Randall E. Morris

Comparison of the effects of tacrolimus and cyclosporine on the pharmacokinetics of mycophenolic acid.

Mycophenolate mofetil (MMF) is almost completely absorbed from the gut and is rapidly de-esterified into its active drug, mycophenolic acid (MPA). The main metabolite is glucuronidated MPA (MPAG), which is excreted into bile and undergoes enterohepatic recirculation. Studies in healthy volunteers treated with cholestyramine show that interruption of the enterohepatic recirculation decreases MPA exposure by approximately 40%. Published data show a difference in mycophenolic acid plasma concentrations between kidney transplant recipients treated with MMF plus cyclosporine (CsA) and those treated with MMF plus tacrolimus (TRL). However, the interpretation of these data is complicated by interpatient differences in variables that may influence MMF pharmacokinetics (e.g., underlying disease, co-medication, and time since transplantation). To understand the influence of TRL and CsA on MMF pharmacokinetics (PK) more completely, the authors eliminated confounding variables in clinical studies by performing drug interaction studies in inbred rats. To achieve a steady state, 3 groups of Lewis rats (n = 8 per group) were treated once daily with oral CsA (8 mg/kg), TRL (4 mg/kg), or placebo on days 0–6 before all rats began once-daily oral treatment with MMF (20 mg/kg) on day 7. Combined treatment with either MMF + CsA, MMF + TRL, or MMF + placebo was continued for 1 week (days 8–14). Thereafter, CsA and TRL treatments were stopped but MMF treatment was continued on days 14–21. Blood was sampled during the 24 hours subsequent to dosing on day 7 (after the first MMF dose), on day 14 (after multiple MMF doses) and on day 21 (after CsA/TRL washout). Rats in the MMF + TRL group and in the MMF + placebo group showed a second peak in the MPA-PK profiles consistent with enterohepatic recirculation of MPA. The MPA-PK profiles for the MMF + CsA–treated animals did not show a second MPA peak. On Day 14, the mean plasma MPA-AUC0–24 hours for the CsA-treated animals was significantly less than MPA exposures for rats in the MMF + TRL– and the MMF + placebo–treated groups. Furthermore, in contrast to results from other investigators, co-administration of CsA and MMF significantly increased MPAG-AUC0–24 hours. Serum creatinines did not differ among rats in the three groups. CsA but not TRL decreased MPA plasma levels and increased MPAG-AUC0–24 hours. These data suggest that CsA inhibits MPAG excretion into bile and offer an explanation for the well-known increased MPA exposure in organ transplant patients caused by conversion from CsA-to TRL-based immunosuppression.

Treatment with rapamycin and mycophenolic acid reduces arterial intimal thickening produced by mechanical injury and allows endothelial replacement

Rapamycin (RPM) and mycophenolic acid (MPA) inhibit immune responses by antagonizing IL-stimulated lymphocyte activation. These 2 drugs, used alone or preferably in combination, also significantly reduced the response of vascular cells to balloon-catheter arterial injury in rats. When rats were treated for 2 weeks with both drugs starting the day of injury, intimal thickening was significantly reduced (P < 0.001) 14 days after injury; however, by 44 days after injury, intimal thickening had progressed to the extent measured in arteries of untreated control rats. When RPM and MPA were administered for 3 days before and 13 days after injury, arterial intimal thickening was significantly (P = 0.024) reduced and endothelium had regrown in vessels analyzed 44 days after injury. Compared with initiation of treatment on the day of injury, starting the administration of RPM plus MPA before injury appears to limit the activation of cells or actions of factors responsible for the progression of intimal thickening that occurred after the administration of the drugs was terminated. RPM and MPA prevented the development of arterial intimal thickening in a model not dependent upon a rejection response. This direct antiproliferative action on smooth muscle cells by RPM and MPA, in vivo, may prevent the development of arterial intimal thickening associated with chronic rejection.

