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The new immunosuppressive malononitrilamide MNA 279 prolongs skin xenograft survival in a mouse-to-rat model
ELSEVIER
The New Immunosuppressive MNA 279 Prolongs Skin Xenograft Survival in a Mouse-to-Rat Model H.U. Schorlemmer and FL Kurrle
T
HE INABILITY to provide an adequate supply of human organs for clinical transplantation has created a strong interest in the use of nonhuman, especially nonprimate, organs. Alternative sources of organs from nonhuman donors could be more feasible if the strong immunologic barriers could be overcome with improved immunosuppression. In the absence of reliable tolerogenic regimens, the primary focus in immunosuppressive therapy is currently on lowering toxicity and improving specificity. Several studies in xenotransplantation have stressed the role of natural and induced xenophile antibodies in the rejection of xenografts. The novel molecular weight immunosuppressive malononitrilamide MNA 279 [2-cyano-N-(4-cyanophenyl)-3-cyclopropyl-3-oxopropanamide] is a derivative of leflunomide’s primary metabolite A 771726 and is chemically unrelated and distinct in its mechanism of action from any other known immunosuppressant. MNA 279 is well tolerated and has been found to inhibit the cellular and humoral immune responses. It was described as an effective inhibitor of B-cell-mediated autoimmune processes against a variety of models for experimental autoimmune diseases in rodents.lm4 This derivative of leflunomide’s metabolite mediates its immunosuppressive effects by binding specifically to dehydroorotatedehydrogenase (DHODH) and thereby inhibiting the de novo pyrimidine biosynthesis? MNA 279 has been found to block T- and B-cell proliferation and strongly suppress IgM and IgG antibody production,6,7 primarily reducing auto-, allo-, and xenoantibody formation. It was very effective when tested in rodent transplantation studies, where it was shown to prevent and reverse acute heart and skin allograft rejection,‘,’ and control hamster-to-rat or mouse-to-rat xenograft survival.‘O~” Because of the reported ability of MNA 279 to inhibit cellular and humoral immune responses and to reduce autoand alloantibody titers in viv~,~**,‘~we were particularly interested in characterizing its effects on xenoantibody formation and to test its activity in the prevention or control of concordant skin xenograft rejection in a mouse-to-rat model. MATERIALS AND METHODS
lmmunosuppressant Malononitrilamide MNA 279 (HMR 1279) was supplied in pure form as a white powder (Hoechst Marion Roussel, Werk KalleAlbert, Wiesbaden, Germany) and was prepared daily as a homo0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation
Proceedings, 29, 3497-3500 (1997)
-Gz4mtrd -H*R
1279,m
+cyA, IO mg,lrg HMR 1279,26 mgncg
IngIll*
Il.
I IO
dey9 aftar
IS
+HMR
1279,Vl mgIkg
)
, 20
transplantation
Fig 1. Monotherapy of MNA 279 (10,20, or 25 mg/kg per day) given by oral gavage on days 0 to 14, dose-dependently prevented hyperacute skin xenograft rejection in the mouse-torat transplantation model.
geneous suspension in 1% carboxymethylcellulose (1% CMC) at varying concentrations. Animals were treated with the drug by oral gavage and at the concentrations and schedules given in the text. Control animals received the vehicle solution (1% CMC) only. Drug treatment normally started either on the same day as the transplantation or shortly before the expected rejection crisis. Animals Rats of the inbred Lewis (LEW, RT-I’) strain, with body weights of about 160 g, and adult BALB/c mice were obtained from Charles River-Wiga (Sulzfeld, Germany). All animals were housed in temperature- and light-controlled rooms in our own animal unit and were fed a standard rat and mouse diet and drinking water ad libitum. Xenotransplantation
Model
In this concordant xenotransplantation model of mouse-to-rat skin grafts, LEW rats were used as recipients and BALB/c mice as donors, as described previously.” Tail-skin grafts (pieces of 0.5 X From the Research Laboratories of Hoechst Marion Roussel (HMR), DG-Rheumatology/Immunology, c/o Behringwerke AG, Marburg, Germany. Address reprint requests to H.U. Schorlemmer, PO Box 1140, D-35001 Marburg, Germany.
