Safety, efficacy, and analysis of key parameters after prophylactic administration of a sustained-release formulation of azithromycin in lung cancer surgery

Introduction

The number of elderly patients, along with various preoperative complications, has been increasing worldwide (1-4). As lung cancer patients often have other complications such as respiratory diseases (e.g., chronic obstructive pulmonary disease) probably caused by smoking, such underlying diseases, besides lung cancer, could affect both the incidence of postoperative complications as well as long-term prognosis (1-3,5,6). The prophylactic use of antibiotics has traditionally included cephem antibiotics for a few days in the field of general thoracic surgery (7,8). However, Since the number of older patients with chronic lung disease or impaired lung function has increased, cephem antibiotics might not be sufficient for preventing postoperative infectious complications, especially postoperative pneumonia (8). Therefore, novel strategies are urgently required in the use of prophylactic antibiotics.

Azithromycin (AZM) is a macrolide antibiotic that targets gram-positive bacteria as well as gram-negative rods and mycoplasma (9-11). The effects of this drug last for 7 days after administration. AZM has been indicated for respiratory tract and skin infections (9-11).

Recently, a single-administration type, sustained-release formulation of AZM, Azithromycin-2 g SR (AZM-SR) has been found to accumulate at high concentrations in the target organs, show broad spectrum for respiratory infection, and ensure simple and secure compliance by single administration (12). Moreover, AZM modulates immune reactions by suppressing inflammatory cytokine release (13-17). Recently, AZM has been reported to reduce lung inflammation and MUC5B gene expression, which is responsible for encoding a major core protein secreted from the respiratory tract that is correlated with inflammation in the respiratory system (13,15).

Considering the above findings, we hypothesized that AZM-SR could be an ideal drug formulation for prophylactic administration in general thoracic surgery. Hence, the purpose of this study was to examine the efficacy of single-dose AZM in general thoracic surgery. The risk factors related to postoperative pneumonia were investigated. Moreover, we determined the levels of AZM in the resected lung specimens and performed bacterial culture of the resected lung specimens. In addition, the efficacy of AZM in modulating inflammatory cytokines in clinical patients was investigated. We present the following article in accordance with the STROBE reporting checklist (available at http://dx.doi.org/10.21037/apm-21-383).

Methods

Patient background and study design

This trial was a non-blinded clinical pilot study. The patients who would receive surgery for lung cancer were enrolled in this study between 2013 and 2015. Under informed consent, Azithromycin-2 g SR (AZM-SR) was administered after the evening meal, 1 day prior to the surgery. Regular antibiotic prophylaxis was not administered. The primary endpoint was any adverse event caused by AZM-SR, which would help in assessing the safety of AZM-SR during the perioperative period. The secondary endpoints were the incidence of postoperative infection including postoperative pneumonia, the need for re-administration of antibiotics, and postoperative mortality at 30 days post-surgery.

Exclusion criteria included patients with allergy to macrolides, QT prolongation syndrome, severe renal dysfunction, fructose intolerance, severe liver dysfunction (Child-Pugh C), and current treatment with other antibiotics.

Postoperative pneumonia was defined by the presence of a new or progressive infiltrate on chest radiography or computed tomography during the patient’s hospitalization, together with any of the following: new-onset purulent sputum, change in the character of chronic sputum, fever ≥38 °C, a new rise in C-reactive protein (CRP) value or white blood cell count, positive blood cultures or isolation of the pathogen from sputum, and transtracheal aspirate (18) (Figure 1).

Figure 1 Scheme of the study protocol. Eligible patients who agreed to participate in this clinical trial by written informed consent were enrolled in this study. All participants took the single dose of sustained-release AZM on the night before the operation. Lung tissue samples without any tumor lesion were obtained during surgery. After the operation, no additional antibiotics were administered. *Antibiotics were re-administered only to patients who showed signs of infection. The postoperative course was observed carefully, and clinical data were collected in the medical records. RT-PCR, real time-polymerase chain reaction; AZM, azithromycin.

