Immunosuppression

Immunosuppression is a phenomenon that is marked by reduction in efficacy or activation of immune response. Immunosuppression could be induced by immunsuppressants drugs or could be as a result of any other condition like HIV, chemotherapy etc.

Immunosuppression regime is followed when a person has undergone organ transplant or to treat autoimmune diseases like myasthenia gravis, systemic lupus erythematosus and sometimes also used to treat non-autoimmune inflammatory disease like allergic asthama. Due to difference in human leucocyte antigen haplotypes body between donor and recepient, immune system attacks the new organ with leucocytes, so in order to prevent rejection immunosuppressants are given. Cortisone was 1st immunosuppressant to be discovered.

As majority of immunosuppressive drugs are non-selective in their action, they show a wide array of toxicity or side effects. Some generally noticed side effects are hypertension, dyslipidemia, hyperglycemia, stomach ulcers, liver and kindney injury (Gummert et al., 1999).

Immunosuppressants also exhibits drug-drug interaction and affect the metabolism and action of other drugs.

Cyclosporine

Introduction

Cyclosporine (abbreviated as CsA) is a neutral, lipophilic, cyclic undecapeptide which was isolated from fungus Tolypocladium inflatum gams (Rezzani, 2004). Cyclosporine’s imunnosupressive properties were discovered by Novartis in 1972. Cyclosporine was initially approved in 1983 to be used as a preventive measure in organ transplantation (Starzl et al., 1981).

Fig . showing molecular structure of Cyclosporine(taken from Zijlstra, et al.,2007).

Now immunosuppressive properties of cyclosporine are being used to treat psoriasis, severe atopic dermatitis, pyoderma gangrenosum, chronic autoimmune urticaria, uveitis, primary biliary cirrhosis, nephrotic syndrome and occasionally used in rheumatoid arthritis and related diseases.

Cyclosporine immunosuppressive action is by inhibition of activation of T-cell, which will be discussed in detail later in the essay. CsA has also got anti-fungal, anti-parasitic and anti-inflammatory properties.

Cyclosporine also affects mitochondria. In low concentrations, Cyclosporine prevents the mitochondrial permeability transition pore (MPTP) from opening, thus inhibiting cytochrome c release, thereby preventing apoptosis. Exact mechanism is unknown. But in high concentration CsA induces apoptosis by forming MPTP, releasing cytochrome c and causing activation of caspases which causes DNA degradation.

Due to several adverse drug reactions associated with cyclosporine it is used as immuno-modulator, which will be discussed later. Cyclosporine is insoluble in water so suspension and emulsions are used for oral administration and for injection.

Pharmacology

T and B cell activation in response to an antigen is a complex process. It involves binding of antigen to lymphocyte receptor with co-stimulatory signals (E.g. B7) provided by surface receptors such as CD28. Binding of antigen to lymphocyte results in activation of signaling pathway which causes lymphocyte proliferation and cytokines release.

Showing mechanism of action of cyclosporine (image taken from Expert review, 2000)

Fig A showing the cascade of molecular events that occurs when T cell receptor gets the signal. In T cell cytoplasm, a series of tyrosine kinase are activated which phosphorylated tyrosine residues on CD-3 and other tyrosine kinase which will further initiate downstream signaling pathway. Several important pathways are activated like Phospholipase C which hydrolyses phosho-inositol and releases IP3. IP3 binds to its receptor and causes release of calcium from Endoplasmic reticulum. Increased concentration of calcium inside the cell causes the activation of Calcineurin, which in turn dephosphorylates nuclear factor activated T cell proteins (NFAT) which translocates to nucleus and binds to IL-2 promoter and causes transcription of IL-2 genes.

Signaling pathways results in release of calcium from calcium channels which activate calmodulin dependent serine phosphate (Calcineurin). Activated calcineurin dephosphorylates its substrates like nuclear factor activated T cell proteins (NFAT). This results in unmasking of nuclear import signal and causes the translocation of NFAT to nucleus. NFAT combined with AP1 is required to activate promoters such as interleukin (IL-2) for T cell activation.

Once inside the cytoplasm, cyclosporine (CsA) binds to immunophilin known as cyclophylin. CsA- immunophilin complex binds to calcineurin and blocks its function, which has a serine/threonine phosphatase activity.

Showing mechanism of action of cyclosporine (adapted from Expert review, 2000)

Showing when T cell receptor gets signal in the presence of Cyclosporine. Normal downstream signaling occurs. Cyclosporine inside the T cell binds to cyclophilin, this complex then interacts with calcineurin and

prevents its activation by calcium. Thereby downstream process of dephosphorylation of NFAT is blocked and it NFAT can no longer enter nucleus. Preventing the production of IL-2 mRNA.

Since Calcineurin is blocked it fails to dephosphorylate the NFAT and thereby transport of NFAT to nucleus is prevented. Hence IL-2 genes could not be transcribed and T cell activation is prevented.

