University of Groningen Optimizing levofloxacin dose in the treatment of multidrug-resistant tuberculosis Ghimire, Samiksha

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University of Groningen

Optimizing levofloxacin dose in the treatment of multidrug-resistant tuberculosis

Ghimire, Samiksha

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2

Pharmacokinetics/dynamics-based optimization

of levofloxacin administration

in the treatment

of multi-drug resistant tuberculosis

Samiksha Ghimire,

Natasha van ‘t Boveneind-Vrubleuskaya,

Onno W. Akkerman, Wiel C.M. de Lange,

Dick van Soolingen, Jos G.W. Kosterink,

Tjip S. van der Werf, Bob Wilffert, Daniel J. Touw,

Jan Willem C. Alffenaar

Journal of Antimicrobial Chemotherapy. 2016 Oct; 71 (10): 2691–2703

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ABSTRACT

The emergence of multi- and extensively-drug resistant tuberculosis (TB) has com-plicated TB treatment success. Among many factors that contribute to the devel-opment of resistance, low drug exposure is not the least important. This review summarizes the available information on pharmacokinetic properties of levofloxacin in relation to microbial susceptibilities, in order to optimize the dose and make general treatment recommendations.

A total of 37 (32+5) studies on adult and pediatric MDR-TB patients were pulled in. Among 32 studies; 19 on susceptibility of M. tuberculosis isolates to levofloxacin by minimum inhibitory concentration (MIC), 1 by minimum bactericidal concen-tration (MBC), 1 by mutant prevention concenconcen-tration, 4 on pharmacokinetics of levofloxacin, and 7 others were included. Like wise, out of 5 studies on children; 2 provided with pharmacokinetic parameters of levofloxacin, 1-reviewed

cerebro-spinal fluid concentrations and 2 dealt with background information.

In adult MDR-TB patients, standard dosing of once daily 1000 mg levofloxacin in TB treatment did not consistently attain the target concentration i.e. (fAUC)/MIC>100 and fAUC/MBC>100) in 80 % of the patients with MIC and MBC of 1 mg/L, leaving them at a risk of developing drug resistance. However, with a MIC of 0.5 mg/L, 100 % of the patients achieved the target concentration. Similarly, pediatric patients failed consistently in achieving given pharmacokinetic-pharmacodynamic targets due to age related differences, demanding a shift towards once daily dosing of 15–20 mg/kg. Therefore, we recommend therapeutic drug monitoring for the patients with strains having MIC of ≥0.5 mg/L and suggest revising the cut-off value from 2 mg/L to 1 mg/L.

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2 INTRODUCTION

TB is one of the world’s deadliest communicable diseases (1–3). The WHO estimated that 9.6 million people developed TB and 1.5 million people died from this disease in 2014, 390,000 of whom were HIV positive(4).The emergence and spread of MDR-TB and XDR-TB have threatened the success of TB programs globally (5,6). The WHO has recommended fluoroquinolones along with injectable aminoglyco-sides in the treatment of MDR-TB (7–9). Yet, the treatment success rates of MDR-TB vary between 36 % and 79 %, thereby increasing the prevalence of XDR-TB in countries burdened with MDR-TB (6,10,11). To tackle the growing problems of drug resistance; either new drugs have to be developed or the dose of currently used therapy should be optimized, or both (2,12–14). Recent advances to treat MDR-TB in-clude approval of two new drugs, delamanid and bedaquiline (15,16) and other new drugs in the development pipeline shed light of hope on the present situation (17).

Higher success rates have been observed in more individualized pro-grams including therapeutic drug monitoring (TDM) (18–21). Among many recognized risk factors for development of drug resistance, par-ticularly low drug exposure has been subject of debate (6,22). Low drug exposure could indeed explain a large proportion of therapy failure (1,3,23). In addition, studies have revealed that in TB patients with other comorbidities such as HIV and diabetes, drug absorption and other pharmacokinetic parameters such as protein binding, distribu-tion, metabolism, and elimination may be altered resulting in lower successful treatment outcomes and twice higher death rates (24–28). In such situations, TDM can help clinicians to make informed dosing decisions (2,3,22,29,30).

Fluoroquinolones and injectable aminoglycosides are important drugs in the combination treatment regimen of MDR- TB (31). Of the available fluoroquinolones, levofloxacin and moxifloxacin are the preferred choices due to their strong activity against M. tuberculosis, once daily dosing and limited adverse effects. Although moxifloxacin has the advantage of a two stage resistance mechanism (12,32), levo-floxacin has multiple advantages in that it has less QT related toxicity, has equivalent efficacy with moxifloxacin for treating MDR- TB (similar

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PK/P D b as ed o pt imiza tio n o f le vo flo xacin admini stra tio n in t he t re at m en t o f MD R-TB

treatment success rate, sputum conversion rate, rate of adverse drug reactions). Finally, levofloxacin is more widely available (33) especially in low- and middle-income countries.

In a recent study, it was observed that development of drug resistance against fluoroquinolones occurred in 12 % while receiving standard dosages (23,24). Pharmacokinetic variability could well explain the acquired drug resistance (34). Since, TDM could help physicians to attain given PK/PD targets for levofloxacin, we conducted a literature search on the PK/PDs of levofloxacin to support TDM in order to reach a given target.

