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Biltstraat 1063572 BJ Utrechtthe Netherlandswww.virology-education.com

11 September 2010

3rd International Workshop onClinical Pharmacology of Tuberculosis

Drugs

11 September 2010, Boston , USA

Program & Abstracts

BOSTON, MA USA 11 September 2010

3 rd

BOSTON, MA USA 11 September 2010

3 rd

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Acknowledgements

This workshop has been made possible by our sponsors. Financial support for the 3rd Int. Workshop on Clinical Pharmacology of Tuberculosis Drugs has been provided by:

Silver Level Sponsor

Principal Sponsor

Supporter

This Workshop is organized by

Biltstraat 106, 3572 BJ Utrecht, the Netherlands Phone: + 31 30 230 7140 Fax: +31 30 230 7148 Email: [email protected] / www.virology-education.com

3rd International Workshop on Clinical Pharmacology of Tuberculosis Drugs – 11 Sep. 2010 – Boston MA, USA

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Content

Acknowledgement Workshop sponsors p. ii

Content Index p. iii

General Information p. iii

Organizing Committee, Scientific Committee p. iv

Program p. v

Invited Speakers p. vii

Abstracts Oral Presenters p. 01

Abstracts Poster Presenters p. 14

Author Index p. 25

General Information Badge Policy

All registered delegates are provided with an identity badge. Please wear it at all times to ensure admission to the meeting.

Certificate of Attendance Delegates who require a certificate of attendance should inform the Workshop Secretariat. The certificates can be picked up during lunch on Friday.

Conference Materials Presentations will be posted on www.virology-education.com shortly after the workshop.

Date and Venue The date of the meeting is Saturday 11 September, 2010 The meeting is held in the Theatre Ballroom at the Courtyard Boston Downtown/Tremont hotel.

Language The official language of the workshop is English.

Social Program A workshop dinner is scheduled for Saturday evening 11 September for those delegates who have pre-registered.

Workshop Secretariat

The registration desk is located near the plenary meeting room. The conference organizers can be addressed for all questions concerning the logistics of the meeting.


http://www.virology-education.com/

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Organizing Committee

Rob Aarnoutse Radboud UMC Nijmegen, Nijmegen, The Netherlands

Gheraint Davies University of Liverpool, Liverpool, United Kingdom

Helen McIlleron University of Cape Town, Cape Town, South Africa

Eric Nuermberger Johns Hopkins School of Medicine, Baltimore, USA

Charles Peloquin University of South Florida, Gainesville, USA

Annette Shadiack Global Alliance for TB Drug Development, USA

Helen Winter Global Alliance for TB Drug Development, USA

Scientific Committee

Bill Burman Denver Public Health, Denver, USA

Jan Gheuens Gates Foundation, Seattle, USA

Stephen Gillespie Royal Free Hospital, UCM School, London, United Kingdom

Saye Khoo University of Liverpool, Liverpool, United Kingdom

William MacKenzie CDC/CCID/NCHHSTP, Atlanta, USA

David McNeeley Tibotec, Yardley, USA

Ikushi Onozaki WHO - Stop TB Department, Geneva, Switzerland

Andrew Vernon CDC TBTC, Atlanta, USA


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Program - Saturday 11 September 2010

08.30 h Opening of the Workshop

Session 1: Pharmacokinetics & Pharmacodynamics of approved TB drugs Chairs: Dr Annette Shadiack & Dr Helen McIlleron

08.45 h

Invited lecture: PK and PD of TB drugs: what do we know and what are the implications ?

G. Drusano

09.15 h

The anti-tuberculous activity of pyrazinamide is driven by AUC/MIC in murine tuberculosis

Z. Ahmad Parry

O_01

09.30 h

Time-kill kinetics of anti-tuberculosis drugs, and emergence of resistance, in relation to metabolic activity of Mycobacterium tuberculosis

J. de Steenwinkel

O_02

09.45 h

Discussion

10.00 h Coffee break

Session 2: Pharmacokinetics & Pharmacodynamics of new TB drugs Chairs: Dr Rob Aarnoutse & Dr Jan Gheuens

10.30 h

Invited lecture: The rocky road from discovery to clinical pharmacology – case study on TMC207

K. Andries

11.00 h

Invited lecture: The place of clinical pharmacology in the decision process to move forward in early drug development - case study on PA-824

A. Ginsberg / N. Erondu

11.30 h

PA-824 exhibits time-dependent bactericidal activity against persisting Mycobacterium tuberculosis

Z. Ahmad Parry

O_03

11.45 h

Population PK-PD modeling of Phase 1 data of PNU-100480 to guide doses for Phase 2a study

V. Kumar

O_04

12.00 h

The penetration of fluoroquinolones in pulmonary lesions of TB infected rabbits

V. Dartois

O_05

12.15 h Round table discussion.

Development of individual drugs versus a regimen: how do they come together ?

13.30 h Lunch


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Session 3: Pharmacokinetic- Pharmacodynamic modelling & Population Pharmacokinetics

Chairs: Dr Eric Nuermberger & Dr Helen Winter

14.30 h Invited lecture: PK/PD modeling and population PK in the optimization of TB treatment

T. Gumbo

15.00 h

Treatment failure and emergence of resistance during non-compliance in an experimental tb model.

S. Srivastava

O_06

15.15 h

Population pharmacokinetics of rifampicin in south african tuberculosis patients and the influence of drug transporter polymorphisms

E. Chigutsa

O_07

15.30 h

A population pharmacokinetic model for rifampicin auto-induction

P. Denti

O_08

15.45 h

Discussion

16.00 h Coffee break

Session 4: Drug-drug & drug-disease interactions and safety/tolerability of TB drugs - The effects of age, weight and gender

Chairs: Dr Charles Peloquin & Dr Gheraint Davies

16.30 h Invited lecture: Drug interactions in TB-HIV co-infected patients – Research questions for the near future.

S. Khoo

17.00 h

Influence of age and nutritional status on the pharmacokinetics of anti-tuberculosis drugs in children with tuberculosis

G. Ramachandran

O_09

17.15 h

Weight and sex are important determinants of antituberculosis drug concentrations in patients with HIV associated tuberculosis

H. McIlleron

O_10

17.30 h

Population pharmacokinetics of rifampicin in childhood tuberculosis and the need for dose adjustment

S. Zvada

O_11

17.45 h

A surprising effect of weight on ethambutol (EMB) pharmacokinetics (PK).

T. Gumbo

O_12

18.00 h

Cumulative dose Amikacin predicts hearing loss in MDR-TB patients

J-W. Alffenaar

O_13

18.15 h

Discussion

18.30 h Closure of the Workshop

18.30 - 19.15 h Poster viewing & Drinks

19.15 h Departure for Workshop dinner (for those who pre-registered)


Program vii

Invited Speakers

Pharmacokinetics & Pharmacodynamics of approved TB drugs: What do we know and what are the implications?

Dr. George Drusano received his medical training at the University of Maryland School of Medicine, graduating cum laude. He served as Chief Resident at the University of Maryland Hospital and entered the Faculty after completing a Fellowship in Infectious Diseases. He is Co-Director of the Ordway Research Institute in Albany, NY, Director of Clinical Pharmacology Studies, Albany Medical College. Dr. Drusano is a Past-President of the International Society for Anti-infective Pharmacology. He has developed theoretical and experimental modeling systems for determining proper drug dose and schedule. Dr. Drusano’s main interests are in the area of anti-infective drug effect. He and his team have performed investigations with drugs that affect bacteria, viruses, and fungi. The approach taken by the team is defined by both bench investigations as well as by clinical studies inspired by the bench investigations. In specific, the hollow fiber system allows the performance of Phase I/II clinical trials on the benchtop, allowing identification of the proper drug dose and schedule to achieve the desired endpoint. His laboratory is supported by 3 RO1’s and a P01 from NIAID and a Grant from the Bill and Melinda Gates Foundation.

The rocky road from discovery to clinical pharmacology – case study on TMC207

Dr. Koen Andries joined the team of Dr. Paul Janssen in Janssen Pharmaceutica (Johnson and Johnson) in 1982 after studying Veterinary Sciences and obtaining his PhD at the University of Ghent. Using whole cell assays in search of antiviral compounds, his team discovered nanomolar uncoating inhibitors of rhinoviruses (R77975 - pirodavir), fusion inhibitors of respiratory syncytial virus (R170591) and, in close collaboration with Dr. Rudi Pauwels who later founded Tibotec NV, several non-nucleoside reverse transcriptase inhibitors of HIV [TIBO’s, alpha-APA’s, and DAPY’s such as R165335/TMC125 (intelence - launched) and R278474/TMC278 (rilpivirine –NDA submitted for approval)]. TMC125 and TMC278 both have high intrinsic activity against wild-type HIV-1 and against HIV strains harbouring resistance mutations. Dr. Andries led the team that discovered R207910/TMC207, one of the more promising new anti-tuberculosis drugs, and its unique mechanism of action. He is Distinguished Research Fellow at Tibotec and Distinguished Professor at the University of Antwerp.

The place of clinical pharmacology in the decision process to move forward in early drug development – case study on PA824

Dr. Ngozi Erondu Dr. Ann Ginsberg


Program viii


PK/PD modeling and population PK in the optimization of TB treatment

Dr. Tawanda Gumbo was born in Zimbabwe, and attended the University of Zimbabwe Medical School. Residency (Internal Medicine/International Health Track) and Fellowship (Infectious Diseases) were at Case Western Reserve University (MetroHealth) and the Cleveland Clinic Foundation from 1993-1999. In 2000 he joined the Albany Medical College, and later the Ordway Research Institute in Albany, New York, where he worked to establish the hollow fiber model of tuberculosis in the laboratory of George Drusano, MD., who mentored him in antimicrobial PK/PD and modelling techniques. In 2006 he moved to the University of Texas Southwestern Medical Center at Dallas and established his laboratory in the Division of Infectious Diseases. The laboratory has elucidated the role of efflux pumps in survival of Mycobacterium tuberculosis and examined molecules that could exploit these to enhance antituberculosis therapy. Work has also included the establishment of a mathematical model for the relationship between antimicrobial drug exposure and resistance emergence, PK/PD of drugs used to treat Mycobacterium avium infection, and recently the establishment of new susceptibility breakpoints of first-line antituberculosis drugs. In 2007 he received the NIH Director’s New Innovator Award to focus on efflux pump inhibition to accelerate therapy and reduce duration of antituberculosis therapy. In 2008 he was promoted to Associate Professor. In 2009 he was made a Mellon Visiting Scholar at the University of Cape Town in South Africa. Recently he contributed two chapters in the 12th edition of the pre-eminent pharmacology text "Goodman and Gilman’s The Pharmacological Basis of Therapeutics" namely, "Principles of Chemotherapy of Infectious Diseases" and "Chemotherapy of Tuberculosis, Mycobacterium avium Complex, Leprosy." He is a published poet and novelist.

Drug interactions in TB-HIV co-infected patients – research questions for the near future

Dr. Saye Khoo is a Reader in Pharmacology at the University of Liverpool and Honorary Consultant Physician in Infectious Diseases. Research interests centre on the pharmacology of HIV treatment failure. This includes the role of therapeutic drug monitoring, population pharmacokinetic modelling, investigation of drug interactions, molecular characterisation of drug metabolism and disposition pathways and the role of host genetic variability in influencing drug exposure and response. There is also a focus on HIV therapy in resource-poor settings, and the pharmacology of anti-tuberculous therapy. Dr Khoo chairs the Mersey, Cheshire & N Wales HIV Managed Care Network, and is actively involved in service development through the Mersey & Cheshire Sexual Health Network, and the Regional Sexual Health Task Group for NW England. He currently serves on the British HIV Association Treatment Guidelines Committee, the joint British HIV Association/British Infection Society Opportunistic Infections Guidelines Committee, and the British Thoracic Society Guidelines Joint Tuberculosis committee advising on the prevention and management of TB in patients with renal impairment. Other recent activities are: PENTA Pharmacology Steering Group, Editor, Journal of Antimicrobial Chemotherapy (2003 - 2007), HEFCE Clinical Senior Lectureship Award Referees Panel.

