SDZ RAD Research Articles

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Everolimus was approved for the prevention of organ rejection of kidney transplants in adult patients in April 2010 by the US Food and Drug Administration. Therapeutic drug monitoring of everolimus is recommended for all patients receiving this immunosuppressant. The goal of this study was to improve and revalidate our previously published high-performance liquid chromatography tandem mass spectrometry method for the measurement of everolimus and to assess the performance of the recently introduced isotopically labeled internal standard (IS). For this method, the following innovative changes were incorporated: (1) sample preparation includes the addition of water to hemolyze red blood cells; (2) a separate extraction column is added to the system; (3) different settings of the switching valve are used to minimize dead volume; (4) a more sensitive mass spectrometer is used (API 5000) for drug detection; (5) isotopically labeled everolimus is used as the IS. The addition of water to blood before adding ZnSO4 with methanol improves absolute recovery of everolimus from 77.3% ± 6.18% to 82.3% ± 6.3%. The use of the more sensitive mass spectrometer permitted the use of a smaller sample volume and lowered the lower limit of quantification from 1.0 to 0.5 ng/mL. Between-day precision for quality control samples is below 9%, and the accuracy ranged from 94.8% to 106.4%. Neither carryover nor matrix effects were observed. A comparison study showed very good agreement between the results obtained by our laboratory and those obtained by a reference laboratory (r = 0.93). The performance of the new IS, [13c2d4] RAD001, was compared with that of SDZ RAD 223-756. Everolimus concentration results obtained using the isotopically labeled IS agreed more closely with the results from the reference laboratory (using 13c2d4) as compared with everolimus concentration results obtained using the analog (SDZ RAD 223-756). This revised methodology together with the comparison study data proved that our novel method for the measurement of everolimus is sensitive, precise, reliable, and robust.

Mycophenolic Acid Inhibits Hepatitis C Virus Replication and Acts in Synergy With Cyclosporin A and Interferon-α

Chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation. Clinical evidence suggests that particular immunosuppressive agents can have an influence on HCV recurrence. Cyclosporine A (CsA) specifically inhibits HCV replication through blocking the viral RNA polymerase enzyme NS5B. In this study, we investigated the effect of mycophenolic acid (MPA) and other immunosuppressants on HCV replication. MPA and other compounds were tested in vitro using an HCV-replication model containing a luciferase reporter gene. At clinically relevant concentrations (1.0-6.0 microg/mL), MPA inhibited HCV replication to approximately 75%. CsA and interferon (IFN)-alpha also showed inhibition in a dose-dependent manner. In these short-term (18 hours) experiments, MPA did not inhibit cell proliferation or induce cell death, which could have accounted for the antiviral effect. In contrast to the antiviral activity of MPA against West Nile virus, the effect of MPA on HCV replication was guanosine independent. When combined, MPA and CsA showed significant synergistic inhibition of replication, reaching maximum inhibition of approximately 90% at the highest doses. Synergistic effects were observed with suboptimal concentrations of IFN-alpha with MPA or CsA. The kinetics of HCV inhibition by MPA, CsA, and IFN-alpha were clearly distinct, with earliest effects seen with IFN-alpha. No specific inhibitory effects were observed with tacrolimus or rapamycin. The immunosuppressive drug MPA is as potent as CsA as an inhibitor of HCV replication. MPA was shown to have a distinct anti-HCV mechanism of action, independent of cell proliferation and guanosine depletion.

Sensitive, High Throughput HPLC-MS/MS Method With On-line Sample Clean-up for Everolimus Measurement

