The Specific Monocarboxylate Transporter-1 (MCT-1)... : Transplantation (original) (raw)
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Immunosuppressive agents used in clinical transplantation have been beneficial in improving graft survival rates; however, an issue with these compounds has been the increased incidence of adverse effects, especially nephrotoxicity, and chronic rejection (1). This has motivated a search for new immunosuppressive agents having efficacy against chronic rejection and exhibiting minimal side effects. A group of small molecular weight compounds have recently been identified that are potent inhibitors of in vitro lymphocyte proliferation that show a different activity profile to existing immunosuppressives; having no effect on cytokine production or expression of early activation markers (2). Further investigation revealed that these compounds inhibited the monocarboxylate transporter (MCT-1) that facilitates the transport of lactate, pyruvate, and ketone bodies across cell membranes (2, 3). As far as we are aware, this association of MCT-1 with lymphocyte proliferation is a novel observation.
AR-C117977 and AR-C122982, which bind to MCT-1 inhibiting lactate transport (2), have been used to establish the potential immunosuppressive utility of MCT-1 inhibitors. The present study describes the effect of inhibiting MCT-1 on in vitro and in vivo alloimmune responses as shown in transplantation models in the rat. In a companion paper, Bueno et al. show that inhibition of MCT-1 also prolongs allograft survival in the mouse (4).
MATERIALS AND METHODS
Compounds
AR-C117977 and AR-C122982 were synthesized at AstraZeneca, R&D Charnwood, UK. CsA for in vitro studies was obtained from Sigma (Poole, UK). For graft versus host response (GVHR) studies AR-C117977 was prepared in vehicle (5% Tween 20 [v/v] sterile saline) giving doses of 3, 10 or 30 mg/kg. Subcutaneous (s.c) injections were performed once daily from day 0 to 6. For rat cardiac transplantation, suspensions of AR-C117977 and AR-C122982 were prepared in vehicle (saline containing 5% [w/v] Tween 20 and 0.6% [w/v) carboxymethyl cellulose) giving doses of 3, 10, or 30 mg/kg. Subcutaneous injections were performed once daily from day 0 to 9. Oral dosing was administered twice daily by gavage from day 0 to 9 at 3, 10, 30, or 100 mg/kg. CsA (Sandimmun® 100 mg/ml; Novartis, Basel, Switzerland) was prepared in Intralipid® (200 mg/ml, Pharmacia & Upjohn, Lund, Sweden) to a final concentration of 4 mg/ml. Rats were dosed orally once daily for either 10 (0–9) or 40 (0–39) days at 5 or 10 mg/kg.
Animals
Mixed Lymphocyte Response (MLR), GVHR, and Obliterative Bronchiolitis (OB) Studies
Male DA (RT1avl), DA/Lewis (RT1avl/l), Lewis (RT1l), and Brown Norway (RT1n) rats were obtained from Harlan (UK).The rats were housed for at least 1 week before use and given water and chow ad libitum. All experiments were performed in strict accordance with the Animals Scientific Procedures Act UK, 1986.
Transplantation Studies
Isogenic rats were obtained from Mollegaard Breeding & Research Centre Ltd (Denmark) and were maintained as above. Male DA (RT1av1), PVG (RT1c) and WF (RT1u) were used. Recipients weighed 180–240 g and donors 100–160 g. Approval was given for the study design by the Animal Research Ethics Committee of Lund University. All procedures were performed in accordance with the Good Laboratory Practice code published by the National Board of Health and Welfare in Sweden.