METHODS

All available articles evaluating the use of levofloxacin in treatment of TB in humans were retrieved through PubMed using a specific search strategy described below. Articles concerning the in vitro activity and/ or efficacy of levofloxacin against M. tuberculosis, and clinical studies on pharmacokinetic monitoring strategy and parameters of levoflox-acin were reviewed. Articles that provided information on the in vitro activity and/or efficacy of levofloxacin against M. tuberculosis were divided into two parts. The first part focused on the MIC to determine the optimal dose for killing the dominant population of M. tuberculosis, whereas the second part focused on the MBC. MIC is the lowest con-centration of drug that inhibits >99 % of the colonies growing on drug free control. MICs at which 50 % and 90 % of the isolates are inhibited are known as MIC50 and MIC90, respectively. MBC is defined as the lowest drug concentration that decreased the bacterial population by 2 or more log10 units within the same period of incubation (35) whereas mutant prevention concentration (MPC) is a measure of the

susceptibility of the mutant sub-population (36). Search Terms

A PubMed search [October 2014] of relevant articles was conducted to retrieve the information on TDM for levofloxacin used to treat TB in adult MDR-TB patients. Likewise, an extended PubMed search was performed [February 2016] on pediatric MDR-TB patients due to wide

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availability of literature published recently. The following search terms

were used with filters turnedon to full text, English: (“Mycobacterium tuberculosis”[Mesh] OR “Tuberculosis”[Mesh] OR tuberculosis [tw]) AND (“Levofloxacin”[Mesh] OR levofloxacin [tw] OR lfx [tw]); (“My-cobacterium tuberculosis”[Mesh] OR “Tuberculosis”[Mesh] OR tuber-culosis [tw]) AND (“Levofloxacin”[Mesh] OR levofloxacin [tw] OR lfx[tw]) AND (“Pharmacokinetics”[Mesh] OR pharmacokinetics[tw] OR concentration[tw] OR pharmacodynamics[tw] OR therapeutic effect*[tw]); Pharmacokinetics AND Pharmacodynamics AND Levo-floxacin AND Tuberculosis. Likewise, for pediatric section, the search term used was “Pharmacokinetics AND Children AND Levofloxacin” with filters turned on to full text and English.

The reference lists of identified articles were manually searched for pertinent articles not identified in the electronic search.

Selection Criteria

Identified articles were included if the activity of levofloxacin against susceptible and MDR-TB by in vitro, in vivo, and clinical studies was evaluated. In vitro studies were included if susceptibility testing meth-ods of M. tuberculosis to levofloxacin were adequately described. This drug susceptibility testing included different methods such as Radio-metric BACTEC, Microplate Alamar Blue Assay (MABA), proportion methods and broth dilution- and micro dilution methods in different media. Case reports and articles describing diseases caused by species of Mycobacteria other than Mycobacterium tuberculosis were excluded. Additionally, articles on bioanalytical procedures and assays for the measurement of plasma concentrations of levofloxacin were excluded. Methods of data collection

Two reviewers collected data independently from the selected literatures and compared to avoid differences.

Data Analysis Strategy

Data were extracted from included articles and were tabulated (Ta-ble 1, 2 and 4). For comparison of results (from Table 2 and 4), mean values were estimated from the given median values, range and size of the sample in different studies by using the formula described by

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Hozo et al. (37). The AUC for free drugs (unbound to plasma protein) was calculated by multiplying the individual AUCs with percentage of protein free drug fraction (38,39); protein binding for levofloxacin being 40 % — range, 24–40 %, as published in the literature (38). The free fractions of this variable (fAUC) were divided by corresponding MIC values. For categorizing MIC values as susceptible and resistant, in vitro studies on more than 100 strains of M. tuberculosis were com-pared to avoid any bias (Table 1).

RESULTS

In total, 248 articles were retrieved from the initial search. After re-moving duplicates, 190 articles were selected for screening based on title and abstract, after which only 40 full text relevant articles were assessed for evaluation. Ultimately, 37 articles were included describing in vitro susceptibility testing of levofloxacin against MDR-TB strains, and clinical studies on pharmacokinetic monitoring strategy and pa-rameters both on adult and pediatric MDR-TB patients (See Figure 1). Out of 32 studies evaluating the use of levofloxacin in the treatment of adult MDR-TB, 19 described the susceptibility of M. tuberculosis isolates to levofloxacin by MIC, one article by MBC, and one by MPC. Four studies revealed the pharmacokinetics of levofloxacin, and 7 pro-vided background information. Since only one study presented data for clearance (CL) and volume of distribution (Vd); these parameters were thus excluded from comparison.

Among 5 articles on children, 2 presented with the pharmacokinetics of levofloxacin in plasma, 1 in CSF of MDR-TBM patients and 2 pro-vided background information.

In vitro studies

MIC

The search strategy on articles exploring in vitro susceptibility testing is presented in Figure 1. Most studies have performed drug susceptibility testing against M. tuberculosis by determining the MIC using different available methods in different culture mediums. Table 1 shows the

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susceptibilities of drug resistant M. tuberculosis strains to levofloxa-cin. Different concentrations of levofloxacin were tested in order to determine the MIC for both resistant- and susceptible strains. Final concentrations of levofloxacin tested ranged between 0.002–512 mg/L, following two- folds dilutions. In most of the studies, susceptible strains had an MIC of ≤1 mg/L (38,40–42). The MIC50 was 0.5 mg/L (42–45) and the MIC90 >1 mg/L (38,40–42). Eight articles (n >100) that pre-sented MIC values were compared (see Table 3). The inhibitory activity of levofloxacin against the strains studied is summarized in Table 3. Minimum bactericidal concentration (MBC)

Mor et al. (35) studied the inhibitory and bactericidal activities of levofloxacin against three strains of M. tuberculosis in vitro. The MIC of levofloxacin for all three strains was 0.5 mg/L, whereas MBC was 1 mg/L for two strains and 0.50 mg/L for one strain, resulting in a MBC/

MIC ratio of 2 or 1.

