simultaneously. In the isolate intermediately resistant to ciprofloxacin and susceptible to moxifloxacin no gyrA mutations were found. The characteristics of the patients from whom resistant M. tuberculosis isolates were 8b obtained are shown in Table 1. The two patients with ciprofloxacin resistant M. tuberculosis had no history of previous fluoroquinolone use and had not been treated for TB before. The patient from whom the M. tuberculosis isolate with intermediate resistance to ciprofloxacin was obtained, was a 40-year-old HIV-positive woman with a CD4 count of 3 cells/mm3. The other patient was a 20-year-old HIV-negative man with a CD4 count of 478 cells/mm3. There was no known link between these two patients.

Discussion Fluoroquinolone resistance in M. tuberculosis is uncommon in Tanzania, despite the widespread use of these antibacterial drugs in the treatment of bacterial infections. The proportion of M. tuberculosis isolates with fluoroquinolone resistance was less than 1% in our study population and there was no association with previous fluoroquinolone exposure in the two isolates with fluoroquinolone resistance. Both fluoroquinolone resistant isolates were susceptible to rifampicin and isoniazid. Fluoroquinolone resistance in M. tuberculosis is associated with mutations in the gyrA and, to a lesser extent, gyrB genes of the mycobacterial genome but these mutations are not always present in resistant isolates [11,32]. Cross-resistance between the different fluoroquinolones is common in M. Tuberculosis [33]. It is hypothesized that resistance to fluoroquinolones with strong bactericidal activity against M. tuberculosis (such as moxifloxacin) is generated through a stepwise process of additive mutations, whereas resistance to ciprofloxacin and ofloxacin requires a single mutation only [11]. This could explain that the M. tuberculosis isolate with intermediate resistance to ciprofloxacin in our study was susceptible to moxifloxacin (and did not have mutations in gyrA), and that the isolate with resistance to ciprofloxacin was intermediately resistant to moxifloxacin. We found a A90V gyrA mutation in the latter isolate but wild type sequences were also detected, suggesting a single strain with both wild type and mutant bacterial populations, as previously observed [30], or a mixed infection with two strains [30]. Considering that our aim was to determine the rate of fluoroquinolone resistance

150 Fluoroquinolone resistance in northern Tanzania and relate this to previous fluoroquinolone use, we decided to categorize the M. tuberculosis isolate with intermediate resistance to ciprofloxacin as fluoroquinolone resistant. However, the clinical relevance of intermediate fluoroquinolone resistance in M. tuberculosis remains controversial [31]. Low fluoroquinolone resistance rates were also found in two other studies from the African continent [12,14]. In Tunisia, 0.8% fluoroquinolone resistance was found in a hospital- based study but the association of fluoroquinolone resistance in M. tuberculosis with previous fluoroquinolone exposure was not investigated [12]. In Rwanda, the fluoroquinolone resistance rate was 0.6% in a nationwide study. The four patients with fluoroquinolone resistant M. tuberculosis in this study all had a history of previous fluoroquinolone use (three as part of a second-line TB treatment regimen) [14]. There are a few possible explanations for the low fluoroquinolone resistance rate and the absence of an obvious association with previous fluoroquinolone exposure in our study. As Devasia et al. (2009) showed, the risk of fluoroquinolone resistance in M. tuberculosis isolates increases particularly after fluoroquinolone exposure of more than 10 days [23]. In our patient 8b population, fluoroquinolone use was restricted to brief treatment episodes (5-10 days) only. Moreover, at the time this study was conducted, there was no second-line treatment for MDR TB available and thus there was no risk of long-term fluoroquinolone exposure as part of a failing second-line TB treatment regimen in M. tuberculosis. In most regions where high (i.e. >15%) fluoroquinolone resistance rates in M. tuberculosis were observed, its occurrence was associated with resistance to rifampicin and isoniazid [15,20,22,34,35]. In our study, the rates of isoniazid, rifampicin, and isoniazid plus rifampicin (MDR) resistance were low: 8.9%, 0.3% and 1.0%, respectively, and similar low rates of MDR TB were found in other studies from Tanzania. This study has some limitations. Since the prescription and sale of antibacterial drugs to patients in Tanzania is not automatically registered, it was difficult to obtain reliable information about previous fluoroquinolone use. Furthermore, it is unknown how long it takes for M. tuberculosis to develop fluoroquinolone resistance after fluoroquinolone exposure. Devasia et al. (2009) assessed fluoroquinolone exposure over a period of 12 months prior to TB diagnosis and found that exposure that had occurred more than 60 days before TB diagnosis was associated with the highest risk of fluoroquinolone resistance [23]. We assessed fluoroquinolone use over a period of 6 months (180 days) prior to TB diagnosis only, because we expected that fluoroquinolone use longer than 6 months ago would be difficult for patients to recall. In conclusion, our findings indicate that the occurrence of fluoroquinolone resistance in M. tuberculosis isolates obtained from Tanzanian patients is limited and not related to previous, brief episodes of fluoroquinolone exposure. This is an encouraging finding in light of the possible incorporation of the fluoroquinolones in first-line TB treatment regimens.

Acknowledgements The authors gratefully acknowledge the assistance of the Tuberculosis Reference Laboratory at the National Institute for Public Health and the Environment (RIVM, Bilthoven, The Netherlands).

151 chapter 8b

Funding This work was funded by the African Poverty Related Infection Oriented Research Initiative (APRIORI), a research network sponsored by the Netherlands-African partnership for capacity development and clinical interventions against poverty-related diseases (NACCAP) (WOTRO/ NACCAP 07.05.203).

References

1. Moadebi S, Harder CK, Fitzgerald MJ et al. Fluoroquinolones for the treatment of pulmonary tuberculosis. Drugs 2007;67:2077-99. 2. World Health Organization. Guidelines for the programmatic management of drug-resistant tuberculosis. WHO/HTM/TB/2006.361. Geneva: WHO, 2006. [Online] Available from: http://whqlibdoc. who.int/publications/2006/9241546956_eng.pdf (11 April 2011, date last accessed). 3. Alvirez-Freites EJ, Carter JL, Cynamon MH. In vitro and in vivo activities of gatifloxacin against 8b Mycobacterium tuberculosis. Antimicrob Agents Chemother 2002; 46: 1022-5. 4. Gillespie SH, Billington O. Activity of moxifloxacin against mycobacteria. J Antimicrob Chemother 1999; 44: 393-5. 5. Ji B, Lounis N, Maslo C et al. In vitro and in vivo activities of moxifloxacin and clinafloxacin against Mycobacterium tuberculosis. Antimicrob Agents Chemother 1998; 42: 2066-9. 6. Van den Boogaard J, Kibiki GS, Kisanga ER et al. New drugs against tuberculosis: problems, progress, and evaluation of agents in clinical development. Antimicrob Agents Chemother 2009; 53: 849-62. 7. ClinicalTrials.gov. Controlled Comparison of Two Moxifloxacin Containing Treatment Shortening Regimens in Pulmonary Tuberculosis (REMoxTB). University College, London.http://www.clinicaltrials. gov/ct2/show/NCT00864383?term=tuberculosis+moxifloxacin&rank=3 (4 March 2011, date last accessed). 8. Oliphant CM, Green GM. Quinolones: a comprehensive review. Am Fam Physician 2002; 65: 455-64. 9. Dooley KE, Golub J, Goes FS et al. Empiric treatment of community-acquired pneumonia with fluoroquinolones, and delays in the treatment of tuberculosis. Clin Infect Dis 2002; 34: 1607-12. 10. Bernardo J, Yew WW. How are we creating fluoroquinolone-resistant tuberculosis? Am J Respir Crit Care Med 2009; 180: 288-9. 11. Ginsburg AS, Grosset JH, Bishai WR. Fluoroquinolones, tuberculosis, and resistance. Lancet Infect Dis 2003; 3: 432-42. 12. Soudani A, Hadjfredj S, Zribi M et al. First report of molecular characterization of fluoroquinolone resistant Mycobacterium tuberculosis strains in a Tunisian hospital setting. Clin Microbiol Infect 2010; 16: 1454-1457. 13. Agrawal D, Udwadia ZF, Rodriguez C et al. Increasing incidence of fluoroquinolone-resistant Mycobacterium tuberculosis in Mumbai, India. Int J Tuberc Lung Dis 2009; 13: 79-83. 14. Umubyeyi AN, Rigouts L, Shamputa IC et al. Limited fluoroquinolone resistance among Mycobacterium tuberculosis isolates from Rwanda: results of a national survey. J Antimicrob Chemother 2007; 59: 1031-3. 15. Grimaldo ER, Tupasi TE, Rivera AB et al. Increased resistance to ciprofloxacin and ofloxacin in multidrug- resistant mycobacterium tuberculosis isolates from patients seen at a tertiary hospital in the Philippines. Int J Tuberc Lung Dis 2001; 5: 546-50. 16. Wang JY, Lee LN, Lai HC et al. Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure. J Antimicrob Chemother 2007; 59: 860-5.

