Protein Binding of Rifapentine and Its 25-Desacetyl Metabolite in Patients with Pulmonary Tuberculosis Eric F. Egelund, Marc Weiner, Rajendra P. Singh, Thomas J. Prihoda, Jonathon A. L. Gelfond, Hartmut Derendorf, William R. Mac Kenzie and Charles A. Peloquin Antimicrob. Agents Chemother. 2014, 58(8):4904. DOI: 10.1128/AAC.01730-13. Published Ahead of Print 19 May 2014. These include: SUPPLEMENTAL MATERIAL REFERENCES CONTENT ALERTS Supplemental material This article cites 14 articles, 4 of which can be accessed free at: http://aac.asm.org/content/58/8/4904#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Downloaded from http://aac.asm.org/ on August 12, 2014 by Univ of Florida Updated information and services can be found at: http://aac.asm.org/content/58/8/4904 Protein Binding of Rifapentine and Its 25-Desacetyl Metabolite in Patients with Pulmonary Tuberculosis Eric F. Egelund,a Marc Weiner,b Rajendra P. Singh,c Thomas J. Prihoda,d Jonathon A. L. Gelfond,e Hartmut Derendorf,a William R. Mac Kenzie,f Charles A. Peloquina College of Pharmacy, University of Florida, Gainesville, Florida, USAa; Departments of Medicine, University of Texas Health Science Center San Antonio and South Texas Veterans Health Care System, San Antonio, Texas, USAb; Clinical Pharmacology Modeling and Simulation, GlaxoSmithKline, King of Prussia, Pennsylvania, USAc; Department of Pathology, University of Texas Health Science Center, San Antonio, Texas, USAd; Departments of Epidemiology and Biostatistics, University of Texas Health Science Center, San Antonio, Texas, USAe; Division of Tuberculosis Elimination, Centers for Disease Control and Prevention, Atlanta, Georgia, USAf T he microbiologically active portion of the total concentration of antibiotic is the fraction not bound to plasma proteins. It is also this free fraction that is distributed into the extravascular space (1). In healthy volunteers, rifapentine and its microbially active 25-desacetyl metabolite have been reported to be 98% and 93% protein bound in healthy volunteers, respectively (2, 3). However, rifapentine protein binding has not been determined in patients with active tuberculosis, an inflammatory state, or malnutrition. The MIC of rifapentine is 0.03 to 0.06 ␮g/ml and of 25-desacetyl-rifapentine is 0.125 to 0.25 ␮g/ml against susceptible strains (4). Based on in vitro studies, up to 38% of the activity of rifapentine is based on the metabolite (3). In a prior pharmacokinetic study, lower rifampin total concentrations were found among African patients of black race than among non-African other patients with tuberculosis (5). In the present study, we determined the free, active fractions and concentrations of rifapentine and its metabolite in 41 patients with tuberculosis. Blood samples were obtained for pharmacokinetic analyses in a phase 2 trial that compared the safety of daily rifapentine (10 mg/kg of body weight/dose) to that of rifampin (approximately 10 mg/kg/dose) together with isoniazid, pyrazinamide, and ethambutol (6). A pharmacokinetic study, a component of the tuberculosis treatment trial, was conducted (7). For the present study, a convenience sample of patients in the pharmacokinetic study group was selected who had an adequate volume of blood in pharmacokinetic samples and who also underwent intensive pharmacokinetic sampling. For intensive pharmacokinetic sampling, blood was drawn into heparin-containing tubes at 7 time points (baseline and 1, 2, 6, 9, 12, and 24 h after drug ingestion) and plasma was frozen at ⫺80°C, shipped on dry ice to the analysis laboratory, and stored at ⫺80°C until assayed. In this exploratory research to develop a technique to evaluate free fractions of rifapentine, samples were selected that corresponded to times of peak (Cmax) and trough (C24) concentrations of rifapentine. Rifapentine and 25-desacetyl rifapentine total plasma concentrations were determined by a validated high-pressure liquid chromatography (HPLC) method (8). For free drug concentration determinations, plasma samples