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.

Care of asthmatics on discharge from hospital: a hospital audit.

The objective of this study was to audit the care of patients after discharge from hospital, following admission for acute severe asthma, using the British Thoracic Society guidelines (1) as a standard. Discharge prescriptions and questionnaires sent to patients at home were analyzed for 51 patients who had been admitted to a teaching hospital with acute severe asthma. Main outcome measures were Peak flow measurements, written instructions, prescription of steroids, and outpatient follow-up. Of the 34 patients responding to the questionnaires, 15 (44%) had no peak flow meter, 23 (68%) had no written instructions, 13 (38%) had no supply of oral steroids at home, and 32 (94%) were prescribed a beta-agonist regularly of whom 12 (35%) were not on an inhaled steroid. Four (9%) patients were not followed up as outpatients; appointments ranged from 2 to 56 days following discharge. In over 60% of asthmatic patients discharged from hospital the guidelines recommended by the British Thoracic Society were not followed. The method used is an inexpensive, efficient way of auditing hospital practice.

Application of new fluorescent thiol reagent to diagnosis of homocystinuria.

A new fluorescent thiol reagent, dansyl-aminophenylmercuric acetate (DAPMA), was applied to the diagnosis of homocystinuria, a disorder which can be associated with vascular disease at an early age. DAPMA was added to urine containing metabisulphite and the resulting fluorescent derivatives were extracted on a cyclohexyl silica column and separated by thin-layer chromatography. 102 coded samples were tested. The derivative of homocysteine was easily identified in samples from 4 children with homocystinuria but was absent from all samples from normal subjects and patients with unrelated disorders. Other thiols (cysteine, acetylcysteine, mercaptolactate, thiosulphate, and thiocyanate) were also identified in urine from healthy fasting subjects.

Blue rubber bleb naevus disease: an uncommon cause of gastrointestinal tract bleeding.

A 23 year old woman presented with facial pain, a right parotid tumour and iron deficiency anaemia. She had several cutaneous venous swellings and tumours with a similar appearance were found in the large bowel. Histological examination of the parotid tumour and angiography of the skin and gut lesions confirmed that they were venous in origin. The aetiology, classification, and complications of disorders of the venous system and the importance of using a tourniquet to examine the peripheral veins is discussed.

Adenine phosphoribosyltransferase deficiency: a simple diagnostic test.

1. A simple method for diagnosing adenine phosphoribosyltransferase (APRT) deficiency using urine is described. 2. T.l.c. of 1 microliter urine from a child with APRT deficiency was performed and adenine was easily detected by its brilliant blue phosphorescence at liquid nitrogen temperature. 3. Four physicochemical characteristics of adenine were recorded: RF value, and the colour, decay time and pH sensitivity of phosphorescence. 4. Adenine was not detected in the urine of 116 subjects used as controls. These included healthy individuals and patients with inherited metabolic disorders, diseases of purine metabolism and of the kidney and urinary tract. Some of them were taking a variety of drugs including purine derivatives. 5. The test correctly diagnosed three cases of APRT deficiency out of 10 urine samples tested blind.

Metabolism of azathioprine to 6-thioguanine nucleotides in patients with pemphigus vulgaris.

Azathioprine metabolism, to red cell 6-thioguanine nucleotides (6TGN), was studied in four patients with pemphigus vulgaris. Throughout treatment blood samples were taken for red cell 6TGN assay and differential white cell counts. Metabolite steady-state occurred in three patients in 2, 2 and 4 months respectively. In the fourth patient red cell 6TGN concentrations increased slowly over 3 years. There was a significant negative correlation between 6TGN concentrations and the white blood cell count (rs = -0.92, P less than 0.0005) in this patient.

Does 2-mercaptoethane sulphonate (mesna) prevent cyclophosphamide and azathioprine induced immunosuppression? In vitro studies.

The effect of 2-mercaptoethane sulphonate (mesna) on the inhibition of the human MLR by 4-hydroxycyclophosphamide or azathioprine was studied. 4-Hydroxycyclophosphamide (34 microM) completely inhibited the MLR and this inhibition was unaffected by 122 microM of 2-mercaptoethane sulphonate or its disulphide. At high concentrations (mM) 2-mercaptoethane sulphonate inhibited the MLR reaching 85% at 24 mM. 2-Mercaptoethane sulphonate (15-122 microM) had no effect on azathioprine (36 microM) inhibition. The results of these in vitro studies suggest that 2-mercaptoethane sulphonate does not interfere with cyclophosphamide or azathioprine induced suppression of cellular immunity.

The effect of folate supplements on 6-mercaptopurine remission maintenance therapy in childhood leukaemia.

The effect of folic acid supplements on 6-mercaptopurine remission maintenance therapy in lymphoblastic leukaemia (ALL) was investigated in a retrospective longitudinal study of 10 children. Red cell concentrations of 6-thioguanine nucleotide, a cytotoxic metabolite of 6-mercaptopurine, were measured and the peripheral neutrophil count was used as an index of myelosuppression. During the control period of the study there were significant correlations between 6-mercaptopurine dose and 6-thioguanine nucleotide concentration (rs = 0.59, P less than 0.0005) and between 6-thioguanine nucleotide concentration and the peripheral neutrophil count at 14 days (rs = 0.58, P less than 0.0005). These relationships were absent when the same children were subsequently taking folate supplements. Also when taking folate supplements the children tolerated significantly more 6-mercaptopurine (P less than 0.005) for a significantly longer time (P less than 0.005) before neutropenia developed. There was no significant difference in red cell 6-thioguanine nucleotide concentration in the absence and presence of folate supplements. These findings suggest that folate supplements may interfere with remission maintenance therapy in ALL.

