Pharmacokinetics of oral antimycobacterials and dosing guidance for Mycobacterium avium complex treatment in cystic fibrosis

Abstract

Background:

Treatment failure of Mycobacterium avium complex (MAC) pulmonary disease occurs in about 30% of people with cystic fibrosis (CF) and may be a result of abnormal drug concentrations.

Methods:

Prospective, cross-over, single-dose PK study of 20 pancreatic insufficient individuals with CF and 10 healthy controls (HC). CF subjects received simultaneous doses of oral azithromycin, ethambutol, and rifampin in the fasting state and with food and pancreatic enzymes, separated by two weeks. HC received fasting doses only. A non-compartmental model was used to estimate PK parameters of drugs and metabolites.

Results:

Azithromycin maximum concentration (C_max) was higher and rifampin C_max was lower in fasting CF subjects compared to HC, while other PK measures, including those for ethambutol, were similar. Addition of food and enzymes did not improve the C_max of the antimycobacterial drugs. Nineteen of 20 of CF subjects had one or more abnormal C_max z-scores in either or both the fasting or fed state, when compared to HC.

Conclusion:

PK profiles of azithromycin and ethambutol were similar between CF and HC, except azithromycin C_max was slightly higher in people with CF after a single dose. Rifampin PK parameters were altered in persons with CF. Addition of food and enzymes in CF subjects did not improve PK parameters. Standard dosing guidelines should be used as a starting point for people with CF initiating MAC therapy and therapeutic drug monitoring should be routinely performed to prevent the possibility of treatment failure due to abnormal drug concentrations.

Clinical Trial Registration:

ClinicalTrials.gov Identifier: NCT02372383

Introduction:

Nontuberculous mycobacteria (NTM) are pathogenic organisms in the cystic fibrosis (CF) population, with prevalence rates up to 22%.^1–8 Mycobacterium avium complex (MAC) is the most commonly isolated NTM in the United States’ CF population.^1,9 NTM infections cause significant morbidity and mortality with associated lung function decline and systemic symptoms of fever, fatigue, and weight loss.^6,9–11 Treatment is especially burdensome and up to 32% of people with CF and MAC can experience treatment failure and remain chronically infected, resulting in accelerated clinical decline.¹¹

One potential reason for treatment failure is subtherapeutic drug concentrations^12,13. Formal pharmacokinetic (PK) trials of the oral antimycobacterial drugs in CF have not been performed and no data are available to support current treatment guidelines in people with CF.¹⁴ Currently recommended dosages and administration guidelines of the antimycobacterial drugs are based on data from healthy volunteers and people with tuberculosis.^9,12,15–17 People with CF have altered PK for other antibiotics due to impaired gut absorption from pancreatic insufficiency, delayed gastric emptying causing reduced bioavailability, increased volume of distribution, alteration in metabolism and clearance, and increased renal elimination.^13,18–20

The primary objective of this study was to determine the PK profiles of the three most commonly prescribed oral antibiotics used to treat MAC, azithromycin, ethambutol, and rifampin, in people with CF compared to healthy controls (HC) to inform dosing and treatment guidelines. We also sought to determine whether taking the antimycobacterials with food and supplemental pancreatic enzymes improves drug concentrations by improving intestinal absorption in people with CF and if specific clinical variables affect drug concentrations.

Materials and Methods:

Single-dose PK measurements were obtained in 20 subjects with pancreatic-insufficient CF, 16 years and older, and 10 adult HC. HC were limited to adult subjects > 18 years of age due to institutional human research requirements. CF subjects were excluded if they had a positive NTM culture in the previous 5 years to avoid partial treatment of NTM. Subjects were excluded if they had elevated liver enzymes greater than five times the upper limit of normal at screening or a history of surgical bowel resection or lung transplantation. Additionally, subjects were excluded if they were taking concomitant medications known to interact with any of the antimycobacterial drugs, including ivacaftor (e-Table 1). This study was approved by the Colorado Multiple Institutional Review Board (14–1043). Informed consent was obtained from the participants and their parents or legal guardians if applicable.

