Rapamycin (sirolimus) was first found to inhibit metabolic processes in yeast.1, 2 These inhibitory effects extended to mammalian cells,4 particularly activated T lymphocytes,3, 4 revealing potent immunosuppressive properties and leading to its approval for prevention of kidney transplant rejection.5 However, the doses used in early trials led to a high incidence of side effects, including slow wound healing, hyperlipidemia, and low white cell and platelet counts.6 The liver trial was marred by complications resulting in a black box warning by the FDA. Most liver transplant programs were therefore hesitant to use the drug. More recently, much lower doses of rapamycin than initially used (loading dose of 15 mg, followed by 5 mg/day) have been found to control rejection and reduce side effects.7-9 In three large, single-center studies with a combined total of 623 patients, the incidence of complications was as low as 1.1%-1.2%. Currently recommended treatment regimes start at 2 mg daily and aim for levels of 4-10 ng/mL. At these lower doses, rapamycin may even improve survival in liver transplant recipients, because of its antiproliferative activity, especially in patients with hepatocellular carcinoma (HCC).10 Lower rates of fibrosis, cytomegalovirus infection, and weight gain after liver transplantation are added advantages.10 Even hyperlipidemia, still a relatively common side effect at currently recommended low doses, can be controlled effectively by omega-3 fatty acids and statins.11
Calcineurin-inhibitor–associated nephrotoxicity provided the rationale for the switch to rapamycin in the study in this issue from Northwestern University in Chicago.12 The results provide evidence that rapamycin may also facilitate immunosuppression (IS) minimization or withdrawal, a holy grail for transplantation.13 With the aim of eventual discontinuation of IS, the AWISH study, sponsored by the Immune Tolerance Network, has followed patients as their IS has been slowly and cautiously reduced. However, the numbers of patients achieving operational tolerance has been disappointing.14 In the Chicago cohort, FoxP3 expression was induced, thereby increasing T-regulatory cell (Treg) numbers and decreasing cytotoxic T-cell activity, perhaps leading to eventual operational tolerance.
Rapamycin forms a drug-receptor complex that specifically blocks mammalian target of rapamycin (mTOR).15 mTOR is a well-conserved serine/threonine kinase that interacts with several proteins to form two multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), both of which have distinct relationships to up- and downstream effectors and to each other (Fig. 1). These complexes influence the metabolic and proliferative processes of many cell types, not just rapidly dividing immune cells activated during graft rejection.16 The mTOR component of mTORC1 is exquisitely sensitive to inhibition by rapamycin, whereas mTOR in mTORC2 is more resistant. mTORC1 is required for T-helper cell (Th)1 and Th17 differentiation and, when activated, inhibits Treg differentiation. In the presence of transforming growth factor beta, stimulation of FOXP3− T cells through T-cell receptor and CD28 promotes expression of the FOXP3 gene through the cooperation of nuclear factor of activated T cells and mothers against decapentaplegic homolog 3. As described by Levitsky et al., this process is mimicked by rapamycin, which shifts the balance of the immune response toward suppression at the expense of Th1 and Th17 activation, as evidenced by increased FOXP3+ Tregs.12
The metabolic effects of mTORC1 and mTORC2 activation18 are also influenced by rapamycin treatment, perhaps providing significant additional clinical benefits, including reduced steatosis and weight gain. Inhibition of hepatic mTORC1 significantly impairs sterol regulatory element-binding protein function, making mice resistant to the hepatic steatosis and hypercholesterolemia induced by a high-fat and high-cholesterol diet. Rapamycin also promotes catabolism by blocking mTORC1 phosphorylation of the Unc-51-like kinase 1/autophagy-related protein 13/focal adhesion kinase family interacting protein of 200 kDa complex and restoring autophagy,19 perhaps explaining the weight loss observed in some rapamycin-treated patients.
Inhibition of mTORC1 by rapamycin activates negative feedback loops that block phosphoinositide 3-kinase signaling, preventing G1- to S-phase transition. Subsequent cell division and cell proliferation is blocked, thereby limiting cell proliferation20 as well as the allogeneic T-cell activity that characterizes graft rejection. The combined antiproliferative and metabolism-altering properties of rapamycin may therefore be important in preventing tumor regrowth post-transplantation and may explain the lower incidence of HCC and skin malignancies observed in transplant recipients taking this drug.
Calorie restriction is known to extend lifespan21 and its effect is apparently mediated through mTOR,22 with superoxide-based signals playing a role.23 The effect of calorie restriction on longevity is highly conserved, because rapamycin also increases murine lifespan, even when administered late in life.24 However, as we have noted above, rapamycin treatment is also associated with insulin resistance (IR), hyperlipidemia, and IS, thus making it important to identify competing downstream mechanisms of the rapamycin/mTOR interaction that may affect aging, as some groups are all ready doing with some success.25, 26
As well as suppressing graft rejection, rapamycin and its analogs have multiple effects with exciting implications for their therapeutic use. By inducing Tregs, rapamycin may prevent the reemergence of autoimmune disease post-transplantation. It may also prevent weight gain, reduce the incidence of malignancy, and increase longevity. However, the negative effect of rapamycin treatment on metabolism, including induction of glucose intolerance and IR, also need to be considered. Regulatory processes are critical for deciding on the balance of efficacy and side effects required for approval of any drug. Occasionally, data prove to be inaccurate or incomplete, and drugs may need to be removed from the market. However, mistaken assumptions and poor study design may also lead to an incorrect interpretation of a drug's potential benefit and result in its failure to be approved or correctly utilized. Regulatory agencies should be just as eager to identify these oversights and have mechanisms in place to resurrect drugs once new supportive evidence for their beneficial use is found. The potential for rapamycin to prevent hepatoma recurrence affects over 1,000 patients in the United States every year (almost one fifth of liver transplant recipients), and promotes graft tolerance in thousands of patients, if the Levitsky hypothesis bears out. To demand stringent new double-blind registration trials is unrealistic, because the drug's patent life is about to expire. A new paradigm must be developed by the U.S. Food and Drug Administration, together with the physician and scientific communities, to realize the extended therapeutic benefit of this and other drugs for the benefit of all patients.