A new HPLC-MS/MS method for everolimus measurement was developed that includes the following features: small sample volume, short run time, fast, simple and cost-efficient sample preparation, assessment of performance of two internal standards (IS), SDZ RAD 223-756 and ascomycin and comparison of the method with an HPLC-MS/MS reference method. The authors established a multiple reaction monitoring positive ion HPLC-MS/MS method with on-line extraction and sample cleanup. This procedure includes: an API 2000 triple quadrupole mass spectrometer with turbo-ion spray, built-in Valco switching valve, an HPLC system; guard column; a Nova-Pak C18 analytical column; washing solution, methanol:30 mM ammonium acetate pH 5.1 (80:20); eluting solution, methanol:30 mM ammonium acetate pH 5.1 (97:3); flow rate 0.8 mL/min; and a run time of 2.8 minutes. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of everolimus (m/z 975.5-->908.5), and two ISs: SDZ RAD 223-756 (m/z 989.8-->922.8) and ascomycin (m/z 809.5-->756.5). The LLOQ was 1.0 microg/L for everolimus using either IS. Between day precision ranged from 3.1% to 5.7% for SDZ RAD 223-756 and 6.0% to 8.6% for ascomycin using spiked blood with everolimus concentrations 2.0 to 25.0 microg/L. Absolute recoveries using spiked samples over the range of 2.5 to 25 mug/L averaged 77.3% (SDZ RAD 223-756) and 76.8% (ascomycin). No matrix effect on everolimus was demonstrated based on the mean observed signal detection of 98.6% (SDZ RAD 223-756) and 105% (ascomycin). Comparison of everolimus concentrations obtained using this method with two internal standards with a reference laboratory demonstrated that the mean everolimus concentration obtained with ascomycin was statistically different (lower) than results with the reference method and the method that used SDZ RAD 223-756 as the internal standard gave equivalent results compared with the reference method.

New Immunosuppressants: Immunosuppression and Immunomodulation

Immunosuppressive therapy can be used to prevent graft rejection and to treat autoimmune diseases. Recent advances in the understanding of this immune response have focused on the development of new immunosuppressive medications and new approaches to induction of immunological tolerance and reduction of late graft losses. In this overview, preclinical and clinical studies of the new immunosuppressive agents and their analogs are reviewed from the discovery of cyclosporine. More recently, certain classical immunosuppressants tacrolimus and sirolimus were well used to prevent acute rejection of transplanted organs and to ensure long-term survival of the allografts. However, some immunosuppressants have specific and significant toxic effects, so that drug combination therapy has been of great interest in addition to the introduction of novel small molecule agents, including mycophenolate mofetil; sirolimus analogs, SDZ RAD; 15-deoxyspergualin (DSG) and its analogs, FTY720; malononitrilamide analogs, FK778 and leflunimide; Sanglifehrins A; PG490-88; FK330 and 4-amino-analog of tetrahydrobiopterin of nitric oxide synthase inhibitors; genistein, baohuoside- 1 and apigenin of flavonoid family; Prostaglandin E2; CYP3A4, CYP3A5, and P-glycoprotein; vitamin E analogs, α-tocopheryl (PEG-1000) succinate (TPGS). A newer immunomodulation concept and their new drugs will also be described. Keywords: immunosuppressant, immunoregulation, transplantation, rejection, autoimmune diseases, fk, pg and baohuoside

A rapamycin derivative (everolimus) controls proliferation through down-regulation of truncated CCAAT enhancer binding protein β and NF-κB activity in Hodgkin and anaplastic large cell lymphomas

AbstractThe immunosuppressive macrolide rapamycin and its derivative everolimus (SDZ RAD, RAD) inhibit the mammalian target of rapamycin (mTOR) signaling pathway. In this study, we provide evidence that RAD has profound antiproliferative activity in vitro and in NOD/SCID mice in vivo against Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) cells. Moreover, we identified 2 molecular mechanisms that showed how RAD exerts antiproliferative effects in HL and ALCL cells. RAD down-regulated the truncated isoform of the transcription factor CCAAT enhancer binding protein β (C/EBPβ), which is known to disrupt terminal differentiation and induce a transformed phenotype. Furthermore, RAD inhibited constitutive nuclear factor κB (NF-κB) activity, which is a critical survival factor of HL cells. Pharmacologic inhibition of the mTOR pathway by RAD therefore interferes with essential proliferation and survival pathways in HL and ALCL cells and might serve as a novel treatment option. (Blood. 2005;106: 1801-1807)

Differential effects of everolimus and cyclosporine A on intimal alpha-actin-positive cell dynamics of carotid allografts in mice.