Ethical approval and informed consent

The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the institutional review board at Nagasaki University Hospital (Approval number; 12121779-7) and was registered in the University Medical Information Network (UMIN) Center (Clinical trial number; UMIN000010368). After the detailed explanation of study-related information and documents, only the patients from whom written informed consent was obtained were enrolled in this study.

Bacterial culture

Bacteria were cultured from lung specimens that were excised under sterile conditions. Each lung specimen was collected and stored in an anaerobic culture bottle for culture on HK semisolid medium. If bacterial growth was confirmed, further cultures were performed using chocolate/blood agar medium, bromothymol blue lactate/chromo agar medium, and Colombia agar medium. Bacterial strain detection was performed by genomic sequencing if 16S-rRNA amplification was detected among culture-positive cases.

AZM concentration in excised lung tissue

The evaluation of AZM concentrations in the excised lung was performed by using the liquid chromatography/tandem mass spectrometry (LC/MS/MS) protocol as previously described (19). A lung specimen, 1 cm² in size, was obtained from excised lung tissue (not the tumor lesion) at the time of surgery and was stored at –80 °C. The weighed lung sample was homogenized, and the concentration of AZM was determined by LC/MS/MS as previously described (19).

RNA extraction and quantitative PCR

RNA extraction from resected lung specimens and qPCR were performed as previously described (20). Briefly, the excised lung tissue sample, 1 cm in size, was stored in RNAlater^®, and total RNA was extracted by using a PureLink RNA micro kit (Life Technologies, CA, USA). One microgram of total RNA was reverse-transcribed into cDNA using a SuperScript VILO cDNA synthesis kit (Life Technologies, Carlsbad, CA, USA).

The expression of MUC5B, tumor necrosis factor-alpha (TNF-α), and IL-6 was measured using a cDNA template with LightCycler 480 probes Master Mix (Roche Diagnostics) and a LightCycler480 PCR System (Roche Diagnostics, Mannheim, Germany). To normalize the results for variability in concentration and integrity of RNA and cDNA, the GAPDH gene was used as an internal control for each sample. PCR primers and TaqMan probes are listed in the Supplemental Table.

Statistical analysis

Data are reported as mean ± standard deviation (SD). All statistical analyses were performed using JMP Pro software (version 14.2.0; SAS Institute, Cary, NC, USA). Comparisons between groups were performed using the Wilcoxon signed-rank test and t-test, and nominal scale analysis involved Pearson χ². We also used Spearman rank correlation to further investigate the relationship between AZM concentration in the lung tissue and other variables. A total of 20 hypotheses were tested, and Bonferroni correction was used for counteracting the multiple testing problem. Values of P<0.0025 were considered as significant.

Results

Patient characteristics

Fifty patients were enrolled in this study and received a single dose of AZM-SR on the day before the operation. The patients’ age was 70±9.0 years [mean ± standard deviation (SD)], and the body mass index was 22.7±2.9 kg/m². Twenty-five (50%) patients were male, and 27 (54%) were ex- or current smokers with an average pack-years of smoking of 15 (range, 0–104). The comorbidity and past history were as follows: 12 cases of hypertension, 11 cases of previous malignancy, 10 cases of respiratory diseases, and 5 cases of thyroid diseases. All of the average data of the parameters of preoperative blood examination were within the normal range. The surgical procedure consisted of lobectomy in 42 cases, segmentectomy in 7 cases, and wedge resection in 1 case. Thirty-three cases received the operation under complete video-assisted thoracoscopic surgery conditions. Operation time and bleeding amount during surgery were 226.5±59.0 min and 100±195.9 g, respectively. Pathological stage I and adenocarcinoma were the most common types of lung cancer (41 cases and 42 cases, respectively) (Table 1).

