Brequinar Plasma Concentration

Basiliximab + early initiation

Tannoureen Restaurant Side Effects. Clinical experience from 2 phase III studies conducted to date indicates that daclizumab does not increase the incidence of adverse events when administered with standard cyclosporine-based dual or triple therapy to renal transplant recipients. The incidence of overall adverse events considered to be possibly or probably related to treatment is similar in the daclizumab and placebo treatment groups in both studies (222). For Simulect, severe acute (onset within 24 h) hypersensitivity reactions including anaphylaxis have been observed both on initial exposure and/or after reexposure after several months. In the case of severe hyper-sensitivity reaction, therapy with Simulect is permanently discontinued (208). The side effects of 0KT3 may be life threatening, given that it is associated with markedly increased susceptibility to infection and leads to hypertension, hypotension, chest pain, dizziness, fainting, trembling, headache, and stiff neck (231).

2.3 Agents that Block Nucleotide Synthesis

2.3.1 Azathioprine History. Several antimetabolites synthesized for cancer therapy were incidentally found to have immunosuppressive activity. In 1951 Elion for the first time synthesized 6-mercaptopurine (6-MP) as an inhibitor of nucleic acid base metabolism that significantly increased the life expectancy of leuke-mic children (232). To increase its efficacy, a large number of derivatives of 6-MP were synthesized and examined for their activities and metabolic fate. One of these was azathioprine, aprodrug of 6-MP, which temporarily protects 6-MP from catabolism and releases 6-MP inside leukemic cells. Though initially used in leukemia, 6-MP was found to suppress antibody formation and allograft rejection. The breakthrough came in 1959 when Schwartz and Dameshek (233) could prevent rabbits from producing antibodies to human serum albumin by treating them for 2 weeks with the antimetabolite 6-MP. Next, Calne et al. observed that bilaterally nephrotectomized dogs living on solitary renal allograft survived for by treating them with derivatives reto 6-MP (234).Among several drugs pro-

Brequinar Structure
Figure 12.12. Structure of azathioprine (9).

vided by Hitchings and Elion, azathioprine was found to have the best therapeutic index. The first renal transplant recipient to receive azathioprine was an adult transplanted with an unrelated kidney in March 1961 (235). Finally, its clinical use was recommended in 1968 and since then it has remained the keystone of immunosuppressive treatment for renal transplantation. It is also used to treat severe cases of rheumatoid arthritis, systemic lupus, polymyositis, Crohn's disease, ulcer-ative colitis, and other autoimmune disorders. Chemical Structure. Chemically, azathioprine is 6-(1-methyl-4-nitro-5-imi-dazyl) thiopurine (9; Fig. 12.12). Pharmacokinetics. Oral azathio-is well absorbed, and both azathioprine and its metabolite 6-mercaptopurine (6-MP) distribute throughout the body and are able to cross the placenta. Azathioprine is converted by hepatic xanthine oxidase to 6-MP, which is further metabolized to several compounds including 6-thiourate. These metabolites are excreted in the urine. The plasma half-life of azathioprine is <15 min, whereas the half-life of its active derivative 6-MP is 1-3 h. Pharmacology. Azathioprine exerts its immunosuppressive and toxic properties through the release of 6-MP as the main metabolite in vivo (235, 236). The metabolic studies of azathioprine revealed that, after absorption, it is nonenzymatically cleaved by sulfhydryl-containing compounds (e.g., cys-teine, red blood cells glutathione, etc.) to 6-MP. The latter is then enzymatically converted to ribonucleotide and thioinosinic acid. Interestingly, it is this thioinosinic acid that eventually interferes with the conversion of inosinic acid to guanylic and adenylic acids and gets itself converted to thioguanylic acid, which in turn affects the synthesis of DNA and polyadenylate-containing RNA (237, 238). The antiproliferative activity of azathioprine allows the drug to affect the dividing B- and T-lymphocytes during their proliferation cycle. Because T-cell stimulation by antigens causes cell proliferation, the predominant im-munosuppressive activity of azathioprine is to block mitosis of activated cells by interfering with the nucleotide synthesis.

Earlier clinical transplant experiments demonstrated that the drug alone was relatively successful in preventing rejection, but soon it became evident that adjunctive maintenance corticosteroids were more effective. Studies suggested that low dose maintenance corticosteroids when used in combination with azathioprine could be as effective as the more generally accepted higher dose (239). Later randomized trials in renal transplant recipients of high dose vs. low dose corticoste-roids in combination with azathioprine supported the concept, in that the low dose group exhibited fewer corticosteroid-related problems. Thus, low dose corticosteroids in combination with azathioprine became the common maintenance immunosuppressive regimen for kidney transplant patients (240). In fact, this was the breakthrough that allowed kidney transplantation to become a routine clinical approach.

