0041-1337/92/6302-0303$03.00/0
`TRANSPLANTATION
`Copyright iCll992 by WilliamB & WilkinB
`
`Vol. 63,303-308, No.2, February 1992
`Printed in U.S.A.
`
`THE EFFECT OF A NEW IMMUNOSUPPRESSIVE DRUG,
`BREQUINAR SODIUM, ON HEART, LIVER, AND KIDNEY
`ALLOGRAFT REJECTION IN THE RAT!
`
`DONALD V. CRAMER,2 FRANCES A. CHAPMAN, BRUCE D. JAFFEE,3 ELIZABETH A. JONES,3
`MICHAEL KNOOp,4 GABRIELLA HREHA-EIRAS, AND LEONARD MAKOWKA
`
`Transplantation Biology Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California; and DuPont
`Merck Pharmaceutical Company, Wilmington, Delaware
`
`Brequinar sodium (BQR) prevents cell proliferation
`by virtue of its inhibition of de novo pyrimidine biosyn(cid:173)
`thesis. BQR is capable of inhibiting immune responses
`in vitro and is effective in suppressing the development
`of contact sensitivity and adjuvant arthritis in rodent
`models. Based on the anti proliferative and immunosup(cid:173)
`pressive capacity of BQR, we have evaluated the effi(cid:173)
`cacy of BQR in preventing allograft rejection utilizing
`experimental models of heterotopic heart and kidney
`and orthotopic liver transplantation in an MHC and non(cid:173)
`MHC mismatched ACI_LEW rat strain combination.
`The immunosuppressive activity of BQR is illustrated
`by its ability to inhibit the development of delayed-type
`hypersensitivity to DNFB in mice. When BQR was ad(cid:173)
`ministered orally throughout the sensitization and elic(cid:173)
`itation phases of the DNFB contact sensitivity response,
`it was found to be a potent immunosuppressant with an
`EDllo value of 0.5 mg/kg. This immunosuppressive activ(cid:173)
`ity is also seen in vitro, where BQR is capable of inhib(cid:173)
`iting the mixed lymphocyte response between allogeneic
`ACI and LEW rat strains with an ICllo of 150 ng/ml.
`The immunosuppressive activity of BQR is highly ef(cid:173)
`fective in prolonging heart, liver, and kidney allograft
`survival in the rat. Cardiac allografts are not rejected
`during the period of drug treatment at dosage levels of
`12 to 24 mg/kg orally three times weekly. The grafts
`survive until the drug is discontinued (30 days post(cid:173)
`transplantation), and the grafts are then rejected ap(cid:173)
`proximately 14 days later. Liver and kidney allografts
`are permanently accepted by approximately 50 to 90%
`of the recipient rats following 30 days of treatment with
`BQR at 12 mg/kg. The tolerance that is induced to the
`liver grafts extends in the majority of animals to greater
`than 250 days and is specific for the donor ACI strain.
`Challenge of long-term liver graft survivors with donor
`cardiac grafts is associated with permanent survival of
`donor, but not third-party, heart grafts. Combination
`therapy consisting of suboptimal doses of BQR and CsA
`demonstrates that the combination of these two immu(cid:173)
`nosuppressive drugs results in an increased efficacy in
`prolonging graft survival.
`The results of these allograft experiments demon(cid:173)
`strate that this new immunosuppressive agent is highly
`effective in preventing allograft rejection in the rat. The
`
`1 Presented at the 17th Annual Meeting of the American Society of
`Transplant Surgeons, May 29-31,1991, Chicago, IL.
`2Address correspondence to: Dr. Donald Cramer, Transplantation
`Biology Laboratory, Department of Surgery, Cedars-Sinai Medical
`Center, 150 N. Robertson Blvd., Beverly Hills, CA 90211.
`3 DuPont Merck Pharmaceutical Company.
`• Present address: Department of Surgery, Berlin, Germany.
`
`antiproliferative activity of BQR is effective for inhib(cid:173)
`iting T-Iymphocyte-mediated immune responses, and
`Brequinar sodium should be an important addition to a
`poly therapeutic approach in the treatment of organ
`graft rejection.