MLR
Each concentration of AR-C117977 and CsA was dissolved in ethanol and dispensed into the wells of a 96 well flat-bottomed microculture plate (Corning, NY). The ethanol was allowed to evaporate before addition of cells as described to follow. Single-cell splenocyte suspensions were prepared from DA and Lewis rats, respectively; washed three times in RPMI (Invitrogen, Paisley, Scotland); supplemented with 5% fetal calf serum (PAA, Pasching, Germany), 50 μM 2-mercaptoethanol (Sigma), antibiotics (penicillin 100 μ/mL and streptomycin 100 μg), and 2 mM glutamine(Invitrogen); and enriched for T cells by passing over nylon wool (Robbins, Sunnyvale, CA). The final cell concentration for the Lewis responder cells was 3–4×106 mL. The DA splenocytes were irradiated with 3000 rads in a Gammacell 3000 (Nordion, Ontario, Canada) and re-suspended at 3–4×106/mL. Then, 100 μL of both cell suspensions were added to each well of the culture plate pretreated with compound as described. Control wells received 100 μL of both suspensions but no compound (positive control) or 100 μL of Lewis responder cells alone (negative control). The plates were incubated at 37°C for 96 hr. Wells were pulsed with 0.5 μCi 3H- thymidine (GE Healthcare, Amersham, UK) for the last 6 hr of culture. Plates were harvested onto glass fiber filter mats (Wallac Milton, Keynes, UK) and incorporated radioactivity was determined by liquid scintillation counting in a MicroBeta plate reader (Wallac). The mean counts from the triplicate wells were calculated and the % inhibition of the MLR by compound was ascertained using the following formula:
Inhibition of 50% of response (IA50) was calculated from a dose response curve.
GVHR
GVHR was based on the method of Ford, Burr & Simonsen (5). In brief, spleens were removed from DA and DA/Lewis rats and made up to single cell suspensions of 5×107 to 1×109/mL dependent on yield. DA/Lewis rats received 100 μL of each suspension into their hind paws, (DA cells into the right and DA/Lewis into the left). Groups of 6 DA/Lewis rats were dosed daily s.c with compound. Control rats received vehicle alone. On day 7, the rats were sacrificed and the popliteal lymph nodes removed and weighed. Additional experiments were performed whereby the rats were dosed daily up to day 17 or 20 and sample groups sacrificed on days 11, 14, 18 or days 12, 14, 18, 21 for CsA or AR-C117977, respectively. Increase in weight was calculated by subtracting the weight of the left lymph node receiving DA/Lewis cells from the right lymph node receiving DA cells. The mean increase of weight in each group of rats was calculated and expressed as a percentage inhibition of weight increase in control rats (formula below). An effective dose of compound giving 50% inhibition (ED50) was ascertained from a dose–response curve.
Cardiac Transplantation
All procedures were performed under aseptic conditions. Rats were anesthetized with chloral hydrate 360 mg/kg. The donor heart was flushed with cold Perfadex® (Pharmacia & Upjohn, Lund, Sweden), and the caval and pulmonary veins were ligated before removal. The heart was heterotopically transplanted to the right neck vessels of the recipient, the aortic root being anastomosed to the common carotid artery and the pulmonary artery to the jugular vein with nonsuture cuff technique (6). In retransplants, a second heart graft was placed in the groin and anastomosed to the femoral vessels. Allograft function was monitored twice daily, rejection being defined as the cessation of palpable heartbeat.
Histological Evaluation of Chronic Allograft Atherosclerosis
Retransplants were performed in animals with graft function >100 days, without immunosuppression. At 150 days after the primary transplantation, both grafts were removed for histological evaluation. The grade of acute rejection was determined according to the 2005 Revision of the 1990 Working Formulation (7). For the evaluation of chronic allograft coronary disease the total number of arteries and the proportion with lesions in the cardiac graft were determined for each graft. The grade of vascular lesion was determined for each vessel in the graft as described by Lurie et.al (8). A mean grade of vascular lesion including all vessels was calculated for each graft.