Using a cross-over study design, clinically stable CF subjects were randomized to receive simultaneous, single doses of oral azithromycin (10mg/kg, max 500mg), ethambutol (15mg/kg, max 2500mg), and rifampin (10mg/kg, max 600mg) in two states: fasting or fed, separated by a two-week washout. In the fed state, CF subjects received a 500–600 kcal macronutrient-controlled meal (20% protein, 35% fat, 45% carbohydrate) plus a meal-dose of pancreatic enzymes (1,500–2,500 lipase units/kg). HC received the same simultaneous doses once, in the fasting state only, per standard dosing guidelines. If applicable, subjects held histamine-2 blockers and proton pump inhibitors for 3 days prior to each PK study day and held chronic azithromycin therapy for a minimum of 2 weeks prior to the first PK day and through completion of all PK study visits.

Blood samples were drawn at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8 and 12 hours after dosing and collected in heparinized containers. Plasma was extracted via centrifugation, transferred into polypropylene tubes, and stored at −80 degrees Celsius prior to analysis. Serum samples were sent for complete blood count, high-sensitivity C-reactive protein (hs-CRP), and creatinine.

Azithromycin, ethambutol and rifampin plasma concentrations, along with their major metabolites (N’-desmethyl azithromycin and descladinose azithromycin; 2,2’-[ethanediyldiimino] bis-butanoic acid; 25-desacetyl rifampicin and N-demethyl rifampicin, respectively) were measured via liquid chromatography-tandem mass spectrometry mass spectrometry (see Supplement, e-Figure 1, and e-Table 2 for methodologic details).

Statistical Analyses:

Non-compartmental analysis was used to summarize the following PK parameters: maximum concentration (C_max), time of maximum concentration (T_max) and area under the curve from 0 to 12 hours (AUC₁₂). Additional details are included in the online supplement. Comparisons across groups were made using Fisher’s exact tests and Wilcoxon and Signed rank tests. PK parameters were associated with clinical variables including age and the following hepatic function, renal function, and systemic biomarkers of inflammation laboratory tests: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, white blood cell count (WBC), absolute neutrophil count, and hs-CRP, using a Spearman’s rank-based correlation coefficient. Using the C_max values from HC subjects, z-scores were calculated for the CF subjects separately for each drug. The z-scores were dichotomized as either normal (within +/− 2 standard deviations (SD) from the HC mean) or abnormal (< −2 or > +2).

Results:

Demographics and clinical characteristics of the CF subjects and HC are shown in Table 1. Twenty-one CF subjects were enrolled; one was removed from the study due to poor venous access and inability to complete the study visit. The remaining 20 CF subjects and 10 HC completed all study visits and are included in the analysis. CF subjects were younger than HC and had higher circulating hs-CRP, WBC, and ALT. At the time of enrollment, no CF subjects were on CF-transmembrane conductance regulator (CFTR) modulator therapies.

Table 1:

Subject Demographics, Clinical Characteristics and Antibiotic Dosing.

N (%) Median (range)	CF subjects (n = 20)	Healthy Controls (HC) (n = 10)	P-valuea
Age (years)	18.3 (16.1 – 24.8)	25.3 (18.2 – 30.7)b	<0.01
Gender (female)	7 (35%)	6 (60%)	0.26
BMI (kg/m2)	21.9 (18.0 – 26.6)	23.0 (18.9 – 25.1)	0.17
CF genotype
F508del/F508del	9 (45%)	--	--
F508del/min	11 (55%)	--	--
% predicted FEV1c	95.0 (67–127)	--	--
Fasting hs-CRP (mg/dL)	0.8 (0.2 – 9.01)	0.2 (0.0 – 5.1)	0.03
Fasting WBC (103/ uL)	7.2 (5.8 – 11.3)	5.6 (3.4 – 10.1)	0.01
Fasting absolute neutrophil count (103/ uL)	3.8 (2.5 – 7.7)	3.0 (1.4 – 7.2)	0.08
Fasting creatinine (mg/dL)	0.9 (0.7 – 1.1)	1.0 (0.8 – 1.2)	0.21
AST U/L	43.5 (22.0 – 94.0)	30.0 (26.0 – 55.0)	0.07
ALT U/L	37.5 (22.0 – 93.0)	26.0 (20.0 – 47.0)	0.01
Actual Fasting Dose (mg)
Azithromycin	500 (500 – 500)	500 (500 – 500)	--
Ethambutol	900 (800 – 1200)	1000 (800 – 1200)	0.54
Rifampin	600 (600 – 600)	600 (600 – 600)	--