were thawed at room temperature and placed in a water bath at 37°C for 30 min. A volume of 1 ml of 4904 aac.asm.org sample was transferred to a Centrifree YM-30 Millipore tube with a molecular mass cutoff of 30,000 Da. The samples were then centrifuged for 25 min at 37 ⫾ 5°C at 1,799 ⫻ g. An API 4000 LC-tandem mass spectrometry (LC/MS/MS) system was used to measure free drug concentrations. Each analysis consisted of a 9-point calibration curve, at least 5 quality controls, and the samples to be analyzed. The lower limit of quantification for both rifapentine and metabolite was 3.13 ng/ml. The calibration curve ranged from 3.13 ng/ml to 800 ng/ml. Rifampin was used as an internal standard. The HPLC autosampler was set to 15-␮l injections and connected to the API 4000 mass spectrometer. Chromatograms were analyzed with Analyst Software 1.4.2 for LC/MS/MS. Patient covariates potentially associated with free drug concentration were evaluated by univariate analysis and combined into a repeated-measure analysis of covariance (ANCOVA) model with multiple covariates using backward elimination (Tables 1, 2, 3, and 4). The ANCOVA model included classification by region (African and non-African; all of the African patients were of the black race, and the non-African patients were classified by country of enrollment), study site (Africans from 2 sites and non-Africans from 5 North American sites), race (black versus others), assay batch number (n ⫽ 4), other demographic data (age, sex, and ethnicity), weight, body mass index (BMI), HIV infection status, drug dose, administration of drug with or without food, albumin concentration, alanine aminotransferase level, ratio of the C-reactive protein (CRP) level to the upper of limit of normal for each reference laboratory, time on treatment at the time of pharmaco- Antimicrobial Agents and Chemotherapy Received 13 August 2013 Returned for modification 18 December 2013 Accepted 12 May 2014 Published ahead of print 19 May 2014 Address correspondence to Marc Weiner, weiner@uthscsa.edu. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.01730-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.01730-13 p. 4904 – 4910 August 2014 Volume 58 Number 8 Downloaded from http://aac.asm.org/ on August 12, 2014 by Univ of Florida Rifapentine is highly protein bound in blood, but the free, unbound drug is the microbiologically active fraction. In this exploratory study, we characterized the free plasma fraction of rifapentine in 41 patients with tuberculosis. We found a lower total rifapentine concentration but significantly higher free rifapentine levels in African patients of black race compared to non-Africans. These data support larger pharmacokinetic/pharmacodynamic studies to confirm these findings and assess free rifapentine in relation to microbiological and clinical outcomes. Protein Binding of Rifapentine in TB Patients TABLE 1 Percent free rifapentine adjusted for other significant factors and covariatesa Factor in the final ANCOVA model n Region and race A: African of black race B: non-African others 20 21 Batch effect C: Batch 1 D: Batch 2 E: Batch 3 F: Batch 4 F value (df1, df2)b Valuesc P value(s) for comparison of groupsd 17.86 (1, 33) 1.02 (0.88, 1.18) 0.65 (0.56, 0.74) 0.0002 8 6 14 13 16.19 (3, 33) 0.59 (0.49, 0.71) 0.68 (0.54, 0.85) 1.29 (1.11, 1.50) 0.84 (0.72, 0.98) Rifapentine, mean total concn (ln), slope 41 12.07 (1, 33) ⫺0.32 (0.09) 0.002 BMI, slope (SE) 41 4.95 (1, 33) ⫺0.03 (0.01) 0.03 Albumin, slope (SE) 41 7.03 (1, 33) ⫺0.27 (0.10) 0.01 ⬍0.0001 0.32 (C vs D), ⬍0.0001 (C vs E), 0.0084 (C vs F), ⬍0.0001 (D vs E), 0.13 (D vs F), 0.0005 (E vs F) kinetic sampling, mean of Cmax and trough total rifapentine drug concentration, and a repeated measure of timing (nominal) at Cmax and trough concentration (24 h after drug administration). A similar analysis was performed for the 25-desacetyl metabolite. Model terms with P ⱕ 0.05 were retained in the final model. Because the variable of African patients of black race versus other