Table 1 Patient baseline demographics and characteristics for the overall population
Full table

Clinical postoperative outcome

Postoperative pneumonia was observed in 5 cases (10%), 3 of which had technical issues including bronchial stenosis. In two cases, a slight bronchial stenosis, due to bronchial kinking following lung resection, was observed during radiological examination. The remaining one showed a strong adhesin between the lung and chest wall, which required extensive dissection. When abnormal shadow was found in the remaining lungs following the postoperative period in these three cases, it was difficult to distinguish pneumonia from atelectasis or lung hematoma. However, we included these three cases as pneumonia according to our definition of postoperative pneumonia. All cases were treated by re-administration of antibiotics and recovered. Arrhythmia (atrial fibrillation, paroxysmal supraventricular tachycardia) and chylothorax were observed as postoperative complications; these cases were treated with conservative treatment and recovered well. Re-administration of antibiotics was conducted in nine cases (19%), including lung hematoma, chylothorax, and bronchial stenosis. In this study, antibiotics were re-administered under the consultation of an infectious disease physician; Tazobactam/Piperacillin was the most commonly used antibiotics (5 cases). The peak value of postoperative C-reactive protein (CRP) was 8.0±5.98 mg/dL. The AZM concentration in lung tissue was 5.9±5.9 µg/g. There was no adverse event related to AZM administration, and postoperative mortality was 0% (Table 2).

Table 2 Postoperative outcome and data
Full table

Bacterial culture of lung tissue

Five patients showed bacterial growth from the resected lung specimens, which did not show any significant correlation with postoperative pneumonia (Table 3).

Table 3 Univariate analysis of the results in patients who experienced postoperative pneumonitis, patients who needed re-administration of antibiotics, and those who tested positive for bacterial lung tissue culture
Full table

Inflammatory cytokine analysis

The relative gene expression of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and MUC5B normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in the resected specimens was 0.37±0.52, 0.00034±0.018, and 0.0044±0.15, respectively.

Correlation analysis for lung AZM concentrations

Correlations between AZM concentrations in lung tissue and clinical/laboratory findings were assessed statistically. As a result, we observed a negative correlation between AZM concentration and relative IL-6 mRNA levels (IL-6/GAPDH); no other significant correlation was detected (Figure 2).

Figure 2 Correlation between the AZM concentration in the lung tissue and clinical/laboratory findings. Analysis of the correlation between the AZM concentration in the lung tissue and variables. Relative mRNA expression, (A) IL6/GAPDH, (B) MUC5B/GAPDH, (C) TNF-α/GAPDH; (D) age; (E) height of the patients; (F) weight of the patients; (G) pack-year index; (H) preoperative eGFR; (I) preoperative AST; (J) preoperative ALT; (K) preoperative ALP; (L) maximum postoperative CRP value; (M) operation time; (N) bleeding amount. AZM, azithromycin; IL-6, interleukin-6; TNF-α, tumor necrosis factor-alpha; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; eGFR, estimated glomerular filtration rate; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; CRP, C-reactive protein.

Analysis for postoperative pneumonia, re-administration of antibiotics, and bacterial detection in lung tissue culture

Correlation analysis was performed between clinical/laboratory data and postoperative pneumonia (n=5), re-administration of antibiotics (n=9), and bacterial detection by lung tissue culture (4 positive cases). In the analysis for postoperative pneumonia, significant differences were observed in heart disease as a comorbidity; however, no other significant correlation was observed including AZM concentrations in lung tissue and the relative mRNA levels obtained by polymerase chain reaction (PCR) analysis. No significant correlation was observed in the analysis of re-administration of antibiotics and tissue culture (Table 3).

Discussion

In this study, we have focused on the prophylactic use of AZM-SR in general thoracic surgery and illustrated the feasibility of this novel concept. Moreover, we evaluated the levels of multiple cytokines associated with inflammation and mucosal immunity, which have reinforced this concept.

IL-6 mRNA levels and AZM concentrations showed negative correlation, demonstrating the anti-inflammatory effect of AZM in general thoracic surgery, while that of TNF-α and MUC 5B showed no correlation. The immunomodulatory effect of AZM has been investigated in lung disease patients (14,21). In the acute phase of immunomodulation, AZM decreases the production of pro-inflammatory cytokines such as IL-6, TNF-α, interleukin-8 (IL-8), granulocyte-macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinases (MMPs) (22-25). Our data demonstrate a negative correlation between AZM concentrations in lung tissue and IL-6 mRNA levels, which is consistent with previous reports. Moreover, especially in lung transplant patients, AZM has been used for its anti-inflammatory effect (26). Our finding of the negative correlation between AZM and IL-6 concentrations could help inform future use of AZM in general thoracic surgery, especially for high-risk patients with impaired respiratory function. Although other cytokines showed no correlation in this study, further investigations may show correlation between AZM and other cytokines in the respiratory tract.