BMI – body mass index; FEV₁ – forced expiratory value in 1 second; hs-CRP – high sensitivity C-reactive protein; AST – aspartate transaminase; ALT – alanine transaminase

^aFisher’s exact test or Wilcoxon test

^bHC limited to adults > 18 years due to institutional human research requirements

^cn = 19

Azithromycin PK parameters across groups

Azithromycin PK results for CF subjects in the fasting and fed state as well as HC are summarized in Table 2 and e-Figure 2A. Median (min, max) azithromycin C_max was higher in fasting CF subjects compared to HC (2.0 [0.1 – 5.3] mg/L vs. 1.1 [0.6 – 2.1] mg/L, p =0.05). Median T_max occurred at 2 hours for all groups. Calculated AUC₁₂ was similar across groups. The ranges of C_max and AUC₁₂ were wider in CF subjects compared to controls. E-Figures 3A and 4A show median and individual time-serum concentrations, respectively. Addition of food and enzymes in CF subjects did not significantly alter PK of azithromycin. Among the two major metabolites measured, the median (min, max) C_max of descladinose azithromycin in CF fasting subjects was significantly higher than HC (294 [34, 1612] mg/L vs. 83 [3, 576] mg/L, p=0.03). Addition of food and enzymes in CF subjects significantly lowered descladinose azithromycin C_max and AUC₁₂. Female gender was associated with a mild increase in T_max in the fed state, otherwise, no tested clinical variables or hepatic, renal, or inflammation laboratory tests were positively or negatively associated with differences in azithromycin C_max, T_max or AUC₁₂ in CF subjects (e-Tables 4A and 4B).

Table 2. Azithromycin Pharmacokinetics Parameters.

Derived from non-compartmental models, results shown in median (min, max).

Azithromycin PK parameter	CF Fasting (n = 20)	CF Fed (n = 20)	Healthy Control (HC) (n = 10)	CF Fasting vs. HC (P-value)	CF Fasting vs. CF Fed (P -value)
Cmax (mg/L)	2.0 (0.1, 5.3)	2.2 (0.1, 4.1)	1.1 (0.6, 2.1)	0.05	0.70
Tmax (h)	2.0 (1, 4)	2 .0 (1, 4)	2.0 (1, 3)	0.89	0.64
AUC12 (mg*h/L)	5.8 (0.4, 13.1)	5.2 (0.6, 8.6)	4.5 (2.2, 9.4)	0.51	0.60

C_max - maximum concentration; T_max – time of maximum concentration; AUC₁₂ – area under the curve from 0 to 12 hours

Ethambutol PK parameters across groups

Ethambutol PK results for CF subjects in the fasting and fed state as well as HC are summarized in Table 3 and e-Figure 2B. Median ethambutol C_max and AUC₁₂ were not significantly different in fasting CF subjects compared to HC. Median T_max occurred at 2 hours for both groups. The ranges of C_max and AUC₁₂ were wider in CF subjects compared to controls. E-Figures 3B and 4B show median and individual time-serum concentrations, respectively. The major ethambutol metabolite was undetectable in serum in all subjects. Female gender was associated with a mild increase in T_max in the fed state, otherwise, no tested clinical variables or hepatic, renal, or inflammation laboratory tests were positively or negatively associated with differences in ethambutol C_max, T_max or AUC₁₂ in CF subjects (e-Tables 4A and 4B).

Table 3. Ethambutol Pharmacokinetics Parameters.

Derived from non-compartmental models, results shown in median (min, max).

