Azathioprine

Azathioprine (INN,, abbreviated AZA) is an immunosuppressive drug used in organ transplantation and autoimmune diseases and belongs to the chemical class of purine analogues. Synthesized originally as a cancer drug and a prodrug for mercaptopurine in 1957, it has been widely used as an immunosuppressant for more than 50 years.

Azathioprine acts as a prodrug for mercaptopurine, inhibiting an enzyme that is required for the synthesis of DNA. Thus it most strongly affects proliferating cells, such as the T cells and B cells of the immune system.

The main adverse effect of azathioprine is bone marrow suppression, which can be life-threatening, especially in people with a genetic deficiency of the enzyme thiopurine S-methyltransferase. It is also listed by the International Agency for Research on Cancer as a Group 1 carcinogen (carcinogenic to humans).

Azathioprine is produced by a number of manufacturers under different brand names (Azasan by Salix in the U.S., Imuran by GlaxoSmithKline in Canada, the U.S., Australia, Ireland and the United Kingdom, Azamun in Finland and Imurel in Scandinavia and France, among others). It is on the World Health Organization's List of Essential Medicines, a list of the most important medication needed in a basic health system.

Medical uses
Azathioprine is used alone or in combination with other immunosuppressive therapy to prevent rejection following organ transplantation, and to treat an array of autoimmune diseases, including rheumatoid arthritis, pemphigus, systemic lupus erythematosus, Behçet's disease and other forms of vasculitis, autoimmune hepatitis, atopic dermatitis, myasthenia gravis, neuromyelitis optica (Devic's disease), restrictive lung disease, and others. It is also an important therapy and steroid-sparing agent for inflammatory bowel disease (such as Crohn's disease and ulcerative colitis) and for multiple sclerosis, which are immune-mediated as well.

In the United States it is currently approved by the Food and Drug Administration (FDA) for use in kidney transplantation from human donors, and for rheumatoid arthritis. Other uses are off-label.

Transplantation
Azathioprine is used to prevent rejections of kidney or liver allografts, usually in conjunction with other therapies including corticosteroids, other immunosuppressants, and local radiation therapy. The administration protocol starts either at the time of transplantation or within the following two days.

Rheumatoid arthritis
Being a disease-modifying antirheumatic drug (DMARD), azathioprine has been  used for the management of the signs and symptoms of adult rheumatoid arthritis. Nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids may be combined or continued (if they were already in use) with azathioprine, but the combination with other DMARDs is not recommended.

Crohn's disease
Azathioprine has been used in the management of moderately to severely or chronically active Crohn's disease, to maintain clinical remission (absence of disease activity) in corticosteroid-dependent patients, and to provide benefit in people with fistulizing Crohn's disease. The onset of action is slow and it may require several months to achieve clinical response.

Azathioprine treatment is associated with an increased risk of lymphoma, however it is unclear if this is due to the drug or a predisposition related to Crohn's disease. Lower doses of azathioprine are used as a therapy in children with refractory or corticosteroid-dependent Crohn's disease, without causing many side effects.

In Crohn’s disease treatment with azathioprine shortly after diagnosis was no more likely to result in corticosteroid-free remission than standard care or placebo.

Others
Azathioprine is sometimes used in systemic lupus erythematosus patients who require a maintenance dose of 15 mg or higher of prednisone and those who experience recurrent flares.

It is used as an adjuvant in the oral steroid therapy of pemphigus and myasthenia gravis, as a "steroid-sparing" agent for reducing the dose of cortisteroids.

It was shown to be very effective in eczema and atopic dermatitis in researches, even though it is not commonly used. The British National Eczema Society lists it as a third-line treatment for severe to moderate cases of these skin diseases.

It was widely used for the treatment of multiple sclerosis until the first half of the 1990s. Concerns about increased risk of malignancy has led to a decreased use, yet it is still used in maintenance treatment for patients who frequently relapse.

Adverse effects
Nausea and vomiting are common adverse effects, especially at the beginning of a treatment. Such cases are met with taking azathioprine after meals or transient intravenous administration. Side effects that are probably hypersensitivity reactions include dizziness, diarrhea, fatigue, and skin rashes. Hair loss is often seen in transplant patients receiving the drug, but rarely occurs under other indications. Because azathioprine suppresses the bone marrow, patients can develop anaemia and will be more susceptible to infection; regular monitoring of the blood count is recommended during treatment. Acute pancreatitis can also occur, especially in patients with Crohn's disease.

