Serum concentrations of clarithromycin and rifampicin in pulmonary Mycobacterium avium complex disease: long-term changes due to drug interactions and their association with clinical outcomes

Subjects

Nine outpatients diagnosed with pulmonary MAC disease at the Chemotherapy research
institute, Kaken hospital (Kaken hospital) from 2012 to 2014 were enrolled in this
study. Patients under 20 years of age, over 80 years of age, or with hepatic or renal
dysfunction were excluded. All patients were required to fulfill the JSTB and JRS
criteria. Doses of CAM, RFP, and EB were based on the recommendations of both societies.
Urine and blood samples were collected on days 1, 15, 29 (for four subjects, sample
collections were continued on days 57, 85, 113, 141, 169, 225, 281, 337, and 365).
Urine samples were collected on the morning of the first day before a medical examination
and the subjects were administered the drugs after lunch with blood collected two
hours later. On days 15, 29, 57, 85, 113, 141, 169, 225, 281, 337, and 365, the subjects
took the drugs at home after breakfast and then two hours later, urine and blood samples
were collected before a medical examination in the hospital. The urine samples were
collected at almost the same time as on the first day. Serum and urine samples were
frozen and stored at –80 °C until analysis.

This study was conducted in compliance with the ethical guidelines for clinical studies.
It was approved by the Ethical Review Committee at Tokyo University of Science (approval
and study number: 12001) and Kaken hospital (approval and study number: 9) and registered
in the University hospital Medical Information Network-Clinical Trial Registry (November
15, 2012, ID: UMIN000009343). All patients provided written informed consent before
entry into the study.

Reagents

The internal standard roxithromycin, CAM, RFP, DR, and F were purchased from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan), while 6?OHF and prednisolone, which
served as an internal standard for analysis, were purchased from SIGMA-Aldrich Japan
Co. (Tokyo, Japan). M-5 was kindly provided by Taisho Toyama Pharmaceutical Co., Ltd.
(Tokyo, Japan).

Measurement of serum drug concentrations and urinary 6?OHF and F concentrations

The serum concentrations of CAM, M-5, RFP, and DR were measured using the liquid chromatography-tandem
mass spectrometry method described by Oswald et al. 15], with modifications. The liquid chromatography-tandem mass spectrometry system used
consisted of an LC-20 AD high-performance liquid chromatography (HPLC) system (Shimadzu
Co., Kyoto, Japan) coupled with an API 3200 mass spectrometer (AB Sciex Japan, Ltd.,
Tokyo, Japan). The separations were performed on the reverse phase column Atrantis
T3 (5 ?m, 2.1?×?150 mm, Nihon Waters Co. Ltd., Tokyo, Japan) and using the solvent
mixtures (A) 10 mM ammonium acetate–acetonitrile (20:80) and (B) 10 mM ammonium acetate-acetonitrile
(80:20) with the following gradient: 40 % B increased to 85 % B over 4 min, decreased
to 40 % B over 1.25 min, increased to 85 % B over 0.85 min, and equilibrated back
to 40 % B over 0.9 min.

Urinary 6?OHF and F concentrations were measured using the HPLC-UV method described
by Hu et al. 16], with modifications. The HPLC system used consisted of an L-2130 pump and an L-2455
UV detector (HITACHI High-Tech Science Co. Ltd., Tokyo, Japan). The detection wavelength
was fixed 245 nm. The separations were performed on the reverse phase column PEGASIL
ODS SP100 (5 ?m, 4.6?×?250 mm, Senshu Scientific Co., Ltd., Tokyo, Japan), and using
the solvent mixtures (A) acetonitrile and (B) acetonitrile-water (17.5: 82.5), with
the gradient starting at 100 % B and decreasing to 63.6 % B over 80 min.

Statistical analyses

The serum concentration for each drug and the ratio of urinary 6?OHF/F are presented
as the mean?±?standard error (SE). Data on day 15 were compared with each initial
value using paired Student’s t-test during the initial period of study (the day 1–29).
The significance level for each comparison was set as P 0.05. Serum RFP concentrations (mean?±?SE) were plotted against the ratio of urinary
6?OHF/F (mean?±?SE) from the same time point and the relationship between the values
was examined using the Pearson correlation coefficient. All statistical analyses were
performed using Excel 2010 (Microsoft Co., Redmond, WA, USA).

Clinical outcomes

The clinical outcomes of four subjects who continued in this study for 1 year were
evaluated by examination of sputum cultures and chest images at the indicated time
points. The cultures were scored as “– (negative)”, “1+”, “2+”, “3+”, or “4+”. Culture
negative conversion was defined as three consecutive negative cultures; with the time
of conversion defined as the date of the first negative culture. The subjects were
monitored and relapse was recorded during the observation period (until day 365) and
post-observation period (after day 365), as the first day of a positive culture after
negative scores. Chest images were evaluated by a pulmonologist, and classified as
“Improved”, “Unchanged”, and “Worsened” compared to the previous assessment.

CAM susceptibility test

CAM susceptibility tests were performed using the minimum inhibitory concentration
(MIC) method for 3 of the 4 subjects who continued in this study for 1 year. If sputum
culture conversion was not achieved after 169 days, isolates from the last positive
cultures collected were tested. According to the JSTB and JRS guidelines for chemotherapy
of pulmonary nontuberculous mycobacterial disease-2012 revised version 13], strains with MIC of CAM (?4 ?g/mL) were considered susceptible to CAM, while those
with an MIC ?32 ?g/mL were considered resistant. Values of 8–16 ?g/mL were considered
intermediate.