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PK/P D b as ed o pt imiza tio n o f le vo flo xacin admini stra tio n in t he t re at m en t o f MD R-TB Ta bl e 1: I n v itr o s us ce pt ib ili ty t es tin g o f M. tu be rc ul os is iso la tes t o l ev ofl oxacin (LFX) b y MI C Ref er en ce Str ai n N D os e m g M etho d Ra ng ea (m g/L) MB C (m g/L) MI C (m g/L) M IC 90 (m g/L) (33) CI 90 (MD R-TB) 750 d ai ly AC M 2 C C ND ND ND (51) CI 169 D R-TB , (7 LFX R) NA IPM o n LJ (ILJ) a nd REMA

2 (ILJ) 0.13–16 (REMA) Cut-o

ff ≥0.50 ND 4– ≥16 (LFX R s tra in s) ND (78) CI 102 (XD R a nd MD R-TB) NA APM Su scep tib le a t ≤1.0; r esi s-ta nt a t ≥2.0 ND 1 C C ND (40) CI 21 (MD R-TB an d DS-TB) NA PM 0.125–4 ND 0.5 ND (41) CI 57 (19 O FX R an d 38 O FX S ) NA APM 0.5, 1.0, 4.0, 8.0 2(cr itic al co ncen tra tio n) ND 0.5 ND (79) CI 68 (38 O FX R an d 30 O FX S NA BAC 0.125- 32 ≥2 (c ut o ff MI C r esi s-ta nce) ND 0.125–1 (O FX S) 2.0 – 8.0 (S M in g yrA) >32 (D M g yr A) ND (80) CI 62 (18 HLR , 42 LLR 2 LFX S ) NA AC M 1 (LLR) a nd 10 (HLR) ND For m at ion of co lo nies ND (46) CI 162 NA MI C 0.5, 1 a nd 2 ND 1 m g/L (f or 131 CI) ND (81) CI 8 NA MAB A 0.1–10 ND <1.0 ND (47) CI 420 SDA P 0.06–128 C ut-o ff MI C >1.0 Su scep ti-bi lit y ra te 98.6 % 0.5– ≤8; 14 (f or FQ R s tra in s) ND

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Ref er en ce Str ai n N D os e m g M etho d Ra ng ea (m g/L) MB C (m g/L) MI C (m g/L) M IC 90 (m g/L) (48) CI 141 (62 FS, 33 MD R, 46 ODR P) NA SDA P 0.03–4 C ut-o ff MI C >1.0 ND FS=1.0 ; MD R = 1– ≥4 ; O D RP= 1.0 (43) CI 101 (MD R-TB) NA M-MT T me tho d PM w ith MB 7H11 0.13–4 4.0 Cut-o ff MI C >1.0 ND M IC 50 a nd M IC 90 0.50 a nd 1.70 (49) CI 243 MB 7H11 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 Cut-o ff MI C ≥2.0 ≥2.0 (f or 5 r esi sta nt stra in s) M IC 50 a nd M IC 90 0.25 a nd 0.5 (82) CI 55 NA MB 7H11 0.5, 1, 2, 4, 8, 16, 32, 64, 128 ND ≤ 0.5 (f or 45 stra in s); = 1(f or 5 s tra in s); 2 (f or 5 s tra in s) M IC 50 a nd M IC 90 0.5 a nd 2 (44) CI 250 NA APM 0.125–128 ND ≤0.125- 8 M IC 50 a nd M IC 90 0.5 a nd 1 (50) CI 135 NA BB 0.25, 0.5,1.0,2 Su scep tib ili ty ra te 99.9 % a t ≤1, o ne pa tien t MI C = 16 ND (45) CI 40 (MD R- TB) NA A DM 0.03–32 C ut-o ff MI C ≥1.0 ND 0.25–16 M IC 50 a nd M IC 90 0.5 a nd 8

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PK/P D b as ed o pt imiza tio n o f le vo flo xacin admini stra tio n in t he t re at m en t o f MD R-TB Ref er en ce Str ai n N D os e m g M etho d Ra ng ea (m g/L) MB C (m g/L) MI C (m g/L) M IC 90 (m g/L) (83) FQ r esi sta nt an d s us cep -tib le MTB . 19 APM 0.5- 2 ND 1 ND (38) CI 90 (24 MD R-TB) M idd le -br oo k 7H10 m edi um 0.002–512 ND 0.125–0.5 Suscep tib le EC O FF ≤0.5 fA UC 0–24 /MI C 45(45–222) f or 90 CI (p ro tein bin din g 40 %) a U ni ts f or MI C a nd MB C h av e b een exp res se d in m g/L t hr oug ho ut t he t ab le a nd t he t exts. ACM= A bs ol ut e C on cen tra tio n M et ho d; C C= Cr itic al co ncen tra tio n; N A= N ot a pp lic ab le; ND= N ot D et er min ed; MTB=M yco bac ter ium tu ber cu losi s; LFX =L ev oflo xacin; O FX= Oflo xacin; O FX R= Oflo xacin r esi sta nt; O FX S= Oflo xacin s ucep tib le; B AC = B ACTEC M GIT 960 S ys -tem; S M= s tra in s w ith sin gle m ut at io n; D M= s tra in s w ith do ub le m ut at io ns; LLR= lo w-le ve l r esi sta nt; HLR=hig h-le ve l r esi sta nt; HRS= H ig hl y resi sta nt s tra in s; S D AP= S er ia l di lu tio ns o n a ga r p la tes; FS= f ul ly s us cep tib le; O D RP= o th er dr ug r esi sta nt p at ter ns; AD M=A ga r Di lu tio n M et ho d, 7H11 m edi um; APM= A ga r P ro po rt io n M et ho d; A CM= A bs ol ut e C on cen tra tio n M et ho d; RMP S = R ifa m picin s us cep tib le; RMP R = R ifa m picin r esi sta nt; RB AC = R adio m et ric B ac te c TB 460; B B= B ACTEC 7H12 B ro th; PM = p ro po rt io n m et ho d; MB= M idd le br oo k; O FX= Oflo xacin; g yrA= D N A g yra se A; D R= Dr ug r esi sta nt; IPM= I ndir ec t p ro po rt io n m et ho d; LJ = L ow en stein J en