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17. Riantawan P, Punnotok J, Chaisuksuwan R et al. Resistance of Mycobacterium tuberculosis to antituberculosis drugs in the Central Region of Thailand, 1996. Int J Tuberc Lung Dis 1998; 2: 616-20. 18. Bozeman L, Burman W, Metchock B et al. Fluoroquinolone susceptibility among Mycobacterium tuberculosis isolates from the United States and Canada. Clin Infect Dis 2005; 40: 386-91. 19. Park IN, Hong SB, Oh YM et al. Impact of short-term exposure to fluoroquinolones on ofloxacin resistance in HIV-negative patients with tuberculosis. Int J Tuberc Lung Dis 2007; 11: 319-24. 20. Huang TS, Kunin CM, Shin-Jung LS et al. Trends in fluoroquinolone resistance of Mycobacterium tuberculosis complex in a Taiwanese medical centre: 1995-2003. J Antimicrob Chemother 2005; 56: 1058-62. 21. Ginsburg AS, Hooper N, Parrish N et al. Fluoroquinolone resistance in patients with newly diagnosed tuberculosis. Clin Infect Dis 2003; 37: 1448-52. 22. Xu P, Li X, Zhao M et al. Prevalence of fluoroquinolone resistance among tuberculosis patients in Shanghai, China. Antimicrob Agents Chemother 2009; 53: 3170-2. 23. Devasia RA, Blackman A, Gebretsadik T et al. Fluoroquinolone resistance in Mycobacterium tuberculosis: the effect of duration and timing of fluoroquinolone exposure. Am J Respir Crit Care Med 2009; 180: 365-70. 8b 24. World Health Organization. WHO report 2010. Global tuberculosis control. WHO/HTM/TB/ 2010.7. Geneva: WHO, 2010. [Online] Available from: http://whqlibdoc.who.int/publications/2010/ 9789241564069_eng.pdf (11 April 2011, date last accessed). 25. Van den Boogaard J, Semvua HH, Boeree MJ et al. Sale of fluoroquinolones in northern Tanzania: a potential threat for fluoroquinolone use in tuberculosis treatment. J Antimicrob Chemother 2010; 65: 145-7. 26. Van den Boogaard J, Lyimo R, Irongo CF et al. Community vs. facility-based directly observed treatment for tuberculosis in Tanzania’s Kilimanjaro Region. Int J Tuberc Lung Dis 2009; 13: 1524-9. 27. Kibiki GS, Mulder B, Dolmans WM et al. M. tuberculosis genotypic diversity and drug susceptibility pattern in HIV-infected and non-HIV-infected patients in northern Tanzania. BMC Microbiol 2007; 7: 51. 28. Ministry of Health and Social Welfare. United Republic of Tanzania. Manual of the National Tuberculosis and Leprosy Programme in Tanzania. Fifth edition. Dar es Salaam, 2010. 29. Kamerbeek J, Schouls L, Kolk A et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997; 35: 907-14. 30. Van Ingen J, Simons S, De Zwaan R et al. Comparative study on genotypic and phenotypic second- line drug resistance testing of Mycobacterium tuberculosis complex isolates. J Clin Microbiol 2010; 48: 2749-53. 31. Springer B, Lucke K, Calligaris-Maibach R et al. Quantitative drug susceptibility testing of Mycobacterium tuberculosis by use of MGIT 960 and EpiCenter instrumentation. J Clin Microbiol 2009; 47: 1773-80. 32. Jacoby GA. Mechanisms of resistance to quinolones. Clin Infect Dis 2005; 41 Suppl 2: S120-6. 33. Von Groll A, Martin A, Jureen P et al. Fluoroquinolone resistance in Mycobacterium tuberculosis and mutations in gyrA and gyrB. Antimicrob Agents Chemother 2009; 53: 4498-500. 34. Dewan P, Sosnovskaja A, Thomsen V et al. High prevalence of drug-resistant tuberculosis, Republic of Lithuania, 2002. Int J Tuberc Lung Dis 2005; 9: 170-4. 35. Ramachandran R, Nalini S, Chandrasekar V et al. Surveillance of drug-resistant tuberculosis in the state of Gujarat, India. Int J Tuberc Lung Dis 2009; 13: 1154-60. 36. Chonde TM, Basra D, Mfinanga SG et al. National anti-tuberculosis drug resistance study in Tanzania. Int J Tuberc Lung Dis 2010; 14: 967-7.

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9 General discussion

General discussion

Introduction The overall objective of this thesis was to address pharmacological aspects in the treatment of tuberculosis patients in Tanzania. Pharmacokinetic and pharmacodynamic problems affecting tuberculosis therapy were therefore investigated. The combined treatment of TB and HIV, pharmacokinetics of TB drugs in plasma and saliva, the effect of type II diabetes mellitus on the pharmacokinetics of TB drugs, TB drug induced hepatotoxicity and fluoroquinolones use and resistance were described in the respective articles. All studies presented in this thesis were conducted in Kilimanjaro Region, Tanzania.

Pharmacokinetics and pharmacodynamics in TB treatment TB therapy must be given with multiple drugs for a prolonged period, making it necessary to monitor drug toxicity, drug interactions, and patients’ adherence. The currently used combination drug regimens produce cure rates that exceed 95% in carefully monitored clinical trials. When there is insufficient patient adherence during the multiple months of the treatment period, failure is common. In addition, the TB drugs exhibit inter-individual differences in 9 their activity and these differences are largely determined by their pharmacokinetic and pharmacodynamic properties. An understanding of the relationships among these properties is considered key for a good response and rational use of anti-tuberculosis drugs. The success of antimicrobial therapy is determined by complex interactions between an administered drug, a host, and an infecting agent. We advocate that combination antimicrobial regimens, dosages and dosing intervals of individual antimicrobial agents should be designed while considering the pharmacokinetic and pharmacodynamic properties of the individual drugs. This means that it should be realized that the dose of drugs results in drug concentrations in various biological matrices; subsequently, these concentrations result in desirable or undesirable (eg. concentration-dependent adverse effects) responses. Thus, the concentration achieved is an important intermediary link between the dose administered and the eventual response to the drug. Several pharmacokinetic and pharmacodynamic parameters have been defined to describe the antibacterial activity of various classes of antimicrobial agents [1,2]. Evolving data suggest that the total exposure to TB drugs or the ‘area under the concentration versus time curve (AUC0-24) is most relevant to the efficacy of the first-line TB drug [3-8]. Ideally, AUC0-24 is combined with the minimum inhibitory concentration (MIC) of the mycobacteria to yield

AUC0-24/MIC, a pharmcodynamic parameter or index that is supposed to best predict response.

Drug interactions between anti-TB and antiretroviral drugs There are four main concerns in combining TB treatment with antiretroviral drugs. One is when to start the drugs. Second, TB treatment involves taking many tablets, as do most ARV regimens. Higher pill burden often results in lower adherence. Third is the immune

157 chapter 9

reconstitution inflammatory syndrome (IRIS). There can be a transient worsening of clinical status of TB patients 2-3 weeks after initiation of ARVs. Fourth is the interaction with TB drugs (especially rifampicin) and antiretroviral drugs as well as their overlapping drug toxicities, which are described in this thesis book. The rifamycins (rifampicin, rifabutin and rifapentine) induce the hepatic cytochrome P450 and uridine diphosphate glucuronosyltransferase (UGT) enzymes and thus can potentially lower blood levels of other drugs that utilize this pathway of metabolism, including many ARVs [9]. Rifampicin is a potent enzyme inducer whereas rifabutin acts as a more moderate enzyme inducer. Other commonly used drugs to treat tuberculosis, such as isoniazid, ethambutol, and pyrazinamide, generally do not have significant interactions with antiretroviral drugs when they are co-administered with rifampicin. Isoniazid inhibits some metabolic enzymes, but the effects of this drug are outweighed by the (inductive) effects of rifampicin when the two drugs are combined. The reviews in chapter 2a and 2b describe details of drug interactions between first-line TB drugs and antiretrovirals used in resource-limited settings. In this discussion, the important findings of these two articles are highlighted. Furthermore, additional information for special groups like pregnant women, children and MDR-TB patients is given. 9 The nucleoside reverse transcriptase inhibitors (NRTIs) are not metabolized by the liver and therefore, their levels are not significantly impacted by medications that induce or inhibit hepatic cytochrome P450 enzymes. One exception occurs with concomitant use of zidovudine and rifampicin, since both zidovudine and rifampicin are cleared by glucuronidation. The interaction between zidovudine and rifampicin resulted in a 47% decrease in the AUC concentration of zidovudine [10]. Nevertheless, no dose adjustment was recommended [11]. For Non Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) efavirenz can be used with rifampicin. Rifampicin lowers nevirapine levels by 20 to 58% and most experts do not recommend using this combination [11] unless the patient is pregnant when efavirenz cannot be given. Although limited data exist with the combinations of rifampicin and etravirine or rifampicin and rilpivirine, the coadministration of these drugs should be avoided because of the potential significant decrease in plasma etravirine and rilpivirine concentrations. Standard doses of protease inhibitors cannot be given with rifampicin as > 90% decreases in trough concentrations of the protease inhibitors occur which makes them ineffective [9,12,13]. Most protease inhibitors are given with low-dose ritonavir (100-200mg per dose of the other protease inhibitor). However, low-dose ritonavir does not overcome the effects of rifampicin, as even after boosting the doses, low serum protease inhibitor concentrations were observed. Therefore, standard protease inhibitor regimens, whether boosted or not, cannot be given with rifampicin. In chapter 2b, the hypothesis is evaluated that a fixed dose combination of emtricitabine, tenofovir and efavirenz (AtriplaR) can be considered as an effective and safe antiretroviral drug in TB-HIV management for adult and children above 3 years of age. In pregnancy, treatment for HIV and TB is challenging as pregnancy alters the distribution and metabolism of a number of drugs, including antiretroviral drugs [14]. Efavirenz is contraindicated in pregnancy and in women of childbearing potential who are not on adequate contraception. The only NNRTI drug that can be administered with rifampicin in pregnancy is nevirapine. However, nevirapine is associated with an increased risk of hepatotoxicity in

158 General discussion women. In such women who are pregnant, HAART regimens will be difficult to administer during TB treatment in areas where rifabutin is not available. Therefore, if nevirapine is used with rifampicin, close clinical, hepatic and virologic monitoring should be performed [15-17]. Of note, the serum concentrations of protease-inhibitors are decreased during the latter stages of pregnancy [18,19]. There are no published data on drug-drug interactions between anti- tuberculosis and antiretroviral drugs among pregnant women. HIV-infected patients with multidrug-resistant tuberculosis, needs fast initiation of antiretroviral therapy to decrease the alarmingly high death rate among them. However, most of the drugs used to treat multidrug-resistant tuberculosis (the “second-line drugs”: e.g. kanamycin, amikacin, capreomycin fluoroquinolones, ethionamide, cycloserine, para-amino salicylate) were developed nearly 40 years ago. These second-line TB drugs are characterized by very diverse metabolic pathways. Unfortunately, there are only limited published studies of possible drug-drug interactions between second-line anti-tuberculosis drugs and antiretroviral drugs [20]. Studies of second line TB drugs are a major priority in this era.