Rapamycin Inhibits Arterial Intimal Thickening Caused By Both Alloimmune And Mechanical Injury: Its Effect On Cellular, Growth Factor, And Cytokine Responses In Injured Vessels

The effect of rapamycin (RPM) on the extent of arterial intimal thickening was determined in rat recipients of orthotopic femoral artery allografts or in rats that had undergone balloon catheter injury to carotid arteries. In untreated rats, neointima comprised approximately 50% of the arterial wall area in both models. Although treatment of allograft recipients for 40 days with 1.5 mg/kg/day RPM was ineffective, a dose of 6 mg/kg/day (days 0-7) followed by 3 mg/kg/day (days 8-39) reduced intimal thickening by 98% (P < 0.0001). The higher RPM dose reduced T cell and macrophage infiltration significantly and decreased the expression of IL-2 receptor, class II Ag, and mRNAs for growth factors and cytokines. Treatment with 1.5 mg/kg/day RPM (days 0-13) after balloon-catheter injury reduced intimal thickening by 45% (P = 0.0254) and substantially decreased macrophage infiltration and expression of class II Ag in the adventitia. Within the neointima, however, mRNAs for platelet-derived growth factor-alpha, basic fibroblast growth factor, and transforming growth factor-beta were still expressed. In summary, we have shown that RPM inhibits not only the vascular response to injury caused by allograft rejection, but also the response to balloon catheter injury. This new information is important to our understanding of: (1) the fundamental processes responsible for intimal thickening regardless of the cause of vascular injury, (2) mechanisms of action of RPM that explain its effects on the response to very different types of vascular injury, and (3) the potentially diverse therapeutic applications of drugs, like RPM, that inhibit the actions of both immune and nonimmune cytokines and growth factors.

Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide

Among all the new immunosuppressive molecules being investigated either preclinically or clinically, four stand out: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide (and its malononitriloamide analogs). Each drug has distinct mechanisms of immunosuppressive action, and in the past year significant advances have been made in our understanding of the actions of these drugs at the molecular and even atomic levels. Data from recent clinical trials demonstrate that these drugs very effectively suppress graft rejection or autoimmune diseases, validating the pivotal role played by each of their distinct molecular targets in the normal functioning of immune cells.

Effects of rapamycin on growth factor-stimulated vascular smooth muscle cell DNA synthesis. Inhibition of basic fibroblast growth factor and platelet-derived growth factor action and antagonism of rapamycin by FK506.

Rapamycin (RPM) is a potent and effective immuno-suppressant which we have shown previously to inhibit intimai thickening in rat allograft and balloon-injured arteries. In this report, we have examined the effects of RPM on growth factor-induced vascular smooth muscle cell (VSMC) DNA synthesis. RPM potently inhibited platelet-derived growth factor (PDGF) (IC50=5×10-9 M) and basic fibroblast growth factor (bFGF) (IC50=8×10-10 M)-induced VSMC DNA synthesis. In contrast, only the highest concentrations of FK506 and CsA significantly altered PDGF- or bFGF-induced VSMC DNA synthesis. Addition of RPM (10-9 M) at as late as 46 hr after growth factor addition still effectively suppressed bFGF- or PDGF-induced DNA synthesis by 76% and 54%, respectively. The extent of the antagonism of RPMs inhibition of bFGF-induced VSMC DNA synthesis by FK506 was inversely proportional to RPM concentration and directly proportional to FK506 concentration.

Rapamycin Reverses Chronic Graft Vascular Disease in a Novel Cardiac Allograft Model

BACKGROUND Chronic graft vascular disease (CGVD) in cardiac allografts has been defined as a slowly evolving vasculopathy unresponsive to conventional immunosuppression. We compared 4 rodent models of CGVD to evaluate the reproducibility of CGVD in heart allografts. Rapamycin (Rapa) and cyclosporine (CSA) were then used to treat CGVD. METHODS AND RESULTS Hearts were harvested and placed heterotopically into allogenic recipients. CGVD scores of PVG allografts from ACI recipients treated with CSA on days 1 through 10 were significantly elevated on day 90 (n=16) compared with other models (immunosuppression used): (1) Lewis to F344 recipients (CSA), (2) Brown Norway to Lewis (FK506), and (3) DA to Wistar-Firth (methylprednisolone, azathioprine, CSA). Although delayed (day 60 to 90) CSA treatment had no effect (n=6), delayed Rapa (3 mg. kg-1. d-1 IP) reversed CGVD in PVG grafts (0.22+/-0.19 on day 90, n=6). ACI isografts showed no evidence of CGVD (n=6) at day 90. Immunohistochemistry of PVG grafts revealed perivascular infiltrates consisting of CD4(+) T cells and limited numbers of macrophages persisting up to day 90. Flow cytometry demonstrated increased levels of anti-donor antibody at day 90, which was significantly inhibited by Rapa treatment. CONCLUSIONS PVG grafts developed a significant increase in CGVD without evidence of ongoing myocardial rejection. This CGVD appeared to be mediated by both cellular and humoral mechanisms, given CD4(+) perivascular infiltrates and increased levels of anti-donor antibody. The anti-CGVD effectiveness of Rapa during a period in which there was little myocardial cellular infiltrate supports a novel mechanism of effect such as smooth muscle or B-cell inhibition.