OB Model
Tracheas from donor BN and Lewis rats were removed aseptically. Lewis recipient rats were anesthetized with Fluothane and two incisions made, one either side of the midline on the dorsal thorax. Skin pockets were prepared and a trachea was placed in each, isograft on the left, allograft on the right. The incisions were closed by suture. Groups of six rats were dosed subcutaneously with AR-C117977 at 30 mg/kg/day or with 5% Tween 20/saline vehicle only. The grafts were removed on day 17 after transplantation. After dissection, the grafts were placed in 10% buffered formalin and processed into paraffin wax. Longitudinal sections, 4 μM, close to the midline of the tracheas were cut and stained with hematoxylin and eosin (H&E).
Pharmacokinetic Analyses
AR-C117977 Subcutaneous Dosing
Blood samples were taken prior to dosing on days 3, 6, and 9 from both high and low responder combinations. Samples were also taken 90–100 days after transplant to measure compound levels in the circulation prior to retransplantation. Nontransplanted PVG rats from the AR-C117977 dosed groups were also sampled between day 12 and day 36.
AR-C117977 Oral Dosing
Blood samples were taken from nontransplanted PVG rats in each dose group. Peak samples were taken on days 0 and 9, at 1 hr and 3 hr after dosing; trough samples prior to dosing on days 3, 6, and 9. Further samples of blood and plasma were taken on days 12 and 15 and stored at –20 °C before analysis.
Calculation of Minimal % Receptor Occupancy
MCT receptor is located on erythrocytes therefore binding of drug to the receptor results in higher whole blood concentrations compared to the corresponding plasma concentrations. The plasma and blood concentrations were related by the equation: [blood]=[plasma]+Bmax*[plasma]/(Kd+[plasma]). Where the Bmax is the maximal binding capacity of the receptor in blood (∼200 ng/ml of drug) and Kd is the equilibrium binding (dissociation) constant for binding of drug to the receptor. The binding site occupancy level (%) therefore equals 100*[plasma]/(Kd+[plasma]).
Sample Analysis
Plasma and blood samples were analyzed using mass spectrophotometry (supplemental information; available for viewing online only).
Toxicity
As an evaluation of toxicity, the postoperative change in body weight was monitored. Additionally tissue samples were taken at 100 days after commencement of treatment in nontransplanted animals for histological examination.
Statistical Analysis
In the GVHR model, differences between dosed and control rats were compared using one-way analysis of variance followed by the Dunnett Multiple Comparison Test (Graphpad, San Diego, CA). Graft survival was plotted according to Kaplan-Meier and differences between groups were compared using log-rank statistics.
RESULTS
AR-C117977 Inhibition of the Rat MLR and Rat GVHR
AR-C117977 and CsA inhibited the rat one-way MLR (Fig. 1a). AR-C117977 was a 100-fold more potent than CsA, (IA50=3.4×10−10 M versus 3.6×10−8 M, respectively), but it did not exhibit total efficacy (60%) compared with CsA (100%). In the GVHR, AR-C117977 dosed s.c. gave good efficacy (>90% inhibition at 30 mg/kg) and potency (ED50=1.3 mg/kg; Fig. 1b).
Effects of AR-C117977 on lymphocyte responses. (a) Inhibition of the one-way mixed lymphocyte response by AR-C117977 and CsA. Spleen cells from Lewis rats were mixed with irradiated spleen cells from DA rats. Compound concentrations tested in range 10 E−11 to 10 E−6. (b) Inhibition of the rat graft vs. host response by AR-C117977. Rats were dosed daily with 3, 10, or 30 mg/kg of AR-C117977 s.c. (days 0–6) and local draining popliteal lymph nodes removed and weighed on day 7.
AR-C117977 Induced Long-Term Graft Survival in a Low Responder Strain Combination
PVG to DA is a low responder combination, with minimal requirements for immunosuppression (9). Median graft survival in nontreated DA rats was 8 days (range, 7–8 days). Low doses of AR-C117977 and CsA (3 mg/kg/day and 5 mg/kg/day respectively) administered for 10 days both maintained long-term graft survival >100 days (Table 1).