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3rd International workshop on Clinical Pharmacology of Tuberculosis Drugs

Abstracts Oral Presentations

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Abstract: O_01 The anti-tuberculous activity of pyrazinamide is driven by AUC/MIC in murine tuberculosis Z. Ahmad1, C.A. Peloquin1, A. Minkowski1, J.H. Grosset1, E.L. Nuermberger1, Z. Ahmad Parry1 1Johns Hopkins University School of Medicine, Department of Medicine 1550 Orleans Street Rm 123 CRB-11, Baltimore, USA Background: Although pyrazinamide (Z) is a key sterilizing anti-TB drug, the pharmacodynamic (PD) parameter which correlates best with its activity under in vivo conditions is still unknown. We performed a dose fractionation study in mice to identify the same. Material & Methods: Single-dose pharmacokinetic (PK) studies were conducted in BALB/c mice at doses from 7-900 mg/kg with or without rifampin (R). WinNonlin was used for PK analysis and simulation. Four wks after aerosol infection with M. tuberculosis H37Rv, treatment with Z in total dosages ranging from 24 to 1296 mg/kg, divided into thrice daily, twice daily, and every 24, 48 and 144 h dosing was given, some doses were also given with R. Results: The mean lung CFU count was 7.26 log10 at treatment initiation. Doses < 50 mg/kg/day were inactive, whereas dose-dependent activity was observed at doses ≥ 50 mg/kg/d and maximal effects were observed with 150 mg/kg/d. For any total dosage level, the activity was generally independent of individual dose size and dosing frequency in the linear range. Although the MIC is 10 µg/ml at pH 5.5, regimens in which the Cmax was below this value were active. Whether the MIC “in vivo” was estimated to be 0.2, 1 or 5 µg/ml, CFU counts correlated best with fAUC/MIC (e.g., R2= 0.87 at MIC 1 µg/ml), followed by fCmax/MIC (R2=0.58) and free drug time above MIC (R2= 0.31). AUC0-

24 values of 65 and 119 µg-h/ml were associated with 1 and 2 log killing, respectively. fAUC/MIC remained most closely associated with activity, but the target values necessary for a given bactericidal effect were lower in the presence of R owing to synergism. Conclusions: The bactericidal activity of Z is driven by AUC/MIC, even in combination. Compared with a recent study in the in vitro

hollow fiber system, the AUC target necessary for comparable bactericidal effect is lower in mice suggesting more favorable conditions for PZA action. Abstract: O_02 Time-kill kinetics of anti-tuberculosis drugs, and emergence of resistance, in relation to metabolic activity of Mycobacterium tuberculosis J.E.M. de Steenwinkel1, G.J. de Knegt1, M.T. ten Kate1, A. van Belkum1, H.A. Verbrugh1, K. Kremer2, D. van Soolingen2, I.A.J.M. Bakker-Woudenberg1

1Erasmus Medical Centre, Medical Microbiology and Infectious diseases, Rotterdam, The Netherlands; 2National Institute of Public Health and the Environment Centre for Infectious Disease Control, National Tuberculosis Reference Laboratory, Bilthoven, The Netherlands Background: The pharmacodynamics of tuberculosis (TB) treatment should be further explored, to prevent emergence of resistance, treatment failure and relapse of infection. Optimization of dosing schedules of currently-used anti-TB drugs resulting in more efficient use of available drugs could be a more realistic short-term goal next to the evaluation of novel anti-TB drugs. For this, insight in the dynamics of the activity of the anti-TB drugs is of critical importance. The diagnostic drug susceptibility tests guiding TB therapy investigate metabolically active Mycobacterium tuberculosis (Mtb) isolates at static conditions and as such are not informative with respect to the time-kill kinetics of anti-TB drugs and the emergence of resistance in metabolically low active or even dormant mycobacterial cells. Material & Methods: In vitro the killing capacity of rifampicin, isoniazid, ethambutol and amikacin regarding the degree of killing, killing rate, and selection of resistant mutants, was investigated in highly active Mtb cells at low density versus low active Mtb cells at high density. Results: Isoniazid showed a rapid and high killing capacity towards highly active mycobacteria at low density, but due to the emergence of resistance could not eliminate the Mtb. Efflux pump-mediated isoniazid resistance

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was predominant. Rifampicin revealed a relatively slow and time-dependent killing capacity, but achieved elimination of all mycobacteria. Ethambutol was not bactericidal. Amikacin showed a high and extremely rapid killing activity that was not time-dependent, and could eliminate all mycobacteria. Exposure of low active Mtb populations at high density to isoniazid, rifampicin or amikacin led to the emergence of resistant mutants. Compared with the highly active mycobacteria, elimination of the low active mycobacteria required a 64-fold increased isoniazid concentration and a 4-fold increased rifampicin concentration, whereas amikacin was equally effective irrespective of the metabolic state of the mycobacteria. Conclusions: The anti-TB drugs differ significantly regarding their time-kill kinetics. In addition, the metabolic state of Mtb significantly affects their antimicrobial susceptibility, with the exception of amikacin. Optimization of dosage of anti-TB drugs is required to achieve maximum drug concentrations at the site of infection in order tot maximize reduction in Mtb load and to minimize the emergence and/or selection of resistance. No conflict of interest

Abstract: O_03 PA-824 exhibits time-dependent bactericidal activity against persisting Mycobacterium tuberculosis Z. Ahmad Parry1, C.A. Peloquin1, A. Minkowski1, A. Ginsberg1, J.H. Grosset1, E.L. Nuermberger1

1Johns Hopkins University School of Medicine, Department of Medicine 1550 Orleans Street Rm 123 CRB-11, Baltimore, USA Background: PA-824 is in Phase II trials for treatment of TB. PA-824 has time-dependent bactericidal activity against actively multiplying M.tb in mice. To shorten the duration of TB therapy, a new drug also must kill slowly or non-multiplying bacilli with phenotypic tolerance to existing drugs. The goal of the present study was to describe the pharmacodynamics (PD) of PA-824 against persistent bacilli.

Material & Methods: Beginning 2 wks after aerosol infection with 3.5 log10 CFU of M. tuberculosis H37Rv, mice received high-dose rifampin (R) 40 mg/kg, isoniazid (H) 10 mg/kg, and pyrazinamide (Z) 150 mg/kg/d for 3 wks to select for persisters. Thereafter, treatment included PA-824 monotherapy in total doses of 144, 288, 576, 1152, 2304 and 4608 mg/kg, divided into 4, 8 12, 24 or 48 doses over 24 d. Lung CFU were counted after treatment. WinNonlin was used for standard pharmacokinetic (PK) analyses and PD analysis using an inhibitory effect sigmoid Emax model, estimating 7.5% free drug. Human PK/PD simulations used published Phase I data. Results: RHZ pre-treatment reduced the lung CFU count from 6.62±0.11 to 4.46±0.16 log10. Thereafter, limited re-growth (to 5.30 log10 CFU) occurred without treatment. Maximal killing (≥2.6 log reduction) was observed with PA-824 at 96 mg/kg q12h and 192 mg/kg qd. CFU counts correlated well with free drug time above MIC (T>MIC) (R2= 0.84) and fAUC/MIC (R2= 0.75) but not fCmax/MIC (R2=0.26). T>MIC values of 28% and 45% were associated with 1 and 2 log killing, respectively. At a given T>MIC, higher AUC/MIC was associated with a greater effect. Conclusions: PA-824 has time-dependent bactericidal activity against slowly or non-multiplying persisters in this murine model. As the T>MIC target for a 2 log kill (0.083 log CFU/day) is attainable in humans with doses ≥200 mg, PA-824 may be useful to shorten TB therapy. Abstract: O_04 Population PK-PD modeling of Phase 1 data of PNU-100480 to guide doses for Phase 2a study V. Kumar1, J. Liu1, R.S. Wallis2, W. Jakubiec3, A.M. Silvia2, D. Paige4, T. Zhu1, G. Nucci1 1Pfizer Global Research and Development, Clinical Pharmacology, Groton, USA; 2Pfizer Global Research and Development, Clinical, Groton, USA; 3VA CT Healthcare, Biology, West Haven, USA; 4Pfizer Global Research and Development, Project Management, Groton, USA Background: PNU-100480 is currently under development for MDR and XDR Tuberculosis. It is rapidly absorbed after oral administration and

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metabolized to two active metabolites: PNU-101244 (sulfone) and PNU-101603 (sulfoxide) with the later contributing significantly to its activity. Whole blood bactericidal activity (WBA) was used as an ex-vivo biomarker assay in the early phase studies to evaluate the bacterial killing. The aim of this analysis was to develop a population pharmacokinetic (PPK) and Pharmacokinetic/pharmacodynamic (PPK/PD) model to characterize the relationship between the total active moiety (APNU) i.e. concentrations of PNU-100480 and sulfoxide metabolite with WBA in healthy volunteers. This model was subsequently used to select doses for a Phase 2a study. Materials & Methods: Data were available from 2 Phase 1 studies in healthy volunteers. The first in human study (n=19) evaluated single escalating doses from 35 to 1500 mg. In the multiple dose tolerance study (n=41) 4 doses/ dosing regimens were evaluated for 14 days. Twice daily doses of 100, 300, and 600 mg were administered along with a 1200 mg QD dose. A mixed-effects modelling approach was implemented using NONEM-VI with the first –order conditional estimation with interaction method (FOCEI). A PPK model was developed using APNU concentrations and sequentially a sigmoid inhibitory (Imax) model was used to model the ex-vivo WBA counts to the observed APNU concentration. Selection of the appropriate structural, variance, and error models was based on the likelihood ratio test (p-value of 0.05), goodness of fit plots, and visual predictive check. Finally, the model parameters from the PPK and PPK/PD model were used to simulate the WBA counts at various doses and regimens for the selection of doses for Phase 2a study. Results: APNU concentration-time data were adequately described by one-compartment PK model with lagged first-order absorption and first-order elimination. Inter-individual variability (IIV) was characterized using exponential error models and residual variability was modelled using additive error model on log transformed concentrations. IIV , expressed as percentage coefficient of variation (CV%), in apparent clearance and volume of distribution was low, 17 and 18% respectively. Presence of food was found to decrease the rate of absorption from 3.52 to 0.52 hr-1 but had no effect on the extent of absorption. A direct sigmoid Imax response model with additive residual error was found to

best characterize the APNU - WBA relationship. Imax and IC50 were well characterized and estimated to be 1.77 log CFU and 647 ng/ml, respectively. Population estimate of hill coefficient was found to be 1.47 (15%). The visual predictive check showed that overall, the model provided an appropriate fit to the data. Results from the simulations using PNU-100480 as monotherapy suggested that a total daily dose of 800 - 1200 mg would likely provide the optimum efficacy. Conclusions: A PPK and PPK/PD model was developed to characterize APNU pharmacokinetics and bacterial killing. The proposed direct inhibitory PK/PD model adequately described the Phase 1 WBA data. Subsequent simulations using the PK/PD model guided the dose selection for the phase 2a study in TB patients. No conflict of interest