The PKC inhibitor AEB071 may be a therapeutic option for psoriasis

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

Stanford experience with obliterative bronchiolitis after lung and heart-lung transplantation

BACKGROUND Obliterative bronchiolitis (OB) is the main chronic complication after heart-lung (HLTx) and lung transplantation (LTx), limiting the long-term success of both transplant procedures. METHODS Since 1981, 135 HLTxs and 61 isolated LTxs were performed in 184 patients at Stanford University. RESULTS The overall prevalence of OB in patients surviving longer than 3 months postoperatively was 64% after HLTx and 68% after LTx. The actuarial freedom from OB was 72%, 51%, 44%, and 29% at 1, 2, 3, and 5 years, respectively, after HLTx and LTx. An analysis of potential risk factors revealed that the frequency and severity of acute rejection episodes (p < 0.001) and the appearance of lymphocytic bronchiolitis on biopsy (p < 0.05) were significantly associated with the development of OB. With regard to diagnosis of OB, pulmonary function tests show early reductions of the forced expiratory flow between 25% and 75% of the forced vital capacity with subsequent decreases in the forced expiratory volume in 1 second. The sensitivity of transbronchial biopsies has increased to 71% since 1993. Current treatment consists of augmented immunosuppression. Concurrent acute rejection episodes or active OB on biopsy have been treated aggressively with high-dose steroid pulses. Analysis of data from 73 patients with OB after HLTx and LTx revealed actuarial 1-, 3-, 5-, and 10-year survival of 89%, 71%, 44%, and 17% versus 86%, 77%, 63% and 56% in patients without OB (p < 0.05 by log-rank analysis). The main complication and cause of death in patients with OB was superimposed respiratory tract infection, which was treated aggressively. CONCLUSIONS Early diagnosis of OB using pulmonary function tests or transbronchial biopsy is possible and important, because immediate treatment initiation has led to acceptable survival rates, with nearly 50% of affected patients still alive 5 years after transplantation. Current experimental research on OB suggests that immune injury is the main pathogenetic event of airway obliteration in animal models; rapamycin and leflunomide are new immunosuppressive agents that may have the potential to prevent and treat airway obliteration.

Effects of cyclosporin, FK506, and rapamycin on graft-vessel disease.

Graft-vessel disease (GVD) limits the long-term survival of heart-transplant patients, and this effect has not been altered by use of cyclosporin for immunosuppression. We compared the effects of the immunosuppressants cyclosporin, FK506, and rapamycin on GVD in a rat-heart transplantation model. Allografted hearts from rats treated with 1 mg/kg FK506 for 50 days showed the same degree of myocardial rejection but a significantly worse (p less than 0.05) grade of GVD compared with grafted hearts from rats treated with 1.5 mg/kg cyclosporin for the same time. 2 mg/kg FK506 for 50 days prevented cellular rejection but GVD was as severe as that found with 1 mg/kg FK506. Moderate GVD was present in two of five allografted hearts after treatment with 4 mg/kg FK506. 1.5 mg/kg rapamycin for 50 days was an effective inhibitor of rejection and GVD. Based on our results in rats, the possibility that GVD may occur in human heart-transplant recipients treated with FK506 cannot be excluded.

Therapeutic monitoring of mycophenolic acid: A consensus panel report

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