patients was significant in the multivariate analyses, the baseline demographic and clinical characteristics of these significant groups were tabulated for comparisons between groups by the Wilcoxon rank sum test for comparing medians and the Student t test for comparing means. A total of 94 samples from 43 patients were available for anal- TABLE 2 Free rifapentine concentration adjusted for other significant factors and covariatesa Factor in the final ANCOVA model n F value (df1, df2)b Valuesc P value(s) for comparison of groupsd Region and race A: African of black race B: non-African others 20 21 17.78 (1, 33) 0.091 (0.078, 0.105) 0.058 (0.050, 0.066) 0.0002 Batch effect C: Batch 1 D: Batch 2 E: Batch 3 F: Batch 4 8 6 14 13 16.28 (3, 33) 0.053 (0.043, 0.064) 0.061 (0.049, 0.076) 0.115 (0.099, 0.133) 0.075 (0.064, 0.088) 41 54.50 (1, 33) 0.67 (0.09) ⬍0.0001 Timing, sample at: Cmax Troughe 41 41 153.4 (1, 40) 0.106 (0.095, 0.118) 0.049 (0.044, 0.055) ⬍0.0001 Albumin, slope (SE) 41 7.03 (1, 33) ⫺0.27 (0.10)e 0.01 BMI, slope (SE) 41 5.01 (1, 33) ⫺0.03 (0.01)e 0.03 Rifapentine, mean total concn (ln), slope ⬍0.0001 0.30 (C vs D), ⬍0.0001 (C vs E), 0.008 (C vs F), ⬍0.0001 (D vs E), 0.14 (D vs F), 0.0004 (E vs F) n ⫽ 41 patients; estimates of the free rifapentine concentration by ANCOVA of data transformed to ln, adjusted for other factors in the final model, and back transformed to the original scale. b Degrees of freedom for the numerator (df1) and degrees of freedom for the denominator (df2). c Values represent adjusted mean (95% CI) free rifapentine concentrations (␮g/ml) unless otherwise indicated. Slope (standard error [SE]) data are for a 1-unit change in ln or the raw value (as labeled) of the parameter of ln of the response. d Data represent P values determined by the Fisher least-significant-difference test for pairwise comparisons of data groups. e Trough, sample at 24 h after drug administration. a August 2014 Volume 58 Number 8 aac.asm.org 4905 Downloaded from http://aac.asm.org/ on August 12, 2014 by Univ of Florida n ⫽ 41 patients; estimates of percent free rifapentine by ANCOVA of data transformed to ln, adjusted for other factors in the final model, and transformed back to the original scale. Abbreviations: ANCOVA, analysis of covariance; BMI, body mass index; CI, confidence interval; ln, logarithm to the base e. b Data represent degrees of freedom for the numerator (df1) and degrees of freedom for the denominator (df2). c Values represent adjusted mean percentages (95% CI) of free rifapentine unless otherwise indicated. Slope (standard error [SE]) data are for a 1-unit change in ln or the raw value (as labeled) of the parameter of ln of the response. d Data represent P values determined by the Fisher least-significant-difference test for pairwise comparisons of data groups. a Egelund et al. TABLE 3 Percent free desacetyl rifapentine adjusted for other significant factorsa Factor in the final model by analysis of covariance n F value (df1, df2)b Adjusted mean % (95% CI) of free desacetyl rifapentine Region and race A: African of black race B: non-African others 20 21 7.81 (1, 36) 4.47 (3.56, 5.61) 2.88 (2.30, 3.59) Batch effect C: batch 1 D: batch 2 E: batch 3 F: batch 4 8 6 14 13 16.61 (3, 36) 2.80 (2.01, 3.91) 5.43 (3.70, 7.99) 6.22 (4.79, 8.08) 1.75 (1.33, 2.30) P value(s) for comparison of groupsc 0.0083 ⬍0.0001 0.01 (C vs D), 0.0005 (C vs E), 0.03 (C vs F), 0.56 (D vs E), ⬍0.0001 (D vs F), ⬍0.0001 (E vs F) a yses and were analyzed in 4 batches. Two patients were eliminated from the analyses because their samples were in a semisolid gel state after thawing at room temperature. In batch 1, samples from 3 time points per patient from 8 patients were assessed for total rifapentine Cmax and trough concentration and a third sample after the Cmax (“nonpeak”) (see Table S4 in the supplemental material). After a matched-pairs analysis of Cmax and nonpeak samples, the 3 subsequent batches analyzed only Cmax and trough concentration rifapentine samples. Patients were