Ethambutol PK parameter	CF Fasting (n = 20)	CF Fed (n = 20)	Healthy Control (HC) (n = 10)	CF Fasting vs. HC (P-value)	CF Fasting vs. CF Fed (P-value)
Cmax (mg/L)	4.2 (1.1, 6.8)	4.3 (1.0, 5.7)	3.0 (2.3, 4.7)	0.23	0.13
Tmax (h)	2.3 (1, 4)	2.0 (1, 4)	2.3 (1, 4)	0.77	0.34
AUC12 (mg*h/L)	18.7 (5.6, 31.2)	18.1 (4.1, 31.0)	17.0 (12.9, 25.1)	0.66	0.02

C_max - maximum concentration; T_max – time of maximum concentration; AUC₁₂ – area under the curve from 0 to 12 hours

Rifampin PK parameters across groups

Rifampin PK results for CF subjects in the fasting and fed state as well as HC are summarized in Table 4 and e-Figure 2C. Median rifampin C_max and AUC₁₂ in fasting CF subjects were significantly reduced compared to HC (12.5 mg/L vs. 16.6 mg/L, p=0.04; 66.2 mg*h/L vs. 91.1 mg*h/L, p=0.02, respectively). Median T_max occurred at 2 hours for fasting CF subjects and HC. The ranges of C_max and AUC₁₂were wider in CF compared to controls. E-Figures 3C and 4C show median and individual time-serum concentrations, respectively. With addition of food and enzymes in CF subjects, rifampin C_max significantly decreased further to 11.2 mg/L and T_max increased to 2.5 hours. The median C_max, T_max and AUC₁₂ of the two major rifampin metabolites were not different between CF fasting subjects and HC. addition of food and enzymes in CF subjects significantly reduced C_max and AUC₁₂ and increased T_max of the N-demethyl rifampicin metabolite and reduced C_max and increased T_max of the 25 desacetyl rifampin metabolite (e-Table 3). No tested clinical variables or hepatic, renal, or inflammation laboratory tests were positively or negatively associated with differences in rifampin C_max, T_max or AUC₁₂ in CF subjects (e-Tables 4A and 4B).

Table 4. Rifampin Pharmacokinetics Parameters.

Derived from non-compartmental model, results shown in median (min, max).

Rifampin PK parameter	CF Fasting (n = 20)	CF Fed (n = 20)	Healthy Control (HC) (n = 10)	CF Fasting vs. HC (P -value)	CF Fasting vs. CF Fed (P -value)
Cmax (mg/L)	12.5 (6.5, 21.9)	11.2 (4.7, 14.5)	16.6 (10.2, 27.0)	0.04	<0.01
Tmax (h)	1.5 (1, 3)	2.5 (1, 6)	1.5 (1, 4)	0.80	0.02
AUC12 (mg*h/L)	66.2 (34.5, 126)	68.6 (26.1, 100.1)	91.1 (64.5, 131)	0.02	0.07

C_max - maximum concentration; T_max – time of maximum concentration; AUC₁₂ – area under the curve from 0 to 12 hours

Single dose peak concentrations for CF subjects compared to HC subjects

The mean (SD) values from the HCs used to calculate z-scores for the CF subjects were 1.2 (0.4) for azithromycin, 3.2 (1.0) for ethambutol and 17.0 (5.4) for rifampin. CF subjects frequently had abnormal C_max values with z-scores that were either greater than +2SD or less than −2SD compared to the HC values (Figure 1). Overall, 16 CF subjects had at least one high C_max z-score greater than +2SD and 6 CF subjects had at least one low C_max z-score below −2SD. In total, 19 of 20 CF subjects (95%) had one or more abnormal C_max z-scores (outside +/− 2SD) when compared to HC. Specifically, related to low C_max z-scores, three CF subjects had a C_max z-score below −2SD for one drug and one had a C_max z-score below −2SD for two drugs in the fasting state (total four CF subjects (20%) in fasting state). In the fed state, one CF subject had one low C_max z-score, one had a low C_max z-score for two drugs, and two had a low C_max z-score for all three drugs (total four CF subjects (20%) in the fed state). There were no differences in clinical characteristics or in hepatic, renal, or inflammation laboratory values among the CF subjects with low C_max z-scores.

Figure 1: Summary of CF subjects (fasting and fed) C_max z-scores for azithromycin, ethambutol, and rifampin compared to HC.