Abstract: O_05 The penetration of fluoroquinolones in pulmonary lesions of TB infected rabbits V. Dartois1, L.E. Via2, B. Prideaux3, A. Goh1, M. Stoeckli3, C.E. Barry2 1Novartis Institute for Tropical Diseases, Pharmacology, Singapore, Singapore; 2NIH NIAID, Tuberculosis Research Section, Bethesda, USA; 3Novartis Institutes for Biomedical Research, Analytical Sciences, Basel, Switzerland Background: Most drugs exert their effect in defined target tissues where they must be distributed. Target site concentrations may be substantially different than plasma concentrations. This differential penetration might be quite acute in TB disease where lesion diversity in size, location, structure and cellular/acellular content is remarkable. Due to varying levels of vascularization and presence of physical barriers such as necrosis and calcification, the extent of sequestration of the bacilli, and therefore the ability of drugs to access them, is suspected to differ between lesion types and lesion compartments. Sub-therapeutic drug levels within TB lesions may contribute to the long treatment duration and the development of drug resistance. We previously showed that among the four major TB drugs

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rifampicin, isoniazid, pyrazinamide and moxifloxacin (MXF), MXF had the ability to significantly accumulate within rabbit granulomas following a single oral dose. In contrast, exposure of the three first line agents was lower in lesions than in plasma. To determine whether drug levels within granulomas correlate with drug efficacy, we have further investigated the distribution of MXF and four other fluoroquinolones which are known to have similar MIC against M. tuberculosis but variable efficacies in TB mouse models and in the clinic. Material & Methods: Rabbits were infected with Mtb HN878 or M. bovis and the infection was allowed to develop for 6 to 8 weeks prior to drug administration. Plasma, lung, and lesion samples were collected from infected rabbits treated with a 25mg/kg single dose of MXF, levofloxacin (LVX), sparfloxacin (SPX), ciprofloxacin (CPX) and clinafloxacin (CLX). The doses were given 0.5, 1.5, 2.3, 3.5, or 5 hours prior to necropsy for MXF, and 2h prior to necropsy in a second study where all five fluoroquinolones were administered simultaneously. Histology analysis of typically sampled lesions revealed solid, coalescing, caseous and liquid granulomas. Drugs were quantified in plasma and tissue homogenates by mass spectrometry. Imaging mass spectrometry, using deuterated MXF as internal standard, provided 2-dimensional ion maps of each fluoroquinolone in lesions and surrounding tissue. Results: The relative accumulation of MXF in granulomas versus plasma was confirmed following single dose administration and at steady-state. Interestingly, MXF was clearly concentrated within the cellular layers at the periphery of granulomas but was not able to reach the necrotic center of caseous lesions. Comparative analysis of fluoroquinolone penetration in rabbit lesions showed markedly different lesion-to-plasma ratios for the five compounds, with MXF and LVX having the highest absolute drug levels in granulomas. The results are in agreement with mouse efficacy data, and with available clinical efficacy data for CPX, LVX and MXF. Conclusion: Collectively, our results indicate that (i) MXF does not distribute evenly within caseous granulomas, (ii) compounds belonging to the same class can display dramatically different abilities to distribute into lesions, and (iii) the amount of drug present inside

granulomas appears to correlate with efficacy. Ongoing studies in TB patients undergoing lung resection will provide some insights with regard to the location and types of lesions which are most resistant to drug dispersal. No conflict of interest

Abstract: O_06 Treatment failure and emergence of resistance during non-compliance in an experimental tb model. S. Srivastava1, A. Wozniakowski1, J. Pasipanodya1, T. Gumbo1 1UT Southwestern Medical Center, Internal Medicine, Dallas, USA Background: Compliance is the reason why directly observed therapy is implemented globally. Currently, it is required that patients take 100% of their doses. However, the true rates and patterns of non-compliance (NC) associated with failure of therapy and emergence of drug resistance are unknown. Material & Methods: In 3 experiments, standard doses of isoniazid (INH), rifampin (RIF), and pyrazinamide (PZA) were administered in hollow fiber systems (HFS), to create different half-lives of drugs in each HFS, for 28 days. Two experiments were performed with media at pH 5.8, and one at normal pH, each inoculated with Mycobacterium tuberculosis H37Rv (Mtb). First, three HFS each were had therapy administered daily to achieve NC rates of 0, 20, 40, 60, 80, 100% in a start-stop-start-stop (SSSS) fashion. The experiment was performed twice. In the next experiment, 60% and 80% NC was administered based on 3 dosing schedules: start-stop (SS), SSSS, or randomly based on random numbers generated from a computer. In addition, some HFS received no therapy (100% NC), or were treated daily for 28 days (0% NC), and in a third group the whole 28 day drug exposure administered during the first 20% of 28 days. In addition, the inoculum in all HFS was spiked with a RIF resistant (RIFR) isogenic strain of Mtb, to make 0.5% of total inoculum. Systems were sampled for resistance to each drug as well as for total Mtb burden.

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Results: NC accounted for 70.5% of total variance of Mtb population (P<0.0001). 0-40% NC was associated with similar Mtb kill rates. However, 60% NC was associated with slower rates of kill up to day 14 (p=0.001), after which it stopped killing. All NC>60% led to failure of therapy. However, no INH resistance (INHR) or RIFR emerged, but resistance to PZA (PZAR) occurred at NC>60%. The worst dose schedule for microbial kill was always SS, while the most forgiving strategy was randomly missed doses. In the HFSs where the entire drug exposure was given in only 20% of the time there was sustained kill for the 28 days, but the kill rates were slower than the 100% compliance (p<0.0001). RIFR isolates did not increase to 1% in any system, despite all having been spiked with 0.5% RIFR. INHR isolates emerged on day 21 in all 20% NC regimens, but the INHR subpopulation was similar for different patterns of NC, and did not achieve 1% proportion of total. PZAR subpopulation was highest with the SS and lowest with random NC. PZAR subpopulation was >1% of total population. Conclusion: First, poor outcomes only occurred at considerably high NC rates of ≥60%. Second, the pattern of NC matters, with randomly missed doses the most forgiving. Third, clinically meaningful INHR and RIFR did not occur in any of the 3 experiments despite extensive NC. This suggests that while NC may be associated with INHR and RIFR, it may not be sufficient on its own to lead to resistance, and other factors may also be important. No conflict of interest

Abstract: O_07 Population pharmacokinetics of rifampicin in South African Tuberculosis patients and the influence of drug transporter polymorphisms E. Chigutsa1, M.E. Visser2, D. Egan3, N.H.G. Holford4, P.J. Smith1, A. Owen3, H. McIlleron1 1University of Cape Town, Medicine, Cape Town, South Africa; 2University of the Western Cape, School of Public Health, Cape Town, South Africa; 3University of Liverpool, Department of Pharmacology and Therapeutics, Liverpool, United Kingdom; 4University of Auckland, Faculty of Health Sciences, Auckland, New Zealand

Background and Objectives: Rifampicin is a substrate of drug transporters that include p-glycoprotein and OATP1B1, which are transcriptionally regulated by nuclear receptors, PXR and CAR. We sought to determine the impact of polymorphisms in the genes coding for these proteins on rifampicin pharmacokinetics. Material & Methods: Fifty-seven tuberculosis patients being treated with a fixed dose combination of rifampicin, pyrazinamide, isoniazid and ethambutol were recruited. The patients underwent pharmacokinetic sampling at least one month after starting treatment. Between 4-8 blood samples were drawn from each patient over a 7 hour period. Twenty-four patients were sampled on two occasions. Rifampicin plasma concentrations were measured using liquid-chromatography with tandem mass spectrometry. Patients were genotyped for ABCB1, SLCO1B1, PXR and CAR polymorphisms using real time PCR. A non-linear mixed effects population pharmacokinetic model was developed using NONMEM VII to evaluate the effect of several covariates including genetic polymorphisms, sex, weight and dose on rifampicin pharmacokinetics. Results: A transit absorption compartment model was used to account for highly variable absorption as well as an absorption delay. Oral clearance (CL/F) and volume (V/F) were allometrically scaled for weight and the typical values were 11 L/h/70kg and 47 L/70kg respectively. The first order absorption rate constant was 1.2 /h and the mean transit time (MTT) was 1.4 h. The model supported both between subject and within subject variability in CL/F, V/F and MTT. Females had a smaller V/F and longer MTT than males. Individuals receiving higher doses (600 mg or 750 mg daily) were found to have a 34% shorter MTT than patients receiving 450 mg. The allele frequency of SLCO1B1 rs4149032 polymorphism was 0.70. Patients heterozygous and homozygous for the variant allele had reductions in rifampicin bioavailability of 20% and 28% respectively. The polymorphism explained 21% of the between subject variability in CL/F and 22% of variability in MTT. Simulations revealed that an increase in the rifampicin dose of approximately 30% for patients with the polymorphism resulted in plasma concentrations similar to those of wild-type individuals, with a typical peak concentration above the recommended minimum

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of 8 mg/L. ABCB1, PXR and CAR polymorphisms did not significantly alter rifampicin pharmacokinetics but low frequency of these variants precluded a robust analysis. Conclusion: The SLCO1B1 rs4149032 polymorphism had a significant effect on rifampicin exposure and may explain why several patients had subtherapeutic plasma concentrations. Should our findings be confirmed, an increased dose of rifampicin would be desirable for carriers of this polymorphism. Larger studies are required to confirm the effects of other polymorphisms affecting the disposition of rifampicin. No conflict of interest

Abstract: O_08 A population pharmacokinetic model for rifampicin auto-induction P. Denti1, W. Smythe1, U.S.H. Simonsson2, R. Rustomjee3, P. Onyebujoh4, P. Smith1, H. McIlleron1 1University of Cape Town, Division of Clinical Pharmacology, Cape Town, South Africa; 2Uppsala University, Department of Pharmaceutical Biosciences, Uppsala, Sweden; 3TB Research Unit: Clinical and Biomedical, South African Medical Research Council, Durban, South Africa; 4World Health Organization, Special Programme for Research and Training in Tropical Diseases, Geneva, Switzerland Introduction: Rifampicin, apart from being the backbone drug of 1st-line anti tubercular treatment, is a potent inducer of numerous drug metabolizing enzymes (e.g. Cytochrome P450 family) and drug transporters (e.g. P-Glycoprotein). Thus rifampicin enhances the clearance of many co-administered drugs, including key antiretrovirals (e.g. protease inhibitors and nonnucleoside reverse transcriptase inhibitors), and rifampicin itself. After several days of exposure to rifampicin, its clearance (CL) significantly increases due to this autoinduction. Inadequately accounting for this can lead to suboptimal concentrations of rifampicin, and increase the risk of treatment failure or the development of resistance. The phenomenon of autoinduction is believed to reach steady state after approximately one week. However, this has not been quantitatively described.

