Immunosuppression

A discussion of immunosuppression for hepatic transplantation must begin with a recapitulation of a few principles. First, although early studies in dogs showed that liver allografts can be vigorously rejected, hepatic allograft rejection is much easier to control than rejection of any other solid organ in all species including man. The explanation for the virtually non-existent hyperacute rejection ,even in the face of performed cytotoxic antibody and the rare occurrence of uncontrollable acute rejection, remains a mystery. Death from acute or chronic rejection is very uncommon, while sepsis accounts for the death of approximately 20percent of patients during the first postoperative year. Therefore , a dynamic balance must be struck between the risk of rejection and infection. The following discussion of immunosuppression will present an overview of the main immunosuppressants employed in transplantation.

Glucocorticoids

Glucocorticoids were the first effective immunosuppressants used in organ transplantation. They were successfully used to reverse a rejection episode in a living related renal transplant recipient who had been immunosuppressed by whole body irradiation. The effect on the immune response was thought to result primarily from an anti-inflammatory action. More recent information suggests that modulation of T-cell activation and monocyte function may also be important. These effects can be caused by inhibition of the "chemical communication of lymphocytes" including interleukin-1 (IL-1) and interleukin-2 (IL-2) production. Steroids also cause neutrophilia which results from increased release of neutrophils from the bone marrow, and prolongation of the half life of neutrophils.  Monocyte differentiation into macrophages is also inhibited by steroids. These agents are used both for maintenance therapy and for treatment of rejection episodes. It is customary to administer 1.5 to 2 mg/kg of solumedrol immediately postoperatively, and to taper the dose to 0.5 to 0.3 mg/kg per day during the first week.  Further changes of steroid doses are made on an individualized basis. Initial and maintenance steroid therapy is reduced in infants and children.

Cytotoxic Agents

These agents include azathioprine (Imuran) and cyclophosphamide (Cytoxan). Azathioprine is well absorbed and rapidly converted to 6-mercaptopurine in the liver. Thereafter it is incorporated intracellularly and competes with the adenosine and guanine in the production of DNA, reducing the synthesis of DNA and RNA in rapidly proliferating cells such as bone marrow. Dosage is 1 to 2 mg/kg/day. Effectiveness and toxicity are monitored by daily white cell counts. Toxicity includes marrow suppression, pruritus, hepatitis and pancreatitis, and predisposition to neoplasia. Less common toxicities are alopecia, stomatitis, and thrombocytopenia.

Cyclophosphamide causes cross linking of DNA strands with subsequent reductions

in cellular metabolic and proliferative capacities. It is usually substituted for azathioprine in instances of azathioprine induced hepatitis. The dosage is 1 to 2 mg/kg. The efficacy of this drug is comparable to azathioprine, but the additional toxicity, particularly hemorrhagic cystitis, limits its general use. The exact mechanism of its immunosuppression has not been explained. The principal limitation of these drugs was the interference with wound healing and myelotoxicity.

Antilymphocyte Globulin (ALG)

ALG has been used clinically both with hepatic and renal allografts to impede the rejection reaction. Antilymphocyte serum was first found to interrupt the immune response and delay graft rejection in 1963. The first clinical trial was reported in 1967 using renal allograft recipients. It has been difficult to assess the efficacy of this agent in clinical transplantation because of difficulties in establishing a uniform dosing schedule that would yield clinical efficacy yet minimize side effects. ALG is prepared from the sera of animals who have been hyperimmunized to human lymphocytes. This is a polyclonal product which contains antibodies to T and B lymphocytes, but also antibodies to non-lymphoid cells. Such polyclonal preparations have antibodies against multiple determinants. Variability in the activity of ALG may differ from lot to lot. Following an injection of ALG there is a profound decrease in the number of T cells and a decrease in the proliferative function of non-specific suppressor cells. Typically 15mg/kg of ALG is administered over four hours, via a central vein. This drug has been used for short term maintenance therapy in patients intolerant to cyclosporine, and has been used for treatment of acute rejection. Side effects include allergic manifestations, thrombocytopenia, increased incidence of infections,leukopenia, serum sickness, and nephritis.