However, after the introduction of cyclo-sporine in 1978, triple-drug therapy with cyclosporin, corticosteroids, and azathioprine became the most frequently used regimen for cadaver kidney recipients (241, 242). These three drugs are believed to complement each other in preventing graft rejection. One advantage of triple-drug therapy is that it allows more flexible immunosuppression, with the possibility of adjusting the dosage of individual components to minimize adverse effects. while maintaining adequate overall immuno-suppression. The superior immunosuppres-sive efficacy of the azathioprine-based triple-drug regimen led to clinical trials with other triple-drug regimens consisting of recently introduced immunosuppressants, to identify the optimal combination of immunosuppressants for a variety of solid organ transplantation settings. Recently, several groups from the United States and Europe reported results of their single center/multicenter randomized trials by replacing azathioprine with other potent immunosuppressants: MMF, tacrolimus, and sirolimus in a variety of organ transplantation models. The results of comparative studies involving an azathioprine-based regimen with other regimens are summarized in Table 12.5. Substituting azathioprine with either MMF or sirolimus or tacrolimus resulted in an improved survival of graft and reduced occurrence and severity of acute rejection episodes in many organ transplantation models. Nevertheless, in another interesting experiment, conversion from azathioprine plus cyclosporin to MMF (20 mg/day) with consecutive reduction of cyclosporine in heart transplant recipients with cyclosporine-im-paired renal function improved renal function to a significant extent (251). Thus, although recent studies point toward superior immuno-suppression by use of a nonazathioprine-based triple-drug regimen, azathioprine still remains the keystone of immunosuppressive treatment.

2.3,1.5 Structure-Activity Relationship. Though it is evident that controlled release of 6-MP plays an important role in azathio-activity, studies from different laboratories suggest that the immunosuppressive effects of azathioprine may not be ascribed to the 6-MP alone. Crawford et al. (252) proposed that the secondary immunosuppressive effects of azathioprine might be attributable to the action of the methylnitroimidazolyl substituent. Based on this hypothesis, several analogs of azathioprine were designed and synthesized by replacing the 6-MP component with nontoxic thiols. In all, 24 such congeners were synthesized, out of which two compounds, Dta and EXb (Fig. 12.13), were found to be more effective than azathioprine in prolonginggraft survival in mice. Toxicity studies with these two compounds showed that these analogs had no toxic effects at doses equivalent to that of azathioprine, which caused severe bone marrow depression. Biological effects that have been attributed to the methylnitroimidazolyl moiety of azathioprine involve interference with the processes such as antigen recognition, adherence, and cell-mediated cytotoxicity (253). These findings suggest that azathioprine may further inhibit cell proliferation by mechanisms

Table 12.5 Comparison of Azathioprine (h a) - B as e d Regimen with Other Potent Immunosuppressive Drugs in Different Organ Transplantation Settings


Transplantation Model

Azathioprine-Based Treatment

Other Immunosuppressants



Renal transplantation Pediatric renal transplant recipients Renal recipients

Renal allograft recipients Simultaneous tn kidney-pancreas vl recipients

Liver allograft recipients

Liver transplantation

Kidney transplantation Pancreas transplantation

Aza + cyclosporine + steroids (n = 161) Cyclosporine + Aza + corticosteroids (n = 6)

Aza + cyclosporine +

prednisone (n = 50) Aza + cyclosporine +

lymphocyte antibodies T steroid (n = 29) Tacrolimus + Aza + antilymphocyte globulin + prednisolone (n = 56)

Aza + cyclosporine +

steroid {n = 26) Aza + cyclosporine + steroid + antilymphoçyte globulin (n = 13)

Sirolimus + cyclosporine + steroids (n = 558) Cyclosporine + MMF + corticosteroids (re = 16)

MMF + cyclosporine + prednisone (n = 62) MMF + cyclosporine + prednisone (re = 22) MMF (n = 74)

lymphocyte antibodies T steroid (n = 28) Tacrolimus + prednisone (n = 61)

MMF + cyclosporine +

steroid (n = 25) MMF + cyclosporine + steroid + antilymphocyte globulin (n = 12)

Use of sirolimus reduced occurrence and severity of acute 114

rejection episodes with no increase in complications.

MMF leads to an improvement in the immunosuppression and 243

renal function in children with ongoing rejection.

MMF-based triple drug regimen results in fewer rejection 244


Graft function was excellent and similar in both groups during 245 the first 6-month observation period.

Trends for most efficacy parameters favored MMF over Aza, 246

and time to renal allograft rejection or treatment failure was statistically significantly longer for MMF. The use of MMF in the treatment of SPK recipients is a useful advance.

Primary immunosuppression with MMF is advantageous over 247

Aza with regard to safety and efficacy.

Both tacrolimus-based dual and quadruple 248

immunosuppressive induction regimens yield similar safety and effectiveness after liver transplantation.