Material & Methods: A cohort of 62 South African HIV infected subjects with pulmonary TB were treated with rifampicin in a fixed dose combination also containing isoniazid, pyrazinamide, and ethambutol, according to the WHO recommendations (weight-based dosing). Most subjects received TB drugs 5 days per week, but due to the introduction of new guidelines, 17% were on a 7 days per week regimen. They underwent pharmacokinetic sampling on the 1st, 8th, 15th and 29th days after starting antituberculosis treatment. Nonlinear mixed-effects modelling using the software NONMEM VII was employed to analyse the data. A one-compartment model with first-order delayed absorption and disposition best described RIF PK. Volume and CL were allometrically scaled with body weight to account for size differences between patients. The CL increase with respect to time after the initiation of TB treatment was described with a sigmoidal function. Results: In our population the typical value for rifampicin CL/F doubles over a period of a month. Our results show that the autoinduction continues well beyond the previously reported period of a week, when only about half of this effect is observed, and that it may take up to a month or possibly more to achieve full autoinduction. Conclusions: The time to achieve full rifampicin autoinduction has been underestimated. Our data, suggest that steady state is only achieved after at least one month. No conflict of interest

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Abstract: O_09 Influence of age and nutritional status on the pharmacokinetics of anti-tuberculosis drugs in children with tuberculosis G. Ramachandran1, A.K. Hemanth Kumar1, L. Sekar2, D. Vijayasekaran3, P.K. Bhavani4, V.V. Banurekha4, N. Poorana Ganga Devi4, N. Ravichandran5, S. Natarajarathinam6, S. Swaminathan4; 1Tuberculosis Research Center, Clinical Biochemistry & Pharmacology, Chennai, India; 2Tuberculosis Research Center, Statistics, Chennai, India; 3Institute of Child Health, Pulmonology, Chennai, India; 4Tuberculosis Research Center, Clinical Research, Chennai, India; 5Government Hospital of Thoracic Medicine, Pediatrics, Chennai, India; 6Government Rajaji Hospital, Pediatrics, Madurai, India Background: Tuberculosis (TB) is an important public health problem and among the 10 major causes of mortality in children. In the Revised National TB Control Program (RNTCP) in India, children diagnosed with TB receive first-line anti-TB drugs with dosages based on body weight. Currently recommended dosages of rifampicin (RMP), isoniazid (INH), pyrazinamide (PZA) and ethambutol are extrapolated from pharmaco-kinetic studies performed in adults and have not been adequately evaluated in children. Children exhibit age-related differences in drug pharmacokinetics because of enzyme maturation and other factors. The effect of malnutrition on blood levels of key anti-TB drugs is not well described. Aim: To study the impact of age and nutritional status on the pharmacokinetics of RMP, INH and PZA in children with TB Material & Methods: This is a multi-centric study being done at four hospitals in south India. Children with TB aged 1 to 12 years receiving anti-TB medications based on RNTCP guidelines for minimum 15 days were recruited to the study. Assessment of nutritional status was done using z scores calculated from the child's weight & height (CDC). On the day of the study, blood samples were collected pre-dosing and at 2, 4, 6 and 8 hours after supervised administration of anti-TB medications. Plasma RMP, INH and PZA were estimated by HPLC and pharmacokinetic variables calculated. Patients are being followed to determine clinical outcomes. Results: Forty children (17 males) have been recruited; the study is ongoing and interim

findings are presented. The mean (SD) age and body weight of the 40 children were 7.2 (3.1) years and 19.1 (7.0) kg respectively; 29 children had extrapulmonary forms of TB. The mean dose mg/kg of RMP and INH was 10.1mg, while that of PZA was 34.6mg. Children were distributed into four age groups, 1-3, 3.1-6, 6.1-9 & 9.1-12 years, and comparison of pharmacokinetics was done among the different groups. Children aged 1-3 years had significantly lower peak concentration and exposure of RMP, INH and PZA compared to the other age groups (p<0.05). Stunted children had significantly lower peak concentration and exposure of RMP and PZA than non-stunted children; p<0.05. Underweight children had significantly lower peak concentration and exposure of PZA; p<0.05. Peak concentration and exposure of INH were significantly higher in males than females; p<0.05. Peak concentration of RMP was sub-therapeutic (<8.0µg/ml) in 30 children. Peak concentration and exposure of RMP and PZA were significantly correlated with mg/kg dose administered (p<0.05) and with each other. Regression analysis showed stunting to be significantly associated with plasma exposure of RMP (p<0.05) after controlling for age and sex. Conclusions: Younger children and those with malnutrition have lower blood levels of key first-line anti-TB drugs. These interim findings have important clinical implications and suggest that the existing dosages of anti-TB drugs, especially in young children may have to be increased in order to achieve optimal blood levels. The impact of acetylator status and sex on blood levels as well as the correlation with TB treatment outcomes are being studied. No conflict of interest

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Abstract: O_10 Weight and sex are important determinants of antituberculosis drug concentrations in patients with HIV associated tuberculosis H. McIlleron1, R. Rustomjee2, M. Vahedi3, T. Mthiyane2, P. Denti1, C. Connolly2, W. Rida4, A. Pym2, P.J. Smith1, P. Onyebujoh3 1University of Cape Town, Division of Clinical Pharmacology, Cape Town, South Africa; 2South African Medical Research Council, TB Research Unit: Clinical and Biomedical, Durban, South Africa; 3World Health Organization, Special Programme for Research Training in Tropical Diseases, Geneva, Switzerland; 4Biostatistics Consultant, Arlington, Virginia, USA Introduction: There is concern that lower drug concentrations may, in part, account for the greater risk of tuberculosis recurrence and acquisition of drug resistance amongst patients with more advanced immune suppression. International guidelines recommend weight-adjusted dosing of the antituberculosis agents assuming a linear relationship between weight and optimal dose. We evaluated the pharmacokinetics of the 1st-line antituberculosis drugs in patients presenting with HIV associated tuberculosis who were dosed according to WHO guidelines (2, 3, 4 or 5 fixed dose combination tablets daily, each containing 150, 75, 400 and 275 mg of rifampicin, isoniazid, pyrazinamide and ethambutol, for patients weighing 30-37 kg, 38-54 kg, 55-70 kg, and >70 kg, respectively) . Patients with < 200 CD4+ lymphocytes/mm3, commenced efavirenz, lamivudine and zidovudine after 2 weeks of antituberculosis therapy, while patients with > 200 CD4+ lymphocytes/mm3 were randomized to the introduction of antiretroviral s after 2 weeks, or to delayed antiretroviral therapy. Materials & Methods: Sixty patients (with median [range] age 32 [18-47] years, weight 55.2 [34.4, 98.7] kg, CD4+ lymphocytes 252 [12, 500]/mm3) underwent intensive pharmacokinetic on the 1st, 8th, 15th and 29th days of antituberculosis treatment. Noncompartmental analysis was used to derive AUC0-12. Multilevel linear mixed-effects (MLME) regression analysis was used to examine the effects of age, sex, baseline weight, drug dose per kilogram of body weight (dose/kg) at baseline, baseline level of

immune suppression (CD4+ lymphocyte count), antituberculosis treatment schedule (5 vs. 7 days/week), concurrent administration of the first dose of antiretrovirals (day 15) and steady state antiretroviral exposure ( day 29) on the AUC0-12 of the respective antituberculosis drugs, and to compare AUC0-12 at day 8, day 15 and day 29 with the baseline AUC0-12 (day 1) adjusted for the other covariates, with random intercepts varying according to individual. Results: In spite of dosing according to weight bands, low weight was an independent risk factor for reduced drug concentrations. After a month of treatment each 10 kg of body weight was associated with a predicted AUC0-12 increase of 14.12% (95%CI: 7.49, 20.76), 14.12% (95%CI: -0.68, 31.12), 6.14% (95%CI: 2.70, 9.58) and 6.04% (95%CI: 0.80, 11.28) for rifampicin, isoniazid, pyrazinamide and ethambutol. The effect was statistically significant for all the drugs except isoniazid (p=0.062). Males had predicted reductions in AUC0-12 of 19.32% (95%CI: 3.55, 35.10) and 14.00% (95%CI: 5.58%, 22.41%) for rifampicin and pyrazinamide respectively. Model-based predictions estimated the rifampicin AUC0-12 to be approximately 2.4-fold higher in a female weighing 75 kg compared to a male weighing 35 kg. For isoniazid and pyrazinamide there was a 1.5-fold increase and for ethambutol, a 1.4-fold increase. Conclusions: While the level of immune suppression and concomitant antiretrovirals had little effect on the concentrations of the 1st-line antituberculosis agents, low weight and male sex were associated with an increased risk of low drug concentrations. Modifications to the standardized dosing approach should be considered. No conflict of interest

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Abstract: O_11 Population pharmacokinetics of rifampicin in childhood tuberculosis and the need for dose adjustment Simbarashe P. Zvada1, *, Ulrika S.H. Simonsson2, Paolo Denti1, Peter R Donald3, H Simon Schaaf3, Pete J. Smith1, Helen M .McIlleron1 1University of Cape Town, Medicine, Cape Town, South Africa; 2Uppsala University, Pharmaceutical Bioscences, Uppsala, Sweden; 3University of Stellenbosch, Paediatrics and Child Health, Cape Town, South Africa Introduction: Rifampicin is a key component of antituberculosis treatment. The aim of our study was to develop a population pharmacokinetic model describing rifampicin plasma concentrations in children and propose dose adjustments if necessary. Materials & Methods: We used data from a previously published study amongst 56 South African children (29 males and 27 females) hospitalized for the treatment of tuberculosis. They were treated with daily doses of rifampicin, isoniazid and pyrazinamide for 2 months before pyrazinamide was stopped. Dispersible fixed dose combination tablets were used. The children had venous blood sampled between 0.75 and 6 h after the dose at 1 month and 4 months after starting treatment. A total of 525 rifampicin concentration-time data points were analyzed using NONMEM VI. Various pharmacokinetic models were tested. Potential covariates included fat-free mass, age, sex, and human immunodeficiency virus status. Total body weight was included through allometric scaling on oral clearance and apparent volume of distribution. Exposures from recommended doses were simulated using the final model. Results: The median age for the children was 3.22 years (interquartile range: 1.58-5.38 years) and the median dosage was 9.56 mg/kg (interquartile range: 8.55-10.34 mg/kg). Twenty-two were human immunodeficiency virus infected and 18 had kwashiorkor (13 being females). The best model was a one-compartment model with a series of hypothetical transit compartments and first-order elimination rate constant, with sex as a covariate on apparent volume of distribution. Interindividual variability was supported on oral clearance while

interoccasional variability was significant on oral clearance, relative oral bioavailability and first-order transit absorption rate constant. We estimated typical values of 27.3 liters/hr/70kg for oral clearance and 81.4 liters/70kg for apparent volume of distribution, which are higher than the previously reported adult values. Females had an apparent volume of distribution 45.9 % higher than males. As 48 % of females and only 17 % of males had kwashiorkor, we repeated the analysis excluding all children with kwashiorkor and the volume of distribution in females was just 27.4 % higher than in males. This suggests that, in our dataset, kwashiorkor might in part account for the increased volume of distribution in females. Human immunodeficiency virus status had no influence on the pharmacokinetics of rifampicin. With a dose of 10 mg/kg, our model predicted median area under the curve (AUC) of 13.3, 15.8 and 18.8 mg·h/L in children weighing 5, 10, 20 kg, respectively. This is significantly lower than the median value of 38.8 mg·h/L (5th - 95th percentiles, 20.1-75.3 mg·h/L) for a typical adult weighing 55-70 kg and receiving a 600 mg dose. Even when the dose is increased to 15 mg/kg (as advocated in the draft 2010 WHO/IUATLD dosing guidelines), we estimate that 99.7 %, 97.7 % and 90 % of children weighing 5, 10, and 20 kg, respectively, would have AUC below the median adult value. Conclusions: Daily 10 mg/kg doses of rifampicin did not achieve same exposure in children as in adults. Even though a dose of 15 mg/kg provided improved exposure, our model suggests that young children are still under-dosed. No conflict of interest

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Abstract: O_12 A surprising effect of weight on ethambutol (EMB) pharmacokinetics (PK). Hall RG, Meek C, Swancutt M, G.T. Leff R Texas Tech University HSC UT Southwestern Medical Ctr, Dallas, Texas Background: EMB is utilized for the treatment of tuberculosis (TB) and Mycobacterium avium, with ideal body weight used to dose obese patients. We have demonstrated that TB failure rates increase as patient weight increases, likely because of lower drug concentrations in obese compared to leaner patients. This study evaluated the EMB PKs in normal and obese volunteers. Material & Methods: Adult volunteers with normal renal function were recruited; they were evenly distributed in body mass index groups of < 25, 25-39.9, and Ž 40 kg/m2. Each volunteer received a single fasting dose of 1600 mg of EMB by mouth, and blood collected at 6 time intervals over 24h. Serum samples were analyzed using LC-MS/MS. Population PKs were examined in ADAPT 5 using a 2 compartment model. Results: Thirteen completed the study; the weight range was 59 to 162 kg. The median age was 32 years. The median (range) EMB PK parameter estimates were ka of 0.45 (0.10-0.86), Vc of 355 (46-882) L, Vp of 658 (197-4953) L, k12 of 0.45 (0.10-1.89)/h, k21 of 0.28 (0.03-0.86)/h. The r2 for predicted versus observed concentrations was 0.91. Surprisingly, the ke decreased with weight at a slope of 0.003 L/h/kg (p=0.007), especially driven by the ? ke which had a slope of -0.013 L/h/kg (p = 0.006). Conclusions: The inverse correlation observed between the EMB ke and weight was unexpected and contrary to ? power law. A possible explanation is that the EMB PK profile is driven by distribution and redistribution and not metabolism, which differs between obese and leaner people. Thus, paradoxically, heavier people may be at higher risk of toxicity from the drug while thinner people may be at greater risk of therapeutic failure.