0041-l 345/97/$17.00 PII so041 -1345(97)00995-O
3497
SCHORLEMMER
3498
AND KURRLE
Table 1. Prolongation of Concordant Mouse-to-Rat Skin Xenograft Survival by Various Concentrations of MNA 279
Control
(1% CMC)
Neoral (5 mg) on days O-14
HMR 1279 10 mg on 10 mg on HMR 1279 20 mg on 20 mg on 20 mg on HMR 1279 25 mg on
Mean survival time in days (MST -c SD)
Days of individual skin xenograft rejection
Drug application (oral gavage)
6.1 2 0.9 6.0 ? 0.5
5, 5, 5, 6, 6 7, 7, 7, 7 5, 6, 6, 6, 6 6, 6, 6, 6, 7
days O-9 days O-14
10, 10, 12, 12, 13 13, 14, 14 13, 13, 14, 15, 15 16, 16, 17, 16
12.3 2 1.6 15.2 2 1.7
days O-4 days O-14 days O-29
16, 16, 16, 16, 17 17, 17, 18, 18, 19 21, 22, 22, 23, 23 24, 25, 25, 25, 26 24, 26, 26, 27, 28 29, 30, 30, 30, 31
17.0 f 1.1 23.6 f 1.8 27.7 f 1.9
days O-14
25, 25,
25, 26, 26 27, 28, 29, 29, 30
27.0 t 1.9
1.0 cm) from BALBic mice were transplanted onto the tails of LEW rats. Rejection day was defined as the day when the skin graft turned red-brown and became hard. Transplanted animals were randomly distributed to the treatment groups. Anti-donor-xenospecific
IgM and IgG Antibody Titers
Xenospecific IgM and IgG antibody titers were quantitated in principle as described by Wasowska et ali3 Briefly, the test sera (dilution 1:lO) were incubated with 1 x 10’ purified T cells (by sheep antimouse IgG dynabeads; Deutsche Dynal GmbH, Hamburg, Germany) from BALB/c donor spleens, for 30 minutes at 4°C. The cells were washed three times with phosphate-buffered saline (PBS; pH 7.2) and then stained for IgG or IgM xenoantibodies, 50 pL of either FITC-conjugated goat antibodies specific for the Fc portion of rat IgG (Southern Biotechnology Associates Inc, Birmingham, Ala) or specific for the p-chain of rat IgM (American Qualex, San Clemente, Calif) were added. After 30 minutes at 4°C the cells were washed twice and analyzed by flow cytometry (FAXStar Plus, Becton Dickinson, Mountain View, Calif). Data are expressed as channel fluorescence intensity (median). RESULTS AND DISCUSSION MNA 279 Monotherapy Prevents Hyperacute Skin Xenograft Rejection
Using the mouse-to-rat concordant skin xenotransplantation model, we found that transplanted LEW rats (untreated) rejected mouse skin xenografts at 6.3 f 0.7 days and this was statistically indistinguishable from the 6.1 i 0.9 days of the placebo-controlled animals (LEW rats gavaged with the vehicle solution only). Several investigators have already demonstrated’O,l’ that, in xenotransplantation models, graft recipients treated with CyA (5 or 10 m&g per day) as a single drug by oral gavage showed no prolongation of xenograft survival (6.0 2 0.5 days). This short xenograft survival is in compliance with antibody-provoked graft rejection and in agreement with descriptions from other laboratories8*r0 When xenograft recipients were immediately treated by oral gavage with various concentrations of HMR 1279 (10 to 25 mg/kg per day) as monotherapy from days 0 to 14, this new immunosuppressive agent significantly delayed the onset of hyperacute xenograft rejection and prolonged skin xenograft survival in a dose-dependent
manner (Fig 1, Table 1). Even a short-term application (only 5 days) of MNA 279 still was able to delay xenograft rejection up to 17.0 C 1.1 days, but it seems that a longer application time was superior for skin xenograft survival (Fig 2, Table 1). The mean graft survival time with applications on days 0 to 29 was increased up to about 28 days, but it should be mentioned that here even some of the skin xenografts were rejected when the recipient animals were still under drug treatment. Control of Acute Ongoing Skin Xenograft Rejection by Therapeutic Treatment With MNA 279 To evaluate the effect of the new immunosuppressant
on advanced graft rejection, in another set of experiments, treatment with HMR 1279 was delayed until day 5 after transplantation and shortly before the expected rejection crisis. In this nonvascularized skin xenotransplantation model the vehicle-treated (1% CMC) LEW recipient rats receiving BALB/c grafts rejected at about 6 days, as described previously, whereas MNA therapy (20 or 25 mg/kg) was effective in the reversal of ongoing acute skin xenograft rejection. Also, short-term application of MNA 279 (25 mg,kg per day) on days 5 to 9 was still therapeutically active 100
I
0
4
I
6
12
16
I
20
1
24
26
32
days after transplantation
Fig 2. Even short-term application (only 5 days) of MNA 279 was able to delay xenograft rejection; however, a longer application time was superior for skin xenograft survival.