Management of TB/HIV co-infected patients Due to recent advances in antiretroviral therapy, anti-TB therapy has become more complicated. 9 Many doctors recommend either delaying HIV treatment until the TB has been controlled, or even stopping or changing anti-HIV medication if a person develops TB whilst taking HIV drugs. In our study (Chapter 3) the antiretroviral regimen of once-daily efavirenz 600mg, tenofovir 300mg and emtricitabine 200mg (Atripla® Merck & Co. Inc. USA) was initiated at week 4 after starting TB treatment. The four weeks meant that if you have a low CD4 cell count, and start anti-HIV drugs immediately after starting your TB treatment, you may be at risk of developing what is called immune reconstitution syndrome. This is when your strengthening immune system is stimulated to attack TB again. In this study, IRIS was not observed. HIV infected patients achieve somewhat lower concentrations of the orally administered first line anti-TB drugs [21-23]. This was also observed in our study (chapter 3). The plasma concentrations of all TB drugs tended to be slightly lower during co-administration with efavirenz, tenofovir and emtricitabine (table 3, chapter 3). Considering the drug-drug interactions in TB-HIV co-infection, these are most likely to be caused by rifampicin [24]. Our study is the first to report on the interactions of efavirenz/ tenofovir/ emtricitabine with first line rifampicin-based TB treatment, however, no interactions were observed in the pharmacokinetic analysis. The pharmacokinetic parameters (AUC, Cmax, and Cmin) of efavirenz, tenofovir and emtricitabine were not significantly altered based on geometric mean ratios (table 2, chapter 3). Pharmacokinetic drug-drug interactions between rifampicin and antiretroviral drugs, may lead to loss of antiretroviral efficacy [25-27]. This was not the case in our study as by the end of eight weeks of TB treatment, 19 of 25 patients (76%) had negative sputum smears (confirmed by negative sputum cultures). In addition, their median (IQR) CD4 count increased from 119 (69-203) cells/mm3 to 238 (147-424) cells/mm3 (p<0.01; Wilcox Signed Rank). There are existing overlapping adverse effects profiles between antituberculosis and antiretroviral drugs [28]. In our study, the combination of first-line TB drugs with efavirenz, tenofovir and emtricitabine was

159 chapter 9

well tolerated. There was a 96% percent of patients who experienced adverse effects mostly grade 2 before the start of ART and the rate was 92% after the start of ART. Generally, there were no serious AEs reported and no modifications or discontinuations of treatment done.

Rifampicin modestly decreases the efavirenz exposure [29,30]. AUC0-24h, Cmax and C24h of efavirenz in our study were slightly higher, which is explained by the presence of genetic differences in most of African population [31-33]. Our findings suggest that co-administration of the standard first line TB treatment regimen with efavirenz, tenofovir, and emtricitabine does not alter pharmacokinetic parameters of either of these drugs to a clinically relevant extent, however future studies should look into genetic factors of the patients because the metabolism of efavirenz is extensively influenced by pharmacogenetic factors [33-35].

Pharmacokinetics of Tuberculosis drugs Therapeutic drug monitoring using pharmacokinetic parameters may be useful in tuberculosis management. However, pharmacokinetic data from sub-Saharan Africa are limited, especially data based on intensive pharmacokinetic sampling. In chapter 4, the pharmacokinetic parameters of isoniazid, rifampicin, pyrazinamide and ethambutol using intensive sampling during the full 9 dosing interval were described. It is well known that certain patients, such as those infected with HIV and thus prone to malabsorption, are at higher risk for low drug levels [36-38]. In our study, 35% of patients had Cmax of rifampicin below the reference range (8-24mg/L). It was noted that none of the determinants (age, body weight, sex, HIV, malnutrition, dose per kg body weight)

were significantly associated with the AUC0-24h or Cmax of isoniazid, rifampicin, pyrazinamide or ethambutol. However, numbers were small for determinant assessment. Although the pharmacodynamics of the first-line TB drugs is not completely established yet, suboptimal treatment responses have been associated with low rifampicin concentrations. This is a reason for considering higher doses of rifampicin in African tuberculosis patients. Apart from this, even higher doses of rifampicin may enable treatment shortening in patients from all races [39]

Saliva sampling as alternative for plasma sampling Ideally, the concentration of drug should be measured at the site of action of the drug; that is, at the receptor. However, owing to inaccessibility, drug concentrations are normally measured in whole blood from which serum or plasma is generated. Other body fluids such as saliva [40,41] and cerebrospinal fluid are sometimes used. It is assumed that drug concentrations in these fluids are in equilibrium with the drug concentration at the receptor. Saliva specimen has several advantages, such as easy and inexpensive sample collection and reduction of infection risk and discomfort especially in individuals infected with HIV, in children, and in compliance measurement. In addition, it should be noted that the measured drug concentrations in plasma or serum refer to the total drug concentration, i.e. a combination of bound and free drug that are in equilibrium with each other. Only the free or unbound fraction of the drug in plasma is active and precisely this fraction is available for diffusion to saliva. Therefore, another advantage of measuring drugs in saliva is that these salivary concentrations may reflect the active, free fraction of drug in plasma.

160 General discussion

We conducted a study (chapter 5) to explore the possibilities of using saliva instead of plasma for therapeutic drug monitoring and pharmacokinetic studies with rifampicin in TB patients. As expected, measurable rifampicin concentrations were not detected in all plasma and saliva samples, as rifampicin is having a short elimination half-life. Pharmacokinetic parameters of rifampicin both in plasma and saliva and their ratios are shown in (chapter 5). The table also shows the unbound rifampicin PK parameters that reflect how much rifampicin could diffuse to the saliva; however, the exposure to salivary rifampicin was significantly lower than the protein-unbound exposure in plasma. Furthermore, it was evident from the study that the salivary rifampicin was not able to predict total or protein-unbound plasma concentrations, due to inadequate precision associated with this prediction. This means, unfortunately, that salivary concentrations cannot be used as a substitute for plasma or serum concentrations in therapeutic drug monitoring or pharmacokinetic studies.

Diabetes mellitus and tuberculosis TB is a major public health problem in many low- and middle-income countries, where the number of people with diabetes is also rising rapidly. The growing prevalence of diabetes poses a challenge for TB control as uncontrolled diabetes leads to a greater risk of 9 developing TB [4]. Figure 1 shows percentages of TB cases attributable to diabetes mellitus. The link between tuberculosis and diabetes has been mentioned previously [43]: not only does diabetes contribute to a person’s risk of developing tuberculosis, but it also makes it more difficult to treat those who have both diseases. Lower plasma concentrations of anti-TB drugs have been associated with clinical failure and acquired drug resistance [44-47]. In an unmatched weight study in Indonesia performed during the continuation phase of TB treatment [48], the AUC0–6 h) of rifampicin was 53% lower in TB and DM patients compared with TB only patients. However, when the researchers matched the weights of the patients in a study performed during the intensive phase of treatment, no differences in drug exposure were found between the two groups for rifampicin, pyrazinamide and ethambutol [49]. Thus, TB patients with diabetes mellitus may need a higher dose of TB drugs in view of their higher body weight, but diabetes per se may not affect the pharmacokinetics of TB drugs, at least in Indonesian patients. In our study described in chapter 6, we screened Tanzanian tuberculosis patients with diabetes mellitus to find out the effect of diabetic disease on the concentrations of TB drugs in the intensive phase of TB treatment. We found that exposure to both isoniazid and rifampicin was reduced in diabetic patients with TB compared to TB patients without diabetic. Much effect was seen in isoniazid as Cmax, clearance and volume of distribution were also reduced. The diabetes disease itself most likely explains these effects as it may reduce the absorption of TB drugs. It is known that low serum levels can also be a consequence of malabsorption, inaccurate dosing, altered metabolism, or drug–drug interactions in TB and diabetic patients [50]. On efficacy and safety, one prospective study examined sputum cultures at the completion of 6 months of TB treatment, and the result was that positive cultures were found in 22.2% of patients with diabetes and 6.9% of those without diabetes [51]. A relative risk of death of 1.89 among TB patients with diabetes was observed in a systematic review of treatment outcomes

161 chapter 9

9 Figure 1. Percentage of TB cases attributable to diabetes mellitus. Source: http//www.idf.org/site map 4.3

when compared to non-diabetic patients [52]. Our study (chapter 6) did not measure sputum cultures for the patients, as we performed a purely pharmacokinetic study.

Hepatotoxicity in TB treatment Drug-induced hepatotoxicity has been a long-standing concern in the treatment of tuberculosis infection. The liver has a central role in drug metabolism and detoxification, and is consequently vulnerable to injury. TB drugs induced hepatotoxicity is a serious adverse effect [53,54]. Treatment should be interrupted and, generally, a modified or alternative regimen must be used for those with alanine aminotransferase (ALT) elevation more than three times the upper limit of normal (ULN) in the presence of hepatitis symptoms and/or jaundice, or five times the ULN in the absence of symptoms [55]. The observational study we conducted (chapter 7) showed a low rate (0.9%) of hepatotoxicity. This was the first study of this kind in Tanzanian hospitalized TB patients. Due to different definitions of hepatotoxicity and differences between studied populations, the incidence of anti-TB drug induced hepatotoxicity varies across studies. A study done in South Africa observed 0.5%, hepatotoxicity [56], Malawi reported 2% [57] while Uganda and Congo reported 1% and zero respectively [58,59]. Higher incidences to be reported were 13-15 percent from India and Iran [60]. Several genetic polymorphisms in drug metabolizing enzymes have been associated with anti-TB induced hepatotoxicity [61]. The three key anti-tuberculosis drugs, isoniazid, pyrazinamide and rifampicin, are all potentially hepatotoxic [62]. A meta-analysis has shown

162 General discussion an incidence rate of liver toxicity of 2.6% with isoniazid and rifampicin coadministration, but only 1.1% with rifampicin alone, and 1.6% with isoniazid alone [63]. The influence of acetylation rate on isoniazid hepatotoxicity has raised a lot of attention in past years. In fast acetylators, more than 90% of the drug is excreted as acetyl-isoniazid, whereas in slow acetylators, 67% of the drug is excreted as acetyl-isoniazid and a greater percentage of isoniazid is excreted as unchanged drug into the urine [64]. Early studies suggested that fast acetylators were at higher risk for hepatic injury because they generated more acetyl-isoniazid, which could be further metabolized to other toxic intermediaries [64-66]. Most of the follow-up phenotyping studies have shown that there is either a higher risk of hepatotoxicity for slow acetylators or that no such difference exists. The majority of genotyping studies, performed more recently, suggest that slow acetylators are at an increased risk for isoniazid-induced hepatotoxicity [67]. Slow and intermediate acetylators are highly prevalent in an Africa population [60], which would contradict the low rate of hepatotoxicity found in our study (chapter 7). To minimize the risk of hepatotoxicity, all patients should be thoroughly educated about the symptoms of hepatitis, and advised to report them promptly for early evaluation. Close clinical monitoring is essential. Contrarily, our study suggests that this may be not large problem. This gives more possibilities for increasing the dosages of the several TB drugs, especially rifampicin and pyrazinamide, to 9 increase the sterilizing effect and the prevention of the emergence of drug resistance.