The enzyme thiopurine S-methyltransferase (TPMT) is responsible for various activation and deactivation steps in azathioprine's mechanism of action, and genetic polymorphisms of TPMT leads to decreased methylation and decreased inactivation of 6MP. This can result in dangerous bone marrow suppression, and an assay of TPMT in red blood cells or a TPMT gene test can avoid this complication. TMPT polymorphisms occur with a frequency of about 5%, meaning that about 0.25% of patients have defects on both of their TMPT genes; they are homozygous.

Under FDA rules, like many other immunosuppressants, the use of this drug excludes eligibility for blood donation.

Cancers
Azathioprine is listed as a human carcinogen in the 12th Report on Carcinogens by the National Toxicology Program of U.S. Department of Health and Human Services, asserting that it is "known to be a human carcinogen based on sufficient evidence of carcinogenicity from studies in humans." Since August 2009, the U.S. Food and Drug Administration has required warnings to be placed on packaging with respect to increased risks of certain cancers.

The risks involved seem to be related both to the duration and to the dosage used. People who have previously been treated with an alkylating agent may have an excessive risk of cancers if treated with azathioprine. Epidemiological studies by International Agency for Research on Cancer (IARC) have provided "sufficient" evidence of azathioprine carcinogenicity in humans (Group 1), although the methodology of past studies and the possible underlying mechanisms are questioned.

The various diseases requiring transplantation may in themselves increase the risks of non-Hodgkin's lymphoma, squamous cell carcinomas of the skin, hepatobiliary carcinomas, and mesenchymal tumours to which azathioprine may add additional risks. Those receiving azathioprine for rheumatoid arthritis may have a lower risk than those undergoing transplantation.

Cases of hepatosplenic T-cell lymphoma – a rare type of lymphoma – have been reported in patients treated with azathioprine. The majority occurred in patients with inflammatory bowel disease. Adolescents and young adult males were the majority of cases. They presented with a very aggressive disease course and, with one exception, died of the lymphoma. The FDA has required changes to the labeling to inform users and clinicians of the issue.

Skin cancers
In transplant patients, skin cancer is 50 to 250 times more common than in the general population, and between 60% and 90% of patients are affected 20 years after transplantation. The use of immunosuppressive medication including azathioprine in organ transplantation has been linked to increased rates of developing skin cancer. Azathioprine causes the accumulation of 6-thioguanine (6-TG) in patients' DNA, which might trigger cancer when the patient is later exposed to ultraviolet light. Patients taking azathioprine were found to be abnormally sensitive to UVA light.

Drug interactions
Other purine analogues such as allopurinol inhibit xanthine oxidase, the enzyme that breaks down azathioprine, thus increasing the toxicity of azathioprine. On the other hand, low doses of allopurinol have been shown to safely enhance the efficacy of azathioprine, especially in inflammatory bowel disease non-responders. This may still lead to lower lymphocyte counts and higher rates of infection, therefore the combination requires careful monitoring.

Azathioprine decreases the effects of the anticoagulant warfarin and of non-depolarizing muscle relaxants, but increases the effect of depolarizing muscle relaxants. It can also interfere with niacin (vitamin B3), resulting in at least one case to pellagra and fatal medullary aplasia. It has also been reported to cause vitamin B12 deficiency.

Overdose
Large single doses are generally well tolerated; a patient who took 7.5 g azathioprine (150 tablets) at once showed no relevant symptoms apart from vomiting, slightly decreased white blood cell count and marginal changes in liver function parameters. Main symptoms of long-term overdosing are infections of unclear origin, mouth ulcers and spontaneous bleeding, all of which are consequences of the bone marrow suppression.

Pregnancy and breastfeeding
Azathioprine can cause birth defects. A 2003 population-based study in Denmark showed that the use of azathioprine and related mercaptopurine resulted in a seven-fold incidence of fetal abnormalities as well as a 20-fold increase in miscarriage. Birth defects in a child whose father was taking azathioprine have also been reported. Although no adequate and well-controlled studies have taken place in humans, when given to animals in doses equivalent to human dosages, teratogenesis was observed. Transplant patients already on this drug should not discontinue on becoming pregnant. This contrasts with the later-developed drugs tacrolimus and mycophenolate, which are contraindicated during pregnancy.

Traditionally, as for all cytotoxic drugs, the manufacturer advises not to breastfeed whilst taking azathioprine. However, the "Lactation Risk Category" reported by Thomas Hale in his book "Medications and Mothers' Milk" lists azathioprine as "L3", termed "moderately safe".

Pharmacokinetics
Azathioprine is absorbed from the gut to about 88%. Bioavailability varies greatly between individual patients, between 30 and 90%, because the drug is partly inactivated in the liver. Highest blood plasma concentrations, counting not only the drug itself but also its metabolites, are reached after one to two hours; and the average plasma half-life is 26 to 80 minutes for azathioprine and three to five hours for drug plus metabolites. 20 to 30% are bound to plasma proteins while circulating in the bloodstream.

