Rational design of novel immunosuppressive drugs: analogues of azathioprine lacking the 6-mercaptopurine substituent retain or have enhanced immunosuppressive effects.

Clinical use of the immunosuppressive drug azathioprine is limited by potentially serious toxic effects related to depression of bone marrow function. The immunosuppressive and toxic properties of azathioprine are regarded as being properties of the cytotoxicity of its metabolite, 6-mercaptopurine (6-MP). However, azathioprine has an immunosuppressive effect additional to that attributable to 6-MP alone, and we propose that this is associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic immunosuppressants by replacing the 6-MP component of azathioprine with nontoxic thiols. We have synthesized and tested in vitro 24 such analogues, with two being further tested in vivo. In the human mixed lymphocyte reaction, virtually all compounds showed some degree of activity, 10 compounds being more active than azathioprine. In vivo, two compounds were more effective than azathioprine at prolonging graft survival in mice. In an oral toxicity study in male CD1 mice at doses equivalent to those at which azathioprine caused severe bone marrow depression both analogues had no toxic effects. Our results show that the immunosuppressive effects and bone marrow toxicity of azathioprine are not a consequence of release of 6-MP alone, and with appropriate modification can be separated, an approach which may lead to less toxic immunosuppressive drugs.

Nucleosides of azathioprine and thiamiprine as antiarthritics.

Azathioprine [Imuran; 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)thio]-1H-purine] is a widely used immunosuppressive and antiarthritic drug. For the sake of comparison, the riboside, the 2'-deoxyriboside, and the arabinoside of azathioprine and its 2-amino congener, thiamiprine, were prepared by an enzymatic method. In vitro, the cytotoxicities of these aglycons and their nucleosides were similar (ED50 = 0.8-2 microM), except for the arabinosides, which were nontoxic (ED50 greater than 100 microM). In vivo, their activities were compared in the rat adjuvant arthritis model. The ribosides and 2'-deoxyribosides were less potent than their corresponding aglycons. The safety indexes of these nucleosides were comparable to those of the corresponding aglycons except for the 2'-deoxyriboside of azathioprine, which had an appreciably lower safety index than did azathioprine. Both arabinosides were inactive and nontoxic. All of the aglycons tested (6-mercaptopurine, azathioprine, 6-thioguanine, and thiamiprine) were of similar potency. However, azathioprine had a more favorable therapeutic index than did 6-mercaptopurine. Similarly, thiamiprine was safer than was 6-thioguanine. In this model, the S-(1-methyl-4-nitro-1H-imidazol-5-yl) moiety imparted greater safety to these thiopurines by decreasing toxicity but not affecting potency.

Determination of 6-thiouric acid in human urine.

A simple and rapid method is described for the determination of 6-thiouric acid in the urine of patients receiving azathioprine (Imuran, Burroughs Wellcome). The technique makes use of anion exchange column chromatography to isolate the 6-thiouric acid which is then measured by its absorption at 356 nm.

Synthesis, spectroscopic and magnetic resonance studies of mercury (II) and methylmercury (II) complexes of azathioprine, a biologically active mercaptopurine derivative.

Synthetic and spectroscopic studies of the Hg(II) and MeHg(II) complexes of azathioprine (AZA), a biologically active 6-mercaptopurine derivative, were undertaken. The altered coordination behavior of AZA with respect to the parent mercaptopurine, with sulfur no longer being the primary donor atom, was confirmed. As concluded by the 1H NMR, 13C NMR, and IR spectroscopic data, Hg(II) binds to the N(9) position of deprotonated AZA, while in the MeHg(II) compound, coordination occurs through the N(3) and N(9) positions of the purine ring. The values of the coupling constants 2J (199Hg-1H), 1J(199Hg-13C) for the MeHg(II) compound further support complexation via nitrogen atoms of the purine. Elemental analyses confirmed the compounds to be Hg(AZA)2 (1) and [(MeHg)2(AZA)](NO3) (2); conductivity measurement values show that 1 is a nonelectrolyte and 2 is a 1:1 electrolyte. Furthermore, the FAB-MS of the compounds confirms direct binding of the metal to the ligand, and in the case of the MeHg(II) compound, the successive loss of one and two MeHg(II) moieties can be clearly observed.

