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- in ammatory disorders. 1-4) The anti-in ammatory 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 proin ammatory 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 in ict in ammatory 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 in ammation. 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 in icts in ammation-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

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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.

5. Effects of macrolides and vitamin E on Results hypotonic hemolysis 1. Concentration-response curves of LPC The effects of macrolides and vitamin E on hy- and NaCl for hemolysis potonic hemolysis were examined. The measure- Figure 1 shows the concentration-response ment procedures for the extent of hemolysis were curves for LPC and NaCl solutions; LPC pro- the same as those described above for examining duced hemolysis at concentrations above 3 µM LPC-induced hemolysis, except that hypotonic (Fig. 1A) and NaCl produced hemolysis at con- buffer (10 mM phosphate buffer containing 60 centrations below 75 mM (Fig. 1B). Figure 1 mM NaCl, pH 7.4) was used for hemolysis. also shows that 6 µM LPC and 60 mM NaCl pro- duced 54.2±2.8％ and 56.6±3.9％ of the total 6. Determination of micelle formation hemolysis, respectively. We therefore used 6 µM We examined whether the macrolides or vita- LPC and 60 mM NaCl in the following experi- min E affected the formation of LPC micelles. ments to examine the effects of drugs on LPC- The extent of micelle formation was measured by and NaCl-induced hemolysis. a uorescence method using 8-anilino-1-naphtha- lenesulphonic acid (ANS).14) Isotonic buffer con- 2. Effects of macrolides and vitamin E on taining ANS (10 µM) and LPC (2-20 µM) was LPC-induced hemolysis incubated in the presence of a macrolide (10 Figure 2 shows the effects of macrolides and nM-1 mM) or vitamin E (1 µM-1 mM) for 30 min vitamin E on LPC-induced hemolysis. Vitamin E at 37 °C. The intensity of ANS uorescence was significantly (P < 0.01) attenuated LPC-induced measured using excitation and emission wave- hemolysis at concentrations between 1 µM and 1 lengths of 375 and 480 nm, respectively, with a mM. However, erythromycin, azithromycin, ki- Hitachi model 240-R fluorescence spectropho- tasamycin, midecamycin, rokitamycin, telithro- tometer. The intensity of ANS fluorescence ob- mycin and cethromycin had no effect on LPC-in- tained in isotonic buffer containing LPC (20 µM) duced hemolysis at concentrations between 10 was taken as 100％, while its fluorescence in nM and 10 µM. At higher concentrations (100 LPC-free isotonic buffer was taken as 0％. Nei- µM or 1 mM), these macrolides significantly ther the macrolides nor vitamin E interfered with (P<0.01) attenuated LPC-induced hemolysis, al- the measurement of uorescence intensity. though cethromycin accelerated LPC-induced he-

620 Vol.38，No.10（2012）

100 100 100 Vitamin E Erythromycin Clarithromycin Fig. 2 80 80 80

is (%) 60 is (%) 60 is (%) 60 ** 40 ** 40 ** ** 40 ** **

Hemolys Hemolys Hemolys 20 20 ** 20 ** ** ** ** 0 0 0 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 nM nM M M M mM nM nM M M MmM nM nM M M MmM vehicle vehicle vehicle

100 100 100 Azithromycin Kitasamycin Midecamycin 80 80 80 60 60 60 ** olysis (%) olysis 40 (%) olysis 40 ** olysis (%) olysis 40 m

m ** ** m 20

He 20

He 20 He 0 0 0 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 nM nM M M M mM nM nM M M MmM nM nM M M MmM ehicle ehicle ehicle v v v

** 100 100 100 Rokitamycin Telithromycin Cethromycin ) ) ) 80 80 80 % % % 60 60 60 ** ** 40 ** 40 40 ** emolysis ( emolysis ( emolysis ( emolysis H H

H 20 20 20

0 0 0 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 nM nM M M M mM nM nM M M MmM nM nM M M MmM vehicle vehicle vehicle