We recently demonstrated that donor medial cells are replaced by smooth muscle alpha-actin (SMA)-expressing host cells in murine carotid allografts. In this study, we investigated neointimal cell dynamics including effects of cyclosporine A (CsA) and everolimus (SDZ RAD, Certican). To obtain a functional readout, we measured the effects of these treatments on cardiac allografts. Heterotopic heart allotransplantation was performed between C57BL/6 (B6) and BALB/c (B/c) mice. Orthotopic carotid artery allotransplantation was performed between B6 and B/c mice expressing green fluorescent protein (GFP). Both strains served as donors and recipients. Groups of mice were treated for 4 and 8 weeks (heart and carotid recipients, respectively) with placebo, CsA 20 mg/kg per day, or everolimus 1.0 mg/kg per day using Alzet minipumps. At 2, 4, and 8 weeks, carotid grafts were harvested for histology. In the GFP-B/c to B6 strain combination, everolimus but not CsA significantly reduced neointima formation and accumulation of SMA-positive host (non-GFP) cells at doses that did not fully suppress heart rejection. In the B6 to GFP-B/c strain combination, everolimus and CsA strongly prevented heart rejection under treatment, partly suppressed neointima formation, and completely prevented SMA-positive host (GFP) cell accumulation. Donor-derived cells expressing SMA never appear in the neointima, and such cells are completely recipient derived. Adequate immunosuppression delays or prevents the disappearance of donor cells and the appearance of host cells in the graft, a phenomenon apparently associated with the neointima formation. Even immunosuppression sufficient to prevent heart allograft rejection under treatment completely, diminishes neointima formation only by approximately 50%.

Differential effect of cyclosporine A and SDZ RAD on neointima formation of carotid allografts in apolipoprotein E-deficient mice.

Apolipoprotein E-deficient (apoE-/-) mice spontaneously develop hypercholesterolemia and atherosclerotic lesions. This may be an appropriate background for the development of graft vessel disease. The authors therefore investigated the effects of cyclosporine A (CsA) and SDZ RAD (RAD, everolimus, Certican) on neointima formation of carotid allografts in apoE-/- mice. To ascertain that equipotent immunosuppressive doses were used, the authors also investigated their effects on cardiac allografts using the same wild-type strain combination. Heterotopic heart allotransplantation was performed in the BALB/c to C57BL/6 strain combination. Graft survival was monitored daily. Orthotopic carotid artery allotransplantation was performed from BALB/c to apoE-/- (C57BL/6) mice. Groups of mice were treated for 8 weeks with placebo; CsA 10, 20, and 30 mg/kg/d; or RAD 0.3, 1.0, and 3.0 mg/kg/d using ALZET minipumps. Body weight, CsA blood levels, serum lipids, and histology were examined 8 weeks after transplantation or on the day of rejection. Compared with the placebo group, CsA or RAD did not affect body weight. Both CsA and RAD prolonged the survival of cardiac grafts in a dose-dependent manner. CsA blood levels were not different between wild-type and apoE-/- recipients. CsA increased total cholesterol and low-density lipoprotein, but not high-density lipoprotein dose-dependently and significantly, but RAD did not affect the lipids. RAD but not CsA significantly attenuated neointima formation. These results suggest that RAD at a dose preventing organ rejection may also prevent transplant vasculopathy even in the presence of hyperlipidemia.

The influence of the rapamycin-derivate SDZ RAD on the healing of airway anastomoses.

Among the many immunosuppressive effects of SDZ RAD (40-0(2-hydroxyethyl)-rapamycin), a rapamycin derivative, is the inhibition of fibroblast proliferation. Since the long-term success of lung transplantation is limited by the development of bronchiolitis obliterans, a fibroblast-associated progressive luminal obstruction of the terminal bronchioli, the use of SDZ RAD as immunosuppressive in pulmonary graft recipients may counteract this process. However, reduction of fibroblast activity, posttransplant, may impair the healing of the bronchial anastomoses. The cervical trachea in pigs was denuded, divided and re-anastomosed with Prolene 4-0 single stitches. Control animals (group 1, n=4) were without, and study animals (group 2, n=6) were with SDZ RAD therapy (1.25 mg/kg/day, p.o., 14 days). After 14 days, the pigs were sacrificed. The anastomoses were examined histologically, and breaking strength of tracheal strips of 5-mm width was measured. All animals survived without complications. Serum levels of SDZ RAD were 30.9+/-8.7 ng/ml (recommended level 20-40 ng/ml). All anastomoses healed macroscopically without difference between the two groups. Breaking strength was significantly lower in the treated animals (group 1 vs. group 2: 11.75+/-0.35 vs. 7.69+/-1.39 N, P=0.01). Histology did not show a significant change in histoarchitecture between the groups. Although SDZ RAD significantly reduced the breaking strength of the tracheal anastomosis, no obvious histological differences between treated and untreated animals could be detected. Since this model does not reflect the clinical situation, further investigations are necessary to reveal the effect of SDZ RAD on airway wound healing in concert with a contemporary clinically used multidrug immunosuppressive regimen in allograft recipients.