Abstract: O_13 Cumulative dose Amikacin predicts hearing loss in MDR-TB patients J.W.C. Alffenaar1, M. van der Meer1, J.G.W. Kosterink1, D. van Soolingen2, T.S. van der Werf3, R. van Altena4 1University Medical Center Groningen, Hospital and Clinical Pharmacy, Groningen, The Netherlands: 2National Institute of Public Health and the Environment, National Mycobacteria Reference Laboratory, Bilthoven, The Netherlands; 3University Medical Center Groningen, Pulmonary Diseases and Tuberculosis, Groningen, The Netherlands: 4University Medical Center Groningen, Tuberculosis Center Beatrixoord, Groningen, The Netherlands Introduction: Amikacin (AMK) is a second line, injectable drug for the treatment of tuberculosis (TB) and is administered in a dose of 15-30 mg/kg/day with a maximum of 1000 mg once daily to thrice weekly. Hearing loss and nephrotoxicity are the commonly observed adverse events and may aggravate with prolonged and higher dosage. In earlier studies the Cmax to MIC ratio of 10 was found the most relevant pharmacokinetic (PK) parameter to predict the efficacy of amino glycoside treatment. At the Tuberculosis Centre Beatrixoord AMK peak levels are monitored and the dose adjusted if needed. To evaluate the hearing loss in patients receiving TDM-guided dosing of AMK, we retrospectively evaluated medical charts of TB patients treated between in the period of 1998-2008. Materials & Methods: Patients receiving AMK as part of their TB treatment for at least 3 days were eligible for evaluation if at least a peak and trough level were recorded at steady state. Cmax was obtained 30 min after a one hour infusion and Cmin was obtained immediately before infusion. Samples were determined by fluorescence polarization immunoassay (TDx, Abbott laboratories). The Middlebrook 7H10 agar dilution method was applied for drug susceptibility testing (DST) of the isolates at the RIVM. Clinical data were extracted from the patients’ medical charts: age, sex, weight, length, ethnicity, co-morbidity, type of diagnosis, localisation of TB, MIC, DST results, dose and duration treatment, serum values (albumin, creatinine, ureum), adverse events (hearing loss and renal dysfunction). The Cmax/MIC ratio was calculated as efficacy predicting parameter. Data are presented as mean (range).

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Results: Seventy two patients with a mean age of 33.8 (13-74) years met the inclusion criteria. Although males (n=57) and females (n=35) received the same dose of 8.4 vs 8.8 mg/kg (P= 0.23), the C was higher for females (28.3; 18.9-43.0) than for males (24.6; 7.9-35.0) (P = 0.021). C was < 3 mg/L in all but one patient (6.8 mg/L). The mean MIC value for AMK was 2.0 (range 0.25-5.0). The mean C/MIC ratio was 26.3 (3-104). The Spearman correlation coefficient showed a significant correlation of R 0.27 (P = 0.022) between the C of AMK and body weight and a higher correlation using ideal body weight (R 0.47; P < 0.005). Hearing loss was observed in 13 patients. These patients received a higher mean cumulative dose of 50826 (1200-181857) (P= 0.023) mg during a shorter mean period of 93 (3-453) days (P=0.015) than patients that did not experience adverse effects [40488 (5400-64528) mg ; 187 (6-473) days]. Conclusions: In contrast with earlier studies only the cumulative dose and not treatment duration was correlated with hearing loss. TDM potentially contributed to the reduction in toxicity by using a lower dose/kg body weight for an adequate C/MIC ratio. However, this should be confirmed in a prospective study.

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3rd International workshop on Clinical Pharmacology of Tuberculosis Drugs

Abstracts Poster Presentations

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Abstract: P_01 Towards a guinea pig model for preclinical TB drug testing Z. Ahmad1, M.L. Pinn1, S. Tyagi1, C.A. Peloquin2, E.L. Nuermberger1, J. Grosset1, P.C. Karakousis1 1Johns Hopkins University School of Medicine, Medicine/ Infectious Diseases, Baltimore, USA; 2University of Florida, College of Pharmacy, Gainesville, USA Background: Recently, there has been significant interest to develop an alternative animal model to the mouse, which will more closely simulate the histopathology of human TB lesions, for preclinical TB drug screening. We compared the bactericidal and sterilizing activity of human-equivalent doses of the standard anti-TB regimen against acute and chronic TB infection in mice and guinea pigs. Materials & Methods: Pharmacokinetic studies were performed to determine human-equivalent doses of rifampin (R), isoniazid (H), and pyrazinamide (Z) in guinea pigs. The bactericidal and sterilizing activities of a predominantly twice-weekly regimen of RHZ were compared in guinea pigs and mice using acute and chronic infection models in both species. Post-treatment relapse rates were also assessed at various intervals. Results: Guinea pigs tolerated twice-weekly dosing with human-equivalent doses of RHZ (H 60 mg/kg, R 100 mg/kg, and Z 300 mg/kg). The bactericidal activity of RHZ was significantly greater in guinea pigs than in mice during both acute and chronic infection. Post-treatment relapse rates in guinea pig lungs were 0% (0/10) both after 5 months and after 6 months of treatment in the acute infection model, and 0% (0/10) after 3 months of therapy in the chronic infection model. In contrast, mouse lungs were still culture-positive after 6 months and 3 months of treatment in the acute and chronic infection models, respectively. Conclusions: Responses to equivalent doses of standard TB chemotherapy are significantly greater in guinea pigs than in mice, suggesting that caseation necrosis does not interfere with the sterilizing activity of these drugs. These results have important implications for the use of alternative animal models for preclinical screening of novel antituberculous drugs. No conflict of interest

Abstract: P_02 Weight not diabetes mellitus predicts failure of anti-tuberculosis therapy in treated tuberculosis patients with concurrent diabetes mellitus J. Pasipanodya1, T. Gumbo1, S. Weis2 1UT Southwestern Medical Center, Internal Medicine, Dallas, USA; 2University of North Texas Health Sciences Center at Fort Worth Internal Medicine, Fort Worth, USA Background: Patients with diabetes mellitus (DM) fail anti-tuberculosis therapy at higher rates than those without. These patients often have the metabolic syndrome, which consists of being overweight, DM, dyslipidemia and hypertension. We recently demonstrated that weight alone had a profound effect on failure of anti-tuberculosis therapy. We were interested in determining if the failure of therapy in DM was driven by the diabetes itself or high weight. Materials & Methods: We reviewed a computerized database of medical records of pulmonary TB patients seen between January 1986 and December 2007 in Tarrant County, Texas, USA. The outcomes measured were; time-to-smear and culture conversion and the corresponding 2-month conversion rates, treatment failure, relapse and death. The relationship between weight and outcome in TB-DM versus TB patients was examined using the fractal geometry 3/4 power law. Results: There were 117 TB patients with DM and 967 TB patients without DM in the study. Median time to culture conversion in diabetics was 37 days versus 31 days in those who did have diabetes mellitus. The hazard ratio was 1.23 (95% CI 1.03, 1.47). Similarly, sputum smear clearance was also slower in diabetics but it did not reach statistical significance; hazard ratio of 1.19 (0.96, 1.46). Proportions of patients converting smears and cultures at 2 months and at the end therapy were similar between TB-DM and TB only. The plots of (weight/weighttypical)

0.75 against the probability of relapse in TB-DM and TB patients and demonstrated that in both groups majority of the variability related to TB relapse for this cohort could be explained by changes in weight with an r2=0.99 for TB alone and r2=0.96 for TB-DM.

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Conclusion: An overwhelming proportion of failure of therapy in DM is explained by patient weight, not the DM per se. No conflict of interest

Abstract: P_03 Antituberculous activity of pyrazinamide in an animal model of caseous TB granulomas Z. Ahmad Parry1, M.L. Pinn1, S. Tyagi1, E.L. Nuermberger1, J.H. Grosset1, P.C. Karakousis1 1Johns Hopkins University School of Medicine, Department of Medicine 1550 Orleans Street Rm 123 CRB-11, Baltimore, USA Background: Pyrazinamide (Z) has unique sterilizing activity against M.tb in humans, allowed shortening of the TB treatment to 6 months. Older studies using lower Z doses have suggested that Z has no activity in the guinea pig model of TB infection on the basis of gross pathology. We hypothesized that Z has sterilizing activity against M.tb in guinea pigs, and evaluated reduction in M.tb colonies against a range of Z doses in a chronic infection characterized by caseating TB granulomas, like in humans. Methods: Z was tested over a range of doses ranging from 75.0-600.0 mg/kg, as well as in combination with rifampin (R) 50 mg/kg, in guinea pigs. Equivalent doses were used in mice. Treatment (5 times weekly) was initiated 28 days after low-dose aerosol infection with M. tuberculosis CDC1551. Animals were sacrificed after 4 weeks of therapy, and lungs were examined for gross pathology, histopathology and CFU enumeration. Results: Guinea pigs and mice were infected with 1.57±0.27 and 1.23±0.43 log10 CFU and at treatment initiation, had lung bacterial burdens of 6.41±0.18 and 5.71±0.28 log10 CFU, respectively. Isoniazid reduced lung CFU by ~1.0 log in both species. Over the same time period, Z given at ½ and ¼ of the human-equivalent doses was either inactive or moderately active in both species. At human-

equivalent dose, Z reduced the lung bacterial burden by ~1.0 log in both species. Doubling the dose resulted in a bacillary kill of 1.7 and 3.0 logs in mouse and guinea pig lungs, respectively. In both species, Z showed significant synergy with R, as in humans. At all doses tested, gross pathology was not significantly different among treated and untreated groups in both species. Conclusion: Z has strong antituberculous activity in guinea pig. As in humans and mice, Z exhibits strong synergy with R in guinea pigs. No conflict of interest

Abstract: P_04 Auc of ethambutol (emb) is not predicted by weight based dosing J.W.C. Alffenaar1, R. Ruslami2, J.G.W. Kosterink1, T.S. van der Werf3, R. van Crevel4, R.E. Aarnoutse5 1University Medical Center Groningen, Hospital and Clinical Pharmacy, Groningen, The Netherlands; 2Padjadjaran University, Pharmacology, Bandung, Indonesia; 3University Medical Center Groningen, Pulmonary diseases and Tuberculosis, Groningen, The Netherlands; 4Raboud University Medical Center, Internal Medicine, Nijmegen, The Netherlands; 5Raboud University Medical Center, Clinical Pharmacy, Nijmegen, The Netherlands Introduction: EMB is a first line anti-TB drug that is administered in a dose of 15 – 25 mg/kg/day. Toxicity is dose-dependent and occurs more frequently at doses over 25 mg/kg/day. The most important PK parameter for efficacy is the area under the time-concentration curve (AUC). EMB is a water soluble agent and fat tissue does not contribute to the volume of distribution. As the influence of weight and body composition (obese patients) may alter the pharmacokinetics of EMB, weight based dosing may result in increased toxicity if standard recommendations for dosing in mg/kg are followed. To improve the accuracy of weight based dosing in relation to AUC we tried to detect weight parameters that correlate the best with the AUC. Materials & Methods: Based on pharmacokinetic data from two earlier studies (PADI and HDR) we tested the correlation