Graft survival demonstrated 12.5% graft losses in the Aza 249

group vs. no kidney transplant losses in MMF group.

Patients treated with MMF required less frequent and less 250

intensive treatment for acute rejection. However, its short-and long-term side effects should be further investigated.

Azathioprine Structure
Figure 12.13. Structure of azathioprine analogues (9a & 9b).
Figure 12.14. Structure of brequinar sodium (10).

independent cf its effect on purine synthesis (254, 255). The drug inhibits the primary immune response with little effect on secondary responses, and is thus useful in preventingacute rejection but not in reversing the process, once started. Side Effects. Major side effects associated with azathioprine are nausea, vomiting, mouth ulcers, or anorexia (60%).More serious. but less common. is bone marrow suppression, with reversible leucopenia occurring in up to 20% of the patients. This adverse reaction is usually dose dependent and can lead to further complications including infections and bleeding. Other unwanted side effects include headache, muscle aches, rash, pancreatitis, and, rarely, hepatotoxicity. The reported increased risk of lymphoproliferative disease and skin and urogenital cancers has not been confirmed but may be up to 4%. Aza-thioprine, however, does not appear to be excreted into breast milk but, in principle, its introduction during lactation should be avoided. Hepatotoxicity occurs in 2-10% of transplant patients receiving azathioprine. Metabolism of azathioprine is inhibited by Allopurinol, which potentiates the effect of azathioprine and increases the risk of myelo-suppression. Combining it with other myelo-suppressives may also increase the risk (256, 257).

2.3.2 Brequinar History. Like other antiproliferative agents, brequinar was originally developed in 1985 as an antitumor agent (258). Later in 1993 it was found to exhibit potent and selective immunosuppressive activity (259). Chemical Structure. The chemical structure of synthetic brequinar 6-fluoro-2-(2'-f3uoro-l,l/-biphenyl-4-yl)-3-methyl-4-quino-

linecarboxylic acid sodium salt (10) is shown in Fig. 12.14. Pharmacokinetics. Brequinar is a water-soluble derivative with an oral bioavailability of 90%, and reaches peak concentrations in the plasma within 2-4 h of oral administration (260). The drug circulates in the peripheral blood tightly bound to serum proteins, with a half-life of approximately 15 h in humans and 17 h in rats. Once the drug is absorbed, it is distributed rapidly to peripheral organs, including liver and kidney (261). The areas under the curve (AUCs) for plasma levels of the drug increase linearly with the dose of the drug, and the plasma clearance is 19.2 ± 7.7 mL/min/mm2 (260, 262). The extended half-life and the low plasma clearance allow its administration at an interval of 24-48 h. Brequinar is metabolized in the liver by the P450 cytochrome oxidase system, and is excreted primarily in feces (66%) and, to a lesser extent, in urine (23%).The oral administration of brequinar to rats for 30 days did not affect cyclosporine pharmacokinetics (263). The levels of the compound in the blood can be directly measured using high pressure liquid chromatography (264).The high level of bioavailability, the relative ease with which the compound can be administered, and the prolonged half-lifeare all features of the pharmacokinetics of brequinar that make the compound attractive for use in the clinical setting. Pharmacology. Brequinar inhibits pyrimidine biosynthesis and noncompetitively blocks the activity of the enzyme dihydrooro-tate dehydrogenase (DHODH) (265, 266).

This enzyme is critical for the formation of uridine and cytidine, which are required for the synthesis of DNA and RNA (267). Studies have shown that brequinar sodium also has the ability to inhibit protein tyrosine phosphorylation and src-related protein tyrosine kinases, thereby suggesting that the activity of brequinar sodium may not be solely the result of the inhibition of pyrimidine nucleotide synthesis; inhibition of protein tyrosine phosphorylation may also be involved (268).

Although brequinar displays antitumor effects over a wide range of concentrations, it displays antilymphocyte effects over only a relatively narrow range (22-185. nmol/L) (269).In vitro, brequinar not only inhibits the proliferation of lymphocytes in a mixed lymphocyte culture but also a wide variety of cellular immune responses including alloantigen and mitogen-induced proliferation (270,271).

In vivo, brequinar sodium has been found to be effective in suppressing graft-vs.-host responses and allograft rejections, which can be attributed to the potent inhibitory effect on Tand B-cell-mediated responses. Its efficacy as a primary immunosuppressive agent is evident by the effective inhibition of rejections in several models of vascular allografts in rodents. In rat transplant models, brequinar mono-therapy has been shown to prolong the allo-graft survival of hearts, livers, and kidneys. A dose cf 12 mg/kg brequinar administered three times a week for 30 days prolonged the survival of heart allografts to 45.5 ± 12.26 days, compared with a survival rate of 7.0 ± 0.69 days for untreated controls. The same brequinar protocol produced long-term survival (>230 days) in 12 out of 26 rat recipients of orthotopic liver (272). Furthermore, the oral administration of 4 mg/kg brequinar three times per week prolonged the survival of heterotopic cardiac allografts in nonhuman primates to 20.0 ±21.5 days, compared to a survival rate of 8.0 ±0.5 days for the controls (272).