Each line represents the C_max z-scores from one CF subjects across all drugs and fasting and fed states. The shades area represents the typical C_max z-scores from HC subjects (within +/− 2 standard deviations (SD) from the mean). Overall, 16 CF subjects had at least one high C_max z-score greater than +2SD and 6 CF subjects had at least one low C_max z-score below −2SD. In total, 19 of 20 CF subjects had one or more abnormal C_max z-scores (outside +/− 2SD) when compared to HC.

Conclusion:

In this study of the three most commonly prescribed oral antibiotics used to treat MAC in people with CF, the PK profiles of azithromycin and ethambutol were overall similar between CF and HC, and rifampin PK were altered in CF compared with HC. Specifically related to peak concentrations, azithromycin C_max in CF subjects is higher and rifampin C_max in CF subjects is significantly lower than in HC. Addition of food and enzymes in CF subjects did not improve PK parameters of the three drugs. Specifically, addition of food and enzymes further lowered C_max and delayed T_max of rifampin, decreased C_max of both rifampin metabolites, and decreased C_max and AUC₁₂ of the primary azithromycin metabolite. Importantly, at the individual level, nearly every CF subject one or more abnormal single dose C_max z-scores when compared to HC subjects.

Our HC population showed similar PK parameters to prior published single-dose fasting PK parameters for ethambutol¹⁶ and rifampin¹⁵. Our HC single dose azithromycin C_max values were higher than prior published data, but similar differences in C_max values between people with CF and HC were detected, though prior reported differences did not reach statistical significance²¹. Current drug package inserts recommend that rifampin be given on an empty stomach, and ethambutol and azithromycin can be given with or without food^22–24. Our data support that people with CF should also follow these guidelines when initiating these antibiotics (Table 5). Notably, the co-administration of food and enzymes with the drugs did not improve PK parameters. This aligns with the general finding that addition of food typically prolongs absorption and lowers C_max^13,15,16. We speculate malabsorption may impact PK for some individuals with CF, as a two of our subjects were found to have one and two low C_max z-scores, respectively, when fasting that improved when the drug was taken with food and enzymes.

Table 5.

Summary of Antibiotic Dosing and Monitoring Recommendations for Patients with CF and MAC Pulmonary Disease.

Drug	Initial Oral Dose	Condition	Peak Drug Measurement
Azithromycin	10mg/kg (max 500mg) once daily	With or without food	2 hours post dose
Ethambutol	15mg/kg (max 2500mg) once daily	With or without food	2 hours post dose
Rifampin	10mg/kg (max 600mg) once daily	Empty stomach	2 hours post dose

A total of 19 of our 20 CF subjects had at least one abnormal drug C_max in either the fasting or fed state (or both) when compared with the HC group. In a clinical scenario, if a low single dose C_max persisted at steady-state, this might result in treatment failure in a person with CF on treatment for MAC pulmonary disease, especially in a case where more than one drug level is low.²⁵ Alternatively, if those with high single dose C_max values persisted at steady-state, one may anticipate more reported toxicities and treatment intolerance. In our CF participants, there were no clinical characteristics or laboratory measurements associated with having low drug levels or altered PK parameters. In this population without NTM, using HC data and published target indices,^{15–17,26,27} we observed wide PK ranges in our C_max values, thus, we recommend that standard-dosing regimens should be employed, yet close therapeutic drug monitoring be used in all people with CF to adjust dosing and dosing condition in the individual patient (Table 5). In order to account for the prolonged half-life of azithromycin and potential auto-induction of rifampin, we recommend waiting at least 2 weeks to check drug levels to allow patient to reach steady-state. Achievement of proposed target serum concentrations is important for clinical efficacy.^28–31 In a retrospective clinical drug monitoring study,³¹ low C_max serum concentrations were found for multiple antimycobacterial drugs in CF subjects, including two subjects with treatment failure. Dose adjustment resulted in a clinical improvement in these subjects. One report described similar findings in a patient with AIDS and tuberculosis who experienced clinical decline in the setting of low anti-tuberculosis drug concentrations measured by C_max and clinical and bacteriologic response following dose adjustment and improved concentrations²⁹. In non-CF patients with MAC infection, Jeong et al³² reported that peak plasma concentrations of azithromycin were associated with favorable microbiologic responses when receiving daily therapy. Koh et al.³³ demonstrated that subtherapeutic drug concentrations of clarithromycin were common but were not associated with treatment outcomes suggesting that drug level monitoring may not be necessary; however, this may not be translatable to people with CF. In summary, we suggest therapeutic drug monitoring may be beneficial in patients with CF, but recognize the need for additional research in this population.