MNA 279 IN SKIN XENOGRAFT SURVIVAL
3499
Table 2. Treatment of Ongoing Hyperecute Skin Xenograft Rejection by Monotherepy With MNA 279 Days of individual skin xenograft rejection
Drug application 25 mg/kg PO
Control (1% CMC) Neoral(10 mg)on
days O-14
Mean survival time in days (MST z SD)
5, 5, 6, 6, 6 6, 7, 7, 8, 8
6.4 t 1.1
6, 6, 7, 7, 7 7, 7, 7, 8, 8
7.0 -t0.7
HMR 1279 17.2 t 1.2
15, 16, 16, 17, 17 18,
Days 5-9
18, 18, 18, 19 22.8 t 1.3
20, 22, 22, 23, 23 23,
Days 5-14
24, 24, 24 26.1 t 1.7
23, 24, 25, 26, 26 26,
Days 5-19
27, 28, 28, 28 27.0 + 1.9
25, 25, 25, 26, 26 27,
Days0-14
28, 29, 29, 30
in the prolongation of skin xenograft survival up to 17 days (Table 2). There was a dose and application-time-dependent prolongation of skin xenograft survival up to about 26 days after stopping drug treatment. Xenograft survival times (17.2 2 1.2 days and 26.1 -t 1.7 days), produced by HMR 1279 (25 mg/kg) given therapeutically by oral gavage either on days 5 to 9 or on days 5 to 19, respectively, were comparable to those of the groups in which treatment was started together with the grafting on day 0 (Fig 3). Even when CyA (20 mglkg per day) was administered as a single drug as late as days 5 to 19 skin xenograft survival failed to improve. In this mouse-to-rat xenotransplantation model, MNA 279 as a monotherapy was always superior to CyA, which is consistent with other transplant studies using CyA for rescue therapy.i4,i5 In animals allowed to undergo acute rejection, MNA 279 is superior to CyA in its ability to reverse such a reaction and to produce significant graft survival.
- . 0
5
10 15 20 days after transplantation
25
30
Fig 3. MNA 279 (25 mg/kg per day) has therapeutic activity on ongoing hyperacute skin xenografl rejection and prolongs application-time-dependent xenograft survival.
lmmunosuppression
of Antimouse
Xenoantibody
Formation
by MNA 279
Lin et all6 described that antibody formation is crucial for xenograft survival, it was essential to investigate the IgM and IgG xenoantibody formation of the MNA 279-treated animals in our skin xenotransplantation model. Prior to xenotransplantation, the LEW rats had no detectable mouse xenoantibodies. Mouse skin recipient control rats rapidly produced high titers of both antimouse IgM and IgG xenoantibodies at the time of rejection. As already described, in this mouse-to-rat xenotransplantation model, treatment with CyA (10 mg/kg per day) did not prolong xenograft survival nor did it suppress antimouse xenoantibody formation as compared with control animals (Fig 4). Treatment with MNA 279 (20 mg/kg per day) resulted in significantly delayed and reduced levels of the xenoantibody titers. Even when MNA 279 treatment was delayed until day 5 after xenotransplantation we observed a prolongation of skin xenograft survival for about 26 days. This prolongation of xenograft survival correlated quite well with the strong prevention or reduction of IgM and IgG xenoantibody production up to this time. Upon cessation of drug treatment, the xenoantibody titers gradually increased and, at the time of rejection, significant levels of xenoantibodies, predominantly of the IgG isotype, were found in the MNA 279-treated recipients. This result is very similar to that described for leflunomide and its active metabolite in xenotransplantation,17~1s and suggests that these drugs can prevent early IgM xenoantibody formation, which occurs with hyperacute rejection in control animals. In contrast, the delayed high xenoantibody titers seen under MNA 279 treatment belonged predominantly to the IgG isotype and seem to be resistant to this drug family. In conclusion, these studies show that the new immunosuppressant, HMR 1279, is very efficient in controlling hyperacute skin xenograft rejection in the mouse-to-rat transplantation model and this effect correlates very well with the strongly suppressed antimouse specific IgM and
As
SCHORLEMMER
AND KURRLE
ACKNOWLEDGMENTS We greatly appreciate the skilled technical assistance of Mr M. Ellrich, Mr J. Gamb, and Mr W. Jahnz. We kindly acknowledge Mrs W. Seyfert-Brand and Mr H.W. Beltz for their flow cytometry work. We also gratefully acknowledge the excellent secretarial help rendered by Mrs C. Fanslau in the preparation of this manuscript. REFERENCES
s
xenoantibodies
;
g
IgM o,““:““:““:““;““;““( 0
5
10
15
20
25
30
25
30
B 8 150. e! z G
xenoantibodies
IgG oI”“:““;““:““:““:““( 0
5
10 15 20 days after transplantation
Fig 4. MNA 279 (20 mg/kg per day) given by oral gavage on days 0 to 14 strongly delayed and reduced antimouse-specific IgM and IgG xenoantibody production.