`
`The introduction of effective immunosuppressive agents has
`made the transplantation of solid organs a viable and important
`therapeutic alternative for end-stage disease of these organs.
`The most useful of these immunosuppressive agents, cyclo(cid:173)
`sporine A, is one of a family of fungal metabolites that has
`proven to be a potent and novel immunosuppressive agent in
`both animal models and clinical transplantation (1-4). While
`CsA has had an important impact on the survival of trans(cid:173)
`planted organs, adverse side effects have been recognized, and
`they represent a serious limitation to the long-term use of this
`immunosuppressive drug (5, 6).
`The pharmacologic agent Brequinar sodium (BQR) is an
`anticancer drug that inhibits cell proliferation by virtue of its
`inhibition of the activity of dihydroorotate dehydrogenase and
`de novo pyrimidine biosynthesis (7). The application for the
`use of BQR in clinical medicine has been for the treatment of
`metastatic tumors and leukemias. Further experimentation
`with this compound, however, has demonstrated that it is
`capable of modulating immune responses and effective in sup(cid:173)
`pressing the development of contact sensitivity and adjuvant
`arthritis in rodent models.5 The drug acts most efficiently in
`the early stages of the development of the adjuvant arthritis
`and the induction of contact sensitization. When compared
`with other immunosuppressive drugs (i.e., CsA or azathioprine),
`BQR is more effective in preventing the development of these
`immunological reactions (8).
`Based on the anti proliferative nature of these compounds
`and preliminary indications of the immunosuppressive capacity
`of BQR, our laboratory has evaluated the efficacy of BQR in
`preventing allograft rejection, utilizing surgical models of heart,
`liver, and kidney transplantation in an MHC and non-MHC
`mismatched rat strain combination (ACI-+LEW). Our results
`have demonstrated that this novel immunosuppressive agent is
`highly effective in preventing allograft rejection in the rat. Our
`preliminary in vitro and in vivo data demonstrating increased
`efficacy of combinations of BQR and CsA suggest that BQR
`may represent an agent that will be an important addition to a
`
`5 Jaffee BD, Kerr JS, Jones EA, Ackerman NR. The effects of
`Brequinar sodium on adjuvant-induced arthritis in Lewis rats. (Sub(cid:173)
`mitted for publication.)
`303
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`TRANSPLANTATION
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`Vol. 53, No.2
`
`poiytherapeutic approach in the treatment of organ graft rejec(cid:173)
`tion.
`
`MATERIALS AND METHODS
`Animals. Rats: Adult LEW (RTl i), BN (RT1"), and ACI (RTl"vi)
`rats, 10-16 weeks of age, were purchased commercially from Harlan
`Sprague-Dawley (Indianapolis, IN). All animals brought into the ex(cid:173)
`perimental colony were certified virus-free, and the colony is monitored
`regularly for accidental contamination with infectious diseases. The
`animals were housed in microisolator cages in isolated animal facilities.
`The animals were fed rodent laboratory chow (Purina Mills, Inc., St.
`Louis, MO) and tap water ad libitum. The animal facilities at Cedars(cid:173)
`Sinai Medical Center are accredited by the American Association for
`Accreditation of Laboratory Animal Care, and the animals included in
`these studies were handled humanely in accordance with animal exper(cid:173)
`imental protocols approved by the Institutional Animal Care and Use
`Committee.
`Mice: The mice used for the contact sensitivity experiments were
`Balb/c females purchased from Charles River Breeding Laboratories
`(Kingston, MA). All mice used in these experiments were 8-10 weeks
`of age and were maintained at the DuPont Merck Pharmaceutical
`Company Experimental Station, Wilmington, DE.