predominately male (n ⫽ 30), and the median age was 31 years (Table 5). All patients from Africa were of black race. The median weight of African patients was 54 kg versus 66 kg for non-Africans (P ⬍ 0.05), while albumin levels were not significantly different between groups. The geometric mean (GM) Cmax values for total rifapentine were 9.87 ␮g/ml and 16.13 ␮g/ml for Africans and non-Africans, respectively (Table 6 and Fig. 1 and 2). The GM (95% confidence interval [CI]) percent free (non-protein-bound fraction of the total) rifapentine values for Africans and non-Africans were 1.39% (1.10, 1.76) and 0.59% (0.50, 0.70), respectively. The GM free rifapentine Cmax values for Africans and non-Africans were 0.14 ␮g/ml and 0.10 ␮g/ml. The GM Cmax values for total 25-desacetyl rifapentine were 8.03 ␮g/ml and 11.82 ␮g/ml for Africans and non-Africans. The GM percent free metabolite values in Africans and non-Africans were 6.23% (4.78, 8.55) and 2.44% (1.82, 3.08). The GM Cmax values for free, unbound 25-desacetyl rifapentine in Africans and non-Africans were 0.42 ␮g/ml and 0.28 ␮g/ml. The percent free rifapentine was significantly greater for Africans of black race than for non-African patients when adjusted for other significant variables by ANCOVA. The percent free rifapentine was inversely associated with mean total rifapentine concentration, albumin concentration, and BMI (Table 1). The mean values of percent free rifapentine adjusted for all other significant variables were significantly different among the assay batches (Table 1; see also Fig. S1 in the supplemental material). The adjusted free mean rifapentine concentration by ANCOVA was TABLE 4 Free 25-desacetyl rifapentine concentration adjusted for other significant factors and covariatesa Factor in the final ANCOVA model n F value (df1, df2)b Valuesc P value(s) for comparison of groupsd Region and race A: African of black race B: non-African others 20 21 5.01 (1, 35) 0.37 (0.29, 0.47) 0.26 (0.20, 0.32) 0.03 Batch effect C: batch 1 D: batch 2 E: batch 3 F: batch 4 8 6 14 13 17.03 (3, 35) 0.24 (0.17, 0.33) 0.47 (0.32, 0.69) 0.54 (0.42, 0.70) 0.15 (0.11, 0.20) Timing, sample at: Cmax Troughe 41 41 4.36 (1, 40) 0.35 (0.29, 0.43) 0.27 (0.22, 0.33) 0.04 41 36.53 (1, 36) 0.84 (0.12) ⬍0.0001 Desacetyl rifapentine, mean total concn (ln), slope ⬍0.0001 0.009 (C vs D), 0.0004 (C vs E), 0.04 (C vs F), 0.56 (D vs E), ⬍0.0001 (D vs F), ⬍0.0001 (E vs F) a Estimates of free 25-desacetyl rifapentine concentration by ANCOVA of data transformed to ln, adjusted for other factors in the final model, and back transformed to the original scale. b Degrees of freedom for the numerator (df1) and degrees of freedom for the denominator (df2). c Values represent adjusted mean (95% CI) free desacetyl rifapentine concentrations (␮g/ml) unless otherwise indicated. Slope (standard error [SE]) data are for a 1-unit change in ln of the parameter of ln of the response. d Data represent P values determined by the Fisher least-significant-difference test for pairwise comparisons of data groups. e Trough, sample at 24 h after drug administration. 4906 aac.asm.org Antimicrobial Agents and Chemotherapy Downloaded from http://aac.asm.org/ on August 12, 2014 by Univ of Florida Estimates of percent free desacetyl rifapentine by ANCOVA of data transformed to ln, adjusted for other factors in the final model, and transformed back to the original scale. Degrees of freedom for the numerator (df1) and degrees of freedom for the denominator (df2). c Data represent P values determined by the Fisher least-significant-difference test for pairwise comparisons of data groups. b Protein Binding of Rifapentine in TB Patients TABLE 5 Demographic and clinical characteristics of the patients in this study Value(s) Non-African other (n ⫽ 21) All patients (n ⫽ 41) 14/20 (70.0) 16/21 (76.2) 30/41 (73.2) 24.5 (22.5, 31.5)b 27.5 (23.7, 31.2)c 42.0 (27.0, 55.0) 42.3 (35.9, 48.7) 31.0 (24.0, 43.0) 35.0 (30.7, 39.4) 53.5 (49.0, 62.3)b 54.8 (51.3, 58.3)c 66.0 (54.0, 79.6) 66.2 (59.3, 73.1) 