Drug-drug interactionCalcium channel antagonists like nifedipine, verampamil and diltiazepam increases the CsA concentration in blood and CsA metabolities reach 5 times higher concentration in plasma than without calcium channel antagonists. So, concomitant use of other medicine should be done very carefully. Phramacokinetic studies have shown that calcium channel antagonists like diltiazem increase the terminal hal life of CsA by 2 hours.

Since CsA is extensively metabolized in liver by cytochrome P450 (drug metabolizing enzymes) so drugs which are inducers of CYP enzymes can reduce the concentration of CsA in plasma and can lead to increased requirement of CsA and in unmonitored situation can lead to organ rejection.

On the other hand when CYP inducers are withdrawn without appropriate reduction in dose can lead to elevated levels of CsA in the blood and causes adverse drug reactions.

As CsA is a substrate of CYP450 3A, it acts as competitive inhibitor of other drugs that are metabolized by CYP 3A enzymes. In vivo studies have shown that CsA interferes with the metabolism of prednisolone and increases the risk of inflammation in transplantation patients.

Inducers and inibitors of P450 like Rifampacin and Ketoconazole changes the bioavailability of the CsA which could not be predicted even on considering the phenomenon of induction and inhibition. This suggests that significant amount of CsA being metabolized by intestines limiting the amount of CsA reaching the systemic circulation.

So, it is evident that combination of CsA with other drugs that could interact with CsA metabolism or with those drugs with whom CsA can compete can result in serious adverse effects.

Adverse drug reactions

CsA has toxic effects on numerous organ systems that comprises of immune system, renal, hepatic and nervous system. However CsA is still being used extensively with therapeutic drug monitoring which involves adjustment of dose or withdrawal of the drug.

Immunological complications

Transplant patients on CsA treatment have lower risk of getting bacterial and fungal infections, this fact can be associated with lower requirement of steroid. However, these patients have increased risk of getting viral infections such as herpes simplex, cytomegalovirus etc.

Renal complications

Renal dysfunction is most commonly observed adverse effect of CsA treatment. CsA causes acute and chronic toxicity of kidney.Acute use of CsA causes afferent glomeruloarteriolar constriction, decreased glomerular filtration rate, alteration in solute transport.

Chronic use of CsA inhibits the renin-angeotensin-aldosterone axis; activates profibrotic genes, maintains intrarenal ischemia, which leads to cycles of injury and repair in the kidney.

Hepatic complications

Since CsA undergoes extensive hepatic metabolism, transplantation patients on CsA have been observed to suffer from impaired hepatic function. Commonly bilirubin or aminotransferases levels were found to be elevated which come to normal with reduction in CsA dose.

Neurological complications

50% of the patients on CsA suffers from neurological conditions induced by CsA. Commonly observed neutoxicity is seizures, they are due to retention of fluid in the body, hypertension, high dose steroids, graft dysfunction and demyelination of neurons. One of the possible reasons behind CsA associated seizures is hypomagnesemia. Hypercholesterolemia can also be linked to seizures. Symptoms of neurotoxicity include tremor, neuralgia paresthesia, confusion, ataxia, cortical blindness and occipital seizures.

Hypertensive complications

High incidence of hypertension is reported in patients on CsA treatment, it causes hypertension by increasing the systemic vascular resistance. Commonly observed symptoms were upset circadian rhythm, nocturnal headaches, frequent nocturnal urination and elevated blood pressure at night. Elevated BP causes accelerated hypertension with retinal hemorrhage. Patients exhibits left ventricular hypertrophy, lacunar stroke and microalbuminuria.

Hypertension induced by CsA can cause injury to blood vessels, hemolysis of vessels and sometimes even seizures.

OTK3 orthoclone:

Otk3 is also known as Muromonab, it is a murine CD3 monoclonal antibody to the T3 antigen of human T cells which functions as an immunosuppressant. It is a biologically purified antibody with IgG2a immunoglobulin with heavy chain of approximately 5000 daltons and light chain of 25000 daltons (rx.med 2004).

The proper name Muromonab is derived from descriptive term murine monoclonal antibody. The CD3 designation identifies specificity of the antibody as the cell differentiation.

Orthoclone OKT3 is a very powerful immunosuppressive drug marketed by Laboratory Cilag and the first monoclonal murine antibody to become available for therapy in humans. It is indicated in acute allograft rejection treatment and its side-effects are strongly linked with its mechanism of action. ORT/OKT3 is an Ig2a immunoglobulin produced by hybridoma technique that recognizes, binds and blocks the CD3 complex of the T-cell receptor.

Two types of side-effects may occur that are either the consequences of over immunosuppression or activation of immune system, since ORT/OKT3 behaves as a foreign

antigen.

It recognizes the cells of mature T-lymphocyte populations that express theCD3 antigen and is effective in reversing acute renal, hepatic, cardiac and combined kidney-pancreas transplant rejection episodes. It has also been shown to be effective in the treatment of rejections resistant to conventional treatment.