Monoclonal Antibodies

These compounds are prepared from mouse hybridomas which produce antibodies that are specifically directed against certain T lymphocyte population. OKT3 is such a murine monoclonal antibody which interfaces with determinants on human thymocytes and mature T cells. These monoclonal antibodies are produced to the T3 antigen receptor complex. T cells are coated with antibody and therefore rapidly removed from the circulation by the reticular-endothelial system. OKT3 treatment of human renal transplant patients results in rapid clearing of circulating T lymphocytes leading to a markedly immunosuppressed state. Its immunosuppressive effect has been demonstrated not only in renal but also in hepatic allografts recipients. The dosage is 5 mg/d for adults, 2.5 mg/d for children, and the therapy is continued for ten days. Side effects include a febrile response usually 30 to 60 minutes after the initial infusion. Bronchospasm, pulmonary edema, and circulatory collapse have been reported.

Another disadvantage in using this agent is a rebound rejection if maintenance immunosuppression is not reinstituted strongly during the last four days of OKT3 treatment.

Cyclosporine

Cyclosporine is presently the primary immunosuppressive agent used in patients with liver transplants. Cyclosporine is a metabolic by-product of the fungus Tolypocladium inflatum and was isolated by Dreyfuss in 1972. It was first shown by Borel to have immunosuppressive activity. The drug was first released in the United States as an immunosuppressive agent in 1983. It is lipid soluble and available in both an oral and intravenous form.

The purpose of this section is to review the clinical background for using cyclosporine, review the pharmacokinetics of this drug, and outline the rationale for our cyclosporine dosing in the postoperative period.

Mechanism of action: Cyclosporine inhibits T helper lymphocytes which prevents the generation of cytotoxic T lymphocytes. It also inhibits maturation of cytosol T cell precursors by preventing the development of IL-2 receptors. It does not directly inhibit B cells.

Pharmacokinetics: Absorption: Following oral administration the absorption of cyclosporine is slow and incomplete. Peak concentrations in blood or plasma are observed one to eight hours after oral dosing and half-lives range from 0.6 to 2.3 hours. The effect of food on the absorption of cyclosporine may be related to the nature of the diet and the time of the drug administration in relation to intake, but no consistent effects have been noted. Because cyclosporine is fat soluble its absorption is very low in patients with severe liver disease. Another consequence of the fat solubility of this drug is malabsorption in patients following liver transplants with external biliary drainage. Therefore, a significant increase in the trough blood concentration of cyclosporine is noted following T-tube clamping in patients with open t-tubes. The likely mechanism is improved absorption because of increased bile flow into the intestine rather than enterohepatic recirculation of cyclosporine.

Patients with gastrointestinal disease have significant decreases in the oral absorption of cyclosporine. Patients with greater than 500 ml of diarrhea per 72 hours have significantly impaired absorption.

Distribution: Cyclosporine is widely distributed in the body. The uptake by the liver is the highest of the solid organs. The relative amount of cyclosporine that is bound to blood and plasma is dependent on the hematocrit, temperature and drug concentration. In patients with a low hematocrit, there is a greater portion of the drug residing in the plasma. Cyclosporine diffuses from plasma to red cells as temperature drops below 37 degrees Celsius. Since cyclosporine is lipophilic, body fat contains a high concentration of the drug. Cyclosporine does not cross the blood brain barrier except in the diseased states. Abnormal transit across this barrier may causes central nervous system toxicity as manifested by seizures.

Metabolism: Cyclosporine is primarily eliminated by the liver. The cytochrome p-450 system of enzymes is responsible for this biodegradation. The metabolism is affected by liver disease, age of the patient, and other drugs.

Excretion: Renal excretion is a minor pathway of elimination in humans and animals. Less than one percent of the administered dose is excreted unchanged in the urine. The major route of elimination is via the liver and biliary systems. The total amount of cyclosporine excreted in the bile over one dosing interval is proportional to the bile output. The biliary system is also the major excretion route of the metabolites.