sen; REMA= micr

o p la te co lo rim et ric m et ho d u sin g r es azur in; LFX R= L ev oflo xacin r esi sta nce; DS= Dr ug s us cep tib le; D R= Dr ug r esi sta nt; HIV= H um an I mm un o-deficien cy v iru s; MI C= minim um in hi bi to ry co ncen tra tio n; MAB A= micr o p la te- b as ed A la m ar B lue a ss ay ; M-MT T= micr o p la te b as ed — (4,5-dim et hy lthi azo l-2-y l) 2,5-di ph en yl tet razo lium b ro mide; RMP=R ifa m picin; MI C= S ta nd ar d minim um in hi bi to ry co ncen tra tio n m et ho d; FQ= Fl uo ro quin olo nes; FS= f ul ly s us cep tib le t o fir st lin e dr ugs; O D RP= o th er dr ug r esi sta nt p at ter ns

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Clinical Studies

i. PK/PD of levofloxacin in adult MDR-TB patients

Mpagama et al. performed an experimental study to measure the plasma concentration of levofloxacin in 25 patients with MDR-TB in Tanzania (52). Patients received 750 mg levofloxacin given orally once daily in a standardized treatment regimen. After 14 days of standardized treat-ment with levofloxacin, drug concentrations were measured 2 hrs post medication (C2). The mean levofloxacin concentration (8.0±2.8 mg/L) was lower than the expected range (8.0–12.0 mg/L) for 13 Tanzanian patients. The C2/MIC ratio was found to be 15.8 (± 14.1). The mean values presented in this section are calculated from the given median values in the literature by using the formula described by Hojo et al. (37). Authors concluded that low plasma concentration of levofloxacin could be due to the single time point of plasma withdrawal (C2) in the dosing interval, treatment schedule (2 weeks) and the dose itself. Levofloxacin 1000 mg once daily dosing has been proven to be optimal over 750 mg in many studies. Despite these limitations, this study provides important information on plasma drug concentrations relative to the quantitative susceptibility in a standard MDR-TB treatment regimen. A summary of PK properties of levofloxacin is shown in Table 2.

Two randomized controlled trials (RCT) and one randomized open label trial were identified that included AUC/MIC ratio as a predictor of efficacy for levofloxacin(23,39,53,54). The body weight of the patients included in the clinical studies ranged from 45–66 kg. Johnson et al. conducted a randomized, open label trial to study early bactericidal activity (EBA) of levofloxacin with a daily dose of 1000 mg in 10 TB patients for 7 days(53). Mean fAUC/MIC and Cmax/MIC ratio were 107.98 and 20.7, respectively. However, due to the small sample size this study only adds to the evidence on efficacy using EBA, but not to that of long-term treatment outcome.

A RCT by Peloquin et al. was completed in 10 patients with TB (54). Patients were randomized to receive 7 days of oral isoniazid (300 mg) (standard positive comparator for early bactericidal activity studies), levofloxacin (1000 mg), gatifloxacin (400 mg), or moxifloxacin (400 mg). Compared to gatifloxacin and moxifloxacin, levofloxacin exhibited the highest maximum plasma concentrations (median, 15.55 mg/L;

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gatifloxacin 4.75 mg/L; moxifloxacin 6.13 mg/L), largest volume of distribution (median, 81litres; gatifloxacin 79 litres; moxifloxacin 63 li-tres) and longest elimination half-life (median, 7.4 h; gatifloxacin 5.0 h; moxifloxacin 6.5 h). The mean fAUC/MIC ratio (protein binding 40 % for levofloxacin, using published MIC value of 1 mg/L) was 107.85 and Cmax/MIC90 was 20.66 (54). In addition, from the available pharmaco-kinetics data on 10 patients, we calculated fAUC0–24/MIC ratio for each patients using MIC values 0.5 and 1 mg/L (Figure 2) and found that 8/10 patients had low serum concentrations and fAUC0–24/MIC ratio of less than 100 at 1000 mg once daily levofloxacin dose (MIC; 1 mg/L). A second RCT by Thwaites et al. (39) studied the pharmacokinetics of levofloxacin and exposure-response relationship for the efficacy of levofloxacin in patients with TB meningitis (TBM). Fifteen patients received levofloxacin (500 mg/12 h) for the first 60 days of therapy in combination with standard TB treatment. The efficacy in levofloxacin

Figure 2: Relationship between MIC and fAUC and proportion of patients with response to treatment.

