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Inhibition of human T-lymphocyte activation by macrolide antibiotic, roxithromycin
Life Sciences, Vol. Printed in the USA
51, pp. PL 231-236
Pergamon Press
PHARMACOLOGY Accelerated
LETTERS
Communication
INHIBITION OF HUMAN T-LYMPHOCYTE ACTIVATION MACROLIDE ANTIBIOTIC, ROXITHROMYCIN
BY
Shin-ichi Konno 1.. Mitsuru Adachi 1, Kazuhito Asano 2, Ken-ichi O k a m o t o 2 a n d T e r u m i Takahashil 1 First D e p a r t m e n t o f Internal M e d i c i n e , and 2 D e p a r t m e n t o f M e d i c a l B i o l o g y , S c h o o l o f M e d i c i n e , S h o w a University, hatanodoi, S h i n a g a w a - k u , T o k y o 142, Japan (Submitted June 16, 1992; accepted July 16, 1992; received in final form October 3, 1992)
ABSTRACT. Effects of macrolide antibiotic, roxithromycin (RXM) on human lymphocytes in culture were studied. The drug showed a dose-dependent inhibition of 3H-thymidine and 35Smethionine uptake responding to T cell mitogens and purified protein derivative of tuberculin (PPD). Activation by PPD, as assessed by 3H-thymidine uptake, was more sensitive to inhibition than the response to T cell mitogens. The drug produced a loss of blasts when added soon after transformation commenced. Immunosuppressive effects of RXM were further characterized by using four different types of metabolized RXM, RU 28111, RU 39001, RU 44981 and RU 45179. The most potent inhibitor of lymphocyte transformation was RU 45170, followed by RU 44981, RU 39001 and RU 28111 have little activity.
INTRODUCTION Studies by several researchers have demonstrated direct adverse effects of macrolide antibiotics on several aspects of the host defense system, including chemotaxis, (1) and cellmediated immunity (2). The effects of erythromycin on immune responses have been extensively studied and demonstrated that it reduced the motility of human peripheral mononuclear leukocytes (PMNL)(3), and inhibits production of oxygen radicals from PMNL (4), among others. In vitro studies with clindamycin by Forsgren et al. (5) phytohemagglutinin-induced lymphocytes transformation was reported to be depressed when the lymphocytes were cultured in the presence of the drugs. Furthermore, the novel immunosuppressive agent, FK-506 isolated from a strain of Streptomyces is known to belong an antibiotic of macrolide family and to have strong immunosuppressive activity (6). From these reports, it is reasonable to postulate that immunomodulating, especially immunosuppressive activity is one of feature of macrolide antibiotics. Recently, roxithromycin (RXM) was synthesized by local modification of macrocyclin (7), and used therapeutically without side effects. However, the immunophamacological effects on human lymphocyte function have not been studied. We have therefore studied the effect of RXM on mitogen- and antigen-induced lymphocyte proliferative responses, the in vitro correlates immune responsiveness.
*To whom correspondence should be addressed. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
PL-232
Roxithromycin
on H u m a n L y m p h o c y t e s
Vol.
51, No.