Skin cancer in renal transplant recipients is associated with increased concentrations of 6-thioguanine nucleotide in red blood cells.

Of 108 renal transplant recipients (53 men and 55 women) treated with azathioprine (0.8-2.9 mg/kg/day) and prednisolone (10 mg daily), 10 men had actinic keratoses, and five of these had squamous cell carcinoma, on light-exposed areas of skin. The time from transplantation to diagnosis of these skin lesions varied from 1.2 to 9.0 (mean 5.1) years. The concentration of the active azathioprine metabolite 6-thioguanine nucleotide was 120-425 (mean 276) pmol per 8 X 10(8) red blood cells in the transplant patients who developed skin lesions and 54-203 (mean 130) pmol per 8 X 10(8) red blood cells in a matched control group of renal transplant recipients. This difference was statistically significant (P = 0.005). There was no statistically significant difference between patients and controls in azathioprine dosage, clinical features of immunosuppression, sunlight exposure or infection with human papilloma virus. The association of raised 6-thioguanine nucleotide concentrations in red blood cells with actinic keratoses and malignant skin tumours in these patients supports chemical carcinogenesis as a possible cause.

Azathioprine suppresses the mixed lymphocyte reaction of patients with Lesch-Nyhan syndrome.

The mixed lymphocyte reaction of Lesch-Nyhan patients (HGPRT deficient) was used to study the immunosuppressive effects of azathioprine and 6-mercaptopurine (6-MP). Mitogen stimulated lymphocytes of these patients are highly resistant to azathioprine and 6-MP. When both stimulator and responder lymphocytes in the MLR were HGPRT deficient, azathioprine (36 microM) was much more inhibitory than 6-MP (100 microM). Azathioprine produced inhibition of 98.2% and 78.5% compared with the values of 63.9% and 30.6% for 6-MP. The difference in inhibitory activity between azathioprine and 6-MP was reduced when normal stimulator lymphocytes were cultured with HGPRT deficient responder lymphocytes in the MLR. These results provide very strong evidence that the nucleotide metabolites of azathioprine and 6-MP are unnecessary for immunosuppression. They also suggest that azathioprine and 6-MP interfere with antigenic triggering of the MLR.

Effect of interleukin-2 on the inhibition of the human mixed lymphocyte reaction (MLR) by azathioprine.

The effect of interleukin-2 on the response of the human MLR to azathioprine was studied. Azathioprine (36 microM) inhibited the MLR by 86% and interleukin-2 (25%) stimulated the MLR by 99%. Only partial relief (16%) of azathioprine (36 microM) inhibition was found in the interleukin-2 treated MLR compared with azathioprine treatment alone. However, [3H]-thymidine incorporation by azathioprine (36 microM) + interleukin-2 treated MLR was 4.3 times greater than that exposed to azathioprine alone. As the residual number of immunocompetent cells after drug exposure is pathologically more important than the fraction suppressed, this result may be of clinical relevance. These results show that the major mode of action of azathioprine is not the inhibition of interleukin-2 production.

High-performance liquid chromatographic assay of 6-methylthioguanine, a major metabolite of 6-thioguanine, in urine.

A flow-fluorimetric high-performance liquid chromatographic assay for 6-methylthioguanine in urine has been developed. This compound is a major catabolite of 6-thioguanine, an important drug in cancer chemotherapy. The metabolite was extracted from alkaline urine with ethyl acetate which was injected onto a reversed-phase high-performance liquid chromatographic system for separation and detection. The method is simple, rapid and sensitive to below 500 ng ml-1 which is below the levels encountered following a therapeutic dose of 6-thioguanine. Another metabolite was chromatographically separated from 6-methylthioguanine and partially characterised.

Strength of the human mixed lymphocyte reaction (MLR) and its suppression by azathioprine or 6-mercaptopurine.

Azathioprine (36 microM) had a significantly (P less than 0.00003) greater inhibitory effect on human MLR responses below 20,000 counts/min than on responses above 20,000 counts/min. In contrast, 6-mercaptopurine (100 microM) had a similar suppressive effect on MLR responses below and above 20,000 counts/min suggesting it has a different mode of action to azathioprine.

Lack of inhibition of purine nucleoside phosphorylase and adenosine deaminase in patients treated with azathioprine.

Deficiency of the purine salvage enzymes purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) are known causes of immunodeficiency. Evidence for inhibition of these enzymes was sought in 16 patients on azathioprine therapy by testing for deoxyguanosine (PNP deficiency) and deoxyadenosine (ADA deficiency) in urine using a novel phosphorescence method. These abnormal nucleosides were not found in urine of azathioprine treated patients or in 30 normal controls but were easily detected in urine from proven cases of PNP and ADA deficiency suggesting lack of in vivo inhibition of PNP and ADA by azathioprine.