Pharmacology:

Used in organ transplant prophylaxis, Orthoclone OKT3 (Ortho Biotech) or OKT-3 binds specifically to the CD-3 complex, which is involved in antigen recognition and cell stimulation, on the surface of T lymphocytes. Immediately after administration CD-3-positive T lymphocytes are abruptly removed from circulation. It has been effective in reversing corticosteroid-resistant acute rejection in renal, liver, and cardiac transplant recipients.

Mechanism of action:

T cell recognizes a substance as foreign and produces a cytotoxic effect when it binds to MHC different from host. T cell recognizes antigen as self or own by its recognition sites. If T cell recognizes cell as non-self, it causes proliferation of T cells and produces cytotoxic response towards antigen.

Terminal variable region of Ti complex of T cell is involved in recognition of foreign antigen. Recognition of antigen as foreign results in the activation of CD3 complex, which causes multiplication of the signal and production of effector T cells. OKT3 binds specifically to CD3 complex and prevents the induction of CD3. Thus T cell proliferation and cell mediated lympholysis is blocked.

OKT3 is excessively powerful immunosuppressive drug which has wide array of adverse reaction, discussed later. Toxicity:

Under in vitro conditions, OKT3 has initially stimulating effect on the T cells on binding to CD3 complex. It causes release of lymphokines such as TNF, interleukin 2, γ-interferon due to activation of T cells. Since OKT3 is a murine monoclonal antibody, host immune system gets immunized against antibody and produces two types of anti-OKT3 CD3 antibody, anti-mouse isotype and anti-idiotype. Anti-idiotypic antibody blocks the binding of OKT3 and nullifies its effect. In some patients increase in the concentration of IgE is also observed which causes anaphylactic reactions.

patients receiving Muromonab CD3 should be managed in facilities for cardio pulmonary restiscitation as this drug is known to have toxic effect on heart muscles.

Patient with fluid overload develop sever pulmonary oedema upon treatment with Muromonab, patients had developed cytokine release syndrome apart from cardio toxicity.

Cytokine release syndrome : it is temporally associated with administration of the first few doses of Muromonab which has attributed to release of cytokines by activated lymphocyte or monocyte, this clinical syndrome has ranged from a more frequent mild flu like illness, patients have also been reported with serious shock like reaction as cardiovascular and central nervous system manifestation.( Goldman et.al 1991).

Common clinical manifestation of the cytokine release syndrome may include fever, chill, headache, tremor, vomiting, diarrhoea and abdominal pain.

Severe pulmonary oedema has also been reported, people suffering with this involved increased pulmonary vascular permeability and reduced left ventricle compliance.

OKT3 has been suspected to increase the coagulation and causes thrombosis leading to organ rejection. Apart from being a potent immunosuppressive agent OKT3 also posses a risk of post transplantation malignancy

Discussion:

In case of Immunosuppression, when we compare biologic with small molecule drug although biologic (OKT3) drug is more potent but has narrow therapeutic window limiting its use. OKT3 and CsA both have adverse effect on a number of organ systems, but we need to see which is more effective. While comparing both the drug it was clear that OKT3 has problem in delivery. As it is a monoclonal antibody, it cannot be taken orally and had to be injected intravenously

and as it is murine antibody host gets sensitized to the antibody and with few days produces anti-OKT3 CD3 antibody which prevents it from binding to CD3 and makes it inefficient. Another serious complication with OKT3 is it causes excessive stimulation of immune system in starting doses which leads to release of lymphokines such as TNF, interleukin 2 and γ-interferon. Cytokine release causes systemic inflammatory response, hypotension, fever and pulmonary edema. These conditions are life-threatening. Since biologic drugs have longer half-life than small molecule drug, flushing biologic drug out of the body is very difficult.

On the other hand, CsA is small molecule which undergoes extensive hepatic metabolism and not suitable for concomitant use with other drugs. As drugs which modulates the activity of CYP enzymes affects the bioavailability of CsA. It has a shorter half-life of 17-24 hours. Removing CsA in case of overdose is comparatively easy by introducing an inducer of CYP enzyme which will lower plasma concentration of CsA. When we compare the adverse effects both drugs have on organ system, OKT3 is better than CsA as due to several reasons. Major side effect of OKT3 is cytokine release syndrome which could be overcome by injecting bolus dose of corticosteroid 1 hour before OKT3 administration; fever could be controlled by administering acetaminophen 1 hour before administering OKT3. Other severe side effects could be controlled by administering anti-histamines I.V before starting OKT3, OKT3 is given in slow infusion rather than bolus dose to control side effects. Furosamide is used to control pulmonary edema. Whereas if we see adverse effects of CsA, there exists no combination regime to control or to reduce the wide spread toxic effect. But still CsA is used widely across the globe because it is cheap and easy to administer.

But as the drug discovery has progressed, a new form of cyclosporine has been developed, Cyclosporin G which has less nephrotoxicity than conventionally used cyclosporine.