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between AUC and mg/kg; mg/m2 for total (TBW) and ideal body weight (IBW). Results: Pharmacokinetic and demographic data of 81 patients, having received EMB in a dose of 750 mg, was analyzed. The mean age was 36 ± 11.5 yrs and mean BMI was 19.2 ± 2.9 kg/m2. A weak correlation was observed between AUC and mg/kg TBW (R=0.36; P=0.01), AUC and mg/m2 (R=0.44; P=<0.001) for total body weight. No correlation was found between AUC and mg/kg or mg/m2 (R=0.22; P=0.053[k1] ) for ideal body weight. Conclusion: Body weight or any other weight parameter showed only a weak correlation with the observed AUC values. This implies that new studies are needed to improve the EMB dosing algorithm to decrease toxicity and assure efficacy. No conflict of interest

Abstract: P_05 Pharmacokinetics of Rifapentine in TB patients receiving 600 mg daily C. Peloquin1, M. Weiner2, M. Engle2, J. Johnson3, P. Nsubuga4, E. Bliven5, N. Padayatchi6, T. Mthethwa6, W. MacKenzie5 1University of Florida College of Pharmacy, Infectious Disease Pharmacokinetics Lab, Gainesville, USA; 2University of Texas Health Science Center, Medical Service, San Antonio, USA: 3Case Western Reserve University School of Medicine, Department of Medicine, Cleveland, USA: 4Uganda-Case Western Reserve University Research Collaboration, ., Kampala, Uganda: 5Centers for Disease Control and Prevention, Tuberculosis Trials Consortium, Atlanta, USA; 6University of KwaZulu Natal School of Medicine, ., Durban, South Africa Background: Rifapentine (RPNT) is the cyclo-pentyl derivative of rifampin, and is used for the treatment of drug-susceptible tuberculosis (TB). Originally, it was approved in the USA at 600 mg twice-weekly in the initial phase, and once-weekly in the continuation phase of treatment. Recent work in a murine model of tuberculosis suggested that higher and more frequent doses of rifapentine result in more rapid bactericidal activity and stable cure. Currently, TBTC Study 29 evaluates 600 mg by DOT five times per week in the initial phase of treatment. A PK substudy includes 60 intensively sampled

patients from Africa and North America, who will form the core of the population PK model for the study. The first 21 of 30 African patients are presented here. Material & Methods: 21 patients (6 female), age 19 to 54 years (median 25) received 600 mg RPNT, or a median 11 mg/kg (range 9-14). The 21 patients were administered their reference PK dose of RPNT while fasting. At steady state, and following at least 3 consecutive daily doses, these patients had blood collected at 0,1,2,6,9,12 and 24 hours post dose. Data were analyzed using non-compartmental techniques (WinNonlin 5.2.1). Results: The median (range) RPNT Cmax was 10.57 mcg/ml (4.44-20.15, CV 41%), with a Tmax of 6 h (2-12, CV 34%) post dose. C24 was 4.96 mcg/ml (1.96-12.57, CV 52%). AUC0-24 was 179 mcg*h/ml (76-362, CV 43%). Parameters estimates: lambda z 0.036 1/h (0.016-0.060, CV 28%), t1/2 19.3 h (11.6-42.3, CV 33%), Vz/F 1.00 L/kg (0.54-1.88, CV 40%), and CL/F 0.033 L/h/kg (0.015-0.094, CV 53%). AUC0-24 co-varied with Cmax and C24 (R^2 0.97, 0.96). The median (range) desacetyl-RPNT Cmax was 8.54 mcg/ml (2.84-32.70, CV 66%) at 9 h (6-24, CV 46%) post dose. C24 was 6.56 mcg/ml (1.66-29.28, CV 81%). AUC0-24 was 161 mcg*h/ml (50-688, CV 71%). Cmax, AUC, Vz/F and CL/F did not vary significantly by age (over the 3.5 decade range), or weight. Females had somewhat larger Vz/F (1.23 vs. 0.86 L/kg), similar CL/F (0.033 vs. 0.034), and longer t1/2 (24.0 vs. 17.8 h). Median RPNT exposure was 41% of previously reported exposure with comparable daily dosage from a murine model of tuberculosis (PLoS Medicine, 2007 (4);e344), and 71% of exposure from TB patients from North Americans receiving once weekly 600 mg doses (TBTC Study 25, AUC0-inf 250 mcg*h/ml, Am J Respir Crit Care Med, 2004 (169);1191). Conclusion: As with rifampin, RPNT absorption and metabolism displayed moderate variability. Decreased RPNT blood concentrations were found compared to historical data of patients from North America. As current study doses were given daily, accumulation would have been expected given the median t1/2 of 19.3 h. Therefore, less complete absorption of RPNT in the current study population of African patients seems likely. No conflict of interest

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Abstract: P_06 The pharmacokinetics of Moxifloxacin in mdr TB contacts in the federated states of Micronesia C. Peloquin1, J. Jereb2, C. Daley3, D. Fred4, S. Mase2 1University of Florida College of Pharmacy, Infectious Disease Pharmacokinetics Lab, Gainesville, USA; 2Centers for Disease Control and Prevention, Division of Tuberculosis Elimination, Atlanta, USA: 3National Jewish Health, Department of Medicine, Denver, USA: 4TB/Leprosy Program, Chuuk, Federal States of Micronesia Background: In Chuuk, Federated States of Micronesia, we studied 32 persons with latent TB infection after exposure to multidrug-resistant tuberculosis (MDR TB). Between December 2007 and January 2009, these adolescent and adult patients were started on once daily moxifloxacin (Moxi) by directly observed therapy. Previous data suggested good absorption of Moxi by TB patients (Antimicrob Agents Chemother. 2008; 52: 852-7). The empiric Cmax target for TB is 3-5 mcg/ml. We investigated Moxi pharmacokinetics (PK) in this patient population. Material & Methods: 32 patients (9 female) age 14 to 62 years (median 30) received Moxi 400 mg, or 5.9 mg/kg (range 3.3-7.9); 16 also received ethambutol (EMB) median 1000 mg (800-1200 mg, or 15 mg/kg (10-18). Blood was collected after 362 days of treatment at 1, 2, and 6 hours post dose. Data were analyzed using non-compartmental techniques (WinNonlin 5.2.1). Results: The median Cmax was 4.37 mcg/ml (range 2.23-7.86, coefficient of variation CV 32%) and the Tmax was 1 h (1-6, CV 70%) post dose. AUC0-6 was 20.39 mcg*h/ml (4.47-36.07, CV 35%). PK parameter estimates had to be extrapolated from the 2 and 6 h data. Rough median estimates for lambda z and t1/2 are 0.08 1/h and 9.0 h. Some t1/2 estimates were unreasonably large, probably due to prolonged absorption during the sampling period. Hence, some AUC0-24 estimates appeared to be inflated, primarily due to the restricted sampling scheme. Cmax, AUC0-6, lambda z, and t1/2 did not vary with age. Cmax and AUC0-6 varied by weight (R^2 0.31, 0.29). PK parameters did not vary with sex or with EMB co-administration. Most

Cmax values, 25 of 32 (78%), exceeded the empiric target of 3 mcg/ml; 4 values exceeded 6 mcg/ml. Compared to the early bactericidal activity (EBA) study cited above, in which adults received 7 mg/kg in the fasted state, here the Moxi Cmax were somewhat lower and Tmax more variable. The current study patients were allowed to eat near the time of dosing. Conclusion: Moxi concentrations showed limited variability, with CV near 35%. The sampling scheme was well suited to the conditions at the study center, but did not allow for a good estimation of lambda z, t1/2, or clearance. Most patients achieved the empiric target serum concentrations of Moxi with once daily dosing. This potentially effective regimen is feasible in a resource poor setting. No conflict of interest

Abstract: P-07 The Pharmacokinetics of Levofloxacin in MDR TB Contacts in the Federated States of Micronesia C. Peloquin1, J. Jereb2, C. Daley3, D. Fred4, A. Loeffler5, S. Mase2 1University of Florida College of Pharmacy, Infectious Disease Pharmacokinetics Lab, Gainesville, USA: 2Centers for Disease Control and Prevention, Division of Tuberculosis Elimination, Atlanta, USA: 3National Jewish Health, Department of Medicine, Denver, USA: 4TB/Leprosy Program, ., Chuuk, Federal States of Micronesia: 5Francis J. Curry Center, ., San Francisco, USA Background: In Chuuk, Federated States of Micronesia, we studied 33 children with latent TB infection after exposure to multidrug-resistant tuberculosis (MDR TB). Between December 2007 and January 2009, these children received once daily levofloxacin (Levo) by directly observed therapy (DOT) for presumed MDR latent TB infection (LTBI). Previous pharmacokinetic (PK) studies of Levo suggested more rapid clearance (CL) in young children compared to adults, possibly requiring twice-daily dosing for certain pathogens (J Clin Pharmacol 2005; 45:153-60). The empiric Cmax target for once-daily Levo in TB patients is 8-12 mcg/ml, but definitive pharmacokinetic/ pharmacodynamic (PD) targets are lacking.

Abstracts 19


Therefore, we investigated Levo PK in this pediatric population. Material & Methods: 33 patients (16 female) age 1 to 14 years (median 8) received a median Levo dose of 250 mg (range 100-500) or 8.6 mg/kg (5-20); 15 also received ethambutol (EMB) 500 mg (200-700), or 17 mg/kg (11-37). Blood was collected after 362 days of treatment at 1, 2, and 6 hours post dose. Data were analyzed using non-compartmental techniques (WinNonlin 5.2.1) Results: The median Levo Cmax was 6.39 mcg/ml (range 1.97-17.03, coefficient of variation CV 47%) and the Tmax was 1 h (1-2, CV 35%) post dose. AUC0-6 was 25.43 mcg*h/ml (9.58-75.26, CV 49%); AUC0-24 was 39.81 mcg*h/ml (15.31-103.39, CV 54%). Initial parameters estimates based on 2 and 6 h samples were: elimination rate constant lambda z 0.19 1/h (0.05-0.28, CV 35%), elimination t1/2 3.70 h (2.44-14.29, CV 56%), Vz/F 1.21 L/Kg (0.54-3.27, CV 38%), and CL/F 0.21 L/h/Kg (0.07-0.48, CV 42%). The limited sampling scheme could have affected the accuracy of these estimates. Cmax and AUC0-6 varied with age (R^2 0.48, 0.52), whereas lambda z, t1/2 and CL did not. Cmax and AUC0-6 varied with weight less than with age (R^2 0.23, 0.18); lambda z and t1/2 did not vary with weight; CL was weight normalized. PK parameters did not vary with sex, or with the presence of EMB. AUC was highly correlated with Cmax, and 14 of 33 (42%) Cmax values exceeded the empiric target of 8 mcg/ml. Compared to a recent early bactericidal activity (EBA) study (Antimicrob Agents Chemother. 2008; 52: 852-7), in which adults received Levo 18 mg/kg in the fasted state, in these children the Levo Cmax and AUC values were lower, and t1/2 were shorter. The current study patients were allowed to eat near the time of dosing. Conclusion: Levo concentrations showed moderate variability, with CV near 50%. Only 14/33 children achieved the empiric target serum concentrations of Levo with once daily dosing. This potentially effective regimen is feasible in a resource poor setting. Higher doses should be studied. No conflict of interest