56.5 (50.8, 66.5) 60.6 (56.4, 64.8) 19.2 (18.5, 20.6)b 19.6 (18.7, 20.4)c 23.0 (19.2, 27.1) 23.2 (21.0, 25.5) 20.0 (18.7, 23.0) 21.4 (20.1, 22.8) No. of patients given indicated rifapentine dose/ total no. of patients (%) 450 mg 600 mg 0/20 20/20 2/21 19/21 2/41 (4.9) 39/41 (95.1) Creatinine (mg/dl) Median (IQR) Mean (95% CI) 0.6 (0.6, 0.7)b 0.6 (0.6, 0.7)c 0.7 (0.6, 0.8) 0.7 (0.7, 0.8) 0.6 (0.6, 0.8) 0.7 (0.6, 0.7) Albumin (g/dl) Median (IQR) Mean (95% CI) 3.6 (3.3, 3.9) 3.6 (3.3, 3.8) 3.6 (3.3, 3.8) 3.5 (3.4, 3.7) 3.6 (3.3, 3.8) 3.5 (3.4, 3.7) ALT (U/liter) Median (IQR) Mean (95% CI) 15 (11, 20)b 23 (9, 37) 21 (15, 32) 26 (19, 32) 18 (12, 26) 24 (17, 32) CRP, ratio to upper limit of normal Median (IQR) Mean (95% CI) 11.1 (5.3, 16.0)b 9.8 (6.9, 12.8)c 0.9 (0.8, 0.9) 1.8 (0.8, 2.9) 1.4 (0.8, 10.1) 5.7 (3.8, 7.7) Treatment duration at time of pharmacokinetic sampling, weeks Median (IQR) Mean (95% CI) 3.9 (3.4, 4.6)b 4.0 (3.6, 4.3)c 5.3 (5.1, 7.1) 5.8, (5.0, 6.5) 4.7 (3.9, 5.3) 4.9 (4.4, 5.4) Race, n Black White Asian 20 0 0 3 16 2 23 16 2 No. of males/total no. of patients (%) Age, yrs Median (IQR) Mean (95% CI) Weight, kg Median (IQR) Mean (95% CI) BMI Median (IQR) Mean (95% CI) a Abbreviations: ALT, alanine aminotransferase; BMI, body mass index; CI, confidence interval; CRP, C-reactive protein; IQR, interquartile range. P ⱕ 0.05 by the Wilcoxon rank sum test. c P ⱕ 0.05 by the Student t test. b greater in Africans of black race than in non-African patients and was positively correlated with the total mean rifapentine concentration (Table 2). The adjusted mean free rifapentine Cmax was 0.11 ␮g/ml, and the trough value was 0.05 ␮g/ml. The mean free rifapentine concentrations adjusted for other significant factors remained different among batches (P ⬍ 0.0001). Some similar findings were found in ANCOVA models of the microbiologically active 25-desacetyl rifapentine metabolite (Tables 3 and 4). Despite a lower total plasma rifapentine concentration in Africans of black race than in non-African patients, African patients had significantly greater free rifapentine and metabolite concentrations resulting from lower drug-protein binding at a rifapentine oral dose of 10 mg/kg. The adjusted mean free rifapentine concentration of 0.091 (95% CI ⫽ 0.078, 0.105) ␮g/ml in Africans August 2014 Volume 58 Number 8 of black race (Table 1B) was approximately twice the reported Mycobacterium tuberculosis MIC (0.03 to 0.06 ␮g/ml) (4, 9). For non-African patients, the adjusted mean free concentration of 0.058 (95% CI ⫽ 0.050, 0.066) was similar to the rifapentine MIC. By ANCOVA, both the adjusted mean free rifapentine concentration and the percent free were inversely associated with albumin concentration and BMI. Given that a decrease in albumin levels and/or an increase in wasting is often associated with advanced human immunodeficiency virus (HIV) or tuberculosis infection (10–12), these patients could benefit at comparable total rifapentine concentrations from relatively greater fractions of microbiologically active free rifapentine and metabolite. For example, at the 25th and 75th albumin percentiles, the mean free rifapentine concentrations adjusted for all other significant factors in the aac.asm.org 4907 Downloaded from http://aac.asm.org/ on August 12, 2014 by Univ of Florida African of black race (n ⫽ 20) Patient characteristica aac.asm.org 5.35 (3.68, 7.86)b 5.06 (3.98, 6.42)c 8.34 (5.49, 9.85)b 7.66 (6.06, 9.66)c 0.04 (0.04, 0.06)b 0.04 (0.03, 0.06)c 17.9 (12.18, 20.42)b 16.13 (13.35, 19.49)c 0.10 (0.07, 0.12)b 0.10 (0.08, 0.12)c 0.07 (0.06, 0.09)b 0.06 (0.05, 0.08)c 10.80 (7.60, 12.94)b 9.87 (8.13, 11.98)c 0.14 (0.12, 0.18)b 0.14 (0.11, 0.17)c Total rifapentine (␮g/ml) b CI, confidence interval; IQR, interquartile range; ln, natural logarithm. Difference between African and non-African groups (Wilcoxon rank sum; P ⱕ 0.05). c Difference between African and non-African groups (Student t test on ln scale; P ⱕ 0.05). a Trough (24 h) Africans of black race (n ⫽ 20) Median (IQR) Geometric