Graft survival of DA to PVG or PVG to DA rat cardiac transplantation
AR-C117977 Induced Long-Term Graft Survival in a High Responder Strain Combination
The reverse, (DA to PVG) is a high responder combination requiring more stringent immunosuppression (9). Untreated PVG rats receiving DA cardiac transplants had a median graft survival of 7 days (range, 6–8 days). Standard dosing with CsA (10 mg/kg per os for 10 days) effectively prevented rejection for 8–15 days after cessation of treatment, giving a median graft survival of 20.5 days.
When animals were dosed daily with AR-C117977 at 3, 10, or 30 mg/kg (s.c days 0 to 9) long-term graft survival of >100 days was induced. Pharmacokinetic analysis had shown presence of drug up to 40 days posttransplantation after 10 days dosing as described below (Fig. 3b). It was not clear if this contributed to the long-term survival observed with AR-C117977 compared with CsA. To investigate this possibility a group of rats received CsA for 40 days after transplantation. The median graft survival of this group was 52 days. Oral dosing of AR-C117977 (100 mg/kg for 10 days) resulted in median graft survival of >100 days (4 of 6 animals). At 30 mg/kg AR-C117977 showed similar efficacy to the standard CsA dosing regimen (Table 1).
Pharmacokinetic properties of AR-C117977. (a) Mean trough concentrations of AR-C117977 dosed s.c. in plasma of PVG rats receiving DA cardiac transplants. AR-C117977 was dosed 3, 10, or 30 mg/kg once daily on days 0–9. Kd=equilibrium binding (dissociation) constant for binding of the drug to the receptor. (b) Mean plasma concentrations of AR-C117977 dosed s.c. in plasma of naïve PVG rats. AR-C117977 was dosed 3, 10, or 30 mg/kg once daily on days 0–9. (c) Mean blood concentrations of AR-C117977 dosed p.o. in plasma of PVG rats receiving DA cardiac transplants. AR-C117977 was dosed 1.5, 5, 15, or 50 mg/kg twice daily on Days 0–9. C0 is trough level and C1 is concentration at 1 hr after dose administration.
Donor-Specific Suppression
In high responder recipients (DA to PVG) with graft survival >100 days, a second heart was transplanted from the same donor strain (DA) or from a third-party donor (Wistar Furth, WF) (Table 2). All DA donor grafts survived >50 days after the time of retransplantation without any further need for immunosuppression, showing evidence of donor-specific suppression. This was confirmed by the rejection of third party grafts within 8 days. In low responder recipients, the donor-specific secondary grafts (PVG) also had long-term function. Animals that had received CsA at the time of the primary transplantation also showed donor-specific suppression and third party grafts were all lost within 14 days (Table 2).
Graft survival after transplantation of a second cardiac graft
Prevention of Chronic Transplant Rejection
Chronic Allograft Coronary Disease
In retransplanted animals, primary and secondary heart grafts were removed for histological evaluation at 150 days after initial transplantation. Isograft transplants acted as controls, and were also removed at 150 days. Arteries from the high responder recipients dosed with AR-C117977 showed fewer lesions of vasculopathy (27.7%) after high-dose treatment (Table 3). The mean grade of the vascular lesions ranged from 0.75 to 0.86, and that of interstitial cellular rejection was 2.7. There were no long-term surviving grafts or re-transplants to be evaluated after CsA treatment. In the low responder recipients, vascular lesions were minimal and similarly graded (10–15%) for both AR-C117977 and CsA dosed groups.
Histological evaluation of chronic allograft coronary disease and cellular rejection in primary cardiac transplants
OB Model
In the rat OB model, AR-C117977 totally blocked fibrotic occlusions in the lumen of BN tracheal allografts transplanted into Lewis recipients (Fig. 2G and H). Tracheal allografts from dosed rats also had intact respiratory epithelium (unlike control allografts; Fig. 2E and F) in which no recognizable epithelium was present, having been replaced to a varying extent with luminal fibrosis). However, AR-C117977 dosed allografts show differences in epithelial phenotype compared with both freshly excised trachea and isografts from control and AR-C117977 dosed animals (Fig. 2A and B and C and D). These differences included a shift in the number of prominent goblet cells as well as a change in balance of goblet cell acidic and neutral mucins, in part representing a metaplastic adaptive response to the graft environment. An accumulation of mucosal mononuclear cells in AR-C117977 dosed allografts was also observed.