24,
1992
M A T E R I A L S AND M E T H O D S Materials Roxithromycin (RXM) and metabolized RXM, RU 28111, RU 39001, RU 44981 and RU 45179 were kindly supplied by Eisai Co. Ltd. (Tokyo, Japan) and Roussel Medica Co. Ltd (Tokyo, Japan) as an aqueous-insoluble powder. The structure of these agents are show in Figure 1. The powder was dissolved in absolute alcohol (1 ml) at 50 mg/ml, and diluted with RPMI-1640 medium (Flow Lab., North Ryde Australia) so as to given a concentration of 1 mg/ml. This solution was then filtered through a 0.22 pm millipore filter (nihon millipore Kogyo, Yonezawa, Japan) and stored as stock solution at 4 o c until used. All dilutions used in this study were prepared from this stock solution by diluting with RPMI-1640 medium. 14C-RXM dissolved in alcohol was kindly gifted by Roussel Medica Co. Ltd This solution diluted with RPMI-1640 medium so as to given a concentration of 500 pg/ml. The following reagents were obtained from the indicated companies and dissolved or diluted in RPMI-1640 medium: concanavalin A (Con A; Sigma Chemical Co., St. Louis, Mo); Purified protein derivative of tuberculin (PPD; Japan BCG Co. Tokyo, Japan); Lymphocyte Separation Medium (Organon Teknika Corp., Durhan, NC); fetal calf serum (Flow Lab); 3H-thymidine and 35S-methionine (Sp. Act., 25 and 200 mCi/m mol respectively, from Amersham International plc, Bucks, UK); heparin (Novo, Ind., Denmark). N-O-CH2-O-CH2-CH2-OCH3 H3C,I~L,,T-CH3 HO~.~L,n,"...t 0H /CH3 H=C~I" ..~' T'CH3 H N....
N-O-CH2-O-CH~-CH=-OCH3 H3C',i,,~,CH3 HO"JL'nl4"'/OH NH =
L/C,
,
I~"0,,,-;~' I~ CH3
. ~ ~
RXM
N-O-CH2-O-CH2-CH2-OCH3 H3C.,I~.CH 3 HO,,..,,&n,'.~OH H3C'1 u~' ]"CH3 O~'~,"~'-.OH CH3
. ./CH " N~r', ~ t~" 0;~,~.2( H
Descladinose RU39001
=
N-O-CH2-O-CH2-CH=-OCH3 H3C-T~'~/CH3 HO~'aH ",,/OH . ./H H,C'l. 7'
I"C,3
°'~E'/.."-o H"O,,~ H CH3 Mono-desmethylRU44981
,
.
," ?~H
oc,,""o".~
C,3 ~ ) O H
H
H'1~O~VHHH s Di-desmethyl RU45179
N.O_H H3C.,.i~ ~CH3 HO,,~n,'-...OH H/CH3 H3C"1 ,. ~' ]~CH3 H ,C~.,L.~I~,~. . . . . O ~ Z H, . 3 ~ . 0~""- 0
ocHH\O".,~
H H ~l\Om~HI Free oxime RU28111
Fig.1 Chemical s t r u c t u r e of roxithromycin (RXM) and metabolized RXM. Preparation of and culture of cell populations Human heparinized peripheral blood (20 ml) of a healthy donor was diluted with 20 ml of 0.9% NaC1 and overlaid onto 10 ml of Lymphocyte Separation Medium. After centrifugation at 1500 rpm for 20 min (KUBOTA KN-70: Kubota Co. Ltd., Tokyo, Japan) at room temperature, the plasma-medium interface, which contained mononuclear cell was collected. The cells in this fraction were washed five times at 600 rpm for 10 min to remove platelets. The resultant cells were suspended in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum at a concentration of 1 x 106 cells/ml and used as PMNL. PMNL were cultured at 1 x 105 cells per microtiter plate well (No. 1-63371; Nunc, Denmark) in triplicate with or without Con A, PHA or PPD at 37 o c in a 5% CO2 incubator (Hirasawa, Co. Ltd., Tokyo, Japan). Additions of agents were made simultaneously except where otherwise indicated.
Vol.
51, NO.
24,
1992
Roxithromycin
on H u m a n L y m p h o c y t e s
PL-233
80
70
10
60
'--- 50 X
o
40 30
20
//
[5 ~ 4 3 0.5
~
1
24
48
0
0
72
96
120
Fig.2 Effects of different concentrations of roxithromycin (RXM) on responses of h u m a n peripheral m o n o n u c l e a r leukocytes (PMNL) to Con A. PMNL were stimulated with 10 ~aglml Con A, in the presence (m) or a b s e n c e (o) of RXM. RXM concentrations (pg/ml) are indicated at the right of the curve.