`Heterotopic cardiac grafts. Abdominal grafts: Intraabdominal heter(cid:173)
`otopic cardiac grafting was performed using a modification of the
`technique described by Ono and Lindsey (9). The donor animals were
`anesthetized with ketamine (100 mg/kg), xylazine (10 mg/kg), and
`atropine (0.05 mg/kg) administered intraperitoneally, and then main(cid:173)
`tained as necessary on methoxyflurane via inhalation. The venae cavae
`and the pulmonary veins were ligated with 5-0 silk, and the pulmonary
`artery and aorta were transected 2-3 mm above their origins in the
`heart. After perfusion of the ventricles and atria with lactated Ringer's
`solution (containing 200 U/ml of heparin), the heart was placed in a
`saline bath at 4"C. Recipient animals were anesthetized as described
`above, a midline incision was made, and the great abdominal vessels
`were dissected free from the left renal vein to the bifurcation. The graft
`was implanted in the abdominal cavity with end-to-side anastomoses
`of the donor to recipient aortas and of the pulmonary artery to recipient
`vena cava in a running continuous suture with 10-0 Novafil on a TE-
`70 needle. Operative times ranged from 30 to 45 min, with a success
`rate of approximately 90%. The grafts were evaluated for function by
`abdominal palpation and all grafts were removed for examination at
`the termination of the experiment. At removal the hearts were fixed in
`10% buffered formalin for 24 hr and then stored for histological
`processing.
`Cervical grafts: Heterotopic cardiac grafts placed in the subcutis of
`the neck were used for examining the specificity of tolerance induction
`in recipients of orthotopic liver grafts that had survived for more than
`120 days. The grafts were harvested as described above and were placed
`in a cervical location in the recipient. The donor aorta and pulmonary
`arteries were anastomosed in a running end-to-side fashion with the
`recipient carotid artery and external jugular vein, respectively. The
`operative times ranged from 30 to 45 min, with a success rate of
`approximately 90%. The grafts were evaluated for function by cervical
`palpation and all grafts were removed for examination at the termi(cid:173)
`nation of the experiment.
`Heterotopic kidney grafts. Donor operation: All surgical procedures
`were performed under inhalation anesthesia. The donor left or right
`kidney was exposed via a midline incision and 200 IU of heparin was
`administered intravenously. The renal artery and vein, aorta, and the
`inferior vena cava were dissected free. A polyethylene catheter was
`inserted into the aorta and the kidney was flushed with 10 ml of a
`preservation solution over a 2-min period. The kidney was excised,
`placed in a container of cold perfusate, and stored by hypothermic
`immersion at 4"C prior to transplantation.
`Recipient operation: Heterotopic kidney transplantation was per(cid:173)
`formed following dissection of the recipient abdominal vessels. The
`donor renal artery and vein were anastomosed to the recipient aorta
`
`and inferior vena cava, respectively, using a continuous 10-0 Novafil
`suture on a TE-70 needle. Circulation was restored and reconstruction
`of the donor ureter was completed with an end-to-end anastomosis
`with an absorbable suture material. A nephrectomy of the recipient
`kidneys was performed after the placement of the donor kidney and
`prior to closure of the abdomen.
`Orthotopic liver grafts. Donor operation: The grafting techniques
`used were modified from those originally described by Kamada et al.
`(10). Male rats of 250-300 gm bodyweight were anesthetized with
`methoxyflurane and the liver was removed through a midline incision.
`The left inferior infraphrenic vein was separated from the superior
`vena cava, double ligated with 6-0 silk, and cut. The caudate lobes of
`the liver were freed to expose the hepatoesophageal plexus, which was
`then ligated and cut. The infrahepatic vena cava was freed from
`surrounding fatty tissue. The preparation of the portal area was com(cid:173)
`menced by double ligation and division of the pyloric vein. To obtain
`the celiac segment, the hepatic artery was separated from the gastro(cid:173)
`duodenal artery, which was double-ligated and divided. After ligation
`and division of the left gastric and splenic arteries, the hepatic artery
`was dissected to the celiac trunk. The bile duct was exposed and a
`stent, approximately 8 mm long, prepared from a 22-gauge i.v. catheter,
`was inserted half its length into the bile duct, and the bile duct was
`now completely cut. The liver was perfused with cold saline (4'C)
`through the portal vein, the right suprarenal vessels were transsected,
`and the liver was removed and stored in a 4"C cold saline bath.
`Recipient operation: After removal of the recipient liver, the donor
`organ was positioned and the anastomosis of the SVC was performed.