Fig. 2 Effects of vitamin E and macrolides on hemolysis induced by 6 µM LPC Each value is the mean±SEM (n＝5 or 6). ＊P<0.05, ＊＊P<0.01, these values are significantly different compared with the control (vehicle) group. molysis at the highest concentration (1 mM). In 3. Effects of macrolides and vitamin E on contrast to these macrolides, clarithromycin sig- hypotonic hemolysis nificantly attenuated LPC-induced hemolysis at Figure 3 shows the effects of macrolides and concentrations ranging from 100 nM to 1 mM vitamin E on hypotonic hemolysis. Vitamin E at a (P<0.01). The protective effect of clarithromycin concentration of 10 µM attenuated the hypotonic on LPC-induced hemolysis was similar to that of hemolysis induced by 60 mM NaCl. At concen- vitamin E. These results suggest that vitamin E trations above 100 µM, however, vitamin E failed and clarithromycin protect the erythrocyte mem- to attenuate hypotonic hemolysis, while 1 mM vi- brane to a similar extent from damage induced by tamin E significantly accelerated hypotonic he- LPC. molysis (P<0.01). All the macrolides had no ef- fect at concentrations below 10 µM, although the effects of higher concentrations (10 µM, 100 µM or 1 mM) on hypotonic hemolysis were signifi- cant (P <0.01 or P <0.05). Specifically, erythro-

621 Jpn. J. Pharm. Health Care Sci.

100 100 100 Vitamin E ** Clarithromycin Fig. 3 Erythromycin ** 80 80 80 (%) (%) s (%) s s 60 i 60 * 60 40 ** ** ** 40 40 Hemolys Hemolysi Hemolysi 20 20 20

0 0 0 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 nM nM M M M mM nM nM M M MmM nM nM M M M mM vehicle vehicle vehicle

100 100 100 Azithromycin Kitasamycin Midecamycin 80 80 80 ** 60 60 60 ** ** olysis (%) olysis (%) olysis olysis (%) olysis 40 ** 40 40 ** m m m He He He 20 20 20

0 0 0 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 icle hicle hicle

nM nM M M M mM nM nM M M MmM h e nM nM MM M mM v ve ve

100 100 100 ** Rokitamycin Telithromycin Cethromycin

) 80 ** ) 80 ) 80 % % % 60 60 60 ** 40 ** 40 ** 40 ** Hemolysis ( 20 Hemolysis ( 20 Hemolysis ( 20

0 0 0 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 nM nM M M M mM nM nM M M M mM nM nM MM MmM vehicle vehicle vehicle

Fig. 3 Effects of vitamin E and macrolides on hypotonic hemolysis induced by 60 mM NaCl Each value is the mean±SEM (n＝5 or 6). ＊P<0.05, ＊＊P<0.01, these values are significantly different compared with the control (vehicle) group. mycin, kitasamycin and midecamycin signicant- 4. LPC micelle formation ly attenuated hypotonic hemolysis in a Figure 4 shows the effects of vitamin E, clar- concentration-dependent manner, while the high- ithromycin and midecamycin on micelle forma- est concentration (1 mM) of clarithromycin, roki- tion by LPC. Fluorescence intensity increased tamycin and cethromycin signicantly accelerated markedly when the concentration of LPC in- hypotonic hemolysis in a manner similar to vita- creased to 4 µM or higher, suggesting that the min E. These results suggest that the effects of critical micelle concentration (CMC) for LPC is clarithromycin, rokitamycin, cethromycin and vi- approximately 4 µM. Vitamin E at 1 µM, 10 µM, tamin E on hypotonic hemolysis are biphasic, and 100 µM and 1 mM produced a concentration-de- that the benecial action of clarithromycin on hy- pendent reduction in the increase in uorescence potonic hemolysis was less potent than its effect intensity induced by 5 to 20 µM LPC (Fig. 4A). on LPC-induced hemolysis. Clarithromycin between 10 nM and 1 mM also reduced the LPC-induced increase in uorescence intensity in a concentration-dependent manner

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(P<0.01, Fig. 4B). In contrast, none of the other 120 A macrolides at concentrations between 10 nM and 100 1 µM modied the increase in uorescence inten- 80 ** nits sity induced by LPC, although higher concentra- ensity (%) 60 ** tions (above 10 µM) of these macrolides signi-

40 ** ** cantly reduced the increase in fluorescence ** * ** ** intensity, with the exception of cethromycin (data 20 ** }** ** Fluorescence int Fluorescence u arbitrary } }** }** ** not shown). A typical example is provided in 0 ** Fig. 4C, which shows the effects of midecamycin 0 5 10 15 20 25 Lysophosphatidylcholine (M) (10 µM to 1 mM) on micelle formation by LPC. 120 B These results suggest that clarithromycin and vi-

100 tamin E affect LPC micelle formation more po- ** ** 80 tently than other macrolides, and that the protec- ** ** nits ensity ensity (%) 60 tive action of both compounds against hemolysis ** ** induced by LPC is due to the prevention of mi- 40 } celle formation. ** 20 } }** arbitrary u arbitrary Fluorescence int Fluorescence }** 0 0 5 10 15 20 25 Discussion Lysophosphatidylcholine (M)