We found no AZM-related side effects in our study, such as elevation of liver enzymes, long QT syndrome, or diarrhea, which demonstrates the safety of AZM-SR in general thoracic surgery and suggests the feasibility of another study with increased number of patients with a double-arm study design.

The results of the bacterial culture of the resected lung specimens showed a few positive cases, which showed no correlation with postoperative infection. Thus, we concluded that AZM-SR showed no negative effect with respect to postoperative infection. The positive bacterial cultures of the resected lung might be due to preoperative colonization, as postoperative bacterial examination showed no positive culture. The effect of AZM-SR in the prevention of postoperative infection should be further investigated. AZM levels in pulmonary tissue have been found to remain high for up to 5 days even after a single oral 500 mg dose, in contrast to plasma AZM levels (27). Higher AZM levels have also been reported to be higher in tissue interstitial fluid than in plasma (19,28). These findings suggest that the higher AZM concentration in the respiratory tract would possess an inhibitory effect on many sensitive respiratory pathogens (27).

Postoperative pneumonia is one of the most common complications in patients with lung cancer undergoing therapeutic surgical resection and has been reported as a poor prognostic factor (29,30). The reported incidence of postoperative pneumonia following lung cancer surgery ranges from 3.6% to 18.7% (29-32). The incidence of postoperative pneumonia in our study was 10% (5 cases), while that with traditional cephem antibiotics in our institution was 6.7% to 9.4% between 2013 and 2015 (data not shown). However, some technical issues were involved in 3 cases, suggesting that this AZM formulation was not inferior to classical prophylactic treatment. A significant relationship was only observed between postoperative pneumonia and heart disease as a comorbidity, which was consistent with previous reports that showed underlying heart disease as a risk factor for postoperative pneumonia (31,33). Although direct correlation between AZM and postoperative pneumonia was not found, the high concentration of AZM in the lung may prevent postoperative pneumonia through an anti-inflammatory effect, as evidenced by the low IL-6 expression in this study. Some patients received re-administration of other antibiotics as prophylactics for lung hematoma or chylothorax; however, the sustained effect of AZM-SR observed in this study suggests that re-administration would be unnecessary.

The use of macrolides has drawn attention to possible cardiovascular events, particularly arrhythmias, mainly with concomitant use of other QT-prolonging drugs or underlying structural heart disease. However, azithromycin may be considered the safest of all macrolides in this respect as serum levels in healthy volunteers are >3,000 times lower than drug concentrations required for cardiomyocyte potassium channel (hERG/IKr)-blockade, action potential prolongation, and QTc prolongation (26). In this study, no AZM-related adverse events, including typical arrhythmia (long QT), were observed. We believe that our exclusion criteria, namely, severe liver dysfunction and long QT syndrome, were appropriate in this study.

This study is not without limitations. First, since we focused on the biological features rather than the statistical aspects in this pilot study, the number of patients (sample size) was not statistically calculated, and no control group was included. Second, we did not show a direct relationship between AZM and postoperative pneumonia. However, we believe that this study demonstrates the safety and efficacy of the prophylactic use of AZM-SR in general thoracic surgery.

In conclusion, prophylactic use of AZM-SR in general thoracic surgery seems safe and feasible. Additional benefits from the anti-inflammatory effect of AZM could be expected with such use. This study lays the foundation for future studies on AZM-SR in matched patient populations and offers a new strategy of prophylactic treatment in general thoracic surgery.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at http://dx.doi.org/10.21037/apm-21-383

Data Sharing Statement: Available at http://dx.doi.org/10.21037/apm-21-383

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/apm-21-383). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by institutional review board of Nagasaki University Hospital (approval No. 12121779-7) and was registered in the University Medical Information Network (UMIN) Center (Clinical trial No. UMIN000010368). Informed consent for this retrospective analysis was taken from all individual participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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