IgG xenoantibody titers in LEW xenograft recipient rats compared with CyA, which demonstrated no significant antihumoral effects. MNA 279 suppressed hyperacute xenograft rejection when it was administered to animals with pre-existing antidonor antibodies, whereas CyA failed to suppress both.
1. Schorlemmer HU, Kurrle R, Bartlett RR: Transplant Proc 28:3043, 1996 2. Schorlemmer HU, Bartlett RR: Int J Tissue Reac 19:64, 1997 3. Schorlemmer HU, Bartlett RR: Inflamm Res 46:S165, 1997 4. Schorlemmer HU, Ruuth E, Kurrle R: Int J Tissue Reac 1965, 1997 5. Williamson RA, Yea CM, Robson PA, et al: J Biol Chem 270:22467, 1995 6. Kurrle R, Bartlett RR, Ruuth E, et al: Transplant Proc 28:3053, 1996 7. Kurrle R, Ruuth E, Bartlett RR, et al: Transplant Proc 29: 1302, 1997 8. Morris RE, Huang X, Cao W, et al: Transplant Proc 27:445, 1995 9. Schorlemmer HU, Schwab W, Ruuth E, et al: Transplant Proc 28:3048, 1996 10. Lin Y, Segers C, Waer M: Transplant Proc 28:3036, 1996 11. Schorlemmer HU, Kurrle R: Transplant Proc 28:3037, 1996 12. Schorlemmer HU, Ruuth E, Kurrle R: Int J Tissue Reac 19:66, 1997 13. Wasowska B, Baldwin WM, Sanfilippo F: Transplantation 53:175, 1992 14. Schorlemmer HU, Seiler FR, Bartlett RR: Transplant Proc 25~763, 1993 15. Williams JW, Xiao F, Foster P, et al: Transplantation 57:1223, 1994 16. Lin Y, Sobis H, Vanderputte M, et al: Transplant Proc 26:3202, 1994 17. Chong ASF, Shen J, Xiao F, et al: Transplant Proc 28:684, 1996 18. Lin Y, Vanderputte M, Waer M: Transplant Proc 28:683, 1996
The New Immunosuppressive MNA 279 Prolongs Skin Xenograft Survival in a Mouse-to-Rat Model H.U. Schorlemmer and FL Kurrle
T
HE INABILITY to provide an adequate supply of human organs for clinical transplantation has created a strong interest in the use of nonhuman, especially nonprimate, organs. Alternative sources of organs from nonhuman donors could be more feasible if the strong immunologic barriers could be overcome with improved immunosuppression. In the absence of reliable tolerogenic regimens, the primary focus in immunosuppressive therapy is currently on lowering toxicity and improving specificity. Several studies in xenotransplantation have stressed the role of natural and induced xenophile antibodies in the rejection of xenografts. The novel molecular weight immunosuppressive malononitrilamide MNA 279 [2-cyano-N-(4-cyanophenyl)-3-cyclopropyl-3-oxopropanamide] is a derivative of leflunomide’s primary metabolite A 771726 and is chemically unrelated and distinct in its mechanism of action from any other known immunosuppressant. MNA 279 is well tolerated and has been found to inhibit the cellular and humoral immune responses. It was described as an effective inhibitor of B-cell-mediated autoimmune processes against a variety of models for experimental autoimmune diseases in rodents.lm4 This derivative of leflunomide’s metabolite mediates its immunosuppressive effects by binding specifically to dehydroorotatedehydrogenase (DHODH) and thereby inhibiting the de novo pyrimidine biosynthesis? MNA 279 has been found to block T- and B-cell proliferation and strongly suppress IgM and IgG antibody production,6,7 primarily reducing auto-, allo-, and xenoantibody formation. It was very effective when tested in rodent transplantation studies, where it was shown to prevent and reverse acute heart and skin allograft rejection,‘,’ and control hamster-to-rat or mouse-to-rat xenograft survival.‘O~” Because of the reported ability of MNA 279 to inhibit cellular and humoral immune responses and to reduce autoand alloantibody titers in viv~,~**,‘~we were particularly interested in characterizing its effects on xenoantibody formation and to test its activity in the prevention or control of concordant skin xenograft rejection in a mouse-to-rat model. MATERIALS AND METHODS