Fluoroquinolone use Fluoroquinolones are fluorine-containing nalidixic acid derivatives characterized by broad- spectrum antimicrobial activity. They are currently used as second-line TB drugs, but the fluoroquinolone moxifloxacin is currently being evaluated as a first-line agent as well. Furthermore, the effectiveness and broad-spectrum activity of fluoroquinolones have made this antibacterial class one of the most widely used in other infections, both in human medicine and veterinary practice. This use of fluoroquinolones against various infections has raised concerns about the risk of mycobacterial resistance development in patients who are co-infected with actively replicating Mycobacterium tuberculosis [68]. This is why we have studied the quantification of the fluoroquinolone sale extent (Chapter 8a). It appeared that fluoroquinolones accounted for 12% of the total sale of antibacterials. It was also observed that fluoroquinolones were widely available in unauthorized drug outlets, which make the 12% result an underestimation since these outlets were not included in the evaluation. It is known that reduced mycobacterial susceptibility to one fluoroquinolone results in reduced susceptibility to all fluoroquinolones [68,69]. In addition, resistance to quinolones can evolve rapidly, even during a course of treatment and after as little as 13 days of fluoroquinolone therapy [70]. In Chapter 8b, the risk of development of resistance was evaluated. We assumed that the risk is higher in patients with previous exposure to fluoroquinolones (chapter 8a), but only 0.7% (2/291) of the isolates were resistant to ciprofloxacin, the most widely used fluoroquinolone as reflected in Chapter 8a. Although fluoroquinolone resistance in M. tuberculosis is not routinely assessed, the proportion of fluoroquinolone resistance among newly diagnosed (i.e., previously un-treated) patients with tuberculosis has ranged from 0.15 to 3.6% in previous reports [71-75]. However, the

163 chapter 9

risk factors for fluoroquinolone resistance in M. tuberculosis have not been fully elucidated. It is important to characterize the extent of fluoroquinolone resistance in M. tuberculosis, as well as the risk factors for resistance, so that this potent class of antimicrobial agents can be used rationally to treat both routine bacterial infections and tuberculosis.

Conclusion Tuberculosis remains an important public health problem in Tanzania and worldwide. Medications are the cornerstone of tuberculosis treatment. However, today’s TB drug regimen takes too long to cure, is too complicated to administer and can be toxic. Tuberculosis and HIV/ AIDS are fatally synergistic. In high-burden countries, people with HIV/AIDS are 20 times more likely to contract TB than those not suffering from HIV/AIDS. The deadly synergy of these two diseases demands first-line treatments that can be fully harmonized to avoid ARV interactions, and can be utilized to treat the growing number of people dually infected with TB and HIV.

Other immunosuppressive conditions and MDR-TB have changed the face of TB and posed challenges for its management. Despite the flaws with and growing resistance to current TB 9 treatments, no new TB drugs have been developed in nearly 50 years. In conclusion, improved tuberculosis management plan is urgently required. Strategies that improve the quality of drug use and efficacy are crucial to fight TB successfully

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41. Pichini S, Altieri I, Zuccaro P, Pacifici R. Drug monitoring in nonconventional biologic fluids and matrices. Clin Pharmacokinet 1996;30:211-28. 42. Goldhaber=Fiebert JD, Jeon CY, Cohen T, et al. Diabetis Mellitus and tuberculosis in countries with high tuberculosis burdens: individual risks and social determinants. Int J Epidemiol 2011;40(2):417-428. 43. Nijland, H. M. J., R. Ruslami, J. E. Stalenhoef, E. J. Nelwan, B. Alisjahbana, R. H. Nelwan, A. van der Ven, H. Danusantoso, R. E. Aarnoutse, and R. vanCrevel. Exposure to rifampicin is strongly reduced in tuberculosis patients with type 2 diabetes. Clin. Infect. Dis. 2006;43:848–854. 44. Sahai J, Gallicano K, Swick L, Tailor S, Garber G, Seguin I, et al. Reduced plasma concentrations of antituberculosis drugs in patients with HIV infection. Ann Intern Med 1997 Aug 15;127(4):289-93. 45. Magis-Escurra C, van den BJ, Ijdema D, Boeree M, Aarnoutse R. Therapeutic drug monitoring in the treatment of tuberculosis patients. Pulm Pharmacol Ther 2012 Feb;25(1):83-6. 46. Weiner M, Benator D, Burman W, Peloquin CA, Khan A, Vernon A, et al. Association between acquired rifamycin resistance and the pharmacokinetics of rifabutin and isoniazid among patients with HIV and tuberculosis. Clin Infect Dis 2005 May 15;40(10):1481-91. 47. Kimerling ME, Phillips P, Patterson P, Hall M, Robinson CA, Dunlap NE. Low serum antimycobacterial drug levels in non-HIV-infected tuberculosis patients. Chest 1998 May;113(5):1178-83. 48. Nijland HM, Ruslami R, Stalenhoef JE, Nelwan EJ, Alisjahbana B, Nelwan RH, et al. Exposure to rifampicin is strongly reduced in patients with tuberculosis and type 2 diabetes. Clin Infect Dis 2006 9 Oct 1;43(7):848-54. 49. Ruslami R, Nijland HM, Adhiarta IG, Kariadi SH, Alisjahbana B, Aarnoutse RE, et al. Pharmacokinetics of antituberculosis drugs in pulmonary tuberculosis patients with type 2 diabetes. Antimicrob Agents Chemother 2010 Mar;54(3):1068-74. 50. Root HF. The association of diabetes and tuberculosis. N Eng J Med 1934;210:1–13. 51. Alisjahbana B, Sahiratmadja E, Nelwan EJ, Purwa AM, Ahmad Y, et al. The effect of type 2 diabetes mellitus on the presentation and treatment response of pulmonary tuberculosis. Clin Infect Dis 2007;45:428–435. 52. Baker MA, Harries AD, Jeon CY, Hart JE, Kapur A, et al. The impact of diabetes on tuberculosis treatment outcomes: a systematic review. BMC Med 2011;9:81. 53. Saukkonen JJ, Cohn DL, Jasmer RM et al. An Official ATS Statement: Hepatotoxicity of Antituberculosis Therapy. Am J Respir Crit Care Med 2006;174(8):935-952. 54. Tostmann A, Boeree MJ, Aarnoutse RE, de Lange WC, van d, V, Dekhuijzen R. Antituberculosis drug- induced hepatotoxicity: concise up-to-date review. J Gastroenterol Hepatol 2008;23(2):192-202. 55. Chitturi S, Farrell G. Drug-induced liver disease. In: Schiff ER, SorrellMF, Maddrey WC, editors. Schiff’s diseases of the liver, 9th ed. Philadelphia: Lippincott, Williams & Wilkins; 2002. pp. 1059–1128. 56. Marks DJ, Dheda K, Dawson R, Ainslie G, Miller RF. Adverse events to antituberculosis therapy: influence of HIV and antiretroviral drugs. Int J STD AIDS 2009;20(5):339-345. 57. Tostmann A, Boeree MJ, Harries AD, Sauvageot D, Banda HT, Zijlstra EE. Short communication: antituberculosis drug-induced hepatotoxicity is unexpectedly low in HIV-infected pulmonary tuberculosis patients in Malawi. Trop Med Int Health 2007;12(7):852-855. 58. Johnson JL, Okwera A, Nsubuga P et al. Efficacy of an unsupervised 8-month rifampicin-containing regimen for the treatment of pulmonary tuberculosis in HIV-infected adults. Uganda-Case Western Reserve University Research Collaboration. Int J Tuberc Lung Dis 2000;4(11):1032-1040.

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59. Perriens JH, St Louis ME, Mukadi YB et al. Pulmonary tuberculosis in HIV-infected patients in Zaire. A controlled trial of treatment for either 6 or 12 months. N Engl J Med 1995;332(12):779-784. 60. Baghaei P, Tabarsi P, Chitsaz E et al. Incidence, Clinical and Epidemiological Risk Factors, and Outcome of Drug-Induced Hepatitis Due to Antituberculous Agents in New Tuberculosis Cases. Am J Ther 2009 [epub ahead of print]. 61. Sun F, Chen Y, Xiang Y, Zhan S. Drug-metabolising enzyme polymorphisms and predisposition to anti- tuberculosis drug-induced liver injury: a meta-analysis. Int J Tuberc Lung Dis 2008;12(9):994-1002. 62. World Health Organization. Treatment of tuberculosis: Guidelines for National Programmes. Third Edition WHO/CDS/TB/2003.313, Geneva, 2003. 63. Steele MA, Burk RF, DesPrez RM. Toxic hepatitis withisoniazid and rifampin. A meta-analysis. Chest 1991;99:65- 71. 64. Huang YS, Chern HD, Su WJ, Wu JC, Lai SL, Yang SY, Chang FY, Lee SD. Polymorphism of the N-acetyltransferase 2 gene as a susceptibility risk factor for antituberculosis drug-induced hepatitis. Hepatology 2002;35:883–889. 65. Mitchell JR, Thorgeirsson UP, Black M, Timbrell JA, Snodgrass WR, Potter WZ, Jollow HR, Keiser HR. Increased incidence of isoniazidhepatitis in rapid acetylators: possible relation to hydrazine metabolites. Clin Pharmacol Ther 1975;18:70–79. 9 66. Yamamoto T, Suou T, Hirayama C. Elevated serum aminotransferaseinduced by isoniazid in relation to isoniazid acetylator phenotype. Hepatology 1986;6:295–298. 67. Aarnoutse R. Chapter 18. Pharmacogenetics of anti-tuberculosis drugs. In: Antituberculosis Chemotherapy. Progress in Respiratory Research, volume 40. Karger, 2011, Editors P. Donald, P. van Helden). 68. Ginsburg AS, Grosset JH, Bishai WR. Fluoroquinolones, tuberculosis, and resistance. Lancet Infect Dis 2003;3:432-42. 69. Mitchison DA. Role of individual drugs in the chemotherapy of tuberculosis. Int J Tuberc Lung Dis 2002;4:796–806. 70. Ginsburg AS, Woolwine SC, Hooper N, Benjamin WH Jr, Bishai WR, Dorman SE, Sterling TR. The rapid development of fluoroquinolone resistance in M. Tuberculosis. N Engl J Med 2003;349:1977–1978. 71. Ginsburg A, Hoope N, Parrish N, Dooley K, Dorman S, Booth J, Diener-West M, Merz W, Bishai W, Sterling T. Fluoroquinolone resistance in patients with newly diagnosed tuberculosis. Clin Infect Dis 2003;37:1448–1452. 72. Bozeman L, Burman W, Metchok B, Welch L, Weiner M, Tuberculosis Trials Consortium. Fluoroquinolone susceptibility among mycobacterium tuberculosis isolates from the United States and Canada. Clin Infect Dis 2002;40:386–391. 73. Umubyeyi A, Rigouts L, Shamputa I. Limited fluoroquinolone resistance among mycobacterium tuberculosis isolates from Rwanda: results of a national survey. J Antimicrob Chemother 2007;59: 1031–103. 74. Park I, Hong S, Oh Y, Lim C, Lee S, Lew W. Impact of short-term exposure to fluoroquinolones on ofloxacin resistance in HIV-negative patients with tuberculosis. Int J Tuberc Lung Dis 2007;11:319–32. 75. Devasia R, Griffin M, Blackman A, May S, Holt E, Smith T, Warkentin J, Mitchel E. Trends in fluoroquinolone resistance and exposure in newly diagnosed tuberculosis: 2002–2006 [abstract]. Am J Respir Crit Care Med 2008;177:A19.