AUC; area under the concentration-time curve, MIC; minimal inhibitory concentration,

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treatment group was compared to that in ciprofloxacin (n=15) and

gatifloxacin (n=15) treatment group. Cerebrospinal fluid (CSF) pene-tration, calculated as a ratio of plasma AUC0–24 to CSF AUC0–24 was greater for levofloxacin (median, 0.74; range 0.58 to 1.03) than for gati-floxacin (median 0.48; range 0.47 to 0.50) and ciprogati-floxacin (median, 0.26; range 0.11 to 0.77) at the doses explored. Although, levofloxacin had a better CSF penetration, the fAUC0–24/MIC of plasma was low i.e. 93 (protein binding 40 %) with the 500 mg twice daily dose assuming MIC of 1 mg/L — an assumption based onMIC90.

ii. PK/PD of levofloxacin in pediatric MDR-TB patients

A prospective crossover intensive pharmacokinetic sampling study investigated the pharmacokinetics of levofloxacin (15 mg/kg) in 22 chil-dren (3 months to 8 years) either on WHO’s MDR-TB treatment regi-men or prophylaxis therapy for 6 months. Authors docuregi-mented lower AUCs and Cmax values in children with mean AUC0-∞ and Cmax at 33.04 mg*h/L and 6.58 mg/L resp(55)(Table 4). The estimated mean AUC0–24/MIC and Cmax/MIC was 43.8 and 6.5 resp. when published MIC90 of 1 mg/L was used. Even with a lower MIC of 0.5 mg/L, children failed to meet the clinical outcomes relating to the ratios of AUC0–24/ MIC>125 (mean AUC0–24/MIC at 87.6 in this case). However, esti-mated Cmax/MIC (13.1) was well above the proposed ratio of 8–10(55). In addition, drug clearance was more rapid in children with a half-life of 3 to 4 h and an elimination rate constant (kel) 0.22 h−_{1 compared to} half-lives of 4 to 8 h with an elimination rate constant (kel) of 0.09 to 0.13 h−_{1 in adults which could be attributed to the age related differences} in elimination of drug in the children(54,56). The authors commented that the lower levofloxacin exposure in this study compared to existing pediatric studies could stem from the differences in drug formulations and methods of administration between the studies.

A review article on CSF penetration of anti-tuberculosis agents found favorable CSF concentrations of levofloxacin in relatively lower doses of 300–800 mg, in comparison to MIC and plasma concentrations, in MDR-TBM children(57). This observation is in line with the study on adult MDR-TBM patients. Despite the good penetration profile, levofloxacin 20 mg/kg is recommended to ensure maximum protec-tion, which correlates with excellent EBA, and with preferably once

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daily dosing scheme (55,57). However, in HIV co-infected MDR-TBM patients, use of levofloxacin and other FQs were not that encouraging, which the author explained could result from the lack of adequate protection from companion drugs (57).

Mase et al. studied 50 children receiving once daily levofloxacin based regimen for MDR-TB or latent tuberculosis infection (LTBI) presumed to be MDR-TB at the dose of 10 mg/kg, age >5 years and 15–20 mg/kg, age ≤5years at two different treatment centers (58). The clinical characteristics and levofloxacin pharmacokinetic parameters were correlated to determine the optimal dosages and inform future

Table 2: Drug concentrations in plasma

Refer-ence Type N Dose (mg) Study type Median body weight (kg) PK monitoring strategy

(52) MDR

TB 25 750 daily Experi-mental C2

(53) TB 10 1000 daily ROT 55 (50.5–60.2) C0, C1, C2, C4, C8, C12, C18, C24 Protein binding 40 % (39) TBM 15 (500/12 h) twice daily as a part of standard treatment RCT Median, 48 (min, max; 31,60) Lumbar puncture 0–8, 8–16, 16–24 hours after drug administration. CSF was paired with blood specimen.

(54) PTB 10 1000

daily RCT 56 (41–66) C0, C1, C2, C4, C8, C12, C18, C24

Protein binding 25 %

b Reference 53 and 54 are about the same study

MDR=Multidrug resistant; TBM= Tuberculosis meningitis; q24 h= every 24 hour; AUC0–24 = area under the concentration time curve; C2 = plasma drug concentra-tion at 2 hours after medicaconcentra-tion administraconcentra-tion; HPLC= High performance liquid chromatography; CSF= Cerebrospinal fluid; LC/MS/MS= High pressure liquid

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dosing strategies. Levofloxacin dosage of 15–20 mg/kg achieved desired Cmax>8 mg/L whereas, AUC0–6, mean ± SD was 30.22±19.36 mg*h/L for children at the Federated States of Micronesia (FSM) center and 37.22±7.76 mg*h/L for those at the Republic of Marshall Island (RMI) center. Based on PK modeling, fAUCss,0–24/MIC was calculated at a steady state using simulated pediatric exposures and typical MICs (0.25, 0.50, 1 and 2 mg/L) with protein binding percentage of 25 for levofloxacin. For MIC <0.5 mg/L, high target attainment was achieved with 15 mg/kg daily dosing, whereas with MIC ≥0.5 mg/L, 20 mg/kg daily dosing would meet the target. Authors recommended revision

Table 2: continued

Analytical procedure Result

MIC

PK-PD parameter Values (mg/L) Test procedure

BA C2±SD (mg/L) 8.0±2.8* 0.75 (0.25–1)** ECOFF ≤ 0.5 MSP C2/MIC15.8±14.1* HPLC AUC0–24 hours (mg/hr/L) 129.1(103.4–358.3)** Cmax 15.6(8.6–43.0)** MIC90