lmmunosuppressant Malononitrilamide MNA 279 (HMR 1279) was supplied in pure form as a white powder (Hoechst Marion Roussel, Werk KalleAlbert, Wiesbaden, Germany) and was prepared daily as a homo0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation
Proceedings, 29, 3497-3500 (1997)
-Gz4mtrd -H*R
1279,m
+cyA, IO mg,lrg HMR 1279,26 mgncg
IngIll*
Il.
I IO
dey9 aftar
IS
+HMR
1279,Vl mgIkg
)
, 20
transplantation
Fig 1. Monotherapy of MNA 279 (10,20, or 25 mg/kg per day) given by oral gavage on days 0 to 14, dose-dependently prevented hyperacute skin xenograft rejection in the mouse-torat transplantation model.
geneous suspension in 1% carboxymethylcellulose (1% CMC) at varying concentrations. Animals were treated with the drug by oral gavage and at the concentrations and schedules given in the text. Control animals received the vehicle solution (1% CMC) only. Drug treatment normally started either on the same day as the transplantation or shortly before the expected rejection crisis. Animals Rats of the inbred Lewis (LEW, RT-I’) strain, with body weights of about 160 g, and adult BALB/c mice were obtained from Charles River-Wiga (Sulzfeld, Germany). All animals were housed in temperature- and light-controlled rooms in our own animal unit and were fed a standard rat and mouse diet and drinking water ad libitum. Xenotransplantation
Model
In this concordant xenotransplantation model of mouse-to-rat skin grafts, LEW rats were used as recipients and BALB/c mice as donors, as described previously.” Tail-skin grafts (pieces of 0.5 X From the Research Laboratories of Hoechst Marion Roussel (HMR), DG-Rheumatology/Immunology, c/o Behringwerke AG, Marburg, Germany. Address reprint requests to H.U. Schorlemmer, PO Box 1140, D-35001 Marburg, Germany.
0041-l 345/97/$17.00 PII so041 -1345(97)00995-O
3497
SCHORLEMMER
3498
AND KURRLE
Table 1. Prolongation of Concordant Mouse-to-Rat Skin Xenograft Survival by Various Concentrations of MNA 279
Control
(1% CMC)
Neoral (5 mg) on days O-14
HMR 1279 10 mg on 10 mg on HMR 1279 20 mg on 20 mg on 20 mg on HMR 1279 25 mg on
Mean survival time in days (MST -c SD)
Days of individual skin xenograft rejection
Drug application (oral gavage)
6.1 2 0.9 6.0 ? 0.5
5, 5, 5, 6, 6 7, 7, 7, 7 5, 6, 6, 6, 6 6, 6, 6, 6, 7
days O-9 days O-14
10, 10, 12, 12, 13 13, 14, 14 13, 13, 14, 15, 15 16, 16, 17, 16
12.3 2 1.6 15.2 2 1.7
days O-4 days O-14 days O-29
16, 16, 16, 16, 17 17, 17, 18, 18, 19 21, 22, 22, 23, 23 24, 25, 25, 25, 26 24, 26, 26, 27, 28 29, 30, 30, 30, 31
17.0 f 1.1 23.6 f 1.8 27.7 f 1.9
days O-14
25, 25,
25, 26, 26 27, 28, 29, 29, 30
27.0 t 1.9
1.0 cm) from BALBic mice were transplanted onto the tails of LEW rats. Rejection day was defined as the day when the skin graft turned red-brown and became hard. Transplanted animals were randomly distributed to the treatment groups. Anti-donor-xenospecific
IgM and IgG Antibody Titers