Azathioprine is a prodrug, a substance that is not an active drug itself but is activated in the body. This happens in several steps; at first it is slowly and almost completely converted to 6-mercaptopurine (6-MP) by reductive cleavage of the thioether (–S–). This is mediated by glutathione and similar compounds in the intestinal wall, the liver and on red blood cells, without the aid of enzymes. 6-MP is metabolized analogously to natural purines, giving thioguanosine triphosphate (TGTP) and thio-deoxyguanosine triphosphate (TdGTP) via thioinosine monophosphate (TIMP) and several further intermediates. On a second path, the sulfur atom of 6-MP and TIMP is methylated. The end products of azathioprine metabolism are thiouric acid (38%) and various methylated and hydroxylated purines, which are excreted via the urine.

Mechanism of action
The purine molecule is the framework for two of the four bases that occur in DNA, adenine and guanine. Consequently, blocking the synthesis of purine also hinders DNA synthesis and thus inhibits the proliferation of cells, especially fast-growing cells without a method of nucleotide salvage ("recycling"), such as lymphocytes. Two types of lymphocytes, T cells and B cells, are particularly affected by the inhibition of purine synthesis.

Azathioprine's active metabolite methyl-thioinosine monophosphate (MeTIMP) is a purine synthesis inhibitor that works by blocking the enzyme amidophosphoribosyltransferase, possibly among others. Additional mechanisms, mediated by other metabolites, have been discovered several decades after the drug's introduction, mainly in the 1990s and 2000s: Thioguanosine triphosphate (TGTP) is incorporated into RNA, compromising its functionality. It also interacts with the GTP-binding protein Rac1, blocking upregulation of the protein Bcl-xL and thus sending activated T cells into apoptosis (a kind of programmed cell death). The closely related thio-deoxyguanosine triphosphate (TdGTP) is built into DNA. Thioinosinic acid impedes later steps of DNA synthesis via enzymes such as adenylosuccinate synthase and IMP dehydrogenase. Moreover, azathioprine blocks the downstream effects of CD28 costimulation, a process required for T cell activation. In vivo data indicate inflammatory bowel disease patients treated with azathioprine have more mononuclear cells that have undergone apoptosis than untreated controls, indicating this mechanism may be responsible for the in vivo response to the drug in this disease.

Physical and chemical properties
Azathioprine is a thiopurine linked to a second heterocycle (an imidazole derivative) via a thioether. It is a pale yellow solid with a slightly bitter taste and a melting point of 238–245 °C. It is practically insoluble in water and only slightly soluble in lipophilic solvents such as chloroform, ethanol and diethylether. It dissolves in alkaline aqueous solutions, where it hydrolyzes to 6-mercaptopurine.

Azathioprine is synthesized from 5-chloro-1-methyl-4-nitro-1H-imidazole and 6-mercaptopurine in dimethyl sulfoxide (DMSO). The synthesis of the former starts with an amide from methylamine and diethyl oxalate, which is then cyclizised and chlorinated with phosphorus pentachloride; the nitro group is introduced with nitric and sulfuric acid.



History
Azathioprine was synthesized by George Herbert Hitchings and Gertrude Elion in 1957 (named BW 57-322) to produce 6-mercaptopurine (6-MP) in a metabolically active but masked form, and at first used as a chemotherapy drug.

Robert Schwartz investigated the effect of 6-MP on the immune response in 1958 and discovered that it profoundly suppresses the formation of antibodies when given to rabbits together with antigens. Following the work done by Sir Peter Medawar and Gertrude Elion in discovering the immunological basis of rejection of transplanted tissues and organs, and Schwartz's researches on 6-MP, Sir Roy Calne, the British pioneer in transplantation, introduced 6-MP as an experimental immunosuppressant for kidney and heart transplants. When Calne asked Elion for related compounds to investigate, she suggested azathioprine, which was subsequently found out to be superior (as effective and less toxic to the bone marrow) by Calne. On 5 April 1962, with regimens consisting of azathioprine and prednisone, the transplantation of kidneys to unrelated recipients (allotransplantation) was successful for the first time. For many years, this kind of dual therapy with azathioprine and glucocorticoids was the standard antirejection regimen, until ciclosporin was introduced into clinical practice (by Calne as well) in 1978.

Ciclosporin has now replaced some of the azathioprine use due to a longer survival time, especially in heart-related transplantations. Moreover, despite being considerably more expensive, mycophenolate mofetil is also increasingly being used in place of azathioprine in organ transplantation, as it is associated with less bone marrow suppression, fewer opportunistic infections, and a lower incidence of acute rejection.