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10a Summary

Summary

TB remains a major global health problem, although efficacious treatment has been available for decades now. It is ranked as the second most mortality-causing infectious disease after human immunodeficiency virus (HIV) disease worldwide. Worldwide data show 9 million new cases and 1.4 million deaths annually. TB and HIV are overlapping epidemics. Both have been declared global emergencies demanding global attention, as HIV is the strongest risk factor for the development of TB. Apart from HIV, diabetic mellitus is another disease that also evolves as epidemic and overlaps with TB, especially on the treatment responses. Clinical pharmacological research has always been translational in the sense that the discipline aims to translate new scientific data on drugs into rational patient care. Research in clinical pharmacology therefore includes studies on drug interactions, drug toxicity, pharmacokinetic data and drug utilization. Since TB treatment involves fixed dose combinations, an understanding of their pharmacokinetic properties and the interactions during co- medication for co-infection cases is key for a rational use of antituberculosis drugs. Therefore, various types of clinical pharmacological studies are incorporated in this thesis. Most of the studies presented in this thesis were conducted in the Kilimanjaro Region of Tanzania. Tanzania is ranked 18 among 22 countries with the highest burden of TB worldwide. In Tanzania, TB cases are about 65,000 per annum. Thirty-seven percent of TB patients in Tanzania 10a are co-infected with HIV, accounting for 60-70% of the increase in the number of TB patients.

In chapter 1 of this thesis, the introduction of TB disease, its transmission and major symptoms are described. The introduction also explores the magnitude of TB disease globally, how TB/ HIV influences each other and detail of tuberculosis treatment. This part of the thesis ends by addressing the pharmacological problems in the treatment of tuberculosis patients. The emphasis is on the pharmacokinetic and pharmacodynamics problems affecting tuberculosis therapy, combined treatment of TB and HIV, PK of TB drugs, PK sampling for rifampicin, how type II diabetic mellitus affects pharmacokinetics of TB drugs, issues of hepatotoxicity and fluoroquinolones use in TB treatment. In chapter 2a, the problem of concurrent treatment of HIV infection and tuberculosis is explored from a comprehensive review of the pharmacological interactions between rifampicin (a cornerstone in the TB treatment) and antiretroviral drugs. The emphasis was given to the antiretrovirals used in the resource-limited setting which include nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors. However, new drugs in HIV treatment were also mentioned and the unavailability of second line regimen in TB/HIV co-infection treatment was highlighted. This part of the thesis ended by giving challenges and research priorities as far as resource-limited settings are concerned. This can be done by maximizing the use of already existing programmes and research data, creating new data by conducting clinical trials and prospective observational studies and by engaging a lobby to make currently unavailable drugs available to those most in need. In chapter 2b, a hypothesis that AtriplaR is a focus drug during combined treatment in TB/ HIV co-infected patients was presented. Comprehensive searches in Pubmed with the key words enabled us to find relevant information on the three drugs (emtricitabine, tenofovir and efavirenz) which are in the fixed dose of AtriplaR. Their interactions with first-line TB drugs

173 chapter 10a

were then tested by reading literature as far as safety and efficacy was concerned. This part concluded that AtriplaR can be considered an effective and safe antiretroviral drug in TB/HIV management for adult and children above 3 years of age. In chapter 3, the effect of rifampicin based tuberculosis treatment on the pharmacokinetics of efavirenz/tenofovir/emtricitabine was evaluated in Tanzanian TB/HIV co-infected patients. Approximately one-third of the 40 million people living with HIV/AIDS worldwide are co- infected with TB. The most powerful component of the first-line TB treatment is rifampicin which is also a strong inducer of the cytochrome P450 enzymes. We conducted a phase II clinical trial to evaluate its effect on the efavirenz/tenofovir/emtricitabine pharmacokinetic parameters. An inducing effect of rifampicin was not found in this study as the area under the

curve AUC0-24h (exposure) of efavirenz, tenofovir and emtricitabine were slightly higher when these drugs were co-administered with TB drugs. On TB drugs the concentrations were slightly low, however equivalence was suggested based on geometric mean ratios. Both TB and HIV drugs were well tolerated as adverse effects observed were mild and no serious adverse event was reported. In chapter 4, an observational study to describe the pharmacokinetic parameters of first- 10a line tuberculosis drugs in Northern Tanzania was conducted. In this study, the geometric mean AUC0-24 h values of isoniazid, rifampicin, pyrazinamide and ethambutol were 11(3.7-22.7), 39.9 (27.4-

68.3), 344 (209-610) and 20.2(13.4-32.0)h*mg/L respectively. Geometric mean Cmax of isoniazid

was 2.8 mg/L, rifampicin 8.9 mg/L, pyrazinamide 38.2 mg/L and ethambutol 3.3 mg/L. Cmax was below the reference range for both rifampicin and isoniazid in 7 and 10 patients respectively.

More than 30% (7/20) of the patients had a low rifampicin Cmax, which is a concern because low rifampicin levels may be associated with worse treatment outcome. A clinical trial with a higher sample size and which can assess concentration effect relationships is recommended. In chapter 5, comparison of rifampicin pharmacokinetic in plasma and saliva was performed. The aim was to assess whether saliva could be an alternative matrix for PK studies and TDM with

rifampicin. It was observed that geometric mean AUC0-24h of rifampicin in saliva (3.1 h*mg/L) was

slightly but significantly lower than the protein-unbound AUC0-24h in plasma (5.3 h*mg/L) and

these were much lower than the plasma AUC0-24h based on total concentrations (32.7 h*mg/L). On performance prediction, the value obtained showed that salivary rifampicin is not able to predict total or protein-unbound plasma concentrations. The study concluded that there is no possibility of using saliva instead of plasma for therapeutic drug monitoring and pharmacokinetic studies with rifampicin. In chapter 6, the effect of type 2 diabetes mellitus on the pharmacokinetics of TB drugs was evaluated. Diabetes compromises the response to TB treatment and we conducted a pharmacokinetic study to evaluate its effect in the plasma concentrations of the first-line TB drugs. Forty Tanzanian adult TB patients (20 TB only, 20 TB and Diabetes) who were in the intensive phase of TB treatment for at least two weeks were recruited. Blood sampling took place at pre dose, 1, 2, 3, 4, 6, 8, 10, 24 hours after observed drug intake. Exposure to isoniazid

and rifampicin was reduced in this study. Cmax, clearance and volume of distribution of isoniazid were also adversely affected. The study concluded that these effects are most likely explained by the diabetes disease. The recommendation was given to increase the doses of these drugs in treating TB-DM patients as it results in improved exposure.

174 Summary

Chapter 7 of this thesis is focussing on antituberculosis drug-induced hepatotoxicity. There is a high prevalence of risk factors for developing liver toxicity in this era of HIV and hepatitis B diseases. However, our study showed that the proportion of patients that developed liver toxicity (1/112) during the intensive phase of TB treatment was low when compared to other studies done in Malawi and Iran. The study ended by saying that liver toxicity is uncommon in the Northern Tanzania region. However, it remains important that TB patients receive clear instructions on possible signs and symptoms of hepatotoxicity and they should be instructed to visit their doctor in case such symptoms occur. In chapter 8a, the issue of fluoroquinolone use was addressed. The survey, which was conducted, revealed that fluoroquinolones were used extensively in the registered pharmacies and accounted for 12% of the total antibiotic sales. This gave an alarming note as some of the fluoroquinolones (e.g. moxifloxacin) are in the second line TB regimen and are being explored as first-line TB drugs. In chapter 8b, the occurrence of fluoroquinolone resistance was assessed. This chapter was aiming to find out whether the resistance is related to previous use of fluoroquinolones (chapter 8a). The result was not as expected since we found very low (<1%) fluoroquinolone resistance. This finding is encouraging; hence, fluoroquinolone-containing TB regimens can be 10a developed to shorten TB treatment duration. In conclusion, improved tuberculosis management is urgently required. Strategies that improve the quality of drug use and efficacy are crucial to fight TB successfully. Therefore, in chapter 9, all the findings reported in the chapters 2-8 are discussed. Pharmacokinetic and pharmacodynamic problems affecting tuberculosis therapy were therefore addressed in perspective. Furthermore, priorities for future research are explored.