1 APM and BRM C15.6(12.2–16.6)**max/MIC AUC0–24/MIC 129.1(121.0–145.0)** LC/MS and LC/ MS/MS AUC0–24 hours (mg/hr/L) Plasma 155 (81.8,284)* CSF 94.1(53.1,208)* OFX MIC determined, correlated to LFX by potency relationship. 1 % PM on LJ medium CSF AUC0–24 /Plasma AUC0–24 0.74 HPLC AUC0–24 hours (mg/hr/L) 129(103–358)** Cmax 15.55(8.55–42.99)** Actual MIC 0.5** MIC90 1** BRM AUC0–24/actual MIC 258 AUC0–24/MIC 90 129

chromatography tandem mass spectrometry; LC/MS= High performance liquid chro-matography mass spectrometry; RCT= Randomized control trial; ROT= Randomized open label trial; BA=Bio assay; BRM= Bactec radiometric method; MSP= MYCOTOB Sensititre plates; MDM= Microtitre dilution method; *= Mean values; ** = Median values; AUC 0–24 hours (mg/hr/L)= Total 0–24 hour area under the concentration time curve (AUC)

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of current dosage for levofloxacin to achieve a target Cmax ≥8 mg/L and advised to use 15–20 mg/kg once daily levofloxacin in children ≥2 years of age (58).

DISCUSSION

The most important finding of this review is that currently recom-mended dosage of levofloxacin in MDR-TB treatment is not sufficient to reach target concentration in majority of patients that have strains of M. tuberculosis with MIC value of >0.5 mg/L. This adds to a serious concern on the development of drug resistance, treatment failure and further extension to XDR-TB. Since, MDR-TB is currently treated with a combination therapy in which fluoroquinolones, including levoflox-acin, are the central drugs in the treatment regimen, optimizing the dose of individual drugs (levofloxacin in this case) would allow for optimal efficacy of combination therapy regimens. Dose of levofloxacin could be optimized by taking into consideration the pharmacokinetic- pharmacodynamic (PK/PD) calculations. The best activity of levofloxa-cin against M. tuberculosis and decreased likelihood of drug resistance

Table 3: Comparison of LFX MIC values from 8 studies with n>100

Reference n MIC (mg/L) 0.06 0.125 0.25 0.5 1 ≥2 4 8 16 >16 (51) 162 162### (46) 162 152 10 (78) 102 9 93 (47) 420 3 17 139 216 39 3 1 2 (49) 243 2 25 106 104 1 1 1 2 1 (50) 135 134### ₁ (48) 141 108# ₃₃## (44) 250 4 240# ₆ Total 1615 5 42 245 324 845 106 35 9 3 1 % 100 0.31 2.60 15.17 20.06 52.32 6.56 2.17 0.56 0.19 0.06 #_{= MIC90;}##_{= MIC90 (1– >4);}###_{= <1}

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Ta bl e 4: Dr ug c onc en tr at io ns in p las ma o f p edi at ri c MD R-TB p at ie nts Ref -er -en ce Ty pe N D os e (m g) St ud y typ e Ag e (y ea rs) PK m on -itor in g stra teg y Ana ly ti-ca l p ro ce -du re Re su lt MI C (m g/L) PK /PD Va lues Tes t p ro -ce dur e (55) MD R-TB pr op hy -laxi s o r tre at m ent 21 15 m g/kg od PCIPSS <8 C0, C1, C2, C4, C6, C8 LC-MS/ MS AU C0-∞ (m g*h/L) m edi an (I Q Rs) 32.92 (25.44–40.88) Cma x m edi an C ma x m edi an (I Q Rs) 6.79(4.69–8.06) 1 a nd 0.5 Pub lishe d M IC 90 es tim at es W hen MI C i s 1 (m ea n±S D) AU C0–24 /MI C 43.8 (13.3) Cma x /MI C 6.5 (2.0) W hen MI C i s 0.5 AU C0–24 / MI C 87.6 C ma x /MI C 13.1 (4) (58) FSM MD R-TB o r pr es um ed LT BI 33 (8MD R-TB , 25 pr es um ed LTB I) 10 m g/kg , ag e>5; 15–20 m g/kg , ag e ≤ 5 0.5 t o 15 C1, C2, C6 H PLC m ean C ma x 6.09 f or a ge >5, 8.0 f or a ge≤ 5 (m ea n±S D) A UC 0–6 (m g*h/L) 29.88±8.09 0.25, 0.5,1 and 2 Ref er r es ul t s ec tio n

RMI Pre- sum

ed t o inf ec tio us MD R-TB 17 12 m g/kg , ag e ≥5; 11–16 m g/kg , ag e <5 1 t o 15 C0, C1, C2, C6 H PLC m ean C ma x 8.13 f or a ge >5, 8.0 f or a ge≤ 5 (m ea n±S D) A UC 0–6 (m g*h/L) 37.22±7.76 Ref er r es ul t s ec tio n PCIPSS= P ros pe ct iv e cr os so ver in ten siv e p ha rm aco kin et ic s am plin g s tud y; I Q R= I nt er qu ar tile ra ng e; A UC 0–24 = a re a un der t he co ncen tra -tio n t im e c ur ve o ver 24 h our s; C ma x = p la sm a dr ug co ncen tra tio n a t 2 h our s a fter m edic at io n admini stra tio n; HP LC= H ig h p er fo rm an ce liq uid chr om at og ra ph y; L TB I=L at en t t ub er cu losi s inf ec tio n L C/MS/MS= H ig h p er fo rm an ce liq uid c hr om at og ra ph y t an dem m as s s pe ct ro m et ry ; FS M=F edera l S ta tes o f M icr on esi a; RMI= t he R ep ub lic o f M ar sh al l I sla nd s

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is well recognized at fAUC/MIC ratio >100 and Cmax/MIC ratio >8–10 (52). Without exception, patients with fAUC/MIC ratio <100 are at a greater risk of drug resistance (39). Therefore, this paper outlines more rational methods for designing the dosages and dosing schedule of levofloxacin in MDR-TB treatment.

