Calcineurin Inhibitors

Cyclosporine

Cyclosporine was first isolated from two strains of imperfect fungi ( Cylindrocarpon lucidum Booth and Trichoderma polysporum Rifai) from soil samples by the Department of Microbiology at Sandoz (Basel, Switzerland) as an antifungal agent of limited activity. Borel and colleagues demonstrated its potent immunosuppressive activity in a variety of in vitro and in vivo experiments. Interestingly, it nearly never made it into clinical trials, as the company saw little point in developing it for transplantation, citing a small market and limited financial return. Calne, then professor of surgery at the University of Cambridge, succeeded in convincing Sandoz to develop the agent for transplantation. It was first used clinically in England in the late 1970s by Calne and his associates in Cambridge. Initially, it was used with other drugs, such as prednisolone or Asta 036.5122 (cytimum , an analog of cyclophosphamide). Cyclosporine revolutionized the field of transplantation, improving outcomes in renal transplantation, making it possible for routine liver and heart transplantation to be performed, and allowing the first clinical trials of pancreas and lung transplants.

Tacrolimus

Tacrolimus (FK506, Prograf) was isolated in 1984 from the fermentation broth of Streptomyces tsukubaensis, a soil organism found at the foot of Mount Tsukuba near Tokyo. This compound was developed by researchers at the Chiba University of Japan. In the first clinical (rescue) trial, tacrolimus was administered to patients who were taking standard immunosuppressive therapy but who faced retransplantation because of ongoing organ rejection, or who had undesirable drug toxicities. The initial clinical trial of tacrolimus as a primary immunosuppressive agent for the prophylaxis of rejection in liver transplant recipients began in the spring of 1990 at the University of Pittsburgh. This work eventually led to multicenter randomized trials in liver and kidney transplantation. Patients treated with tacrolimus had significantly fewer and less severe episodes of acute rejection than did patients given cyclosporine therapy. The phase III trials leading to US Food and Drug Administration (FDA) approval of tacrolimus (in 1994) were conducted first in liver rather than in kidney transplant recipients, in contrast to other immunosuppressive agents. Subsequent clinical trials in kidney transplantation led to FDA approval for kidney transplantation in 1997. Tacrolimus has also shown efficacy as a rescue agent and as a primary maintenance immunosuppressive agent in heart, lung, pancreas, and small-bowel transplantation, and was approved for heart transplantation in 2006.

Cyclosporine and tacrolimus are the current mainstream maintenance immunosuppressive medications used, with a shift toward progressively higher utilization of tacrolimus-based regimens over the past 15 years. In 2013 more than 90% of all new adult and 96% of all new pediatric kidney transplant recipients in the US were receiving tacrolimus as maintenance immunosuppressive therapy before discharge. This transition was based on the outcomes of a number of prospective, randomized trials demonstrating the superior efficacy of various tacrolimus-based regimens compared with cyclosporine-based regimens.

Mechanism of Action

Calcineurin inhibitors exert their immunosuppressive effects by reducing interleukin-2 (IL-2) production and IL-2 receptor expression, leading to a reduction in T cell activation. Tacrolimus inhibits T lymphocyte activation by binding to FKBP-12, an intracellular protein. A complex is then formed of tacrolimus–FKBP-12, calcium, calmodulin, and calcineurin, which inhibits the phosphatase activity of calcineurin. This complex prevents the dephosphorylation and subsequent translocation of the nuclear factor of activated T cells (NF-AT), a nuclear component that initiates gene transcription for the formation of IL-2 ( Fig. 17.1 ). As a result, T lymphocyte activation is inhibited. The mechanism of action of cyclosporine is similar, except that the binding protein is cyclophilin. Tacrolimus is 10 to 100 times more potent than cyclosporine in its immunosuppressive effects.

Pharmacokinetic Properties

The pharmacokinetic characteristics of CNIs show high interindividual and intraindividual variability (i.e., different patients have different pharmacokinetic characteristics, and the same patient may have different pharmacokinetic characteristics at different time points after transplantation), and the drugs have a narrow therapeutic index; therefore therapeutic drug monitoring is necessary to optimize treatment. Because 90% of the agents is partitioned in the cellular components of blood, whole-blood concentrations correlate better with drug exposure (area under the curve [AUC]) than do plasma concentrations.