175

10b Samenvatting

Samenvatting

Tuberculose blijft wereldwijd een groot gezondheidsprobleem, terwijl effectieve behandeling al tientallen jaren beschikbaar is. Het wordt wereldwijd gezien als de infectieziekte met de op één-na-hoogste mortaliteit na het humaan immunodeficiëntie virus (hiv). Data laten zien dat er wereldwijd jaarlijks negen miljoen nieuwe gevallen zijn en 1.4 miljoen gevallen overlijden. Tuberculose en hiv zijn overlappende epidemieën. Beiden zijn wereldwijd verklaard als ziekten waarvoor wereldwijde aandacht nodig is omdat hiv de grootste risicofactor is voor het ontwikkelen van tuberculose. Naast hiv ontwikkelt ook diabetes mellitus zich als een epidemie welkeoverlapt met tuberculose, vooral waar het gaat om behandelingsuitkomsten. Klinisch farmacologisch onderzoek is altijd translationeel geweest omdat deze discipline als doel heeft nieuwe wetenschappelijke data te vertalen in rationale patiëntenzorg. Daarom omvat onderzoek in klinisch farmacologische studies onderzoek naar medicijn-interacties, medicijntoxiciteit, farmacokinetische data en medicijngebruik. Omdat de behandeling van tuberculose een fixed-dose combinatie betreft, is het begrip van farmacokinetische eigenschappen en interacties tijdens comedicatie voor co-infectie de sleutel voor rationeel gebruik van antituberculose medicatie. Daarom zijn verschillende typen onderzoek opgenomen in dit proefschrift. De meeste studies in dit proefschrift zijn uitgevoerd in de regio Kilimanjaro in Tanzania. 10b Tanzania is wereldwijd het achttiende land van de22 landen met de hoogste druk van tuberculose. Er zijn ongeveer 65.000 tuberculose-gevallen in Tanzania. Zevenendertig procent van de tuberculose-gevallen in Tanzania zijn co-geïnfecteerd met hiv, wat ongeveer 60-70% is van de toename in het aantal gevallen van tuberculose.

In hoofdstuk 1 van dit proefschrift worden tuberculose, de transmissie ervan en de voornaamste symptomen geïntroduceerd. De introductie exploreert ook het probleem van tuberculose wereldwijd, hoe tuberculose en hiv elkaar beïnvloeden en de details van tuberculose- behandeling. Dit deel van het proefschrift eindigt met het formuleren van de farmacologische problemen in de behandeling van tuberculose patiënten. De nadruk ligt op de farmacokinetische en farmacodynamische problemen die tuberculosebehandeling beïnvloeden, de combinatie therapie voor tuberculose en hiv, farmacokinetiek (PK) van TB drugs, PK monsterafname van rifampicine, hoe diabetes mellitus II de farmacokinetiek van tuberculose medicatie beïnvloedt, vragen over hepatotoxiciteit en fluoroquinolonen gebruik in de behandeling van tuberculose. In hoofdstuk 2a worden de problemen van gelijktijdige behandeling van hiv infectie en tuberculose geëxploreerd in een uitgebreide review over de farmacologische interacties tussen rifampicin (de hoeksteen van tuberculose behandeling) en antiretrovirale medicatie. De nadruk lag vooral op antiretrovirale medicatie die in ontwikkelingslanden worden gebruikt zoals nucleoside reverse transcriptase remmers, non-nucleoside reverse transcriptase remmers en proteaseremmers. Echter, nieuwe middelen in hiv behandeling worden ook genoemd en de tweedelijns medicatie voor tuberculose/hiv infectie behandeling die niet beschikbaar is wordt belicht. Dit deel van het proefschrift eindigt met het beschrijven van uitdagingen en onderzoeksprioriteiten voor ontwikkelingslanden. Dit kan gedaan worden door maximaal gebruik van bestaande programma’s en onderzoeksdata, door nieuwe data te creëren door klinische trials en prospectief observationeel onderzoek uit te voeren en door te lobbyen voor

179 chapter 10b

het beschikbaar maken van medicatie die momenteel nog niet beschikbaar is voor hen die dat het meeste nodig hebben. In hoofdstuk 2b wordt de hypothese gepresenteerd dat AtriplaR de focus medicatie is voor de combinatie-behandeling voor tuberculose-hiv co-geïnfecteerde patiënten. Een uitgebreide zoekactie in pubmed en google met sleutelwoorden gaf ons de mogelijkheid om relevante informatie te vinden over de drie medicijnen (emtricitabine, tenofovir en efavirenz) die de fixed-dose combinatie vormen van AtriplaR. Deinteractie met eerstelijns tuberculose medicatie werd getest door het bestuderenvan literatuur waar het ging om veiligheid en effectiviteit. Dit onderdeel concludeerde dat AtriplaR overwogen kan worden als een effectieve en veilige medicatie in de behandeling van tuberculose en hiv voor volwassenen en kinderen ouder dan 3 jaar. In hoofdstuk 3 werd het effect van een op rifampicine gebaseerde behandeling van tuberculose op de farmacokinetiek van efavirenz/tenofovir/emtricitabine geëvalueerd in Tanzaniaanse tuberculose-hiv co-geïnfecteerde patiënten. Ongeveer een-derde van de 40 miljoen mensen die wereldwijd met hiv/aids leven is co-geïnfecteerd met tuberculose. De krachtigste component van eerstelijns tuberculosebehandeling is rifampicine welke ook een 10b sterke inductie heeft op cytochroom P450 enzymen. We voerden een fase-II trial uit om het effect te evalueren op de farmacokinetische parameters van efavirenz/tenofovir/emtricitabine. Er werd geen inducerend effect van rifampicine gevonden want het gebied onder de curve

AUC0-24h(blootstelling) van efavirenz, tenofovir en emtricitabine was maar iets hoger als deze medicatie tegelijkertijd met tuberculose-medicatie werd gegeven. De concentraties op tuberculose medicatie waren iets lager, maar equivalentie werd gesuggereerd op basis van de geometrische gemiddelden ratio’s. Zowel tuberculose en hiv medicatie werden goed getolereerd want bijwerkingen die werden gezien waren mild en er waren geen serieuze bijwerkingen. In hoofdstuk 4 wordt een observationeel onderzoek beschreven naar de farmacokinetische parameters van eerstelijns tuberculose medicatie in Noord Tanzania. In dit onderzoek waren

de geometrisch gemiddelden van AUC0-24 h voor isoniazide, rifampicine, pyrazinamide en ethambutol 11(3.7-22.7), 39.9 (27.4-68.3), 344 (209-610) en 20.2(13.4-32.0)h*mg/L respectievelijk.

Meer dan 30% (7/20) van de patienten had een lage rifampicine Cmax, wat zorgelijk is, omdat lage rifampicine waarden geassocieerd kunnen zijn met een slechte behandeluitkomst. Een klinische trial met een grotere steekproef, welke concentratie-effect-relaties kan aantonen, wordt aanbevolen. In hoofdstuk 5 wordt een vergelijking van farmacokinetiek in plasma en speeksel uitgevoerd. Het doel was om na te gaan of speeksel als alternatief kan dienen voor PK onderzoek en therapeutic drug monitoring (TDM=therapeutische medicatie monitoring) met rifampicine. Het

geobserveerde geometrisch gemiddelde van AUC0-24h van rifampicine in speeksel (3.1 h*mg/L)

was iets, maar significant, lager dan de eiwit-ongebonden AUC0-24h in plasma (5.3 h*mg/L) en

was veel lager dan de plasma AUC0-24h gebaseerd op totale concentraties (32.7 h*mg/L). Op voorspellend niveau liet de verkregen waarde zien dat speeksel rifampicine niet in staat is om totale of eiwit-ongebonden plasma concentratie te voorspellen. Het onderzoek concludeerde dat er geen mogelijkheid is om speeksel te gebuiken in plaats van plasma voor TDM en voor studies met rifampicine.

180 Samenvatting

In hoofdstuk 6 werd het effect van diabetes mellitus type 2 op de farmacokinetiek van tuberculose medicatie geëvalueerd. Diabetes compromitteert de respons op tuberculose- behandeling en we hebben een klinische trial uitgevoerd om het effect te evalueren op de plasmaconcentraties van eerstelijns tuberculose medicatie. Veertig volwassen Tanzaniaanse tuberculose patiënten (20 patiënten met alleen tuberculose, 20 patiënten met zowel tuberculose en diabetes) die minstens twee weken in de intensieve fase van tuberculosebehandeling zaten werden gerekruteerd. Bloed werd afgenomen op pre-dosis, 1, 2, 3, 4, 6, 8, 10, 24 uur na waargenomen medicatie-inname. We vonden dat blootstelling aan isoniazide en rifampicine werd gereduceerd in deze studie. Cmax, klaring en volumeverdeling van isoniazide werden ook nadelig beïnvloed. Het onderzoek concludeerde dat deze effecten hoogstwaarschijnlijk worden verklaard door diabetes. De gegeven aanbeveling is om de dosis van deze medicatie te verhogen in tuberculose/diabetes patiënten omdat dat resulteert in verbeterde blootstelling. Hoofdstuk 7 richt zich op door antituberculosis medicatie-geïnduceerde hepatotoxiciteit. Er is een hoge prevalentie van risicofactoren voor het ontwikkelen van levertoxiciteit in deze tijd van hiv en hepatitis B. Echter, onze studie laat zien dat het percentage patiënten dat levertoxiciteit ontwikkelde tijdens de intensieve fase van tuberculose behandeling laag is in vergelijking met andere studies die werden uitgevoerd in Malawi en Iran. De studie eindigde met 10b te zeggen dat levertoxiciteit niet veel voorkomt in Noord Tanzania. Echter, het blijft belangrijk dat tuberculosepatiënten duidelijke instructies krijgen over mogelijke tekenen en symptomen van hepatotoxiciteit en dat ze hun arts moeten bezoeken wanneer zulke symptomen zich voordoen. In hoofdstuk 8a wordt het gebruik van fluoroquinolonen aangekaard. De survey die werd uitgevoerd liet zien dat fluoroquinolonen veel gebruikt worden in geregistreerde apotheken en dat ze 12% van de totale antibiotica verkoop omvatten. Dit was alarmerend omdat sommige van de fluoroquinolonen, bijvoorbeeld moxifloxacine, onderdeel zijn van tweedelijns tuberculosebehandeling. In hoofdstuk 8b werd het voorkomen van fluoroquinolonen resistentie bepaald. Het doel was om na te gaan of resistentie gerelateerd is aan eerder gebruik van fluoroquinolonen (hoofdstuk 8a). Het resultaat was onverwacht omdat we een heel laag resistentieniveau vonden (<1%). Deze bevinding is bemoedigend; tuberculose behandeling met fluoroquinolonen kan worden ontwikkeld om de behandeling van tuberculose te verkorten. Concluderend, verbetering van tuberculose management is hard nodig. Strategieën om de kwaliteit en effectiviteit van medicatie te bevorderen zijn cruciaal in het succesvol aanpakken van tuberculose. In hoofdstuk 8 worden daarom alle resultaten van hoofdstuk 2 tot en met 7 gerapporteerd en bediscussieerd. Farmacokinetische en farmacodynamische problemen die tuberculosebehandeling beïnvloeden worden in perspectief behandeld. Bovendien worden prioriteiten voor toekomstig onderzoek besproken.