Assessment of P M N L activation Cell activation was assessed by determination of the rate of incorporation of 35Smethionine into protein or 3H-thymidine into DNA. One/aCi of radiolabelled precursor in l0 ~tl of medium was added to each culture 8-hr before termination TABLE 1 Effects of RXM on h u m a n peripheral mononuclear lymphocyte DNA synthetic activity induced by mitogen and antigen Irritant
(medium alone)
Dose of RXM (~tg/ml) 0
3H-thymidine uptake (mean cpm + SD) 515.7 +
88.8
% inhibition
--
Con A
0 10 100
18745.3 +___1141.1 15883.3 -+ 1491.5 1287.7-+ 200.5
0 15.3 __+8.0 93.1 ___+1.1
PHA
0 10 100
28001.0 -+ 2234.4 27353.7 -+ 4711.5 22406.3 + 4670.9
0 97.7 + 16.8 80.0 +___16.7
PPD
0 10 100
1376.0__+ 138.0 486.0_+ 159.7 420.0 + 126.4
0 64.7+ 11.6 69.5 + 9.2
PL-234
Roxithromycin
on H u m a n L y m p h o c y t e s
Vol.
51, No.
24,
1992
RESULTS
Effects of RXM on PMNI. activation To examine effects o f RXM on P M N L activation, two different doses o f RXM were added to cell cultures which were simultaneously stimulated with Con A. As shown in Figure 2, RXM caused significant reduction on P M N L incorporation of 3H-thymidine into DNA. This suppression occurred in a dose-dependent fashion with significant suppression noted at 100 pg/ml o f RXM. DNA synthetic activity o f PMNL induced by PHA or PPD stimulation was also inhibited by RXM, added at the commencement o f the culture (Table I). This inhibition was dose dependent, as the same as in the case o f Con A stimulation. The next experiment was carried out to examine whether RXM influenced protein synthetic activity of PMNL. Protein synthesis was measured 24 hr in culture. RXM did not have any significant effect on the low rate o f protein synthesis exhibited by unstimulated lymphocytes, while protein synthesis consequent on PMNL activation induced by Con A was significantly inhibited by RXM (Table II). To examine relative effects on blast cells as compared with small lymphocytes, 100 tag/ml of RXM was added to Con A-stimulated cultures at various times after the start o f culture. The data in Table III show that RXM was most effective at 3H-thymidine uptake when added at, or soon after the start of culture. TABLE II Effect of roxithromycin on the incorporation of 3 5 S - m e t h i o n i n e into protein 24 hr after its addition with or without Con A. RXM concentration (pg/ml) Mitogen None Con A
0
10
744.3 + 194.9 2397.0 +
84.5
100
910.3 + 87.2
920.7 + 39.9
1204.3 + 74.7
770.3 + 76.4
(Results expressed as mean cpm Z SD) TABLE III
Inhibition of transformation Time o f addition after start o f culture (hr) 0 4 16 24 36 38
% inhibition 94.9 + 85.3 + 51.0+ 43.7 + 31.3 + 12.6 +
0.96 0.39 1.09 1.50 1.16 1.45
RXM ( 100 lag/ml) added to cultures at indicated times. 3H-thymidine incorporated, into D N A was measured at 48 hr after the start o f culture.
Vol.
51, No. 24, 1992
Roxithromycin
on Human Lymphocytes
PL-235
Effects of metabolized RXM on PMNL activation The final set of experiments were undertaken to examine effects of metabolized RXM on PMNL activation. As shown in Table IV, the most striking suppressive effects of metabolized RXM was observed as cells cultured in the presence of RU 45179. However, activation of cells were scarcely affected by addition of RU 39001 and RU 28111. T A B L E IV Inhibition of human peripheral mononuclear leukocyte activation by metabolized roxithromycin 3H-thymidine uptakes; mean cpm (SD) Conc. (~tg/ml)
RXM
RU 44981
RU 45179
RU 39001
RU 28111
N.T.
N.T.