In the present study, LPC clearly caused hemo- 120 C

lysis only at concentrations that induced micelle 100 ** formation, suggesting that LPC may cause hemo- ** 80 15) nits

lysis by the formation of micelles. Vitamin E ensity (%) 60 }** significantly attenuated hemolysis induced by LPC at concentrations of 1 µM to 1 mM, whereas 40 }** } clarithromycin attenuated hemolysis over a wider 20 ** Fluorescence int Fluorescence arbitrary u arbitrary range of concentrations (100 nM to 1 mM). These 0 results suggest that both vitamin E and clarithro- 0 5 10 15 20 25 Lysophosphatidylcholine (M) mycin exert a direct cytoprotective action against

LPC-induced cell damage, and that clarithromycin Fig. 4 Effects of vitamin E, clarithromycin and is more effective than vitamin E. Concentrations midecamycin on micelle formation by LPC A． The fluorescence intensity obtained from 10 mM phosphate buffer of clarithromycin at which this action was ob- (pH 7.4) containing LPC alone (○), and LPC with 1 µM (●), 10 µM (□), 100 µM (■) and 1 mM (△) vitamin E. served (1 to 10 µM) are clinically relevant to the B． The fluorescence intensity obtained from 10 mM phosphate buffer (pH 7.4) containing LPC alone (○), and LPC with 10 concentration typically used in low-dose adminis- nM (▲), 1 µM (●), 10 µM (□), 100 µM (■) and 1 mM (△) tration for in ammatory airway diseases. 2, 3, 16, 17) clarithromycin. C． The fluorescence intensity obtained from 10 mM phosphate buffer By what mechanism, then, do vitamin E and (pH 7.4) containing LPC alone (○), and LPC with 10 µM (□), 100 µM (■) and 1 mM (△) midecamycin. clarithromycin protect the erythrocyte membrane Each value is the mean ±SEM (n＝4). ＊P <0.05, ＊＊P <0.01, these 18) values are significantly different compared with the LPC alone (○) from LPC-induced damage? Bierbaum et al. group. found that exogenous LPC micelles liberated sev-

623 Jpn. J. Pharm. Health Care Sci. eral cell surface proteins from erythrocyte mem- The inhibitory effect of clarithromycin on LPC branes to increase sodium permeability, and that micelle formation was weaker than that of vita- this action was inhibited by tetrodotoxin. Further- min E. Nevertheless, clarithromycin more effec- more, the consequent hemolysis was also sup- tively reduced LPC-induced hemolysis than did pressed by tetrodotoxin or sucrose, suggesting vitamin E. Clarithromycin must therefore exert that an osmotic imbalance induced by abnormal additional effects to reduce LPC-induced hemoly- Na＋ uptake was the primary cause of hemolysis sis, unless it also controls micellar formation. It induced by LPC. It is therefore possible that has been reported that vitamin E protects synaptic drugs that prevent osmotic imbalance can inhibit membranes 21) and liposomes 22) from damage in- or reduce the hemolytic effect of LPC. However, duced by bioactive phospholipids via a mem- in the current study, vitamin E and clarithromycin brane-stabilizing action. Membrane stabilization did not reduce hypotonic hemolysis at concentra- decreases membrane fluidity as well as permea- tions that inhibit LPC-induced hemolysis (1 µM, bility.23) Since the membrane stabilizing action of 100 µM and 1 mM for vitamin E, and 100 nM, 1 vitamin E correlates with high lipophilicity,24) the µM, 10 µM and 1 mM for clarithromycin). If the high lipophilic properties of macrolides may play hemolytic action of LPC is a consequence of os- a role in protection against hemolysis induced by motic imbalance, vitamin E or clarithromycin LPC. Table 1 summarizes the effects of macro- should attenuate hypotonic hemolysis. Thus, there lides and vitamin E in terms of their ring size, li- must be other mechanisms through which these pophilicity, and anti-hemolytic action on LPC. two compounds reduce LPC-induced hemolysis. The high lipophilic property of clarithromycin It has been proposed that the hemolytic capaci- may be related to the protective effect of this drug ty of LPC is determined by the chemical structure on hemolysis induced by LPC. Indeed, it has been of the LPC molecule. 19) According to this hypoth- reported that clarithromycin exhibits a membrane esis, vitamin E and clarithromycin might directly stabilizing action on human neutrophils.25) Howev- bind with LPC to lengthen its aliphatic chain, per- er, rokitamycin was not effective, despite it being haps resulting in mixed larger micelles and there- much more lipophilic than clarithromycin.16, 26) by reduced hemolytic activity. If this is true, then Therefore, high lipophilicity alone cannot com- micellar saturation with vitamin E or clarithromy- pletely explain the additional protective effects of cin should reduce or prevent micelles from solu- clarithromycin on hemolysis induced by LPC. bilizing membrane components. In fact, there is Both clarithromycin and erythromycin have a evidence that vitamin E can interact physico- 14-membered ring lactone, but clarithromycin chemically with LPC to form a complex, thereby differs structurally from erythromycin by its attenuating its undesirable effects. 13, 20) In the pres- methyl substituted hydroxy group at position 6 in ent study, both vitamin E (1 µM to 1 mM) and the lactone ring, making it more lipophilic than clarithromycin (10 nM to 1 mM) reduced the erythromycin. These ndings suggest that macro- amount of LPC micelles, as indicated in Fig. 4. lides require both high lipophilicity and a Therefore, the protective effects of vitamin E and 14-membered ring lactone structure to inhibit the clarithromycin likely arise from their counterac- hemolysis induced by LPC. The amphiphilic tion of micelle formation in human erythrocytes. properties of LPC cause LPC molecules and mi-