181

10c Muhtasari

Muhtasari

Ugonjwa wa Kifua Kikuu umeendelea kuwa tatizo kubwa duniani pamoja na kwamba una tiba yenye ufanisi kwa miongo kadhaa sasa. Gonjwa hili linashika nafasi ya pili duniani kwa magonjwa ambukizi yanayoongoza kwa vifo duniani baada ya ugonjwa wa ukimwi. Takwimu za Dunia zinaonyesha kuwa kuna maambukizi mapya yapatayo milioni 9 na vifo milioni 1.4 kwa mwaka. Ugonjwa wa Kifua Kikuu na Ukimwi ni magonjwa yanayoingiliana na yameshatangazwa kuwa magonjwa hatari duniani yanayohitaji kupewa kipaumbele huku ugonjwa wa Ukimwi ukiwa ndio chanzo kikuu cha kuongezeka kwa maambukizi ya Kifua Kikuu. Mbali na ugonjwa wa Kifua Kikuu, Kisukari ni ugonjwa ambao pia unaingiliana na Kifua Kikuu ambapo unapokuwa na magonjwa yote mawili (yaani Kifua Kikuu na Kisukari) tiba ya Kifua Kikuu imeonyesha kutofanya kazi ipasavyo. Utafiti huu umekuwa na jukumu la kuangalia takwimu za kisayansi katika matumizi sahihi ya dawa za kifua kikuu. Hivyo basi utafiti huu uliangalia matumizi ya dawa yanayowezesha kiwango kamili kinachostahili kuuwa wadudu ambukizi, mwingiliano wa dawa za kifua kikuu na dawa zingine kama za ukimwi na kisukari, na madhara yanayoweza kutokana na dawa za kifua kikuu. Kwa vile tiba ya Kifua Kikuu ni muunganiko maalumu wa dawa nne (rifampicin, isoniazid, pyrazinamide na ethambutol), hivyo unahitajika uelewa wa hatua zote za mzunguko na mwingiliano wa dawa hizi mwilini wakati mgonjwa anapotumia. Pia ni muhimu kuwa na 10c kiwango kamili cha dawa hizi katika damu kwa vile usahihi wa matumizi ya dawa za kifua Kikuu ndio msingi mkuu wa matibabu. Taarifa za tafiti za kisayansi zilizowasilishwa katika kitabu hiki, nyingi zimefanyika Tanzania katika Mkoa wa Kilimanjaro. Tanzania iko katika nafasi ya 18 kati ya nchi 22 zilizobeba mzigo wa maambukizi makubwa ya ugonjwa wa Kifua Kikuu Duniani. Katika Tanzania kuna takribani maambukizi 65,000 kwa mwaka. Asilimia thelathini na saba ya wagonjwa wa Kifua Kikuu wana maambukizi ya ugonjwa wa Ukimwi ambapo kiwango hiki cha asilimia Thelathini na saba ndicho kinachoongeza idadi ya wagonjwa wa Kifua Kikuu kwa asilimia 60 – 70.

Katika sehemu ya kwanza ya taarifa hii ya tafiti za kisayansi; utangulizi unaohusiana na ugonjwa wa Kifua Kikuu, jinsi unavyoambukiza na dalili zake kuu zimeainishwa. Pia utangulizi huu umeainisha kiwango cha ugonjwa huu duniani; vilevile namna ambavyo kifua Kikuu kinavyoweza kuongeza makali ya ugonjwa wa Ukimwi na vivyo hivyo Ukimwi unavyoweza kuongeza makali ya ugonjwa wa Kifua Kikuu na taarifa zingine zinazohusu tiba ya Kifua Kikuu. Mwisho wa sehemu hii ya kitabu hiki mkazo umetiliwa kwenye matatizo mbalimbali ya kutibu wagonjwa wa Kifua Kikuu. Katika sehemu ya 2a; matatizo ya kutibu maambukizi zaidi ya moja yaliangaliwa katika kufanya tathmini ya mwingiliano wa dawa ya rifampicin ambayo hutibu kifua kikuu na dawa za kurefusha maisha kwa wenye ukimwi. Vilevile mkazo uliwekwa kwenye dawa za kurefusha maisha zinazotumiwa na nchi zinazoendelea. Hata hivyo zimetajwa pia dawa mpya za tiba ya ukimwi na swala la kutopatikana kwa kundi la pili la dawa kwa wagonjwa walio na maambukizi zaidi ya moja yaani Kifua Kikuu na Ukimwi. Mwisho katika sehemu hii changamoto na vipaumbele vinavyoendana na watumiaji wa dawa kwenye nchi zinazoendelea. Ushauri ukatolewa wa kutumia kikamilifu programu zilizopo na takwimu za kitafiti.kutengeneza takwimu mpya kwa kufanya tafiti za kitatibu, tafiti za kuangalia mwenendo wa ugonjwa na kutafuta njia mbadala itakayowezesha dawa zisizopatikana kwa sasa ziweze kupatikana kwa wale wanaozihitaji zaidi.

185 chapter 10c

Katika sehemu ya 2b matumizi ya dawa ya atriplaR wakati wa tiba ya kifua kikuu na ukimwi yaliwasilishwa. Vilevile kupitia mitandao ya kisayansi kama Pubmed na google tumeweza kutoa taarifa za dawa aina tatu (emtricitabine, tenofovir na efavirenz) ambazo zipo kwenye kiwango maalumu kwenye dawa ya atriplaR. Mwingiliano wa dawa hizi na dawa za tiba ya kifua kikuu ulifanyiwa majaribio. Hitimisho lilionyesha kuwa dawa ya atriplaR ni salama na ni chaguo la kwanza kutibu mtu mwenye maambukizi ya ukimwi na kifua kikuu kwa watu wazima na watoto walio zaidi ya miaka 3. Katika sehemu ya 3 wagonjwa wenye maambukizi ya ukimwi na kifua kikuu walitumika kufanya tathmini ya adhari ya dawa ya rifampicin wakati ikitumika pamoja na dawa za efavirenz/ tenofovir/emtricitabine. Vilevile tulifanya jaribio la kuangalia athari ya kiwango cha dawa kilichopo kwenye dawa za efavirenz/tenofovir/emtricitabine inavyotumika na rifampicin. Madhara ya rifampicin hayakuonekana kwenye utafiti huu kwa kuwa kiwango cha dawa ya efivarenz/tenofovir/emtricitabine kilikuwa kimeongezeka kidogo tofauti na matarajio yetu. Kwenye upande wa dawa za kifua kikuu kiwango kilipungua kidogo lakini hakikuwa na madhara. Dawa zote za Kifua Kikuu na za ukimwi zilitumika vyema bila madhara yeyote. Sehemu ya 4. Tafiti ilifanyika kuangalia kiwango cha dawa kwenye chaguo la kwanza la dawa 10c za kutibu Kifua kikuu (rifampicin, isoniazid, pyrazinamide na ethambutol). Hii ni muhimu ambapo unaweza kulinganisha matumizi halisi ya dawa na kiwango cha dawa kinacholeta ufanisi wa dawa mwilini. Zaidi ya asilimia 30% yaani (7/20) ya wagonjwa walikuwa na kiwango kidogo cha dawa ya rifampicin, hali ambayo inatia wasiwasi kwa vile kiwango kidogo cha rifampicin kinaweza kuleta usugu na mgonjwa kutokupona. Tunashauri tafiti kubwa zaidi ifanyike itakayopima athari za viwango vidogo vya dawa za kifua kikuu. Sehemu ya 5: Kiwango cha dawa ya rifampicin katika damu na mate vililinganishwa. Lengo likiwa ni kujaribu kama mate yanaweza kutumika kupima kiwango cha dawa kama rifampicin badala ya damu. Hii ni kwa sababu kutoa mate ni rahisi kuliko kutoa damu, hivyo kama mate yatatumika kupima viwango vya dawa hata kwa watoto itakuwa rahisi kupimwa. Kwa ujumla matokeo ya tafiti hii yalionyesha kiwango kikubwa kwenye damu ikilinganishwa na mate (5.3mg/L kwa 3.2mg/L). Hii ilichangiwa na uchache wa wagonjwa kwenye tafiti (15 tu), hivyo tunashauri tafiti kubwa zaidi ifanyike ili kuweza kutoa uamuzi wa uhakika kama mate yanaweza kutumika badala ya damu. Katika sehemu ya 6, tathmini yaathari za ugonjwa wa Kisukari kwenye upatikanaji wa kiwango sahihi cha dawa za kifua kikuu ilifanyika. Watu wazima 40 (20 wenye maambukizi ya Kifua Kikuu pekee na 20 wenye maambukizi ya Kifua Kikuu na Kisukari) ambao walikuwa kwenye matibabu ya awali ya tiba ya Kifua Kikuu kwa wiki mbili waliandikishwa kwenye utafiti. Sampuli za damu zilichukuliwa kabla ya kutumia dawa na saa 1,2,3,4,6,8,10,24 baada ya kutumia dawa. Katika kundi la wagonjwa wenye kifua kikuu na kisukari, kiwango cha isoniazid na rifampicin (dawa muhimu katika kutibu kifua kikuu) kilionyesha kupungua kwenye utafiti huu. Pia kiwango cha juu kinachotegemewa kupatikana katika dawa hizi mwilini kiliathiriwa. Madhara haya yanafikiriwa kuwa yametokana na ugonjwa wa kisukari. Ushauri unatolewa kwamba wakati wa kutibu mgonjwa mwenye Kisukari na kifua kikuu kiwango cha hizi dawa muhimu kiongezwe. Sehemu ya 7: Tafiti hii ilikuwa ikionyesha uharibifu wa Ini unaotokana na dawa za kutibu kifua kikuu. Kuna kiwango kikubwa cha visabababishi vya uharibifu wa Ini unaotokana na Matumizi