0
32448.6 (483.1)
N.T.
N.T.
10
22135.3 (1793.1)
31133.7 (1648.2)
27992.3 (927.9)
29986.7 (1390.6)
27242.7 (1170.9)
100
243.0 (72.2)
26712.7 (861.9)
14195.7 (873.6)
29456.0 (1091.8)
30329.3 (680.9)
DISCUSSION The present studies indicate that large amount of RXM (100 lag/ml), but not small amount of RXM (10 pg/ml) can significantly suppress blastic activity of PMNL induced by mitogenic stimulation. It is also revealed that RXM in clinically achieved concentration at about 10 pg/ml (8) can strongly inhibit the low rate of blastic activity observd in PMNL cultured with (Table I). The fact that small amount of RXM ( 10 ~g/ml) appeared to have little effect on mitogen-induced blastic activity of PMNL may be related to stronger stimulus when compared to antigenic stimulation and the greater response induced by mitogens. Studies on effect of RXM on a hybridoma, PC6153 showed that in vitro RXM inhibited proliferation of this cell line (unpublished observation). Since this cell line can proliferate without antigenic and mitogenic stimulation, this result strongly indicates that RXM inhibits the cell proliferation rather than the antigenic recognition phase. RXM added to cell cultures stimulated with Con A at 38 hr after the start of culture gave significantly less inhibition than when added earlier, showing that this drug is not simple inhibiting uptake of thymidine and acts early in the cycle of cell division. It is reported that macrolides orally administered to human and mice were analyzed into several types of metabolized materials by liver cytochrome P450 (9). So, RXM may be also metabolized by liver cytochrome P450 and then most of metabolized RXM, except for two types of metabolized materials such as RU 44981 and RU 45179, are excreted into urine, feces, etc. (8). We previously observed that lymphocytes prepared from mice orally administered with RXM for longperiod loss reactivity to mitogenic stimulation (Konno et al., unpublished). Taken together with these reports and our observation, the results in Table IV may indicate that accumulated-RXM, especially RU 44981 and RU 45179 bring about inhibition of lymphocyte transformation. Further more, the results strongly suggest that RXM will be useful drug in treatment of allergic diseases, since lymphocytes reacted with antigen induced these diseases. REFERENCES 1 A. EYRAUD, J. DESCOTES, J.Y. LOMBARD, A. LASCHI-LOQUERIE, P. TACHON, C. VEYSSEYRE, and J.C. EVREUX, Chemotherapy 32 379-382.(1986). 2 G. BANCK, and A. FORSGREN, Antimicrob. Agents Ch. 16 554-560.(1979).
PL-236
Roxithromycin
on H u m a n L y m p h o c y t e s
Vol.
51, No.
24,
1992
3 M.B. ESTERLY, N.L. FUREY, and L.E. FLANAGAM, J. Invest. Dermator. 70 51 (1978). 4 S. MIYACHI, A. YOSHIOKA, S. IMAMURA, and Y. NlWA, J. Invest. Dermatol. 86 449-453 (1986). 5 A. FORSGREN, G. BANCK, H. BECKMAN, and A. BELLAHRENE, Scand J Infect Dis 24 (suppl): 195-203.(1980). 6 T. KINO, H. HATANAKA, M. HASHIMOTO, M. NISHIYAMA, T. GOTO, M. OKUHARA, M. KOHSAKA, H. AOKI, and H. IMANAKA,.J Antibiot. 40 1249-1255.(1987) 7 J.F. CHANTOT, A. BRYSKIER, and J.C. GASC, J. Antibiot. 39 660-668 (1986). 8 M.KOYAMA, M.TATENO, M.SHIROTSUKA, T. YAMAMOTO, M. HIRAYAMA, K. SAITOH, and K. OKUI, Jpn. J. Chemotherapy 36 164-183.(1988). 9 G. BABANY, D. LARREY, and D. PESSAYRE, Progress in Drug Metabolism vol. 11, G.G. Gibson (ed), 62-98,Taylor & Francis, London (1988).