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Table 1 C ‌ hemical Structure Family, Partition (Octanol/water) Coefficients, and Anti-hemolytic Effects of Macrolides and Vitamin E Octanol/water Anti-hemolytic Chemical Structure Coefficient a） Effect Drug Family (at room temperature, (LPC-induced pH 7~8) hemolysis) Vitamin E α-Tocopherol tocotrienol 476 Strong Macrolides Erythromycin 14-membered 2.48 Weak Clarithromycin 14-membered 46.4 Strong Azithromycin 15-membered 0.39 Weak Kitasamycin 16-membered 2.47 Weak Midecamycin 16-membered 2.74 Weak Rokitamycin 16-membered 1202 Weak Telithromycin ketoride 1.56 Weak Weak or Cethromycin ketoride 5.24 Deterioration a ）The value for α-tocopherol is from Lee et al.32) The values for erythromycin and midekamaycin are from Tosti et al.33) and those for kitasamycin and cethromycin are from Tanaka et al.34) and Kiem et al.35) respectively. The values for clarithromycin,16) azithromycin,36) rokitamycin 26) and telithromycin 37) are from their respective interview forms.

celles to bind to and associate with the cell mem- tested (1 mM), although they exerted anti-hemo- brane.15, 27) Macrolides that are both highly lipo- lytic action at 10 µM or 100 µM. Similar biphasic philic and contain a 14-membered ring lactone action on hypotonic hemolysis has been observed structure, such as clarithromycin, might compete with propranolol, 29) and it is known that high dos- with LPC to penetrate into the membrane matrix, es of α-tocopherol have a lytic effect on erythro- thereby preserving the membrane integrity of cytes.30) These studies demonstrated that at lower erythrocytes. On the other hand, it is possible that concentrations propranolol and α-tocopherol in- the lipophilicity of clarithromycin may decrease teract with membrane proteins or lipoproteins to at the site of in ammation, thereby reducing the attenuate hypotonic hemolysis, whereas at higher inhibitory effects on LPC-induced hemolysis, concentrations they partition into the inner mem- since clarithromycin is a basic drug and the pH of brane of the lipid bilayer and accelerate hypotonic an in ammatory site is typically slightly acidic. It hemolysis. The effect of macrolides on hypotonic has been reported that the pH of the airways of hemolysis may also be biphasic due to interaction patients with asthma is 5.2-7.1, depending on dis- with membrane proteins or lipids. In particular, ease severity.28) Because clarithromycin provides high concentrations of clarithromycin, rokitamy- a greater protective effect on normal tissue than cin and cethromycin may accelerate hypotonic on tissue at sites of in ammation, it will be neces- hemolysis because these lipophilic macrolides sary to examine the drug’s effects on LPC-in- can easily enter the cell membrane.17) In contrast, duced hemolysis under acidic conditions. except for cethromycin, none of the drugs exacer- Vitamin E, clarithromycin, rokitamycin bated LPC-induced hemolysis, even at the highest (16-membered ring lactone derivative) and ce- concentration tested (1 mM), perhaps because of thromycin (ketoride derivative) all accelerated their inhibitory effects on LPC micellar forma- hypotonic hemolysis at the highest concentration tion. Cethromycin did not affect LPC micellar