Effects of AR-C117977XX on OB model. Effects of AR-C117977 on tracheal isografts and allografts, Day 17 after transplantation. (A and B) Representative isograft control non-dosed demonstrating an intact epithelium and epithelialization of the thin fibrous ‘cap’ forming over the cut end of the graft (arrowhead). The isograft lumen is filled with proteinaceous/ mucoid material containing a small number of inflammatory cells. There is no fibrous occlusion of the graft lumen. The higher magnification (B) provides detail of the mucosa showing a relatively normal phenotype with a prominent ciliated epithelium. (C and D) Representative isograft AR-C117977 dosed, morphologically similar to the untreated control isograft. The higher magnification (D) shows a similar ciliated epithelium and slightly more prominent mononuclear cell infiltrate in the mucosa. (E and F) Representative allograft control nondosed. There is complete loss of tracheal epithelium and obliterative fibrosis of the allograft lumen, with evidence of neovascularization and inflammation. (G and H) Representative allograft AR-C117977 dosed. Compared with the control (untreated) allograft, there is an intact epithelium including epithelialization of the thin fibrous cap sealing the cut end of the graft (as seen with the isografts). There is no fibrous obliteration of the graft lumen, which contains proteinaceous/ mucoid exudate. The majority of allografts exposed to the compound (5/6) had a marked mucosal mononuclear cell infiltrate, frequently seen packed in distended vessels (PV). The higher magnification (H) illustrates a ciliated epithelial phenotype although there is evidence of an intraepithelial as well as a mucosal inflammatory cell infiltrate. Scale bars: A, C, E, and G = 500 μm; B, D, F, and H = 50 μm.
Pharmacokinetic Data
In the high responder strain combination the minimum trough plasma concentration (of samples taken before s.c dosing of AR-C117977 on days 3, 6, and 9) was four times greater than the Kd (2 ng/mL), indicating that minimum receptor occupancy was 80%. Trough levels were proportional to dose and increased from day 3 to 9 as a result of accumulation of solid drug at the dose site. Slow dissolution of the drug depot resulted in prolongation of drug exposure. However, by day 100 the plasma levels were <Kd and receptor occupancy was 3, 4 and 24% for 3, 10, and 30 mg/kg doses, respectively (Fig. 3a).
Plasma samples were collected on days 12, 15, 18, 22, and 36 from nontransplanted PVG rats dosed as described previously to determine the exposure time of AR-C117977. At 3 and 10 mg/kg, the plasma concentration was≥Kd for approximately 40 days. At 30 mg/kg, the exposure time was longer (Fig. 3b).
Trough blood levels of AR-C117977 in samples from nontransplanted PVG rats dosed p.o at 1.5, 5, 15, and 50 mg/kg (twice daily), were 33, 43, 79, and 108 ng/mL, respectively. This equates to minimal receptor occupancy of approximately 16%, 21%, 40%, and 55% (Fig. 3c). The blood levels in all groups declined rapidly after cessation of dosing and by day 15 all were <25 mg/mL (receptor occupancy <15%). Plasma samples from the low responder strain combination dosed s.c gave similar results to those reported above (unpublished observations).
Toxicity
The body weights of the transplanted rats were measured daily. Rats dosed s.c or p.o with AR-C117977 had similar or faster weight gain compared to those receiving no treatment. In contrast, rats dosed with CsA showed a slower rate of weight gain postoperatively (data not shown). Seminiferous tubular degeneration of the testes was the main histological finding seen in rats receiving the higher doses of AR-C117977.