186 Muhtasari ya dawa katika zama hizi za ugonjwa wa HIV na Uvimbe kwenye Ini. Hata hivyo utafiti wetu umeonyesha kuwa uwiano wa mgonjwa kupata uharibifu wa Ini ni 1/112 katika hatua za mwanzo za tiba ya TB. kiwango ambacho ni cha chini ukilinganisha na tafiti nyingine zilizofanyika Malawi na Iran. Majumuisho ya tafiti yanaonyesha kuwa uharibifu wa Ini unaotokana na utumiaji wa dawa za tiba ya TB ni mara chache sana kutokea Kaskazini mwa Tanzania. Hata hivyo bado ni muhimu kwa wagonjwa wa TB kupata maelekezo sahihi ya viashiria na dalili za uharibifu wa Ini na wanatakiwa waelekezwe kuwasiliana na Daktari mara tu wanapoona dalili hizo zinajitokeza. Sehemu ya 8a, suala la dawa ya fluoroquinolone lilizungumziwa. Utafiti uliofanyika umeonyesha kwamba dawa hii inatumika sana katika maduka ya dawa yalio na usajili ambapo inachukua asilimia 12% ya dawa zote za magonjwa ya kuambukiza. Hii inatupa tahadhari kwa vile baadhi ya fluoroquinolones kama Moxifloxacin ziko katika chagua la pili ya tiba ya TB. Hivyo katika sehemu ya 8b, usugu wa dawa ya fluoroquinolone ulifanyiwa Tathmini. Ambapo nia ni kuangalia kama usugu huo una uhusiano wowote na matumizi ya awali ya dawa hiyo. Matokeo yake ilikuwa kinyume na matarajio kwa vile usugu wa dawa hii ulikuwa chini ya asilimia 1(<1%). Hii inatia moyo na sasa dawa za aina ya Fluoroquinoline zinaweza kutumika kutibu kufua kikuu bila wasiwasi. Kwa kujumuisha ni kwamba inahitajika hatua za haraka za kuboresha usimamizi wa tiba ya 10c kifua kikuu. Na mikakati ambayo itaboresha kiwango cha dawa na ufanisi wake, vitu ambayo ni muhimu katika mafanikio ya vita dhidi ya TB. Hivyo sehemu ya tisa ni muhutasari wa sehemu zote (1 hadi 8). Matatizo yatokanayo na ufanyajikazi wa dawa mwilini kwenye tiba ya Kifua Kikuu na mapendekezo ya tafiti zaidi yametolewa.

187

List of publications

List of publications 1. Hadija H Semvua, Charles M Mtabho, Quirine Fillekes, Jossy van den Boogaard, Riziki M Kisonga, Liberate Mleoh, Arnold Ndaro, Elton R Kisanga, Andre van der Ven, Rob E Aarnoutse, Gibson S Kibiki1, Martin J Boeree, David M Burger. Efavirenz, tenofovir and emtricitabine combined with first-line tuberculosis treatment in TB-HIV-co-infected Tanzania patients: a pharmacokinetic and safety study. Antiviral Therapy 2013;18(1):105-13. 2. Alma Tostmann, Charles M. Mtabho, Hadija H. Semvua, Jossy van den Boogaard, Martin J. Boeree, Gibson S. Kibiki, Rob E. Aarnoutse. Pharmacokinetics of first line tuberculosis drugs in Tanzanian tuberculosis patients. Journal of Antimicrobial Agents Chemotherapy 2013;57(7):3208. 3. Hadija H. Semvua, Gibson S. Kibiki. AtriplaR/anti-TB combination in TB/HIV patients. Drug in focus. BMC Research Notes2011;4:511. 4. J van den Boogaard, H.H. Semvua, M.J. Boeree, R.E. Aarnoutse and G. S. Kibiki. Assessment of antibacterial sale by using the Anatomic Therapeutic Chemical classification and Defined Daily Dose methodology in Moshi Municipality, northern Tanzania. Tanzania J Health Research 2010;12(3):208-212. 5. Jossy van den Boogaard, Hadija H. Semvua, Martin J. Boeree, Rob E. Aarnoutse and Gibson S. Kibiki. Fluoroquinolone sale in northern Tanzania: a potential threat for fluoroquinolone use in tuberculosis treatment. Journal of Antimicrobial Chemotherapy 2010;65:145-147. 6. Jossy van den Boogaard, Hadija H. Semvua, Jakko van Ingen,3 Solomon Mwaigwisya, Tridia van der Laan,4 Dick van Soolingen, Gibson S. Kibiki, Martin J. Boeree, Rob E. Aarnoutse Low rate of fluoroquinolone resistance in Mycobacterium tuberculosis isolates from northern Tanzania. Antimicrobial and Chemotherapy 2011 Aug;66(8):1810-4. 7. Mgogwe J1, Semvua H, Msangi R, Mataro C, Kajeguka D, Chilongola J. The evolution of haematological and biochemical indices in HIV patients during a six-month treatment period. African Health Sciences 2012;12(1):2–7. 8. Alma Tostmann, Jossy van den Boogaard, Hadija Semvua, Riziki Kisonga, Gibson S. Kibiki4, Rob E. Aarnoutse and Martin J. Boeree. Antituberculosis drug-induced hepatotoxicity is uncommon in Tanzanian hospitalized pulmonary TB patients. Trop med Int health 2010;15(2):268-272.

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Acknowledgements

Acknowledgements In the past five years (2008-2012), in which I had to work with determination within the APRIORI and PanACEA collaborations, I had an opportunity to meet many experts. This made me active and increased the moral in research work. I am sincerely grateful to collaborate with them. To start, I express my sincere gratitude to Prof. David Burger, my promotor, he was of great help in all aspects from the beginning to the completion of the work. The fast response to our daily communication was really encouraging. To Prof. Gibson Kibiki, my co-promotor and director of research, for his tirelessness advise in my research work throughout. Our Wednesday meeting in which I had to present on PhD progress made me work hard and aiming to complete the work before another Wednesday. To Dr. Rob Aarnoutse, my professional co-promotor, who was a technical adviser and supervisor in my research work. His clear knowledge of the topics in this book made me possible to move forward towards the completion of this book. Dr. R. Aarnoutse was hand in hand with Dr. M. Boeree, my other co-promotor, and I am thankful to them and to Dr. Georgette Plemper van Balen, PanACEA project manager, for their financial support through PanACEA. To the KCMC administration (Former Director Prof. J. Shao, former Director of research, Prof. F. Mosha, KCMC Executive Director Prof. Mosha Ntabaya) for allowing me to carry out my studies. To Prof. Dr. Wil Dolmans for keeping an eye on my progress throughout the years. To the Pharmacy Department staff members of KCMC hospital, especially the Good Samaritan Foundation pharmacy shop, for their co-operation and support throughout my studies. To the administration, physicians and nurses at the Kibong’oto national TB referral hospital (Dr. Liberate Mleoh, Dr. R. Kisonga, Dr. S. Mpagama, M. Batuli, T. Mnzava, T. Mmari and many others) for their contribution in most of the research activities. To the TB clinic staff of Mawenzi hospital for their support in enrolling patients. To Prof. Andre van der Ven, director of APRIORI, Henri van Asten, Mieke Daalderop, Carla IJsenbrandt and Clara Blaauw for the opportunity to work within APRIORI and for all administrative assistance. To Dr. Jossy van den Boogaard and Dr. Charles Mtabho, these were my co-researchers and we used to work hand in hand in all research works. Each one was responsible for publication progress. I am thankful to them. To Dr. Marion Sumari (Data Manager KCRI) for her help in first reviewing most of the articles and manuscripts before sending them to the supervisors. All staff members in the KCRI datamanagement unit (Salim for Swahili translation, Sonda for statistical issues, without forgetting Krisanta, Hosiana, Lilian and Glory) for their support. To Dr. Cecile Magis Escurra for working together in the start up of the studies To the technicians of the Department of Pharmacy, Radboud University Medical Centre, The Netherlands for performing all sample analyses to measure TB and HIV drugs. To Angela Colbers for assistance in performing all pharmacokinetic data analyses and reviewing most of the references in the PETE article.

191 Acknowledgements

To the KCMC Biotechnology laboratory staffs, for performing all safety and microbiology analysis, especially to A. Ndaro who was the key laboratory personnel in most of the studies. To my colleagues in the Kilimanjaro Clinical Research Institute PhD room, Jossy van den Boogaard, Charles Mtabho, Ramsey Lymo, Charles Mwanziva, Eva Muro and Stellah Mpagama for their encouragement. To all staffs of the Kilimanjaro Clinical Research Institute for their administrative support. To all co-authors in all my publications for their contribution while working with them. To the members of the Manuscript Committee who reviewed my thesis. To my paranymphs Dr. Marion Sumari and Dr. Alma Tostmann who were at my side during the preparation of the manuscript and will be at my side during the defense. To my family, dear husband Amani Dagila, dear daughter Nuru Dagila, dear son Ahmed Dagila, for their patience, courage, love and support throughout my studies. To my parents Mr. and Mrs. Hamisi Selemani Semvua, my brothes and sisters, for their understanding where I had to miss some of the family activities. Last but not least, to all patients who voluntarily consented to join the study for the benefit of the future community.

192 Curriculum Vitae

Curriculum Vitae Hadija Hamisi Semvua is a Tanzanian woman, married with two children. She was born on 22nd August 1968 in Kilimanjaro Region of Tanzania. Hadija obtained primary school education from Kisutu, Ilala Municipal and she attended ordinary level and advanced level secondary education at Zanaki and Weruweru high school respectively. This was from 1984 -1990. In 1991, she joined Muhimbili University College for Health Sciences where she attained a degree of Bachelor of Pharmacy (B. Pharm) in 1995. From Nov 1995 to Dec 1996 she worked as intern pharmacist at Kilimanjaro Christian Medical Centre (KCMC) hospital and she was permanently employed at the same hospital in 1998 after attaining fully registration as a pharmacist from TFDA in 1997. She worked as hospital pharmacist up to October 2005 where she was selected to join a master course in Public Health at Tumaini University KCM College. In Nov 2006 she was awarded Master of Public Health (MPH) degree and the best performer for the year 2005-2006 in-take. She successfully attended a research methodology course in epidemiology and biostatistics in Nov 2007 and in October 2008 she started her PhD trainee at Kilimanjaro Clinical Research Institute and later registered with Radboud University Nijmegen, the Netherlands. While in the hospital she is a member of pharmacology department and works with Kilimanjaro Christian Medical University College (KCMUC) as assistant lecturer. She is also a member of palliative care and hospital therapeutic committee. She has attended several short courses of clinical trial monitoring, good clinical practice, palliative care coordination and management, writing biomedical papers and teaching methodology. As for research experience, she has been managing pharmacological clinical trials as a coordinator. She has experience in conducting both fieldwork and making different source documents. Her current research work is on infectious diseases, with focus on clinical pharmacological studies in tuberculosis patients. Different pharmacokinetic, pharmacodynamic, safety and tolerability studies were conducted from 2008 to 2012. Hobbies: Praying, reading books, travelling, cooking.

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