`The animal was turned back into the normal position, the portal vein
`stumps were approximated with 8-0 angle sutures, and the venous
`blood supply of the liver was restored. The anhepatic time did not
`exceed 20 min. The infrahepatic inferior vena cava was reconnected
`with 8-0 nylon suture and the common hepatic artery was rearterialized
`according to the method described by Steffen et al. (11). A single 10-0
`nylon stay suture was fixed at the margin of the arterial opening and a
`vascular cuff was prepared from a 24-gauge i.v. catheter. The donor
`celiac artery was pulled over the cuff and fixed with a 6-0 ligature.
`Arterial blood flow was then reestablished. The bile duct anastomosis
`was accomplished as described by Zimmermann et al. (12) by suturing
`the free margin of the donor bile duct to the recipient bile duct with 8-
`o sutures. A two-layer suture of the abdominal wall and the skin with
`3-0 absorbable suture was placed to complete the recipient operation.
`Successfully grafted rats recover and start drinking water in 1-2 hr.
`Pharmacologic agents. Brequinar sodium: This agent was formulated
`fresh daily in distilled-deionized water at concentrations of 1, 2, and 6
`mg/ml and administered orally by gavage three times weekly for
`inhibiting allograft rejection. All mice were treated with the drug
`prepared fresh daily in 0.25% methocel (Dow Chemical Co., Midland,
`MI).
`Cyclosporine A: A commercially available formulation of CsA (100
`mg/kg) from Sandoz Pharmaceutical Corporation (East Hanover, NJ)
`was diluted in olive oil and administered orally by gavage at a dosage
`of 1 mg/kg three times weekly. For the contact sensitivity experiments,
`CsA was administered daily.
`Mixed lymphocyte reaction. The sensitivity of one-way rat mixed
`lymphocyte responses to inhibition with BQR was tested by incubation
`of purified ACI and LEW splenic lymphocytes with increasing concen(cid:173)
`trations of the drug. A total of 2XI0' responder lymphocytes/well were
`cultured with 2X10' irradiated donor, third-party, or syngeneic lymph
`node cells for 5 days in 5% CO2 at 37"C. The culture medium consisted
`of RPM I 1640 medium (GIBCO, Grand Island, NY) containing 5%
`pooled sterile ACI serum, 2 mM I-glutamine, 1xlO-' M 2-mercaptoeth(cid:173)
`anol, 0.1 % nonessential amino acids, 1 mM Na pyruvate, and penicillin/
`streptomycin. Sixteen hr prior to harvesting, the cultures were pulsed
`with 11'Ci of 3H-thymidine. At harvesting the cultures were collected
`on glass fiber mats and the level of thymidine incorporation were
`measured by counting in a beta scintillation counter.
`Contact sensitivity and related methods. Mice were sensitized by
`application of 25 1'1 of 0.5% 2,4-dinitrofluorobenzene (DNFB; Eastman
`
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`February 1992
`
`CRAMER ET AL.
`
`305
`
`Kodak Company, Rochester, NY) in a vehicle of consisting of a 4:1
`solution of acetone and olive oil to the shaved abdominal skin on days
`o and 1. On day 5, the mice were challenged by application to ear skin
`of 20 1'1 of 0.2% DNFB. The ears were measured for thickness with a
`micrometer immediately before challenge and 24 hr later. The differ(cid:173)
`ence in ear thickness is expressed units of 10-' ± 1 SE.
`Statistics. The effect of BQR on the survival of the allografts was
`estimated using Kaplan-Meier curves. The differences in survival be(cid:173)
`tween different groups were assessed using the log-rank test (general(cid:173)
`ized Savage/Mantel Cox). Procedures involving the comparison of
`experimental data from groups of individual animals first had the
`equality of variance examined using the F test (two groups) or Levene's
`test (multiple groups). When normally distributed sample means were
`compared, the Student's t test (two groups) was used. Differences
`between test groups were considered to be statistically significant when
`P<O.01.
`
`RESULTS
`Suppression of mixed lymphocyte responses. The incubation
`of increasing concentrations of BQR in MLR cultures between
`ACI and LEW rats was associated with a sharp inhibition of
`the proliferative reaction (lCw = 150 ng/ml) (Fig. 1). The BQR
`was added to the cultures at the beginning of the experiment
`and was present throughout the entire culture period. We have
`previously demonstrated in comparable experiments that the
`ICw for CsA in rats is 45 I'g/ml and that for FK506 the ICw is
`0.33 ng/ml (13).