The strengths of this study included that it was an intensive, controlled PK study performed in a fed and fasting state within the target disease population and included a concurrent HC group. Limitations of our study include that data were limited to single-dose kinetics, and that steady-state PK may show different results, particularly for rifampin due to potential auto-induction and azithromycin due to prolonged elimination half-life.^22,34 Administration of the three drugs simultaneously was assumed to be acceptable based on literature and historic precedent,^15,16,22 but not specifically tested in this study. Additionally, subjects in this study were excluded if they were on a medication with a known drug-drug interaction with the study medications. People with CF are commonly taking medications including CFTR modulator therapies containing ivacaftor, antifungal drugs, proton-pump inhibitors and histamine-2 blockers which may alter absorption of these oral antibiotics. Individual drug level monitoring may reveal further PK derangements (or normalization) in the setting of co-administration with an inducer or inhibitor (ex. ivacaftor and rifamycins). This study was also done in patients without NTM positive cultures or evidence of NTM pulmonary disease requiring treatment; thus, we could not perform true pharmacodynamic measures using minimum inhibitory concentration data, nor validate these data against treatment outcomes such as infection clearance. Lastly, our results do not provide PK information for young people with CF (< 16 years of age).

In summary, the PK profiles of azithromycin and ethambutol were similar between subjects with CF and HC, except azithromycin C_max was slightly higher in people with CF after the single dose. Overall, rifampin PK were altered in people with CF. Addition of food and enzymes did not improve PK. Importantly, on an individual level, nearly all CF subjects had at least one peak drug level outside the HC normal range. We recommend that standard dosing guidelines⁹ are used when initiating MAC therapy in people with CF as follows: azithromycin (10mg/kg, max 500mg), ethambutol (15mg/kg, max 2500mg), and rifampin (10mg/kg, max 600mg). Medications can be co-administered, but rifampin should preferably be taken on an empty stomach. Peak drug concentrations can be measured 2 hours after dosing, in line with current guidelines for people without CF.⁹ Although current CF Foundation/European CF Society NTM treatment guidelines³⁵ recommend that therapeutic drug monitoring be reserved for people experiencing treatment failure, our data suggest that therapeutic drug monitoring should be performed for all people with CF on MAC treatment to prevent the possibility of treatment failure due to abnormal drug concentrations.

HIGHLIGHTS.

• This was the first intensive, controlled, pharmacokinetic study performed for antimycobacterial drugs in people with cystic fibrosis (CF).

• The pharmacokinetic profiles of azithromycin and ethambutol were found to be similar between subjects with CF and healthy controls, and rifampin pharmacokinetics were significantly altered in people with CF.

• Addition of food and enzymes in CF subjects did not improve PK parameters.

• Nearly all CF subjects had one or more abnormal C_max z-scores when compared to HC.

• We report a summary of antibiotic dosing and therapeutic drug monitoring recommendations to be used for people with CF on treatment for Mycobacterium avium complex pulmonary disease.

Abbreviation List:

AIC

Akaike’s Information Criterion

ALT

alanine transaminase

AST

aspartate transaminase

AUC₁₂

area under the curve from 0–12 hours

C_max

Maximum concentration

BMI

body mass index

CF

cystic fibrosis

CFTR

cystic fibrosis transmembrane conductance regulator

FEV₁

forced expiratory value in 1 second

HC

healthy control

hs-CRP

high sensitivity C-reactive protein

PK

pharmacokinetics

MAC

Mycobacterium avium complex

NTM

nontuberculous mycobacteria

SD

standard deviation

T_max

time of maximum concentration

WBC

white blood cell count