51, pp. PL 231-236
Pergamon Press
PHARMACOLOGY Accelerated
LETTERS
Communication
INHIBITION OF HUMAN T-LYMPHOCYTE ACTIVATION MACROLIDE ANTIBIOTIC, ROXITHROMYCIN
BY
Shin-ichi Konno 1.. Mitsuru Adachi 1, Kazuhito Asano 2, Ken-ichi O k a m o t o 2 a n d T e r u m i Takahashil 1 First D e p a r t m e n t o f Internal M e d i c i n e , and 2 D e p a r t m e n t o f M e d i c a l B i o l o g y , S c h o o l o f M e d i c i n e , S h o w a University, hatanodoi, S h i n a g a w a - k u , T o k y o 142, Japan (Submitted June 16, 1992; accepted July 16, 1992; received in final form October 3, 1992)
ABSTRACT. Effects of macrolide antibiotic, roxithromycin (RXM) on human lymphocytes in culture were studied. The drug showed a dose-dependent inhibition of 3H-thymidine and 35Smethionine uptake responding to T cell mitogens and purified protein derivative of tuberculin (PPD). Activation by PPD, as assessed by 3H-thymidine uptake, was more sensitive to inhibition than the response to T cell mitogens. The drug produced a loss of blasts when added soon after transformation commenced. Immunosuppressive effects of RXM were further characterized by using four different types of metabolized RXM, RU 28111, RU 39001, RU 44981 and RU 45179. The most potent inhibitor of lymphocyte transformation was RU 45170, followed by RU 44981, RU 39001 and RU 28111 have little activity.
INTRODUCTION Studies by several researchers have demonstrated direct adverse effects of macrolide antibiotics on several aspects of the host defense system, including chemotaxis, (1) and cellmediated immunity (2). The effects of erythromycin on immune responses have been extensively studied and demonstrated that it reduced the motility of human peripheral mononuclear leukocytes (PMNL)(3), and inhibits production of oxygen radicals from PMNL (4), among others. In vitro studies with clindamycin by Forsgren et al. (5) phytohemagglutinin-induced lymphocytes transformation was reported to be depressed when the lymphocytes were cultured in the presence of the drugs. Furthermore, the novel immunosuppressive agent, FK-506 isolated from a strain of Streptomyces is known to belong an antibiotic of macrolide family and to have strong immunosuppressive activity (6). From these reports, it is reasonable to postulate that immunomodulating, especially immunosuppressive activity is one of feature of macrolide antibiotics. Recently, roxithromycin (RXM) was synthesized by local modification of macrocyclin (7), and used therapeutically without side effects. However, the immunophamacological effects on human lymphocyte function have not been studied. We have therefore studied the effect of RXM on mitogen- and antigen-induced lymphocyte proliferative responses, the in vitro correlates immune responsiveness.
*To whom correspondence should be addressed. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
PL-232
Roxithromycin
on H u m a n L y m p h o c y t e s
Vol.
51, No.
24,
1992
M A T E R I A L S AND M E T H O D S Materials Roxithromycin (RXM) and metabolized RXM, RU 28111, RU 39001, RU 44981 and RU 45179 were kindly supplied by Eisai Co. Ltd. (Tokyo, Japan) and Roussel Medica Co. Ltd (Tokyo, Japan) as an aqueous-insoluble powder. The structure of these agents are show in Figure 1. The powder was dissolved in absolute alcohol (1 ml) at 50 mg/ml, and diluted with RPMI-1640 medium (Flow Lab., North Ryde Australia) so as to given a concentration of 1 mg/ml. This solution was then filtered through a 0.22 pm millipore filter (nihon millipore Kogyo, Yonezawa, Japan) and stored as stock solution at 4 o c until used. All dilutions used in this study were prepared from this stock solution by diluting with RPMI-1640 medium. 14C-RXM dissolved in alcohol was kindly gifted by Roussel Medica Co. Ltd This solution diluted with RPMI-1640 medium so as to given a concentration of 500 pg/ml. The following reagents were obtained from the indicated companies and dissolved or diluted in RPMI-1640 medium: concanavalin A (Con A; Sigma Chemical Co., St. Louis, Mo); Purified protein derivative of tuberculin (PPD; Japan BCG Co. Tokyo, Japan); Lymphocyte Separation Medium (Organon Teknika Corp., Durhan, NC); fetal calf serum (Flow Lab); 3H-thymidine and 35S-methionine (Sp. Act., 25 and 200 mCi/m mol respectively, from Amersham International plc, Bucks, UK); heparin (Novo, Ind., Denmark). N-O-CH2-O-CH2-CH2-OCH3 H3C,I~L,,T-CH3 HO~.~L,n,"...t 0H /CH3 H=C~I" ..~' T'CH3 H N....