mean (95% CI) Others (n ⫽ 21) Median (IQR) Geometric mean (95% CI) Cmax Africans of black race (n ⫽ 20) Median (IQR) Geometric mean (95% CI) Others (n ⫽ 21) Median (IQR) Geometric mean (95% CI) Free rifapentine (␮g/ml) 0.58 (0.46, 0.76)b 0.58 (0.47, 0.73)c 1.42 (0.81, 1.82)b 1.27 (0.98, 1.63)c 0.61 (0.46, 0.80)b 0.59 (0.50, 0.70)c 1.37 (1.09, 2.01)b 1.39 (1.10, 1.76)c % free rifapentine 0.19 (0.13, 0.41) 0.21 (0.15, 0.30) 0.33 (0.18, 0.50) 0.33 (0.23, 0.48) 0.27 (0.18, 0.43)b 0.28 (0.18, 0.46) 0.53 (0.31, 0.85)b 0.42 (0.26, 0.67) Free 25-desacetyl rifapentine (␮g/ml) 10.89 (6.67, 14.55)b 9.55 (7.11, 12.83)c 6.76 (3.26, 9.30)b 5.96 (4.40, 8.06)c 10.56 (7.82, 14.34) 11.82 (9.23, 15.14)c 8.37 (4.85, 11.74) 8.03 (6.23, 10.34)c Total 25-desacetyl rifapentine (␮g/ml) Downloaded from http://aac.asm.org/ on August 12, 2014 by Univ of Florida 4908 Parameter (n ⫽ 82)a TABLE 6 Free rifapentine concentration, total concentration, and percent free total rifapentine concentration at Cmax and trough 24 h after ingestion of rifapentine 2.19 (1.67, 2.88)b 1.90 (1.42, 2.79)c 5.53 (4.21, 7.26)b 6.38 (3.60, 8.35)c 2.40 (1.91, 3.01)b 2.44 (1.82, 3.08)c 5.19 (2.82, 9.56)b 6.23 (4.78, 8.55)c % free 25-desacetyl rifapentine Egelund et al. FIG 1 Scatterplots of mean free rifapentine (␮g/ml) (y axis) versus mean total rifapentine concentrations (␮g/ml) (x axis) without adjustments (A), percent free rifapentine versus mean total rifapentine concentrations (B), mean free 25-desacetyl rifapentine versus mean total 25-desacetyl rifapentine concentrations (C), and percent free 25-desacetyl rifapentine versus mean total 25-desacetyl rifapentine concentrations (D). Plot symbols represent patients of black race from Africa (black-filled circles), patients of black race from North America (gray-filled circles), and patients of other races from North America (open circles). Solid and dashed lines represent the best fit by linear regression for African patients of black race and others. Antimicrobial Agents and Chemotherapy Protein Binding of Rifapentine in TB Patients axis, right) (A), percent free rifapentine (B), total 25-desacetyl rifapentine (y axis, left) and free 25-desacetyl rifapentine (y axis, right) (C), and percent free 25desacetyl rifapentine divided by the number of subjects classified by race (black versus others) (D). Plot symbols represent patients of black race from Africa (black-filled circles), patients of black race from North America (gray-filled circles), and patients of other races from North America (open circles). The mean is represented by the gray “X” and one standard deviation by the gray bars. The 25th, 50th, and 75th percentiles are indicated by the bottom, middle, and top of the rectangular boxes, respectively. The black whiskers are drawn at either the minimum (maximum) or 1.5 times the IQR below (above) the 25th (75th) percentile depending on which of the two is closer to the median. August 2014 Volume 58 Number 8 aac.asm.org 4909 Downloaded from http://aac.asm.org/ on August 12, 2014 by Univ of Florida FIG 2 Boxplots illustrating total rifapentine (y axis, left) and free rifapentine (y ANCOVA model were 0.12 ␮g/ml and 0.07 ␮g/ml (based on Table 2 data). The free rifapentine concentration was directly associated with the total plasma concentration, but the percent free rifapentine was inversely related to the mean total rifapentine level adjusted for other significant effects. No significant difference was observed in percent protein binding between the times of Cmax versus trough concentration for either rifapentine or 25-desacetyl rifapentine, suggesting similar levels of protein binding in these sequential samples with the individual patient over the concentration-time profile. The free (microbially active) rifapentine concentration in blood is determined by the total rifapentine concentration and by the fraction of drug not bound to plasma proteins. Rifapentine is highly protein bound (Table 1 and Table 6). Rifampin, a rifamycin related to rifapentine, is reported to be