DISCUSSION
The major finding of this study was that inhibition of MCT-1 resulted in significant suppression of alloimmune responses, preventing acute and chronic rejection and also inducing donor-specific suppression in rat cardiac transplants. In the rat MLR assay AR-C117977, in contrast to CsA, did not completely inhibit the proliferative response indicating that a small population of lymphocytes is refractory to the inhibition of MCT-1. This observation was made using a number of different stimuli against both human and rodent lymphocytes (2). Other studies have revealed that AR-C117977 delays lymphocyte proliferation rather than reducing lymphocyte viability (2).
In the rat GVHR model, AR-C117977 inhibited proliferation of the responding lymph node cells measured on day 7 (the maximal GVHR response of control un-dosed rats peaking on day 7). After prolonged dosing of AR-C117977 to day 20, the GVHR of the dosed rats was shown to peak at day 14 indicating that the compound effected a rightward shift of the GVHR response rather than inhibiting it as seen with CsA (data not shown).
To establish if this rightward shift in the response time would have benefit in transplantation, AR-C117977 was tested in rat cardiac transplantation models using both low and high responder combinations. Prolonged survival of the heart allografts in the high responder model, with only 10 days administration of AR-C117977, was markedly superior to the activity of CsA. Because the plasma concentration of AR-C117977 was found to remain above the Kd threshold for more than 40 days after cessation of dosing it was possible that this accounted for the superior activity of AR-C117977 in maintaining the heart allografts compared with CsA. To explore this possibility, rats were dosed with CsA for 40 days; however, none of the grafts survived more than 60 days. In these rats, graft failure was observed 12–21 days after cessation of CsA treatment, which was similar to the graft failure time of 8–15 days observed after stopping CsA treatment on day 9.
The critical factor determining graft survival in AR-C117977-dosed rats appears to be the minimal receptor occupancy maintained during the initial dosing interval. Minimal occupancy levels of <30% did not mediate prolongation of graft survival, whereas graft survival for >100 days was observed for all dose groups where minimal receptor occupancy was >55%. This finding was further supported by AR-C122982, another MCT-1 inhibitor that was less efficacious in maintaining long-term graft survival (Fig. 4).
Correlation of minimum AR-C117977 and AR-C122982 receptor occupancy (%) of MCT-1 receptor (on days 0–9) with median DA to PVG graft survival. AR-C117977 was dosed at 3, 10, 30 mg/kg s.c. and 3, 10, 30, 100 mg/kg p.o. AR-C122982 was dosed at 3, 10, 30 mg/kg s.c. The calculated receptor occupancy (%) and median graft survival for each of these 10 treatment/dose groups are depicted. Graft survival of >100 days was associated with minimum occupancy of >55%.
The possibility that the long-term graft survival, in rats dosed with AR-C117977 s.c., resulted because of prolonged drug exposure, due to slow release from the injection site, was unlikely as rats dosed p.o. with 100 mg/kg AR-C117977 also had graft survival >100 days despite receptor occupancy being <15% by 15 days. Furthermore, had AR-C117977 been present at 100 days to maintain allograft survival the third-party allografts would also be maintained, but this was not the case.
In the low responder combinations PVG to DA, graft failure from acute rejection was prevented and operational tolerance achieved by CsA at 5 mg/kg that is half its standard dose (10). AR-C117977 at 3 mg/kg also maintained graft survival and induced donor-specific suppression in the low responder combinations.
Indefinite graft survival and potential donor-specific suppression found with dosing AR-C117977 in the high responder combination was not observed in CsA treated rats even after prolonged dosing. This was contrary to a previous report that CsA dosed intramuscularly, over 14 days at 15 mg/kg or intermittently over 17 days, induced donor-specific suppression in identical high responder strains with third party skin grafts being rejected (11). In this latter study, the exposure levels of CsA were maintained between 200 and 300 ng throughout 60 days. It is not known whether these dose levels of CsA were achieved in our studies, which may explain our failure to induce donor-specific suppression with CsA.