N-O-CH2-O-CH~-CH=-OCH3 H3C',i,,~,CH3 HO"JL'nl4"'/OH NH =
L/C,
,
I~"0,,,-;~' I~ CH3
. ~ ~
RXM
N-O-CH2-O-CH2-CH2-OCH3 H3C.,I~.CH 3 HO,,..,,&n,'.~OH H3C'1 u~' ]"CH3 O~'~,"~'-.OH CH3
. ./CH " N~r', ~ t~" 0;~,~.2( H
Descladinose RU39001
=
N-O-CH2-O-CH2-CH=-OCH3 H3C-T~'~/CH3 HO~'aH ",,/OH . ./H H,C'l. 7'
I"C,3
°'~E'/.."-o H"O,,~ H CH3 Mono-desmethylRU44981
,
.
," ?~H
oc,,""o".~
C,3 ~ ) O H
H
H'1~O~VHHH s Di-desmethyl RU45179
N.O_H H3C.,.i~ ~CH3 HO,,~n,'-...OH H/CH3 H3C"1 ,. ~' ]~CH3 H ,C~.,L.~I~,~. . . . . O ~ Z H, . 3 ~ . 0~""- 0
ocHH\O".,~
H H ~l\Om~HI Free oxime RU28111
Fig.1 Chemical s t r u c t u r e of roxithromycin (RXM) and metabolized RXM. Preparation of and culture of cell populations Human heparinized peripheral blood (20 ml) of a healthy donor was diluted with 20 ml of 0.9% NaC1 and overlaid onto 10 ml of Lymphocyte Separation Medium. After centrifugation at 1500 rpm for 20 min (KUBOTA KN-70: Kubota Co. Ltd., Tokyo, Japan) at room temperature, the plasma-medium interface, which contained mononuclear cell was collected. The cells in this fraction were washed five times at 600 rpm for 10 min to remove platelets. The resultant cells were suspended in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum at a concentration of 1 x 106 cells/ml and used as PMNL. PMNL were cultured at 1 x 105 cells per microtiter plate well (No. 1-63371; Nunc, Denmark) in triplicate with or without Con A, PHA or PPD at 37 o c in a 5% CO2 incubator (Hirasawa, Co. Ltd., Tokyo, Japan). Additions of agents were made simultaneously except where otherwise indicated.
Vol.
51, NO.
24,
1992
Roxithromycin
on H u m a n L y m p h o c y t e s
PL-233
80
70
10
60
'--- 50 X
o
40 30
20
//
[5 ~ 4 3 0.5
~
1
24
48
0
0
72
96
120
Fig.2 Effects of different concentrations of roxithromycin (RXM) on responses of h u m a n peripheral m o n o n u c l e a r leukocytes (PMNL) to Con A. PMNL were stimulated with 10 ~aglml Con A, in the presence (m) or a b s e n c e (o) of RXM. RXM concentrations (pg/ml) are indicated at the right of the curve.