Xenospecific IgM and IgG antibody titers were quantitated in principle as described by Wasowska et ali3 Briefly, the test sera (dilution 1:lO) were incubated with 1 x 10’ purified T cells (by sheep antimouse IgG dynabeads; Deutsche Dynal GmbH, Hamburg, Germany) from BALB/c donor spleens, for 30 minutes at 4°C. The cells were washed three times with phosphate-buffered saline (PBS; pH 7.2) and then stained for IgG or IgM xenoantibodies, 50 pL of either FITC-conjugated goat antibodies specific for the Fc portion of rat IgG (Southern Biotechnology Associates Inc, Birmingham, Ala) or specific for the p-chain of rat IgM (American Qualex, San Clemente, Calif) were added. After 30 minutes at 4°C the cells were washed twice and analyzed by flow cytometry (FAXStar Plus, Becton Dickinson, Mountain View, Calif). Data are expressed as channel fluorescence intensity (median). RESULTS AND DISCUSSION MNA 279 Monotherapy Prevents Hyperacute Skin Xenograft Rejection
Using the mouse-to-rat concordant skin xenotransplantation model, we found that transplanted LEW rats (untreated) rejected mouse skin xenografts at 6.3 f 0.7 days and this was statistically indistinguishable from the 6.1 i 0.9 days of the placebo-controlled animals (LEW rats gavaged with the vehicle solution only). Several investigators have already demonstrated’O,l’ that, in xenotransplantation models, graft recipients treated with CyA (5 or 10 m&g per day) as a single drug by oral gavage showed no prolongation of xenograft survival (6.0 2 0.5 days). This short xenograft survival is in compliance with antibody-provoked graft rejection and in agreement with descriptions from other laboratories8*r0 When xenograft recipients were immediately treated by oral gavage with various concentrations of HMR 1279 (10 to 25 mg/kg per day) as monotherapy from days 0 to 14, this new immunosuppressive agent significantly delayed the onset of hyperacute xenograft rejection and prolonged skin xenograft survival in a dose-dependent
manner (Fig 1, Table 1). Even a short-term application (only 5 days) of MNA 279 still was able to delay xenograft rejection up to 17.0 C 1.1 days, but it seems that a longer application time was superior for skin xenograft survival (Fig 2, Table 1). The mean graft survival time with applications on days 0 to 29 was increased up to about 28 days, but it should be mentioned that here even some of the skin xenografts were rejected when the recipient animals were still under drug treatment. Control of Acute Ongoing Skin Xenograft Rejection by Therapeutic Treatment With MNA 279 To evaluate the effect of the new immunosuppressant
on advanced graft rejection, in another set of experiments, treatment with HMR 1279 was delayed until day 5 after transplantation and shortly before the expected rejection crisis. In this nonvascularized skin xenotransplantation model the vehicle-treated (1% CMC) LEW recipient rats receiving BALB/c grafts rejected at about 6 days, as described previously, whereas MNA therapy (20 or 25 mg/kg) was effective in the reversal of ongoing acute skin xenograft rejection. Also, short-term application of MNA 279 (25 mg,kg per day) on days 5 to 9 was still therapeutically active 100
I
0
4
I
6
12
16
I
20
1
24
26
32
days after transplantation
Fig 2. Even short-term application (only 5 days) of MNA 279 was able to delay xenograft rejection; however, a longer application time was superior for skin xenograft survival.