Blood levels: Because of cyclosporine's narrow therapeutic window in man, blood level monitoring has have been used in an attempt to optimize immunosuppression and minimize toxicity. The drug is assayed by both high pressure liquid chromatography (HPLC) and radioimmune assay (RIA). The HPLC is more specific for the parent compound, cyclosporine, while the RIA test measures both the parent compound and the metabolites in the serum. Because of the uncertain role that these metabolites play in immunosuppression and drug side effects, and because of the very high level of these metabolites in patients with cholestasis, we rely on the HPLC assay using whole blood as the specimen. A trough level is obtained at a time when the blood or plasma concentration is the lowest. In most cases this is within one hour prior to the next drug dose. We attempt to attain a TDX mono level between 200 and 350ng/dl.

Drug interactions and adverse effects: One of the most important side effects of cyclosporine is dose dependent nephrotoxicity. The most consistent findings are tubular dilation and epithelial cell vacuolation with tubular destruction. Interstitial fibrosis is found in long standing toxicity. Among the mechanisms of nephrotoxicity, reduced renal blood flow appears to the most consistently documented. Some workers have noted that an alpha sympathomimetic effect was ameliorated by renal denervation or by Dibenzalene or prazosin administration. Other mechanisms may include a mediated reduction in the production of renal cortical vasodilator PGI2 relative to vasoconstrictor thromboxane A2.

A number of prescription drugs have been found to interact with cyclosporine to intensify many of its side effects or conversely lower the cyclosporine blood concentration to nontherapeutic ranges. Listed below are the known drugs which adversely interact with cyclosporine.

DRUG	MECHANISM	EFFECT
Ketoconazole	Impaired metabolism	Increased blood conc. and nephrotox
Erythromycin	Impaired metabolism	Increased blood conc. and nephrotox
Cimetidine	Impaired metabolism
Verapamil	Impaired metabolism
Corticosteroids	Impaired metabolism
Dilantin	Induces metabolism	Lower concentr
Rifampicin	Induces metabolism	Lower concentr
Barbiturates	Induces metabolism	Lower concentr
Trimethoprim-sulfa	Decreased levels Potentates nephrotox Increased metabolism	Nephrotoxicity
Amphotericin B		Nephrotoxicity
Aminoglycosides		Nephrotoxicity
Non steroidal infl		Nephrotoxicity

Studies from renal graft recipients have shown that elevated amounts of cyclosporine may cause elevations of serum bilirubin, associated with variable increases in the transaminases. The effects return to normal after the cyclosporine dose is reduced. Other common side effects include: hypertension, tremors, headaches, gum tissue hyperplasia, and increased hair growth.

Cyclosporine and prednisone are the mainstay of contemporary immunosuppressive therapy. Corticosteroid therapy is initiated with 1.5 mg/kg of Solumedrol in the immediate perioperative period, and the dose is slowly tapered to 20 mg/day during the first postoperative week. The dose is further reduced to 15 mg at 3 months and 10 mg/day of prednisone at the end of one year.

Cyclosporine is usually started 24 hours after the transplantation procedure at a dose of 2mg/dose IV twice daily. The dose is adjusted to maintain the serum HPLC level at 150-175 ng/ml. Cyclosporine and steroids may be withheld or reduced in the severely immunocompromised patients who show no signs of acute rejection on biopsy. Oral cyclosporine is begun after the T-tube is clamped and the patient is able to tolerate oral intake. The oral dose is usually 3 times the intravenous dose. We are able to continue cyclosporine in patients with renal dysfunction by using CAVH to eliminate excess water.

Acute rejection usually responds to 2 boluses of methylprednisolone (17.5 mg/kg for each daily bolus). Steroid refractory rejection is treated with ALG or OKT3. We have used azathioprine in patients who could not tolerate cyclosporine, usually secondary to renal impairment. We use approximately 1.0 to 1.5 mg/kg per day.