Assessment of P M N L activation Cell activation was assessed by determination of the rate of incorporation of 35Smethionine into protein or 3H-thymidine into DNA. One/aCi of radiolabelled precursor in l0 ~tl of medium was added to each culture 8-hr before termination TABLE 1 Effects of RXM on h u m a n peripheral mononuclear lymphocyte DNA synthetic activity induced by mitogen and antigen Irritant
(medium alone)
Dose of RXM (~tg/ml) 0
3H-thymidine uptake (mean cpm + SD) 515.7 +
88.8
% inhibition
--
Con A
0 10 100
18745.3 +___1141.1 15883.3 -+ 1491.5 1287.7-+ 200.5
0 15.3 __+8.0 93.1 ___+1.1
PHA
0 10 100
28001.0 -+ 2234.4 27353.7 -+ 4711.5 22406.3 + 4670.9
0 97.7 + 16.8 80.0 +___16.7
PPD
0 10 100
1376.0__+ 138.0 486.0_+ 159.7 420.0 + 126.4
0 64.7+ 11.6 69.5 + 9.2
PL-234
Roxithromycin
on H u m a n L y m p h o c y t e s
Vol.
51, No.
24,
1992
RESULTS
Effects of RXM on PMNI. activation To examine effects o f RXM on P M N L activation, two different doses o f RXM were added to cell cultures which were simultaneously stimulated with Con A. As shown in Figure 2, RXM caused significant reduction on P M N L incorporation of 3H-thymidine into DNA. This suppression occurred in a dose-dependent fashion with significant suppression noted at 100 pg/ml o f RXM. DNA synthetic activity o f PMNL induced by PHA or PPD stimulation was also inhibited by RXM, added at the commencement o f the culture (Table I). This inhibition was dose dependent, as the same as in the case o f Con A stimulation. The next experiment was carried out to examine whether RXM influenced protein synthetic activity of PMNL. Protein synthesis was measured 24 hr in culture. RXM did not have any significant effect on the low rate o f protein synthesis exhibited by unstimulated lymphocytes, while protein synthesis consequent on PMNL activation induced by Con A was significantly inhibited by RXM (Table II). To examine relative effects on blast cells as compared with small lymphocytes, 100 tag/ml of RXM was added to Con A-stimulated cultures at various times after the start o f culture. The data in Table III show that RXM was most effective at 3H-thymidine uptake when added at, or soon after the start of culture. TABLE II Effect of roxithromycin on the incorporation of 3 5 S - m e t h i o n i n e into protein 24 hr after its addition with or without Con A. RXM concentration (pg/ml) Mitogen None Con A
0
10
744.3 + 194.9 2397.0 +
84.5
100
910.3 + 87.2
920.7 + 39.9
1204.3 + 74.7
770.3 + 76.4
(Results expressed as mean cpm Z SD) TABLE III
Inhibition of transformation Time o f addition after start o f culture (hr) 0 4 16 24 36 38
% inhibition 94.9 + 85.3 + 51.0+ 43.7 + 31.3 + 12.6 +
0.96 0.39 1.09 1.50 1.16 1.45
RXM ( 100 lag/ml) added to cultures at indicated times. 3H-thymidine incorporated, into D N A was measured at 48 hr after the start o f culture.
Vol.
51, No. 24, 1992
Roxithromycin
on Human Lymphocytes
PL-235
Effects of metabolized RXM on PMNL activation The final set of experiments were undertaken to examine effects of metabolized RXM on PMNL activation. As shown in Table IV, the most striking suppressive effects of metabolized RXM was observed as cells cultured in the presence of RU 45179. However, activation of cells were scarcely affected by addition of RU 39001 and RU 28111. T A B L E IV Inhibition of human peripheral mononuclear leukocyte activation by metabolized roxithromycin 3H-thymidine uptakes; mean cpm (SD) Conc. (~tg/ml)
RXM
RU 44981
RU 45179
RU 39001
RU 28111
N.T.
N.T.
0
32448.6 (483.1)
N.T.
N.T.