MNA 279 IN SKIN XENOGRAFT SURVIVAL
3499
Table 2. Treatment of Ongoing Hyperecute Skin Xenograft Rejection by Monotherepy With MNA 279 Days of individual skin xenograft rejection
Drug application 25 mg/kg PO
Control (1% CMC) Neoral(10 mg)on
days O-14
Mean survival time in days (MST z SD)
5, 5, 6, 6, 6 6, 7, 7, 8, 8
6.4 t 1.1
6, 6, 7, 7, 7 7, 7, 7, 8, 8
7.0 -t0.7
HMR 1279 17.2 t 1.2
15, 16, 16, 17, 17 18,
Days 5-9
18, 18, 18, 19 22.8 t 1.3
20, 22, 22, 23, 23 23,
Days 5-14
24, 24, 24 26.1 t 1.7
23, 24, 25, 26, 26 26,
Days 5-19
27, 28, 28, 28 27.0 + 1.9
25, 25, 25, 26, 26 27,
Days0-14
28, 29, 29, 30
in the prolongation of skin xenograft survival up to 17 days (Table 2). There was a dose and application-time-dependent prolongation of skin xenograft survival up to about 26 days after stopping drug treatment. Xenograft survival times (17.2 2 1.2 days and 26.1 -t 1.7 days), produced by HMR 1279 (25 mg/kg) given therapeutically by oral gavage either on days 5 to 9 or on days 5 to 19, respectively, were comparable to those of the groups in which treatment was started together with the grafting on day 0 (Fig 3). Even when CyA (20 mglkg per day) was administered as a single drug as late as days 5 to 19 skin xenograft survival failed to improve. In this mouse-to-rat xenotransplantation model, MNA 279 as a monotherapy was always superior to CyA, which is consistent with other transplant studies using CyA for rescue therapy.i4,i5 In animals allowed to undergo acute rejection, MNA 279 is superior to CyA in its ability to reverse such a reaction and to produce significant graft survival.
- . 0
5
10 15 20 days after transplantation
25
30
Fig 3. MNA 279 (25 mg/kg per day) has therapeutic activity on ongoing hyperacute skin xenografl rejection and prolongs application-time-dependent xenograft survival.
lmmunosuppression
of Antimouse
Xenoantibody
Formation
by MNA 279
Lin et all6 described that antibody formation is crucial for xenograft survival, it was essential to investigate the IgM and IgG xenoantibody formation of the MNA 279-treated animals in our skin xenotransplantation model. Prior to xenotransplantation, the LEW rats had no detectable mouse xenoantibodies. Mouse skin recipient control rats rapidly produced high titers of both antimouse IgM and IgG xenoantibodies at the time of rejection. As already described, in this mouse-to-rat xenotransplantation model, treatment with CyA (10 mg/kg per day) did not prolong xenograft survival nor did it suppress antimouse xenoantibody formation as compared with control animals (Fig 4). Treatment with MNA 279 (20 mg/kg per day) resulted in significantly delayed and reduced levels of the xenoantibody titers. Even when MNA 279 treatment was delayed until day 5 after xenotransplantation we observed a prolongation of skin xenograft survival for about 26 days. This prolongation of xenograft survival correlated quite well with the strong prevention or reduction of IgM and IgG xenoantibody production up to this time. Upon cessation of drug treatment, the xenoantibody titers gradually increased and, at the time of rejection, significant levels of xenoantibodies, predominantly of the IgG isotype, were found in the MNA 279-treated recipients. This result is very similar to that described for leflunomide and its active metabolite in xenotransplantation,17~1s and suggests that these drugs can prevent early IgM xenoantibody formation, which occurs with hyperacute rejection in control animals. In contrast, the delayed high xenoantibody titers seen under MNA 279 treatment belonged predominantly to the IgG isotype and seem to be resistant to this drug family. In conclusion, these studies show that the new immunosuppressant, HMR 1279, is very efficient in controlling hyperacute skin xenograft rejection in the mouse-to-rat transplantation model and this effect correlates very well with the strongly suppressed antimouse specific IgM and
As
SCHORLEMMER
AND KURRLE
ACKNOWLEDGMENTS We greatly appreciate the skilled technical assistance of Mr M. Ellrich, Mr J. Gamb, and Mr W. Jahnz. We kindly acknowledge Mrs W. Seyfert-Brand and Mr H.W. Beltz for their flow cytometry work. We also gratefully acknowledge the excellent secretarial help rendered by Mrs C. Fanslau in the preparation of this manuscript. REFERENCES
s
xenoantibodies
;
g
IgM o,““:““:““:““;““;““( 0
5
10
15
20
25
30
25
30
B 8 150. e! z G
xenoantibodies
IgG oI”“:““;““:““:““:““( 0
5
10 15 20 days after transplantation
Fig 4. MNA 279 (20 mg/kg per day) given by oral gavage on days 0 to 14 strongly delayed and reduced antimouse-specific IgM and IgG xenoantibody production.
IgG xenoantibody titers in LEW xenograft recipient rats compared with CyA, which demonstrated no significant antihumoral effects. MNA 279 suppressed hyperacute xenograft rejection when it was administered to animals with pre-existing antidonor antibodies, whereas CyA failed to suppress both.
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