`Inhibition of contact sensitivity. The contact sensitivity re(cid:173)
`sponse to DNFB is a delayed-type hypersensitivity reaction.
`As such, BQR was used to determine the effect of the drug on
`this in vivo T-Iymphocyte-mediated immune response. The
`activity of BQR was compared with that of CsA, an effective
`immunosuppressive drug that can inhibit T-Iymphocyte func(cid:173)
`tion in this assay. The results demonstrate that Brequinar is
`more potent (EDw = 0.5 mg/kg) in this model than CsA (ED5o
`= 60 mg/kg) when the two drugs are administered orally from
`day 0 through day 6 (Table 1).
`Heterotopic cardiac allografts. Abdominal heterotopic cardiac
`allografts were exchanged between ACI donor and LEW recip(cid:173)
`ient animals and the recipients were treated with BQR. The
`treatment at each dosage level began 1 day prior to transplan(cid:173)
`tation and continued until 30 days after surgery. Administra(cid:173)
`tion of BQR in increasing doses was associated with prolonga-
`
`.. Inhibition 01 MLR
`100r---~~----------~~----==l=====~
`
`80
`
`80
`
`40
`
`20
`
`Ob=~~wA~~~~~~UU~~~llW __ ~~W
`0.001
`0.01
`0.1
`1
`10
`100
`eOA (ug/mll
`FIGURE 1. The inhibition of one-way mixed lymphocyte responses
`between ACI and LEW strain rats by Brequinar sodium. The data
`represent the inhibition of MLR in eight animals in three different
`experiments.
`
`TABLE 1. The inhibition of the contact sensitivity response to DNFB
`in mice by Brequinar sodium and cyclosporine A·
`Change in
`Dose
`(mg/kg)
`ear swelling
`1.8±0.8
`
`Treatment group
`
`Vehicle control
`
`% Suppression"
`
`ED ..
`
`Positive control
`
`CsA
`
`56.8±3.0
`
`49.2±3.8
`44.9±3.4
`41.8±3.2
`11.7±2.5
`
`2.0
`10.0
`50.0
`100.0
`
`13.9
`21.7
`27.3
`82.0
`
`60
`
`BQR
`
`44.7
`32.3±3.2
`0.4
`2.0
`72.7
`16.9±3.2
`10.0
`100.0
`1.8±1.1
`20.0
`94.9
`4.6±1.4
`• Balb/c mice were sensitized with 0.5% DNFB on days 0 and 1. On
`day 5, the ears were measured and then challenged with 0.2% DNFB.
`The increase in ear thickness was measured 24 hr following challenge.
`
`0.5
`
`.
`b OT S
`70 uppresslOn =
`
`(positive control-vehicle control)
`. .
`.
`(positive control-vehicle control)
`
`00
`Xl
`
`TABLE 2. The effect of Brequinar sodium on cardiac allograft
`survival in rats: ACI-.Lewis
`
`n
`
`Graft survival time
`(days)
`
`Median survival
`(days ± 1 SO)
`
`Drug
`treatment
`group·
`5,6,6,6,7x7
`None
`11
`7.0±0.69
`6 8,13,15,15,16,16
`15.0±3.06
`6 mg/kg
`8 14,38,45,45,46,47,49,54
`45.5±12.26
`12 mg/kg
`43.0±12.21
`7 12,42,42,43,43,43,49
`24 mg/kg
`• BQR administered p.o. X3/week, days -1 -. +30.
`b Compared with the control group.
`
`P"
`
`0.002
`0.002
`0.001
`
`tion of median graft survival at all levels of the drug tested
`(Table 2). Median graft survival was significantly prolonged
`(P<0.002) at the 12 and 24 mg/kg treatment levels. The selec(cid:173)
`tion of a treatment schedule of three times weekly was based
`on observations in humans suggesting that this was the most
`effective dosage schedule for the antineoplastic effect of the
`drug. At the higher dosage levels (24 mg/kg), all ofthe recipient
`grafts survived the treatment period. Once treatment had
`stopped, the grafts were rejected in approximately 2 weeks.