10
22135.3 (1793.1)
31133.7 (1648.2)
27992.3 (927.9)
29986.7 (1390.6)
27242.7 (1170.9)
100
243.0 (72.2)
26712.7 (861.9)
14195.7 (873.6)
29456.0 (1091.8)
30329.3 (680.9)
DISCUSSION The present studies indicate that large amount of RXM (100 lag/ml), but not small amount of RXM (10 pg/ml) can significantly suppress blastic activity of PMNL induced by mitogenic stimulation. It is also revealed that RXM in clinically achieved concentration at about 10 pg/ml (8) can strongly inhibit the low rate of blastic activity observd in PMNL cultured with (Table I). The fact that small amount of RXM ( 10 ~g/ml) appeared to have little effect on mitogen-induced blastic activity of PMNL may be related to stronger stimulus when compared to antigenic stimulation and the greater response induced by mitogens. Studies on effect of RXM on a hybridoma, PC6153 showed that in vitro RXM inhibited proliferation of this cell line (unpublished observation). Since this cell line can proliferate without antigenic and mitogenic stimulation, this result strongly indicates that RXM inhibits the cell proliferation rather than the antigenic recognition phase. RXM added to cell cultures stimulated with Con A at 38 hr after the start of culture gave significantly less inhibition than when added earlier, showing that this drug is not simple inhibiting uptake of thymidine and acts early in the cycle of cell division. It is reported that macrolides orally administered to human and mice were analyzed into several types of metabolized materials by liver cytochrome P450 (9). So, RXM may be also metabolized by liver cytochrome P450 and then most of metabolized RXM, except for two types of metabolized materials such as RU 44981 and RU 45179, are excreted into urine, feces, etc. (8). We previously observed that lymphocytes prepared from mice orally administered with RXM for longperiod loss reactivity to mitogenic stimulation (Konno et al., unpublished). Taken together with these reports and our observation, the results in Table IV may indicate that accumulated-RXM, especially RU 44981 and RU 45179 bring about inhibition of lymphocyte transformation. Further more, the results strongly suggest that RXM will be useful drug in treatment of allergic diseases, since lymphocytes reacted with antigen induced these diseases. REFERENCES 1 A. EYRAUD, J. DESCOTES, J.Y. LOMBARD, A. LASCHI-LOQUERIE, P. TACHON, C. VEYSSEYRE, and J.C. EVREUX, Chemotherapy 32 379-382.(1986). 2 G. BANCK, and A. FORSGREN, Antimicrob. Agents Ch. 16 554-560.(1979).
PL-236
Roxithromycin
on H u m a n L y m p h o c y t e s
Vol.
51, No.
24,
1992
3 M.B. ESTERLY, N.L. FUREY, and L.E. FLANAGAM, J. Invest. Dermator. 70 51 (1978). 4 S. MIYACHI, A. YOSHIOKA, S. IMAMURA, and Y. NlWA, J. Invest. Dermatol. 86 449-453 (1986). 5 A. FORSGREN, G. BANCK, H. BECKMAN, and A. BELLAHRENE, Scand J Infect Dis 24 (suppl): 195-203.(1980). 6 T. KINO, H. HATANAKA, M. HASHIMOTO, M. NISHIYAMA, T. GOTO, M. OKUHARA, M. KOHSAKA, H. AOKI, and H. IMANAKA,.J Antibiot. 40 1249-1255.(1987) 7 J.F. CHANTOT, A. BRYSKIER, and J.C. GASC, J. Antibiot. 39 660-668 (1986). 8 M.KOYAMA, M.TATENO, M.SHIROTSUKA, T. YAMAMOTO, M. HIRAYAMA, K. SAITOH, and K. OKUI, Jpn. J. Chemotherapy 36 164-183.(1988). 9 G. BABANY, D. LARREY, and D. PESSAYRE, Progress in Drug Metabolism vol. 11, G.G. Gibson (ed), 62-98,Taylor & Francis, London (1988).