Pharmacogenetics

The absorption, bioavailability, and elimination of these drugs are primarily controlled by efflux pumps and enzymes of the cytochrome P (CYP) 450 family. DNA variants of the genes encoding these proteins contribute to the interindividual heterogeneity of the metabolism of CNIs. Cyclosporine and tacrolimus are metabolized by CYP3A4 and CYP3A5, and several single-nucleotide polymorphisms in the two genes have been associated with differences in drug clearance. Homozygotes for a common DNA variant that affects gene splicing (CYP3A5∗3) may require a lower dose to remain within the target blood concentration. The Tactique study randomly assigned 236 patients to either receive tacrolimus at a fixed dosage or dosage according to their CYP3A5 genotype. Dosing tacrolimus according to CYP3A5 genotype had no effect on long-term clinical outcomes, including rates of acute rejection and graft survival. A prospective randomized trial evaluated tacrolimus dosing in patients heterozygous for CYP3A4∗22, an allelic variant that is associated with decreased cytochrome activity. Carriers of the CYP3A4∗22 allele had significantly altered tacrolimus metabolism and reached higher plasma concentrations compared with the control group.

Absorption and Distribution

The gastrointestinal absorption of CNIs is highly dependent on the presence of food, bile acids, and motility. They are rapidly but incompletely absorbed in the gastrointestinal tract, and peak concentrations in whole blood are attained 1 to 2 hours after oral administration. Tacrolimus has low oral bioavailability (average 25%; range 4%–93%). The mean oral bioavailability of tacrolimus is comparable in adult (25%) and pediatric (31%) transplant recipients. The rate and extent of absorption of tacrolimus are reduced in the presence of food, with the peak concentration in whole blood compared with the fasting state decreased by approximately 50% to 75%, and the AUC decreased by 25% to 40% when the drug is taken after a meal. Tacrolimus is highly bound to erythrocytes, in a concentration-dependent manner, with reduced ratios at higher drug concentrations related to binding saturation. Plasma protein binding may be 99%, with most of the drug bound to α ₁ -acid glycoprotein and albumin. Tacrolimus is widely distributed in most tissues, including lungs, spleen, heart, kidney, pancreas, brain, muscle, and liver; tacrolimus crosses the placenta, with umbilical cord plasma concentrations one-third of those in maternal plasma. Tacrolimus also is present in breast milk, but at extremely low levels (<2.5 ng/mL).

Metabolism and Elimination

Cyclosporine is metabolized almost entirely in the liver, mostly through the CYP-450 system. Most of the drug is excreted in the bile, with only trace amounts being excreted in the urine. Tacrolimus is metabolized extensively in the liver as well and, to a much lesser extent, in the intestinal mucosa, with metabolism mediated at both sites by CYP3A4 isoenzymes. Tacrolimus is converted by hydroxylation and demethylation to at least 15 metabolites, with the main metabolite being 13- O -dimethyl-tacrolimus. The mean clearance after intravenous administration of tacrolimus is as follows: 0.040 L/h/kg in healthy volunteers, 0.083 L/h/kg in adult kidney transplant patients, 0.053 L/h/kg in adult liver transplant patients, and 0.051 L/h/kg in adult heart transplant patients. When administered orally, fecal elimination accounts for 92.6 ± 3.07% and urinary elimination accounts for 2.3 ± 1.1% of the administered dose in healthy volunteers.

The main drugs that interact with CNIs when administered simultaneously are either inducers or inhibitors of CYP3A4. CYP3A4 inhibitors potentially increase whole-blood concentrations, whereas CYP3A4 inducers decrease CNI concentrations ( Table 17.1 ).

Special Patient Populations

Three percent of patients require higher dosages (>0.4 mg/kg/d) to reach therapeutic tacrolimus concentrations. This is a reflection of the low bioavailability and, to a lesser extent, the high clearance of the drug. In a nonblinded parallel-group study, the bioavailability of tacrolimus was significantly ( P = 0.01) lower in African American (11.9%) and Latin American (14.4%) patients than in white patients (18.8%). A retrospective study in renal transplant recipients showed that African American recipients required higher dosages of tacrolimus on a milligram-per-kilogram basis.

Children typically require higher tacrolimus dosages on a milligram-per-kilogram basis than adult patients, most likely reflecting the higher mean total body clearance and volume of distribution in children. Clinically relevant differences do not exist between adults and children, however, in terms of the time taken to reach maximal blood concentrations (2.1 hours in children vs. 2 hours in adults), bioavailability (31% vs. 25%), and mean terminal elimination half-life (11.5 hours vs. 12 hours). The mean clearance of tacrolimus in patients with renal dysfunction was similar to that in normal volunteers; tacrolimus pharmacokinetics after a single intravenous administration was similar in seven patients not receiving dialysis and five receiving dialysis.

The mean clearance of tacrolimus in patients with mild hepatic dysfunction (mean Pugh score of 6.2) was not substantially different from that in normal volunteers after a single intravenous and oral dose. The mean clearance was substantially lower in patients with severe hepatic dysfunction (mean Pugh score >10), regardless of the route of administration.

Clinical Studies in Kidney Transplantation