`Orthotopic liver allografts. The administration of BQR to the
`recipients of orthotopic liver grafts was also associated with
`significant improvement in graft survival (Table 3). The graft
`survival induced by treatment with 12 mg/kg of BQR was
`frequently permanent, with approximately 50% of the grafts
`surviving greater than 250 days. In contrast to the heart graft
`recipients, administration of BQR at levels of 24 mg/kg was
`associated with a sharply increased mortality in the graft recip(cid:173)
`ients. These animals died with signs of drug toxicity, primarily
`diarrhea, suggesting that the liver transplantation procedure
`may have been responsible for a decreased ability to excrete
`the drug. The clinical signs of toxicity were associated with
`histopathological evidence of necrosis and atrophy of the gut
`epithelium in the intestinal crypts and hypocellularity of the
`bone marrow (data not shown).
`The effectiveness of BQR when used in suboptimal doses
`
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`Vol. 53, No.2
`
`TABLE 4. The effect of Brequinar sodium on kidney allograft
`survival in rats: ACI~Lewis
`
`n
`
`Graft survival
`time (days)
`
`Median survival
`(days ± 1 SD)
`
`p'
`
`Drug
`treatment
`group·
`Control
`12 mg/kg
`
`9
`6
`
`6.0±0.3
`>99.0±4.90
`
`5,6,6,6,6,6,6,6,6
`>87,>93,>93,
`>99,>99,
`>99
`7,7,10,11,13,15,
`18,29,>92
`a BQR administered p.o. x3/week, days -1 ~ +30.
`b Compared with the control group.
`
`24 mg/kg
`
`9
`
`13.0±26.95
`
`0.003
`
`0.006
`
`TABLE 3. The effect of Brequinar sodium on liver allograft
`survival in rats: ACI ~ Lewis
`Median
`survival
`(days ± 1 SO)
`10.0±0.9
`
`n
`
`Graft survival
`time (days)
`
`p'
`
`Drug
`treatment
`group·
`Control
`
`6 mg/kg
`
`12 mg/kg
`
`26
`
`13.5±83.5
`
`0.36
`
`9l.5±117.2
`
`0.008
`
`8 9,10,10,10,
`10,10,11,12
`8 7,8,9,9,18,
`29,69,>250
`6,6,7,8,8,13,
`14,15,19,19,
`23,26,44,
`139,12 x
`>250
`8 6,6,6,7,11,
`18,23,23
`8 9,10,10,10,
`10,10,11,
`>230
`8 7,7,10,37,
`>220,>220,
`>221, >222
`a BQR and CsA administered p.o. X3/week, days -1 ..... +30.
`b Compared with the control group.
`
`24 mg/kg
`
`CsA 1 mg/kg
`
`BQR 6 mg/kg +
`CsA 1 mg/kg
`
`9.0±7.7
`
`0.49
`
`10.0±77.8
`
`0.70
`
`220.6±110.3
`
`0.09
`
`TABLE 5. Induction of specific tolerance to cardiac grafts in the
`recipients of long-term surviving liver allografts
`Median graft
`survival
`(days)
`>160 days
`8.00±0.50
`
`Liver donor
`strain
`
`Heart donor
`strain
`
`ACI
`ACI
`
`ACI
`BN
`
`Recipient
`strain
`
`LEW
`LEW
`
`n
`
`4
`4
`
`with suboptimal doses of CsA was examined in recipients of
`orthotopic liver grafts. Small doses of CsA (1 mg/kg three times
`weekly) and BQR (6 mg/kg three times weekly) that separately
`do not induce significant prolongation of graft survival were
`administered alone and in combination to liver recipients for a
`period of 30 days (Table 3). The combination of the two drugs
`was highly effective in prolonging graft survival to a median of
`greater than 200 days in a small group of animals.
`Heterotopic kidney allografts. Treatment of kidney allograft
`recipients at 12 mg/kg three times weekly was also associated
`with excellent graft survival (Table 4). All of the recipients in
`this group had permanent survival of their kidney allografts,
`with survival times in excess of 90 days. The kidney graft
`recipients treated with 24 mg/kg of BQR displayed, as seen
`with the liver recipients, an increased sensitivity to the toxic
`side effects of the drug. The median survival for the animals in
`this group was 13 days posttransplantation.