Abstract: P_08 Limited sampling strategies for therapeutic drug monitoring (tdm) of moxifloxaxcin (mfx) in patients with tuberculosis A.D. Pranger1, J.G.W. Kosterink1, R. van Altena2, R.E. Aarnoutse3, T.S. van der Werf4, D.R.A. Uges1, J.W.C. Alffenaar1 1University Medical Center Groningen, Hospital and Clinical Pharmacy, Groningen, The Netherlands; 2University Medical Center Groningen, Tuberculosis Center Beatrixoord, Groningen, The Netherlands; 3Radboud University Nijmegen Medical Center, Clinical Pharmacy, Nijmegen, The Netherlands: 4University Medical Center Groningen, Pulmonary Diseases & Tuberculosis, Groningen, The Netherlands Introduction: MFX is a powerful second-line agent with high in vitro and in vivo activity against Mycobacterium tuberculosis and is required in case of resistance or intolerance to first-line agents. The AUC0-24h to MIC ratio is the most predictive parameter for efficacy. To prevent treatment failure, TDM is recommended based on the inter-individual variability in AUC0-

24h values, the decrease in AUC0-24h values caused by concomitant treatment with rifampicin (RIF), and variable MIC values of 0.25-0.5 mg/L. As obtaining a full concentration-time curve of MFX for TDM is not feasible in clinical practice, the objective of this study is to develop a limited sampling procedure based on population pharmacokinetics. Material & Methods: Intensive pharmacokinetic sampling (t=0,1,2,4,8,12,24h) was performed in 22 TB patients who had used MFX until steady-state. Steady-state total plasma concentrations of MFX 400 mg once daily were determined using a validated LC/MS/MS method. A one-compartment population model with first-order absorption pharmacokinetics without lag time was generated using the MFX dose, the body surface area of the patients and the observed MFX plasma concentrations, using an iterative two-stage Bayesian procedure. The population pharmacokinetic model was cross validated by developing a model based on n-1. Limited sampling models were calculated using Monte Carlo data simulation (n=1000). The correlation between predicted MFX AUC0-24h and observed AUC0-24h was investigated by Bland-Altman analysis. The predictive performance of the final model was prospectively tested with MFX

Abstracts 20


profiles of TB patients receiving 400, 600 or 800 mg once daily. Results: The median AUC0-24h after 400 mg MFX was 24.8 (range: 8.45–72.2) mg*h/L which resulted in a geometric mean AUC0-24h/MIC ratio of 69.8 (12.7–320.5). The M. tuberculosis isolates had a median MIC of 0.25 (IQR: 0.23–0.5) mg/L. Population pharmacokinetic analysis, based on 22 pharmacokinetic profiles, resulted in a population pharmacokinetic model with the following parameters: Cl 18.46 ± 8.02 L/h/1.85 m2, Vd 3.15 ± 0.45 L/kg LBMc, Ka 0.95 ± 0.75 h-1 and F was fixed at 1. The cross validation showed that the median values of the model based on n-1 were Cl 18.39 (IQR: 18.29–18.78) L/h/1.85 m2, Vd 3.16 (IQR: 3.12–3.18) L/kg LBMc, Ka 0.95 (IQR: 0.92–0.99) h-1, which was not significantly different from the parameters of the population pharmacokinetic model (P = 0.23). After a Monte Carlo simulation (n=1000) the clinically most relevant model for prediction of MFX AUC0-24h used the MFX concentration 21 and 22 h post dose (r = 0.87, prediction bias = -2.7% and RMSE = 21%). The Bland-Altman analysis showed a good correlation between predicted and observed AUC0-24h values. The AUC0-24h of MFX of newly diagnosed TB patients showed a median difference of 0.8% for 400 mg (n=3), 7.5% for 600 mg (n=1) and 1.8% for 800 mg (n=5) between the observed and the predicted AUC0-24h. Conclusion: This study showed that MFX AUC0-

24h in TB patients could be predicted accurately using limited sampling and by the developed population pharmacokinetic model. The predicted AUC0-24h, based on two samples 21 and 22 h post dose, can be used to individualize treatment. No conflict of interest

Abstract: P_09 Clarithromycin significantly increases linezolid serum concentrations M.S. Bolhuis1, R. van Altena2, D.R.A. Uges1, T.S. van der Werf3, J.G.W. Kosterink1, J.W.C. Alffenaar1 1University Medical Center Groningen, Hospital and Clinical Pharmacy, Groningen, The Netherlands; 2University Medical Center Groningen, Tuberculosis Center Beatrixoord, Groningen, The Netherlands; 3University Medical Center Groningen, Pulmonary Diseases and Tuberculosis, Groningen, The Netherlands Introduction: Linezolid may be a promising drug for the treatment of multidrug resistant tuberculosis (MDR-TB), as it has high antibacterial activity against Mycobacterium tuberculosis. However, severe time- and dose dependant adverse effects such as anaemia, thrombocytopenia and peripheral and optical neuropathy limit its use. Case studies in non TB patients suggested that rifampicin may decrease linezolid serum concentrations by a P-glycoprotein mediated interaction. Clarithromycin is a potent inhibitor of P-glycoproteins and may therefore result in increased linezolid serum concentrations. As combined treatment of WHO class 5 drugs is advised to assure efficacy this drug-drug interaction between to class 5 drugs may occur in daily practice and its effect may be clinically relevant. Material & Methods: In our hospital we monitor linezolid serum levels on a regular basis in patients receiving linezolid for the treatment of MDR-TB using a validated method on LC/MS/MS. To evaluate this potential drug-drug interaction we reviewed the medical charts of tuberculosis patients treated at Tuberculosis Centre Beatrixoord, The Netherlands between the 1st of January 2008 and the 31st of May 2010, receiving Linezolid and Clarithromycin as a part of their TB treatment. Pharmacokinetic analysis was performed using MWPharm 3.60. Results: Three patients received 300mg linezolid twice daily combined with clarithromycin as part of their anti TB treatment. Considerably increased linezolid serum concentrations were observed and appeared to coincide with the start of clarithromycin. This resulted in an increase in AUC0-12h of 168, 250 and 370 percent after start of clarithromycin 250mg, 750 mg and 1000 mg once daily. We also observed a prolonged

Abstracts 21


absorption phase, with a mean Tmax of 4 hours. Other drug-drug interactions were not observed. No significant changes in liver- or renal function were observed that could have accounted for the sudden rise of linezolid serum concentrations. Based on these observations, the linezolid dose was decreased to 150mg twice a day. Conclusions: Based on our observations, a dose reduction of linezolid and therapeutic drug monitoring should be considered if linezolid is co-administered with clarithromycin in order to prevent potential linezolid induced toxicity. A prospective pharmacokinetic study may help to quantify the interaction that we describe. No conflict of interest

Abstract: P_10 Adverse side effects of Anti TB drugs among newly diagnosed PTB patients attending Mbagathi and Thika District Hospitals in Kenya Magu, D; Gichobi, D²; Mutugi, M², Wanzala, P²; Ndahi, L²; Ndegwa, D; Mutai. J² 1 Institute for Tropical Medicine and Infectious Diseases, Nairobi, Kenya; 2 Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya; 3 Kenya Medical Research Institute, Nairobi, Kenya Introduction: Globally, over 8 millions cases of PTB occur each year and 95% are in developing countries (A Gordon Leitch, 2000).TB affects human lungs although in 1/3 of cases in other organs are involved. It is caused by bacteria belonging to mycobacterium tuberculosis complex. Treatment within the 1st 2 months, isoniazid, rifampin and pyrazinamide are given, followed by isoniazid and rifampin for remaining 4 months (William A Petri Jr, 2001). The objective of the study will be to establish the association between anti TB drugs and adverse side effects among 100 newly diagnosed PTB patients enrolled in the study and selected from OPD chest medical clinic and chest wards at Mbagathi and Thika District Hospitals. Materials & Methods: Recruitment will be after prior consent of the subjects. Inclusion will be diagnosed PTB cases between 18 - 70 years.

Exclusion criteria will be patients suffering from liver, cardiac diseases, renal, diabetes, respiratory, HIV infections, pregnant or nursing women or patients with previous multiple drug resistance. A longitudinal study will be conducted up to 24 weeks and 12 follow up visits of patients will be taken with questionnaires administered. Materials used are disposable syringes, C.P. bottles, Kits for the liver function test, measurement of urea, creatinine. The selected patients will be divided into groups according to the drugs effects during the study period. Group1: Hepatitis. Group2: peripheral neuropathy. Group3: skin rashes. Group 4: Joint pains. Group5: Optic neuritis. Group6: Thrombocytopenia. Group7: Nephrotoxicity. Group 8: Ototoxicity. Observations of treatment: groups will be recorded on 0, 30 and 80th day. After pre-testing modifications will be made in the questionnaires. This will be a longitudinal study and data entry will be done in Microsoft Access cross-checked, cleaned for entry error and analyzed using Statistical Package for Social Sciences (SPSS) version 12.0. Results: Descriptive statistics will be done for different groups and frequencies and percentages to present categorical variables. Using Multivariate and univariate analysis for comparisons, variables with p < 0.05 in the logistic regression will have a significant association with group’s drug effects. Conclusion: Major side effects give rise to health hazards, chemotherapy should be stopped or interrupted if severe drug intolerance or toxicity occurs. TB drugs are toxic and mild side effects are common but most do not warrant drug withdrawal. Recommendation to the policy makers will be formulated based on the findings of the study for the optimization of TB therapy.

Abstracts 22


Abstract: P_11 Steady state gatifloxacin pharmacokinetics in adult tuberculosis patients W. Smythe1, C. Merle2, R. Rustomjee3, M. Gninafon4, M. Bocar5, O.B. Sow6, P. Olliaro7, C. Lienhardt8, J. Horton9, P. Smith1, U.S.H. Simonsson10, H. McIlleron1 1University of Cape Town, Division of Clinical Pharmacology Department of Medicine, Cape Town, South Africa; 2London School of Hygiene & Tropical Medicine, Epidemiology & Population Health Tropical Epidemiological Group, London, United Kingdom; 3Medical Research Council, Clinical and Biomedical TB Research, Durban, South Africa; 4Programme National de Lutte contre la Tuberculose, BP 817, Cotonou, Benin; 5Programme National de Lutte contre la Tuberculose, BP 5899, Dakar-Fann, Senegal; 6Service Pneumo-Phtisiologie, CHU Ignace Deen BP 634, Conakry, Guinea Conakry; 7World Health Organization, World Health Organization, Geneva, Switzerland; 8Institut de recherché pour le Développement, IRD, Paris, France; 9TDR, Tropical Projects, Hitchin, United Kingdom; 10Uppsala University, Department of Pharmaceutical Biosciences, Uppsala, Sweden Background: The potential for shortening of standardized first-line antituberculosis treatment by substitution of ethambutol with gatifloxacin is currently being evaluated. A single dose cross-over study in healthy normal volunteers suggested that the combination of rifampicin, isoniazid and pyrazinamide reduced the elimination rate of concomitantly administered gatifloxacin such that the area under the concentration time curve (AUC) from 0 hours to infinity was increased by approximately 15%. These findings raise concern that pharmacokinetic interactions may lead to a risk of increased exposure to gatifloxacin when daily doses are given in combination with rifampicin, isoniazid and pyrazinamide in the treatment of tuberculosis. This study aims to describe the pharmacokinetic drug interactions in adults with tuberculosis during the first month of treatment with daily doses of gatifloxacin in combination with rifampicin, isoniazid and pyrazinamide using a population pharmacokinetic model. Materials & Methods: Adults (n=170) with pulmonary tuberculosis received once daily doses of 400 mg gatifloxacin together with rifampicin, isoniazid and pyrazinamide for 6 days of the week. Three blood samples per patient were taken after the first dose (pre-steady state) and sampling was repeated after approximately 28 days (steady state) yielding a total of 927 plasma gatifloxacin concentrations. A

pharmacokinetic model incorporating separate typical values of gatifloxacin clearance at single dose and steady state occasion was developed using the first order conditional method with interaction in NONMEM VI. Results: After approximately 1 month of multiple concomitant dosing of gatifloxacin, rifampicin, isoniazid and pyrazinamide an unexpected increase in gatifloxacin clearance of approximately 40 % was observed. Conclusions: After repeated daily doses of gatifloxacin together with rifampicin, isoniazid and pyrazinamide for approximately 1 month, gatifloxacin clearance was increased. Thus the reduced elimination of gatifloxacin after administration with single doses of rifampicin, isoniazid and pyrazinamide (as reported previously) is apparently compensated for by increased clearance after repeated doses. No conflict of interest