bound to albumin, gamma globulin, and fibrinogen (13). Similarly, in this study, we found that percent free rifapentine and free rifapentine concentrations were inversely related to the albumin concentration (Table 1 and Table 2). In a prior study of disopyramide and salicylic acid, albumin and alpha 1-acid glycoprotein were identified as 2 binding proteins, and the drugs were more highly protein bound among Caucasians than among African Americans (14, 15). Although alpha 1-acid glycoprotein was not measured in this study, the alpha 1-acid glycoprotein level increases with age (16). In our study group, the members of the African group were younger than the non-Africans (median ages, 24.5 and 42.0 years), suggesting that a difference in the alpha 1-acid glycoprotein concentration might have accounted for the greater protein binding seen in the non-African group. Thus, differences in rifapentine protein binding between Africans and non-Africans in our study group could have been related to race, age, and nutritional status affecting some protein concentrations or possibly to genetic variants of these proteins affecting binding affinity of rifapentine to plasma proteins or the number of protein binding sites. A potential limitation of this study was the significant difference in percent free rifapentine and free rifapentine concentrations adjusted for all other significant factors and covariates among assay batches. Although a standard ultrafiltration method and a validated assay for rifapentine and metabolite were used and no procedural errors were identified, improvements in the interbatch methodology would allow improved comparisons of samples across assay batches. Another potential limitation of the study was that race and geographic region of patients were not balanced in individual batches. In the statistical analysis by ANCOVA, we simultaneously adjusted for the effects of the batch and of the covariate of “Africans versus non-Africans” to enable us to estimate the effects of each variable independently of the other. Because each batch contained a combination of Africans and nonAfricans, the effect of African patients could be estimated independently of the batch effect and could thereby mitigate the potential effect of the imbalance of the numbers of Africans in 2 of the 4 batches. Another limitation of this study was that it was not designed to evaluate pharmacodynamic effects on treatment outcomes. A convenience sample of available samples was chosen without regard to treatment outcomes, and higher rifapentine exposures than those achieved in this study may be needed to demonstrate a pharmacodynamic effect (S. E. Dorman, R. Savic, S. Goldberg, J. E. Stout, N. Schluger, G. Muzanyi, J. L. Johnson, P. Payam Nahid, E. Hecker, C. M. Heilig, L. Bozeman, P.-J. Feng, R. 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Further, a larger sample size likely will be needed than in this feasibility study, because tuberculosis outcomes depend on multiple factors in addition to free rifapentine exposure: the mycobacterial burden of the disease (lung cavitation and extent of disease); the virulence of mycobacterial isolates; the age, nutritional state, and immunity of the patients; and, potentially, drug exposures other than rifamycin in multidrug induction-phase therapy. This was the first study to examine free, active rifapentine plasma concentrations in patients with tuberculosis. These exploratory results demonstrate that despite a lower total rifapentine plasma concentration, significantly greater free rifapentine and metabolite concentrations were achieved among African patients of black race than among non-African patients. Patients with lower albumin concentration, BMI, and total rifapentine concentration values had greater percentages of free rifapentine. These data support larger pharmacokinetic/pharmacodynamic studies to confirm the findings and to assess the free rifapentine concentration in relation to microbiological and clinical outcomes of tuberculosis treatment.