Protective Effects of Clarithromycin, a Lipophilic 14-Membered Macrolide, on Hemolysis Induced by Lysophosphatidylcholine in Human Erythrocytes
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Regular Article Jpn. J. Pharm. Health Care Sci. 38(10) 617―627 (2012) Protective Effects of Clarithromycin, a Lipophilic 14-membered Macrolide, on Hemolysis Induced by Lysophosphatidylcholine in Human Erythrocytes Kuninori Iwayama 1, Yusuke Takashima 1, 4, Ko-ichi Ohtaki 1, 5, Kikutaro Endoh 2, Yoshiko Tampo 3 and Nobumasa Hayase＊1 Departments of Pharmacology & Therapeutics 1, Life Sciences 2, and Public & Health 3, Hokkaido Pharmaceutical University School of Pharmacy, Department of Hospital Pharmacy, Oji Sogou Hospital 4, Department of Hospital Pharmacy & Pharmacology, Asahikawa Medical University Hospital 5 Received May 7, 2012 Accepted July 29, 2012 We examined the effects of 8 macrolide antimicrobial agents, including 14-, 15- and 16-membered ring lactone and ketoride derivatives, on hemolysis induced by lysophosphatidylcholine (LPC) in human erythrocytes. LPC induced hemolysis at concentrations above the critical micelle concentration (4 µM). Vitamin E (α-tocopherol), used as a reference drug, attenuated the 50％ hemolysis induced by 6 µM LPC when present at concentrations between 1 µM and 100 mM. Clarithromycin significantly attenuated LPC-induced hemolysis at a wider range of concentrations (100 nM to 1 mM), but other macrolides attenuated hemolysis only at high concentrations (100 µM and/or 1 mM). Since vitamin E tends to stabilize membranes due to its high lipophilicity, it appears that the high lipophilicity of clarithromycin is responsible for its protective action against damage induced by LPC. However, rokitamycin was not effective, although it is more lipophilic than clarithromycin, indicating that factors other than high lipophilicity are responsible for the protective effects of macrolide antimicrobial agents against LPC-induced hemolysis. Neither vitamin E nor clarithromycin attenuated hypotonic hemolysis (60 mM NaCl) at concentrations that inhibit LPC-induced hemolysis. On the other hand, both vitamin E and clarithromycin affected LPC micelle formation, suggesting that these drugs directly bind to LPC. We therefore believe that the protective effects of clarithromycin on LPC-induced hemolysis may be related physicochemically to its high lipophilicity and 14-membered ring lactone structure, which help maintain erythrocyte membrane integrity by preventing LPC micelle formation. These drugs likely do not act by a mechanism that protects against osmotic imbalance. Key words ―― anti-inflammatory effect of macrolides, clarithromycin, lysophosphatidylcholine (LPC), hemolysis, lipophilicity necrosis factor alpha and interleukin-8 from bron- Introduction chial epithelial cells. This property affects the mi- Macrolide antimicrobial agents, such as eryth- gration of neutrophils and impairs their genera- romycin, clarithromycin and other 14-, 15-mem- tion of superoxide.5) However, there are limited bered ring macrolides have been shown to be ef- data concerning the direct effect of macrolides on fective for the treatment of chronic airway the cell membrane under inflammatory condi- inammatory disorders. 1-4) The anti-inammatory tions. effect of macrolides may arise from their molecu- The direct cellular effects of macrolides can be lar, biochemical and immunological mechanisms. examined using bronchial epithelial cells. How- For instance, erythromycin inhibits the produc- ever, bronchial epithelial cells have a variety of tion of proinammatory cytokines such as tumor active membrane and cytoplasmic proteins asso- ＊ 7-1 Katuraoka-cho, Otaru-shi, Hokkaido, 047-0264 Japan 617 Jpn. J. Pharm. Health Care Sci. ciated with inflammatory signaling transduction tive action of macrolides against LPC-induced pathways,6) so it is difcult to determine whether cell damage? Human erythrocytes were used as macrolides exert a direct or indirect effect on the model cells, and vitamin E was used as the refer- cell membrane. Human erythrocytes are a conve- ence drug since it is known to attenuate LPC-in- nient cell type in this regard, because they lack a duced hemolysis via a nonspecic mechanism. 13) nucleus and thus nuclear transcriptional regula- tion 7) of the intracellular signaling pathways. Materials and Methods However, although cell membrane damage can be directly detected by hemolysis, it is not obvious 1. Materials how to inict inammatory damage since eryth- Erythromycin stearate, clarithromycin, mideca- rocytes are resistant to oxidative stress,8) which is mycin acetate, kitasamycin and rokitamycin were a primary cause of airway inammation. purchased from Wako Fine Chemicals Co., Ltd. Lysophosphatidylcholine (LPC) is a bioactive (Osaka, Japan). Telithromycin was obtained from phospholipid that accumulates significantly in Roussel Uclaf S.A. (Romainville Cedex, France), bronchoalveolar lavage fluid during airway in- and cethromycin was kindly donated by Abbott ammation and inicts inammation-like damage Laboratories, Inc. (Abbott Park, IL, USA). on erythrocytes.9, 10) It has therefore been assumed Azithromycin hydrate, vitamin