Influence of SDZ RAD vs. MMF on gastric emptying in renal transplant recipients.

SDZ RAD and mycophenolate mofetil (MMF) are increasingly used in the prevention of renal allograft rejection. SDZ RAD, having a macrolide structure, and MMF, known with gastrointestinal side-effects, may have gastric motility modifying properties. Gastric emptying was examined 1 yr after renal transplantation in eight patients taking corticosteroids (CS), cyclosporin A (CsA) and SDZ RAD and six patients treated with CS, CsA and MMF. Comparing the two groups, no significant differences in gastric emptying of solids and liquids were noted. Compared with normal volunteers, solid gastric emptying was faster in the SDZ RAD group and similar in the MMF group. It is concluded that in stable renal transplant recipients treated with MMF, gastric emptying was normal. Because of the impact on drug absorption and gastrointestinal symptoms, further studies are indicated to corroborate the potential prokinetic properties of SDZ RAD.

Mycophenolic acid is a potent inhibitor of the expression of tumour necrosis factor- and tumour necrosis factor-receptor superfamily costimulatory molecules.

The tumour necrosis factor (TNF) ligands CD154, CD70 and TNF receptors CD134 and CD137 are all involved in allograft rejection. Because these molecules are not present on resting T cells, we investigated whether immunosuppressive drugs could inhibit their induction. Expression was induced in vitro on T cells by phorbol 12-myristate 13-acetate and ionomycin or by allogeneic dendritic cells in the presence or absence of cyclosporin A (CsA), tacrolimus (TAC), rapamycin derivative (SDZ RAD), or mycophenolic acid (MPA), and determined by flow cytometry. To study the effect of in vivo exposure to immunosuppressive drugs on these molecules, immunohistochemistry was performed on human lymph nodes from patients treated with TAC or TAC and MMF. The calcineurin inhibitors (CI) CsA and TAC strongly suppressed the induction of CD70, CD137 and CD154, but not of CD134, upon pharmacological stimulation of T cells in vitro. In allogeneic stimulations only CD137 and CD154 were inhibited by CI. SDZ RAD did not inhibit pharmacological induction, but in allogeneic stimulations all the investigated molecules were partially suppressed. Both in pharmacological and in allogeneic stimulations, MPA inhibited induction of all tested molecules on T cells nearly completely at 4 micro g/ml. However, in lymph nodes obtained from patients chronically treated with MMF and TAC no reduction of the expression of these molecules was observed. This was possibly caused by trough levels which were in vivo lower (mean: 2.3 micro g/ml) than the concentration giving complete inhibition in vitro. In conclusion, in contrast to CsA, TAC and SDZ RAD, MPA is a potent inhibitor of the induction of TNF- and TNF-receptor superfamily molecules on T cells. To obtain long-term suppression of these molecules in vivo, a plasma trough level of 4 micro g/ml is indicated.

The rapamycin analogue SDZ RAD attenuates bleomycin-induced pulmonary fibrosis in rats.

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix proteins within the pulmonary interstitium. The new macrolide immunosuppressant SDZ RAD, a rapamycin analogue, inhibits growth-factor dependent proliferation of mesenchymal cells and might therefore be of therapeutic interest for the treatment of fibrotic lung disease. In this study the effect of SDZ RAD on lung-collagen accumulation in the bleomycin model of pulmonary fibrosis in rats was investigated. SDZ RAD (2.5 mg x kg(-1) x day(-1)) or drug vehicle were administered orally by daily gavage. Successful dosing was confirmed by measuring splenic weight. Total lung-collagen content was measured by high-performance liquid chromatographic quantitation of hydroxyproline. In animals given bleomycin and drug vehicle, total lung collagen was increased by 182+/-11% (mean+/-SEM) compared with saline controls at 14 days (p<0.001). The increase in lung-collagen accumulation was reduced by 75+/-12% (p<0.01) in animals given SDZ RAD and was accompanied by a concomitant 56+/-6% (p<0.001) reduction in lung weight. SDZ RAD is currently in clinical trials for the prevention of solid organ graft rejection, another condition characterized by excessive extracellular matrix production. The authors propose that SDZ RAD warrants evaluation as a novel therapeutic agent for fibrotic lung disease.