Brequinar is also capable of suppressing xenograft rejection because it effectively prolonged survival of hamster-to-rat heart xeno-graft. In one experiment, brequinar (3mg/kg/ day for 90 days) prolonged the mean survival time cf hamster heart xenotransplants in LEW recipients (an inbred strain of rats). The survival time was increased to 24.5 ± 42.2 days (from 4.0 ±0.48 days in the control animals); furthermore, four of the hearts continued to beat for more than 90 days (272). The hamster-to-rat heart xenograft is an example of an accelerated xenograft reaction and brequinar is the only agent capable of prolonging survival for this type of xenograft.

One of the most striking features of the immunosuppressive activity of brequinar 'sodium is its ability to synergistically interact with a number of other agents to prevent allograft and xenograft rejection. The combination of brequinar with cyclosporine and/or sirolimus was synergistic, as shown by the median effect analysis (273, 274). In primates, the brequinar-cyclosporine combination was able to prolong graft survival to a significant extent (275). Brequinar in combination with leflunomide or tacrolimus exhibited prolonged graft survival in a heterotopic rat cardiac allotransplantation model (276). Administration of BQR (3 mg/kg) with leflunomide (5 mg/kg) or FK 506 (0.5 mg/kg) exhibited prolonged graft survival in both drug combination groups, with a median survival time of 14 days compared to 5 days for controls. Similarly, brequinar in combination with cyclosporine also inhibited islet xenograft rejection in the pig-to-rat model (277). Thus the drug exhibits a number of characteristics that are considered desirable for inclusion in multidrug anti-rejection protocols.

In a phase I safety and pharmacokinetic study the efficacy of BQR in combination with cyclosporine was examined for the treatment and prophylaxis of rejection in organ transplant patients. The studies were performed in stable renal, hepatic, and cardiac transplant patients receiving cyclosporine and pred-nisone maintenance therapy for immunosup-pression. In all three patient populations, the pharmacokinetics of BQR were characterized by a lower oral clearance (12-19mL/min) than that seen in patients with cancer (approximately 30 mL/min at similar doses) and a long terminal half-life (13-18 h). This slower oral clearance for brequinar could be attributed either to a drug interaction between brequinar and cyclosporine or to altered clearance or metabolic processes in patients with transplants. Steady-state cyclosporine trough levels and the oral clearance of cyclosporine were not affected by brequinar coadministration. Among the three transplant populations, the cardiac transplant patients had lower oral clearance values for brequinar and of cyclosporine; however, the cause of this lower clearance is not yet clear. Safety results indicate that brequinar was well tolerated by this patient population (278). Structure-Activity Relationship. Structure-activity relationship studies of substituted cinchonic acid (279) and of some tetracyclic heterocycles (280) related to brequinar were studied to determine the structural features required for good activity and an optimal pharmacokinetic profile. In the first instance, compounds with various substituents at the 2-, 3-, 4-, and 6-position of the quinoline ring system were synthesized. The compounds were evaluated for their DHODase inhibitory activity as well as in the MLR assay, a standard model of cell-based immunity indicative of potential allograft rejection. The cinchonic acid core was found to be essential for activity, with only small lipophilic, electron-deficient substitution allowed on the benzo ring. The methyl group was optimal at position 3, although bridging with the 2-biphenyl retained potency. Among tetracyclic heterocycles series, a correlation between DHODase and MLR was observed. Compounds with an ethylene bridge or compounds with a thiomethyl-ene moiety represent the best of the tetracy-clic compounds synthesized. Molecular modeling showed the topology of all the tetracyclic ring systems to be similar. The differences in the activity observed with these topo-logically similar compounds have been in some cases attributed to changes in lipohilicity or basicity, or to the projection angle of the pendant aryl ring. Several of the compounds exhibited activity in DHODase and MLR assays comparable to that of brequinar, warranting further investigations for the development of potent and clinically useful immunosuppres-sants. Side Effects. In a phase I safety trial. 45 cancer vatients were administered brequinar by a single daily intravenous infusion for 5 days at a dose range of 36-300 mg/ m2/day; several side effects including transaminase elevations, thrombocytopenia, mu cositis, phlebitis, and dermatitis were recorded (262).Subsequently, in a phase II efficacy study of brequinar, doses as high as 1800 mg/m2 failed to reduce tumor growth in cancer patients (281). Once the adverse side effect appears, the noncompetitive nature of the enzyme inhibition provides the opportunity to reduce or withdraw treatment with the drug with rapid reversal of the drug-related effects.

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