625 Jpn. J. Pharm. Health Care Sci. formation, so this drug accelerated LPC-induced 1991, 99, 670-673. hemolysis at the highest concentration. Further 2) Banerjee D, Honeybourne D, Khair OA, The Effect of Oral Clarithromycin on Bronchial Airway In am- investigations are necessary to clarify the detailed mation in Moderate-to-Severe Stable COPD: A Ran- mechanisms of the effects of macrolides on he- domized Controlled Trial, Treat Respir Med, 2004, 3, molysis induced by LPC. 59-65. 3) Gotfried MH, Macrolides for the Treatment of In conclusion, clarithromycin, a highly lipo- Chronic Sinusitis, Asthma, and COPD, Chest, 2004, philic 14-membered macrolide, directly protected 125, 52S-61S. against erythrocyte membrane damage induced 4) Albert RK, Connett J, Bailey WC, Casaburi R, Cooper JAJ, Criner GJ, Curtis JL, Dransfield MT, by LPC. The anti-hemolytic mechanism of this Han MK, Lazarus SC, Make B, Marchetti N, macrolide might depend on the lipophilic struc- Martinez FJ, Madinger NE, MaEvoy C, Niewoehner ture of its 14-membered ring lactone counteract- DE, Porsasz J, Price CS, Reilly J, Scanlon PD, ing the formation of LPC micelles. Furthermore, Sciurba FC, Scharf SM, Washko GR, Woodruff PG, Anthonisen NR, Azithromycin for Prevention of Ex- the anti-hemolytic effect of clarithromycin is con- acerbations of COPD, N Engl J Med, 2011, 365, sistent with the results demonstrating that the 689-698. clinical efcacy of clarithromycin against respira- 5) Miyatake H, Suzuki K, Taki F, Takagi K, Satake T, Effect of Erythromycin on Bronchial Hyperrespon- tory in ammatory disease is relatively higher than siveness in Patients with Bronchial Asthma, Arz- 2, 3, 31) that of other macrolides, and it is thus expect- neimittelforschung, 1991, 41, 552-556. ed that clarithromycin is superior to erythromycin 6) Krunkosky TM, Fischer BM, Martin LD, Jones N, Akley NJ, Adler KB, Effects of TNF-α on Expres- and azithromycin, which show the same anti-in- sion of ICAM-1 in Human Airway Epithelial Cells flammatory properties. We believe that a direct in vitro. Signaling Pathways Controlling Surface and cytoprotective action of clarithromycin may be Gene Expression, Am J Respir Cell Mol Biol, 2000, 22 contribute to its beneficial effects on airway in- , 685-692. 7) Venkatakrishnan A, Stecenko AA, King G, Blackwell ammatory disorders. TR, Brigham KL, Christman JW, Blackwell TS, Exaggerated Activation of Nuclear Factor-κB and Altered IκB-β Processing in Cystic Fibrosis Bron- Acknowledgments chial Epithelial Cells, Am J Respir Cell Mol Biol, 2000, 23, 396-403. The authors wish to thank Roussel Uclaf S.A. 8) Cimen BMY, Free Radical Metabolism in Human (Romainville Cedex, France) and Abbott Labora- Erythrocytes, Clin Chim Acta, 2008, 390, 1-11. 9) Chilton FH, Averill FJ, Hubbard WC, Fonteh AN, tories, Inc. (Abbott Park, IL, USA) for donations Triggiani M, Liu MC, Antigen-induced Generation of telithromycin and cethromycin, respectively. of Lyso-Phospholipids in Human Airways, J Exp This study was supported in part by an Education Med, 1996, 183, 2235-2245. and Research Grant from Hokkaido Pharmaceuti- 10) Weltzien HU, Cytolytic and Membrane-perturbing Properties of Lysophosphatidylcholine, Biochim Bio- cal University. phys Acta, 1979, 559, 259-287. 11) Feldman C, Anderson R, Theron AJ, Rama G, Cole PJ, Wilson R, Roxithromycin, Clarithromycin, and References Azithromycin Attenuate the Injurious Effects of Bio- active Phospholipids on Human Respiratory Epithe- 1) Miyatake H, Taki F, Taniguchi H, Suzuki R, Takagi K, lium in vitro, Inflammation, 1997, 21, 655-665. Satake T, Erythromycin Reduces the Severity of 12) Herfs M, Hubert P, Poirrier AL, Vandevenne P, Bronchial Hyperresponsiveness in Asthma, Chest, Renoux V, Habraken Y, Cataldo D, Boniver J,

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