First of all, levofloxacin doses of 1000 mg daily (53,54)and 500 mg twice daily (39) were evaluated for their ability to achieve desired fAUC/ MIC ≥100. Levofloxacin 500 mg twice daily had fAUC/MIC ratio of less than 100 when MIC value of 1 mg/L was used. This clearly reveals the lack of drug exposure due to the dose related differences in the mean steady state fAUC0–24 and Cmax values between two different dosing schemes. As a result, patients taking less than a 500 mg twice daily regimen are at risk of developing acquired drug resistance (39,52). Because fluoroquinolones have a concentration dependent killing rate, it is expected that administration of a once daily dose would result in better efficacy compared to administering the same total daily dosage in divided portions (59).

In addition, the study by Peloquin et al. (54) showed that8/10 (80 %) patients receiving 1000 mg levofloxacin had low serum concentrations and fAUC0–24 hrs/MIC ratio of less than 100 (Figure 2), when a MIC value of 1 mg/L was assumed. In contrast, when an actual MIC of 0.5 mg/L was adopted, 100 % of the patients had fAUC/MIC >100. The MIC values exhibited by strains of M.tuberculosis from each individual is termed as actual MIC whereas, 1 mg/L is the established MIC90 value (54). It is noteworthy that an actual MIC difference from 0.5 to 1.0 mg/L can be within the range of inter-reader variability in interpreting a MIC manual readout, or within the range of variability (1 dilution either di-rection), if the assay is repeated on the same Mycobacterium tuberculosis isolate. This emphasizes just how near the majority of isolates are to a MIC that is at the breakpoint of clinical efficacy. Sirgel et al. compared the quinolone resistant determining regions of gyr A genes with MICs of ofloxacin and moxifloxacin for Mycobacterium tuberculosis and demonstrated that MIC of ofloxacin and moxifloxacin were not equally affected by the mutations and were still within or marginally outside the normal wild-type distribution(60). Thus, the conventional qualitative susceptibility testing based on single critical concentration, is not suit-able to distinguish between borderline (low-level), moderate-level and

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high-level resistance. Considerable proportion of isolates with so-called

borderline susceptibility to ofloxacin in combination with inadequate drug concentration (due to individual PK variability) resulted in in-effectiveness of the drug (61). Additionally, patients with borderline resistance strain might benefit from dose adjustment or in-class change (61). Therefore, MIC determination reflects the bacterial population giving the possibility to guide the design of treatment modalities. A well-defined clinical breakpoint should be configured to detect resis-tance in each patient and to individualize therapy.

Despite the fact that MIC testing compares favourably to standard phenotypic DST, detection of MICs is still infrequently performed in TB endemic settings for several reasons (62). Recently, MYCOTB MIC plate has been made available for testing MIC of M. tuberculosis. This test configured for determination of MICs exhibits fair agreement with DST results using the Middelbrook 7H10 agar proportion method (APM) and Bactec MGIT 960, with plea for its implementation in MDR treatment centres and further prevention of XDR-TB (61).

On the other hand, 80 % of the patients had fAUC/MBC <100, with a MBC of 1 mg/l. Thus with increasing MIC (>0.5 mg/L), and given MBC, only around 20 % of patients attained the target plasma concentration for free levofloxacin, leaving the remaining 80 % on the verge of devel-oping acquired drug resistance. This provides a big reason to worry on inter-patient variability in the pharmacokinetics of levofloxacin that leads a proportion of patients to achieve suboptimal drug concentra-tions with the standardized dose and stresses the importance of TDM to improve the treatment success. In conclusion; based on these obser-vations, we advocate that TDM should be performed in patients with strains of M. tuberculosis having a MIC value >0.5 mg/L to determine the optimal dose for killing bacteria and decrease the proportion of levofloxacin treatment failure in 80 % of patients(63). Second, the body weight of patients ranged from 45 to 66 kg. What could be learned from this observation is that body weight could influence achievement of target concentration and for a patient who weighs more than 70 kg, a 1000 mg dose might still not be sufficient. This aspect is supported by Peloquin et al. in a recent study that showed the use of levofloxacin dosages in the range of (17 to 20 mg/kg of body weight) had a good target attainment for MICs from 0.25 to 0.50 (64)

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As individualized treatment on MDR-TB depends upon reliable and valid drug susceptibility testing(65), we reviewed the results on 1,615 strains that were tested for susceptibility to levofloxacin. The MIC of levofloxacin ranged between 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, >16 (Table 3). More than half of the strains tested i.e. 52.32 % exhibited MIC value of ≤ 1 mg/L, whereas, 20.06 % were susceptible at 0.5 mg/L and 15.17 % at 0.25 mg/L. This shows that the majori-ty of isolates are susceptible to a MIC ≤1 mg/L. Based on this; we conclude that levofloxacin is a good drug to start with for a desired response (microbial kill). However, 6.56 % of the strains had a MIC of ≥2 mg/L. For susceptible strains, a standardized dose is preferred and for resistant strains other drugs are prescribed. If strains have a MIC between 1 and 2, we recommend increasing the dose thereby increasing the AUC or Cmax (66). With increasing MIC, eventually microbial killing is lost and levofloxacin is no longer effective (66). Therefore, for patients that have MIC values between 1 and 2, it is