Influence of a novel rapamycin analogon sdz rad on endothelial tissue factor and adhesion molecule expression

Influence of a novel rapamycin analogon sdz rad on endothelial tissue factor and adhesion molecule expression

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography–electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC–tandem mass spectrometry. Whole blood samples (500 μl) were prepared by protein precipitation, followed by C 18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 μg/l ( r 2>0.996, n=9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples ( n=5) at 0.5, 1.2, 20.0, and 75.0 μg/l, was 100.3–105.4% and ≤7.6%, respectively. The assay had a mean relative recovery of 94.8±3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at −80 °C for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze–thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase II and III clinical trials.

Long-term viability of articular cartilage after microsurgical whole-joint transplantation and immunosuppression with rapamycin, mycophenolate mofetil, and tacrolimus.

The survival or rejection of articular cartilage in heterotopic vascularized joint transplants in rats immunosuppressed with rapamycin (SDZ RAD), mycophenolate mofetil (MMF), and tacrolimus was evaluated histologically up to 1 year after surgery. The vascularized knee joint of an ACI donor rat was transplanted to the groin of a Lewis recipient rat. Nonimmunosuppressed allografts were evaluated after 6 weeks and 3 months, and immunosuppressed allografts and control isografts were evaluated after 6 weeks, 3 months, 6 months, and 1 year. No rejection was seen in the control isografts. All allografts without immunosuppression were rejected at 6 weeks and 3 months. Eighteen of 21 knee joint transplants immunosuppressed with SDZ RAD and 17 of 22 knee joint transplants immunosuppressed with MMF were rejected between 6 weeks and 1 year. SDZ RAD and MMF caused significant side effects including compromised wound healing and bone marrow suppression culminating in weight loss and death. Eighteen of 19 knee joints immunosuppressed with tacrolimus showed no signs of rejection up to 1 year after surgery. Long-term intermittent immunosuppression with tacrolimus was significantly superior to SDZ RAD and MMF in preventing rejection of the transplanted articular cartilage of a vascularized knee joint allograft up to 1 year after surgery.

Early Clinical Experience With a Novel Rapamycin Derivative

SDZ RAD (everolimus, Certican is a novel macrolide immunosuppressant that blocks growth factor-driven transduction signals in the T-cell response to alloantigen. After stimulation of the IL-2 receptor on the activated T-cell, SDZ RAD inhibits p70S6 kinase, acting at a later stage in the T-cell mediated response than do cyclosporine and other calcineurin inhibitors (CNIs). Unlike the CNIs, SDZ RAD is a proliferation signal inhibitor, blocking growth factor-driven proliferation of both hematopoietic and nonhematopoietic cells. These activities are complementary to those of cyclosporine and provide a rationale for the addition of SDZ RAD to cyclosporine-based immunosuppression, with the potential for minimizing CNI nephrotoxicity, reducing the incidence of acute rejection, and favoring long-term graft survival. Potential also exists for beneficial effects on other factors that may influence the development of chronic rejection. These factors include comorbid diseases such as hypertension, which may affect transplant vasculopathy, and opportunistic infection with cytomegalovirus (CMV) and other viruses, which may increase the risk of chronic rejection. The synergistic effect of SDZ RAD and cyclosporine has been confirmed in preclinical models, with graft survival being significantly prolonged in rat models of kidney and heart allotransplantation. Clinical experience with SDZ RAD in cyclosporine-based immunosuppression, including low-dose cyclosporine regimens, has also resulted in predictable and favorable clinical outcomes. Low rates of acute rejection, excellent rates of patient and graft survival, lower incidence of CMV infections, better cholesterol, triglyceride and creatinine profiles, and better renal function have been demonstrated with SDZ RAD and lower doses of cyclosporine (Neoral; Novartis) in recipients of renal transplants. These findings, combined with good tolerability rates and an acceptable side-effect profile, indicate that the synergistic profile of SDZ RAD in combination with nontoxic dosages of CNI's and IL2 inhibitors will further improve longterm results in renal transplantation.