The mechanism of AR-C117977-induced donor-specific suppression remains to be elucidated. Current understanding of the events leading to tolerance and knowledge of the effects of AR-C117977 on lymphocyte proliferation offers possible explanations for the donor-specific suppression found.
For example, AR-C117977, by delaying proliferation, may result in a change in balance between the generation of effector and regulatory T-cell populations whereby the effector T-cell pool is unable to reach a critical mass to counter the activity of the regulatory T-cell pool. This could occur if the delay in proliferation of effector T-cells by AR-C117977 favors the generation or proliferation of preexisting regulatory T-cells. Recently it has been reported that administration of rapamycin in combination with IL-2 and an antagonist IL-15 fusion protein, in stringent murine skin and heart allograft models, resulted in tolerance that was a result of tipping the balance between regulatory T-cells and cytopathic effector T-cells (12).
The observation that AR-C117977, unlike CsA, does not inhibit IL-2 production may also explain the induction of donor-specific suppression since it has been shown that IL-2 is important for programming T-cells for apoptosis (13) and the peripheral deletion of allograft specific lymphocytes (14, 15). AR-C117977 induced donor-specific suppression could result from the delay of alloimmune proliferation in the presence of normal IL-2 production predisposing the allograft specific lymphocytes to apoptosis.
Alternatively, AR-C117977 may inhibit the induction or expression of co-stimulatory signaling pathways important in T-cell activation and the generation and induction of memory cells (16). AR-C117977 has not been tested against the ICOS, CD154, CD134 or CD137 signaling pathways, all of which when inhibited prolong graft survival and in some cases induce tolerance (17, 18). Interestingly, a recent publication suggests that blockade of CD134-CD134L and CD28 - B7 signaling significantly prolongs allograft survival (19). Because CD134-CD134L co-stimulatory pathway has been shown to be critical for the development of memory T-cells (20), AR-C117977 blockade of its expression or one of the other costimulatory pathways could prevent the generation of specific memory T-cells to the allograft. Clearly further studies are required to establish the mechanism for the induction of donor-specific suppression caused by regulating T-cell metabolism.
The effects of AR-C117977 on chronic allograft atherosclerosis in the cardiac transplant models have been difficult to interpret. In the high responder groups it has not been possible to compare the AR-C117977-treated group to a positive control as the CsA- treated group did not reach a 100-day survival. In the low responder groups, both AR-C117977 and CsA treated rats gave a similar incidence of atherosclerosis, lower than that seen in the high responder animals dosed with AR-C117977.
To further investigate whether AR-C117977 inhibited the pathological events associated with chronic graft rejection its effects on a model of OB in the rat were evaluated. OB is a major problem in lung transplantation and considered to be caused by chronic allograft rejection (21). The rat OB model has been shown to exhibit a similar histological profile to that seen in clinical OB (22) and is therefore a good model to investigate chronic allograft rejection. Tracheal allografts from rats dosed with AR-C117977 did not exhibit the marked fibrosis and loss of epithelium found in trachea allografts in non-dosed animals. These observations suggest that AR-C117977 is effective in preventing the pathophysiology associated with chronic allograft rejection that is not readily controlled by many existing immunosuppressive drugs.
The rats dosed with AR-C117977 did not show any overt toxicity or weight loss. The only abnormal histological observation from the autopsied tissues was a marked atrophy of the testes. This effect was consistent with the key role of MCT-1 induced lactate transport in spermatogenesis (23). In conclusion, inhibition of lactate transport represents a novel mechanism of immunosuppression and may provide utility in the prevention of rejection after organ transplantation and in the treatment of autoimmune disease.
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Keywords:
Immunosuppression; Monocarboxylate transporter; Alloimmune responses; Donor-specific suppression
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