Oxypurine and 6-thiopurine nucleoside triphosphate formation in human erythrocytes.

A variety of oxypurines and 6-thiopurines could be transformed by intact erythrocytes to their nucleoside triphosphate forms when incubations were extended for up to 24 hrs. The specific nucleotide monophosphate kinases which accomplish these reactions in erythrocytes were not identified but their ability to utilize 6-thioIMP, 6-thioXMP and 6-methylthioGMP as substrates, albeit very slowly, is clearly implied by these results. S-methylation of 6-thiopurines was demonstrated in erythrocytes incubated with physiological amounts of methionine-(CH3-3H). 6-Methylthioguanosine triphosphate and 6-methylmercaptopurine riboside triphosphate were formed in micromolar amounts, probably from the corresponding thiopurine nucleotides by methyl transfer from S-adenosylmethionine.

Glutathione transferase activity with a novel substrate mimics the activation of the prodrug azathioprine.

Azathioprine is a prodrug that is widely used clinically as an immunosuppressive agent. The pharmacological action of azathioprine is associated with the release of 6-mercaptopurine by a reaction involving glutathione. This biotransformation of azathioprine is catalyzed by glutathione transferases (GSTs). The nonenzymatic reaction with glutathione is minimal in comparison with the GST-catalyzed process, but azathioprine is still a slow substrate in comparison with the most effective GST substrates. Novel GSTs with higher catalytic efficiency toward azathioprine could be useful in novel therapeutic applications; therefore, directed evolution of GSTs for enhanced activities is desirable. However, screening for variants having higher catalytic activity with azathioprine is a time-consuming process due to the low activity with this substrate. A new chromogenic and faster substrate, 1-methyl-4-nitro-5-(4-nitrophenylthio)-1H-imidazole (NPTI), has been synthesized and characterized by assays with several GSTs. The novel substrate mimicked azathioprine in the reaction with glutathione catalyzed by alpha class GSTs and, therefore, is a valuable surrogate in the screening of large mutant libraries. NPTI may also find use in the elucidation of the exact mechanism of immunosuppression effected by azathioprine where there is evidence that the imidazole moiety of azathioprine, rather than 6-mercaptopurine, is involved.

Determination of plasma azathioprine and 6-mercaptopurine in patients with rheumatoid arthritis treated with oral azathioprine.

Two specific high-performance liquid chromatography methods for determining plasma concentrations of azathioprine and 6-mercaptopurine after oral administration of azathioprine are presented. It was shown that azathioprine is unstable in the blood samples unless immediately cooled in ice water. The 2-amino analog, guaneran, was used as internal standard for azathioprine, which was extracted from plasma with ethylacetate. A Nucleosil C18 column was used for the separation. The detection limit was 6 nM. For quantification of 6-mercaptopurine, 6-thioguanine was used as internal standard. Plasma was deproteinized with HClO4 and the sample was purified on mercurial cellulose. A Beckman ODS column was used and the detection limit was 5 nM. Pharmacokinetic data from two patients are presented. Unchanged azathioprine was seen until 6 h after an oral dose of 32 mg/m2.

Experimental antitumor activity of new azathioprine analogues.

Two newly synthesized azathioprine (AZA) analogues, 6-(1,2-dimethyl-4-nitro-5-imidazolyl)thiopurine (Met-AZA) and 6-(2-methyl-5-nitro-4-imidazolyl)thiopurine (IZO-AZA), were investigated against KB human tumor cells. In 5 transplantable murine tumor models, including sc Sa180, sc Ca755, ip LL and ip leukemias; L1210 and P388 both drugs were found to be antitumor active in all the experiments carried out regardless of dosing regimen or the route of administration. Similar good activity was shown in the KB, ip Sa180, and Ca755 systems and partly against LL as compared to AZA. However, Met-AZA against ip P388 demonstrated therapeutic advantage following qd 1-9 daily dosing, 0.33 log10 tumor cell kill; therapeutic index (TI = ILSmax/ILS 25) = 2, and ILS = 69% in comparison to AZA and IZO-AZA, TI = 1.3, 1.2, and ILS = 31%, 40%, respectively. Met-AZA is comparable to AZA, while seeming to display greater antileukemic activity than AZA.