important to consider different susceptibility break points than those in use for the selection of doses that suppress the emergence of drug resistance. This very recommendation of increasing levofloxacin dose could raise concerns about the dose related toxicities. Fortunately, the rate of adverse drug reactions of levofloxacin is still one of the low-est among FQs (67). There is insufficient evidence to add clarity on whether adverse effects of FQ’s and levofloxacin in particular, in the treatment of TB are highly dose dependent. The high dosage regimens of FQ’s against severe bacterial infections resulted in greater rates of clinical cure and low frequency of side effects (68,69). In addition, increasing the dose of moxifloxacin in patients with TBM appeared to be safe (70,71). Levofloxacin at 1000 mg once daily dosing has not only shown best early bactericidal activity, but also proven to be clinically tolerant and safe (53,54). Nevertheless, worries about development of dose-dependent toxicity are understandable.

ii. Pediatric MDR-TB patients

The results of studies on pediatric MDR-TB patients demonstrates how age related differences in the clearance of levofloxacin could considerably affect drug exposure thereby affecting the clinical out-comes and how dose extrapolation in children based on established

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pharmacokinetics of levofloxacin in adults could fail to approximate

the drug exposures (55,56). Levofloxacin elimination characterized by t1/2 and clearance is age dependent. This clearly stresses on the need to optimize the levofloxacin treatment, either by increasing the dose or dosing frequency in order to attain given PK/PD targets. Although, FQs have been used with caution in children due to safety concerns, the available data from different studies have not demonstrated any serious arthropathy or other severe toxicity in children (55,72). Further studies (RCTs) are needed to evaluate the safety and pharmacokinetics of levofloxacin especially in HIV infected children and children below 2 years in a high dose, long term MDR-TB regimen (56).Despite the dearth of adequate controlled studies, failure to achieve given phar-macodynamics targets with current levofloxacin dosages in children (55,57,58) strongly, demands for current and future dosing recommen-dations, and shift towards the top end of accepted range for levofloxacin i.e. around 20 mg/kg, with careful monitoring of efficacy and toxicity both in MDR-TB and MDR-TBM pediatric patients.

Above all, it is crucial to mention that, TDM testing could be a chal-lenge in the resource-constrained settings highly burdened by TB. For example, for fAUC/MIC ratio, a series of blood samples (minimum of six or seven samples) need to be collected in order to calculate fAUC. However, obtaining a full time concentration profile might not be feasi-ble in the rural clinics (73). Therefore, limited sampling strategies (LSS) could be applied to estimate the total drug exposure (64). In addition, dried blood spots (DBS) sampling could be introduced as a sampling procedure for the measurement of drug concentrations in the settings where the distance between peripheral, intermediate and central labo-ratories is large. DBS overcomes the problems associated with venous blood sampling such as storage, refrigeration and transportation (74). Samples could be collected in the second week of treatment to perform TDM. Figure 3 describes about TDM incorporation for the successful treatment of MDR-TB.

The limitation of this review is that articles related to the aim were detected only by PubMed search. We did not use other search engines such as Medline, Embase or Cocharane database.

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CONCLUSION

We provide a comprehensive summary of data on levofloxacin con-centrations achieved in the plasma of TB patients and MIC values of levofloxacin for susceptible and resistant M. tuberculosis isolates. A PK-PD approach of determining the dose by considering fAUC/MIC ratio for levofloxacin, could help many clinicians to select the right dose for successful treatment of MDR-TB and hence preventing the emergence of XDR-TB. Based on this review, we recommend firstly TDM for patients with isolates having a MIC value of ≥0.5 mg/L to prevent acquired drug resistance to levofloxacin and secondly the use of standardized 1000 mg once daily dosing rather than in divided por-tions. For patients with body weight ≥70 kg and MIC from 0.25–0.50, levofloxacin dosages in the range of (17 to 20 mg/kg of body weight) are suggested. Although, theoretically MPC is considered more sensitive over MIC in terms of preventing the emergence of resistant mutant during the treatment, MPC concept is relatively new in the field of

Figure 3: Flow chart below shows the in-corporation of TDM for the successful treatment of MDR-TB

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PK/PD. Further work (e.g. a prospective study) is needed to consolidate

its usefulness in MDR-TB treatment. Similarly, in pediatric patients due to higher elimination of levofloxacin, especially for those below 5 years of age, current standard dosing clearly falls short in meeting the given PK/PD targets. To increase the compatibility of levofloxacin exposure with the clinical outcomes, we recommend a personalized therapeutics for levofloxacin with 15–20 mg/kg once daily dosing for children on MDR-TB and MDR-TBM treatment.

Based on these observations, we, advice revising the cutoff value for levofloxacin to around 1 mg/L instead of currently used 2 mg/L. Despite the fact that the DST is still regarded as the gold standard, in-terpretation of results may be complicated by various factors such as characteristics of microorganism, not to mention the requirements for quality laboratory techniques. Values of critical concentrations, which differentiate between wild type and non-wild-type strains, are best used as an epidemiological tool to detect changes in drug resistance rates (38,75). WHO guidelines for the programmatic management of drug-resistant TB, revised in 2014, decreased the critical concentration of levofloxacin to 1 mg/L in Middlebrook 7H10 and 1,5 mg/L in MGIT 960, differing from ofloxacin of 2,0 in both(76). This revision confirms how uncommon DST is performed in any quantitative range, and at the same time supports, while there may be some expected variability among the MIC method and media used, it is far more actionable within the context of individual pharmacokinetic variability than a simple qualitative “S” or “R” readout (61,76,77).

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