`Specificity of the allograft tolerance. The nature of the toler(cid:173)
`ance induced by BQR in the recipients of orthotopic liver grafts
`was examined in animals that had received their liver grafts
`more than 120 days earlier. Small groups of these animals
`received a heterotopic cervical cardiac graft from the original
`donor strain (ACI) or an unrelated third-party strain (BN).
`Animals that received donor-strain hearts permanently ac(cid:173)
`cepted the heart grafts. The third-party hearts were rejected at
`times comparable to those expected for first-set rejection re(cid:173)
`actions (Table 5).
`
`DISCUSSION
`Recent advances in immunosuppressive therapy have allowed
`for the development of organ transplantation as an important
`therapeutic procedure. The most powerful of these new immu(cid:173)
`nosuppressive drugs, cyclosporine A, is responsible for graft
`survival rates for many organs that approach 90% for the first
`posttransplant year. These successes, however, are associated
`with limitations in the total amount of CsA that can be admin(cid:173)
`istered because of toxic side effects, individual patients who fail
`to respond to CsA therapy and require alternative drug treat-
`
`ment, and the lack of effectiveness of CsA in preventing B(cid:173)
`lymphocyte-mediated antibody production. In an attempt to
`provide alternatives for these limitations, we have examined
`the effectiveness of a new immunosuppressive drug, Brequinar
`sodium, for the prevention of transplantation rejection.
`Our results demonstrate that BQR is an effective immuno(cid:173)
`suppressive agent. The compound acts to inhibit dihydroorotate
`dehydrogenase (DHO-DH), an effect that results in a decrease
`of de novo pyrimidine biosynthesis and a reduction in the
`nucleotide pool available for cell replication. Normallympho(cid:173)
`cytes contain relatively low levels of DHO-DH and activated,
`proliferating lymphocytes are very sensitive to the effects of
`BQR (8). BQR is, as an example, a potent suppressor of the
`contact sensitivity response to DNFB in mice. These results
`suggest that BQR acts on the sensitization limb of this T(cid:173)
`lymphocyte-mediated reaction, the portion of the response that
`is proliferation-dependent. The immunosuppressive activity of
`BQR extends to rats, as the drug exhibits a highly effective
`inhibition of the in vitro MLR between ACI and LEW rats.
`The experiments described above demonstrate that BQR is
`highly effective in preventing the rejection of a variety of
`vascularized organ grafts. Treatment of LEW recipient rats
`with BQR can provide a high level of protection from rejection
`of ACI strain heart, kidney, and liver grafts. The recipients of
`heterotopic heart grafts display good graft survival at drug
`levels of 12 to 24 mg (administered three times weekly) for as
`long as the treatment is continued. Once the treatment ceases,
`the heart grafts are rejected approximately 2 weeks later, indi(cid:173)
`cating that this period of treatment and graft acceptance is not
`sufficient to induce long-term tolerance to the graft. The recip(cid:173)
`ients of both kidney and liver grafts also are responsive to
`treatment with BQR, with induction of long-term graft survival
`after treatment with the drug at a rate of 12 mg/kg. The
`recipients of these grafts display some important differences
`when compared with the heart recipients. The administration
`of 24 mg/kg to the kidney and liver recipients results in a
`dramatic decrease in the median survival time of the recipients
`and the onset of clinical and pathological signs of drug toxicity.
`Approximately 65-70% of the drug and its metabolites are
`
`NOVARTIS EXHIBIT 2021
`Par v Novartis, IPR 2016-00084
`Page 4 of 6
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`

`
`February 1992
`
`CRAMER ET AL.
`
`307
`
`cleared via the bile and feces and 25-30% in the urine. The
`animals in the high-dose treatment group appear to be more
`sensitive to bone marrow and gastrointestinal side effects of
`the drug, perhaps due to temporary impairment of liver and
`kidney function following transplantation or to the interference
`with drug metabolism by high circulating levels of the drug.