E (tocopherol ace- that LPC plays a role in inflammation-induced tate), and LPC (palmitoyl C16：0) were pur- cell damage. chased from Sigma Chemical Co. (St Louis, MO, Several reports have discussed the effect of USA), and 8-anilino-1-naphthalenesulphonic acid macrolides on LPC-induced cell damage. Feld- was obtained from Tokyo Kasei Kogyo Co., Ltd. man et al. 11) demonstrated that roxithromycin, (Tokyo, Japan). Other chemicals were purchased clarithromycin and azithromycin attenuate LPC- from Wako Fine Chemicals Co., Ltd. (Osaka, Japan). induced damage to isolated human ciliated epi- LPC was dissolved in distilled water to achieve thelium, indicating that these macrolides exert di- the desired concentrations and then allowed to rect cytoprotective action against LPC-induced stand at 4 °C for at least 12 hours. Clarithromycin damage, which may contribute to their anti-in- was dissolved in dimethylsulfoxide (DMSO) and flammatory properties against airway inflamma- then diluted with distilled water to a nal DMSO tory disorders. Nevertheless, it is uncertain concentration of 0.0002-0.02％. The other macro- whether the cytoprotective effects of these macro- lides and vitamin E were dissolved in ethanol and lides on epithelial cells are due to their direct ac- then diluted with distilled water to a nal ethanol tion, because active membrane and cytoplasm concentration of 0.0002-0.02％. Other drugs were proteins are associated with the inflammatory dissolved in distilled water. All drug solutions process in human ciliated epithelium.12) tested were prepared immediately before use. The present study was designed to answer two DMSO and ethanol did not produce hemolysis at questions: 1) do macrolide antimicrobial agents the concentrations used in the present study (data exert a direct cytoprotective action against LPC- not shown). induced cell damage? and 2) are differences in 2. Blood sampling chemical structure responsible for the cytoprotec- Human blood was supplied the Hokkaido Red 618 Vol.38，No.10（2012） Cross Blood Center (Sapporo, Japan); the blood bance of hemoglobin released into the supernatant was drawn from healthy donors several days be- using a Hitachi model 200-10 spectrophotometer fore use. Each blood sample was washed four at a wavelength of 543 nm. The maximum hemo- times with isotonic buffer (10 mM phosphate buf- lysis obtained with distilled water was taken as fer containing 154 mM NaCl, pH 7.4) to remove 100％, and the minimum (spontaneous) hemoly- the buffy coat and plasma to yield a 40％ (V/V) sis obtained in the isotonic buffer was taken as erythrocyte suspension. 0％. The preliminary experiments revealed that experimental conditions producing around 50％ 3. Hemolysis induced by LPC and hypo- hemolysis, obtained using 6 µM LPC or 60 mM tonic solution NaCl (Fig. 1), were suitable for evaluating the ef- Preliminary experiments were performed to de- fects of drugs on erythrocytes. We therefore used termine the extent of hemolysis in response to 6 µM LPC and 60 mM NaCl for LPC- and NaCl- different concentrations of LPC or NaCl. The induced hemolysis, respectively. 40％ erythrocyte suspension (1 mL) was diluted with isotonic buffer solution (5 mL) and incubat- 4. Effects of macrolides and vitamin E on ed for 30 min at 37 °C. The diluted erythrocyte LPC-induced hemolysis suspension (200 µL) was mixed with isotonic or The effects of drugs (8 macrolides and vitamin hypotonic buffer (5 mL) containing different con- E) on LPC-induced hemolysis were examined. centrations of LPC (2-20 µM) or NaCl (45-75 First, the 40％ erythrocyte suspension (200 µL) mM). This resulting mixture (sample) contained was mixed with isotonic buffer (1 mL) containing about 0.26％ erythrocytes. Each sample was in- each of the drugs individually at various concen- cubated for 30 min at 37 °C and centrifuged at trations, and incubated for 30 min at 37 °C. Sam- 1300×g for 5 min. After centrifugation, the ex- ples containing one of the drugs and LPC were tent of hemolysis was determined from the absor- then prepared to measure the extent of hemolysis. Fig. 1 Extent of hemolysis in response to different concentrations of LPC (A) or NaCl (B) A：Erythrocytes and LPC were incubated in isotonic buffer (10 mM phosphate buffer containing 145 mM NaCl; pH 7.4), and B：erythrocytes were incubated in hypotonic buffer (10 mM phosphate buffer containing 45 to 75 mM NaCl; pH 7.4) for 30 min at 37 °C. Each point is the mean±SEM of five experiments. 619 Jpn. J. Pharm. Health Care Sci. The concentrations of the drugs in the isotonic 7. Statistical analysis buffer solutions were 10 nM, 100 nM, 1 µM, 10 µM, 100 µM or 1 mM, and the concentration of All values are expressed as the means±SEM. LPC was 6 µM. Each sample was further incubat- Statistical analysis was performed using ANOVA ed for 30 min at 37 °C and then centrifuged at followed by the post hoc Dunnett’s t-test for com- 1300×g for 5 min. The extent of hemolysis was paring the control group with each of the drug- measured as described above. Neither the macro- treated groups. A probability value of P < 0.05 lides nor vitamin E interfered with the measure- was considered statistically signicant. ment of hemolysis.