Abstract: P_12 Rapid clinical improvement after start of HRZE given in divided doses after meals G. Warmelink1, R. van Altena1, W.C.M. de Lange1, N.H.T. ten Hacken2, H.A.M. Kerstjens2, T.S. van der Werf2, J.W.C. Alffenaar3 1University Medical Center Groningen, Tuberculosis Center Beatrixoord, Groningen, The Netherlands; 2University Medical Center Groningen, Pulmonary Diseases and Tuberculosis, Groningen, The Netherlands; 3University Medical Center Groningen, Hospital and Clinical Pharmacy, Groningen, The Netherlands Introduction: Although product leaflets advise to take HRZE on an empty stomach to maximize absorption, gastrointestinal intolerance is frequently observed. These adverse effects occur particularly during the first weeks of treatment and may place patients at risk for drug-induced hepatotoxicity, non-compliance and reduced food intake. International guidelines state that in case of gastro-intestinal side effects, the drugs should be taken with meals to reduce gastrointestinal complaints. In our TB center the usual care is to administer HRZE after meals to prevent gastrointestinal intolerance without compromising TB treatment.

Abstracts 23


Materials & Methods: We conducted a retrospective medical record review of 40 patients who were admitted and started TB treatment in our TB Center from April 2009 to July 2010. The standard treatment regimen was used for all patients and isoniazid and rifampicin were administered after breakfast; ethambutol after lunch; and pyrazinamid after the evening meal. Differences in symptoms before and after start of TB treatment were evaluated using the McNemar test. Differences in weight at start and after two weeks of taking anti-TB drugs were evaluated using the Wilcoxon signed-rank test. Drug-induced hepatotoxicity necessitating interruption of anti-TB drugs was classified as failure of the divided dosing scheme. Successful treatment was based on culture conversion; by lack of culture results, treatment success was based on significant clinical and radiological improvement. Results: After 8.2 ± 5.9 days of TB treatment patients were interviewed by the dietitian, using a semi-structured questionnaire, about experienced symptoms before start of TB treatment, and current symptoms present at the day of interview. Diarrhoea before start of TB treatment was reported by 22.5% patients and by none during early treatment (p=0.004). Vomiting before start of TB treatment was reported by 27.5% patients and in 2.5% during early treatment, (p=0.001). Coughing during untreated TB was reported by 27.5% patients and in 20.0% during early treatment, (p=0.001). A mean weight gain of 1.6 ± 1.4 kg (p=0.000) was observed in the first two weeks of treatment. All patients were treated successfully and discharged for further outpatient treatment after 50.2 ± 42.0 days of admission. In none of the study subjects, TB treatment had to be interrupted for hepatotoxicity. Conclusions: Administration of anti-TB drugs after meals resulted in a reduction of gastrointestinal symptoms. Short-term clinical improvement was noted by reduction of coughing and weight gain. A blinded randomized placebo controlled study therefore is urgently needed to assess the effect of intake of anti-TB drugs after meals on gastrointestinal symptoms and clinical performance during early TB treatment. Pharmacokinetics of anti-TB drugs have to be performed to assure adequate drug exposure if the drugs are administered after meals. No conflict of interest

Abstract: P_13 Population pharmacokinetics of pyrazinamide in healthy volunteers and tuberculosis patients in Thailand G. Davies1,2, N. Cheirakul3, N. Saguenwong4, A. Chaiprasert3, C. Chuchuttaworn5, B. Eompokalap4, S. Likanonsakul4, R. Hartkoorn1, Owen A1, N. White2, D. Back1, S. Khoo1 1. Department of Pharmacology and Therapeutics, University of Liverpool, UK; 2. Wellcome -Mahidol- Oxford Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 3. Siriraj Hospital, Bangkok, Thailand, 4. Bamrasnaradura Infectious Diseases Institute, Nonthaburi, Thailand, 5. Central Chest Hospital, Nonthaburi, Thailand Background: Pyrazinamide (PZA) is a key sterilizing drug in modern short-course chemotherapy for tuberculosis. While it has the most robust pharmacokinetics (PK) among first-line agents, its disposition, metabolism and pharmacodynamics are perhaps the least well-understood. Materials & Methods: A two-phase factorial study of the effects of HIV infection and TB disease on the PK and pharmacodynamics (PD) of first-line anti-TB drugs in a WHO pre-qualified fixed dose combination (Rimstar 4-FDC),dosed according to weight, was conducted in Bangkok, Thailand. Healthy volunteers ( 20 HIV+ and 20 HIV- ) and patients with tuberculosis ( 28 HIV+ and 29 HIV-) were sampled at 0, 30/60/90, 120, 240 and 660 minutes on day 1 and the latter underwent repeated similar sampling on day 15. PZA was assayed by HPLC with an isocratic mobile phase and acetazolamide as internal standard, using a HyPURITY C18 column and UV detection at 268 nm. The assay limit of detection was 1 mg/ml with lower and upper limits of quantification 2.5 mg/ml and 80 mg/ml. Population pharmacokinetic analysis employed NONMEM VI version 2, installed/validated with nmqual and embedded into R version version 2.8.1 with the library MIfuns. The first order conditional estimation estimation method with ?-? interaction was used. Models were compared on the objective function value, likelihood ratio test and size of predicted standard errors. Covariate screening was done using visual display of the BLUE random effects and univariate fitting to the basic model. Model checking involved examination of conditional weighted residual plots, log-likelihood profiling of

Abstracts 24


parameters of interest and visual predictive check. Standard errors for the final model were obtained by non-parametric bootstrap. Results: The data were well-described by a multiple-dosing one compartment model with proportional and additive components for residual within-subject error and log-normal random effects for all parameters allowing for covariance between volume of distribution (V/F) and clearance (Cl/F). Inter-occasion variability of parameters was not supported. Variability in absorption was high and a number of alternative models were compared for describing the absorption process including combined zero- and first-order processes, normal mixtures of first order processes and transit models. A sequential zero- and first order model was preferred, with a duration of zero-order input of 0.36h, and the first order component of significantly affected by HIV infection (Ka 3.25 vs 1.86 hr-1). Weight strongly predicted V/F (28.3 L + 0.381 L kg-1) and V/F was 30.5% higher in males. The presence of tuberculosis disease did not affect population mean Cl/F (2.24 L hr-1) but significantly increased the variability of this parameter. Simulation-based diagnostics suggested adequate internal validation of the model. Conclusions: Parameter estimates from this population PK analysis are broadly in agreement with those previously published. However, the effect of HIV on absorption and the increased variability of clearance in patients with tuberculosis have not been previously evaluated directly in a dataset comprising both patients and healthy volunteers. These disease effects may be of clinical relevance and warrant further investigation with regard to their metabolic determinants and possible relation to PZA toxicity.

Abstracts 25

Author Index Author Abstract Title Abstract # Page # Ahmad Parry, Z.

The anti-tuberculous activity of pyrazinamide is driven by AUC/MIC in murine tuberculosis

O_01

3

Ahmad Parry, Z.

PA-824 exhibits time-dependent bactericidal activity against persisting Mycobacterium tuberculosis

O_03

4

Ahmad Parry, Z.

Antituberculous activity of pyrazinamide in an animal model of caseous TB granulomas

P_03

16

Alffenaar, J. W.

Cumulative dose Amikacin predicts hearing loss in MDR-TB patients

O_13

12

Alffenaar, J. W.

Auc of ethambutol (emb) is not predicted by weight based dosing

P_04

16

Alffenaar, J. W.

Limited sampling strategies for therapeutic drug monitoring (tdm) of moxifloxaxcin (mfx) in patients with tuberculosis

P_08

19

Alffenaar, J. W.

Clarithromycin significantly increases linezolid serum concentrations

P_09

20

Alffenaar, J. W.

Rapid clinical improvement after start of HRZE given in divided doses after meals

P_12

22

Chigutsa, E.

Population pharmacokinetics of rifampicin in south african tuberculosis patients and the influence of drug transporter polymorphisms

O_07

7

Dartois, V.

The penetration of fluoroquinolones in pulmonary lesions of TB infected rabbits

O_05

5

Davies, G. R

Population pharmacokinetics of pyrazinamide in healthy volunteers and tuberculosis patients in Thailand

P_13

23

de Steenwinkel, J.

Time-kill kinetics of anti-tuberculosis drugs, and emergence of resistance, in relation to metabolic activity of Mycobacterium tuberculosis

O_02

3

Denti, P.

A population pharmacokinetic model for rifampicin auto-induction

O_08

8

Gumbo, T.

A surprising effect of weight on ethambutol (EMB) pharmacokinetics (PK).

O_12

12

Karakousis, P.

Towards a guinea pig model for preclinical TB drug testing

P_01

15

Kumar, V.

Population PK-PD modeling of Phase 1 data of PNU-100480 to guide doses for Phase 2a study

O_04

4

Magu, D.

Adverse side effects of Anti TB drugs among newly diagnosed PTB patients attending Mbagathi and Thika District Hospitals in Keny

P_10

21

McIlleron, H.

Weight and sex are important determinants of antituberculosis drug concentrations in patients with HIV associated tuberculosis

O_10

10

Pasipanodya, J.

Weight not diabetes mellitus predicts failure of anti-tuberculosis therapy in treated tuberculosis patients with concurrent diabetes mellitus

P_02

15

Peloquin, C.

Pharmacokinetics of Rifapentine in TB patients receiving 600 mg daily

P_05

17

Peloquin, C.

The pharmacokinetics of Moxifloxacin in mdr TB contacts in the federated states of Micronesia

P_06

18

Peloquin, C.

The Pharmacokinetics of Levofloxacin in MDR TB Contacts in the Federated States of Micronesia

P_07

18

Ramachandran, G.

Influence of age and nutritional status on the pharmacokinetics of anti-tuberculosis drugs in children with tuberculosis

O_09

9

Smythe, W.

Steady state gatifloxacin pharmacokinetics in adult tuberculosis patients

P_11

22

Srivastava, S.

Treatment failure and emergence of resistance during non-compliance in an experimental tb model.

O_06

6

Zvada, S.

Population pharmacokinetics of rifampicin in childhood tuberculosis and the need for dose adjustment

O_11

11

rd 3 International Workshop on Clinical Pharmacology of Tuberculosis Drugs – 11 Sep. 2010 – Boston MA, USA

Program & Abstractswww.virology-education.com

Biltstraat 1063572 BJ Utrechtthe Netherlandswww.virology-education.com

11 September 2010

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