`The treatment of liver and kidney graft recipients with the
`drug for a period of 30 days is sufficient to induce a permanent,
`stable tolerance to the graft. Approximately 50% of the liver
`graft recipients and all of the kidney graft animals treated with
`12 mg/kg exhibit long-term graft survival that has extended
`for more than 90 to 250 days. The tolerance induced to liver
`grafts is specific for donor tissues. Recipients of liver grafts
`that had survived for more than 120 days were challenged with
`heart grafts from either the donor strain or an unrelated third(cid:173)
`party BN strain. The third-party grafts were rejected promptly,
`while hearts from the original donor strain have remained
`functional for more than 160 days. We have previously observed
`a similar difference in the ease of inducing tolerance to liver
`grafts (when compared with heart grafts) in studies conducted
`with FK506, suggesting that this phenomenon is the result of
`differences in the organ graft, rather than a feature of the
`immunosuppressive activity of BQR (14, 15).
`In summary, our experimental results have demonstrated
`that Brequinar sodium can induce effective suppression of
`normal immune responses, including MLR in the rat and the
`delayed hypersensitivity reaction seen in skin contact sensitiv(cid:173)
`ity to DNFB in mice. This suppression of normal immune
`responsiveness extends to the prolongation of vascularized
`organ allografts in rats. Treatment with BQR prolongs the
`survival of heart, kidney, and liver allografts in LEW strain
`recipients, with permanent graft survival in the majority of
`kidney and liver graft recipients when treated with the most
`effective doses of BQR.
`
`Acknowledgments. The authors thank Ms. Elaina Cajulis for her
`excellent technical assistance for these studies and Dr. Hong Kai Wang
`for performing the kidney allografts.
`
`ORAL DISCUSSION
`
`DR. BOLLINGER (Durham, NC): You have added to the
`potpourri of new immunosuppressive drugs. One of the chal(cid:173)
`lenges that has yet to be addressed is small intestinal trans(cid:173)
`plantation. The idea that you could transplant the liver, and
`achieve indefinite tolerance of the heart suggests that perhaps
`you might do the same with the intestine. Do you have any
`data on gut transplantation either alone or with the liver
`transplant using Brequinar?
`DR. CRAMER: No, we have done small bowel transplanta(cid:173)
`tion with other compounds, but not yet with Brequinar. Part
`of our reluctance has been that one of the major target organs
`for Brequinar is the gut.
`DR. KAHAN: I was very impressed with the low dose level
`for mouse and human compared with the high rat dose of 12
`mg/kg, yet you had a high bioavailability. It almost seems like
`the drug is more effective with mouse and human cells than it
`is with rat cells, possibly the reverse of cyclosporine. How do
`you explain the fact that you needed such a high dose of drug
`through the oral route if you had a high oral bioavailability?
`Did you do in vitro studies with rat cells to see whether or not
`they were as sensitive to Brequinar as mouse and human cells?
`
`DR. CRAMER: Yes. A series of experiments looking at the
`effect of Brequinar on MLR proliferation have been completed.
`There was a wide range in sensitivity between species. The
`most effective inhibition was with rodents, humans, and mon(cid:173)
`keys. There was less effectiveness when pigs and dogs were
`examined. We think the relationship between in vitro sensitiv(cid:173)
`ity and graft survival may correlate.
`DR. VAN BUREN (Houston, TX): Can you elaborate on the
`drug's toxicity? You indicated that one rapidly dividing group
`of cells, namely, the gut, is potentially a target tissue. Is this
`drug myelosuppressive? Can you give us some idea of the
`window between the toxic and therapeutic doses?
`DR. CRAMER: The toxic side effects were very consistent
`from species to species. We have done pharmacotoxicity work
`in a variety of species. Almost all species exhibited bone marrow
`and gut effects. There was a fair difference in susceptibility to
`the drug between species. Dogs are very sensitive, whereas
`monkeys and humans tend to be relatively insensitive. At this
`point, I can't give you an exact correlation between toxic plasma
`levels and effectiveness of the drug for allograft survival. In
`rats, however, treatment with 12 mg/kg three times a week
`resulted in plasma levels of about 3-4 JLg/ml. That level resulted
`in prolonged graft survival.
`
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`
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`3. Thompson AW. FK-506: profile of an important new immunosup(cid:173)
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