Efficacy of SDZ RAD compared with CsA monotherapy and combined RAD/FTY720 treatment in a murine cardiac allotransplantation model

Objective: The macrolide immunosuppressant RAD and the immunomodulator FTY720 have distinct mechanisms of action. We investigated the efficacy of RAD (everolimus, certican®) alone or in combination with FTY720 on graft survival (GS) and histology in comparison with CsA, using mouse strains with strong MHC disparity. Methods: Heterotopic cardiac grafting was performed using the C57Bl/6 to C3H strain combination. Osmotic mini-pumps filled with CsA or RAD were implanted subcutaneously. FTY720 was administered as a single daily dose by gavage. Peripheral lymphocyte count (PLC) was determined at 1, 4 and 8 weeks or on the day of sacrifice. Body weight was recorded on the day of surgery and weekly. Grafts were histologically evaluated. Main findings: In placebo-treated mice the allografts were rejected after 7 days. Monotherapy with 10 and 30 mg/kg/day CsA achieved 10 and 22.5 days median survival time (MST), while 0.1, 0.3, 1 and 3 mg/kg/day RAD resulted in 10.5, 20, >56 and >56 days MST, respectively. FTY720 lowered the PLC significantly, while the lower CsA dose and RAD did not influence the PLC. Adding FTY720 to the 0.6 mg/kg/day dose of RAD extended GS modestly but reduced significantly the perivascular infiltration and endothelialitis in the grafts compared with RAD monotherapy. Conclusions: Under the conditions of the present experiment RAD was more potent than CsA in extending the GS. Combining FTY720 and RAD was well tolerated with respect to weight gain and lack of clinically detectable infections in the mice. The 2-drug regimens suppressed the inflammatory allo-response better than RAD monotherapy.

Long-term pharmacokinetics of the metabolites of SDZ RAD and cyclosporine in transplant reciplents

Long-term pharmacokinetics of the metabolites of SDZ RAD and cyclosporine in transplant reciplents

Long-term pharmacokinetics of the metabolites of SDZ RAD and cyclosporine in transplant reciplents

Long-term pharmacokinetics of the metabolites of SDZ RAD and cyclosporine in transplant reciplents

The peripheral lymphocyte count predicts graft survival in DA to Lewis heterotopic heart transplantation treated with FTY720 and SDZ RAD

Objective: The new immunomodulator 2-amino-2-(2-{4-octylphenyl}ethyl)-1,3-propanediol hydrochloride (FTY720) lowers the peripheral lymphocyte count (PLC) by inducing migration of circulating lymphocytes to secondary lymphoid organs. This effect is dose-dependent at low (up to 0.1 mg/kg per day) doses in rats. We investigated the correlation between PLC and the later rejection, when FTY720 was combined with RAD. Methods: Heterotopic cardiac grafting was performed using the DA–Lewis strain combination. FTY720 and RAD were administered as single daily doses by gavage alone and in combination starting 3 days before to 28 days after transplantation. Graft survival was monitored daily by palpation. PLC was determined at 1 and 4 weeks, body weight (BW) weekly. Histologic evaluation of grafted hearts was performed after rejection. Main findings: FTY720 at doses of 0.03, 0.1 and 0.3 mg/kg per day prolonged graft survival dose-dependently from 6 (placebo) to 7, 9.5 and 15 days median survival time (MST). RAD at doses of 0.3, 1 and 3 mg/kg per day delayed rejection to 8.5, 18 and 37.5 days MST. Very small FTY720 doses added to the lower RAD doses were effective in maintaining grafts throughout the treatment period and with normal weight gain, as opposed to regimens with 1 mg/kg or more per day RAD, which resulted in delayed weight gain. FTY720 lowered the PLC significantly and dose-dependently. The PLC correlated well with graft survival [Spearman rank correlation ( n=30, rs=−0.75)]. Conclusions: Fully effective FTY720+RAD combination regimens caused no side effects with respect to the rats’ general well-being or weight gain and were better tolerated than equiactive RAD monotherapy, suggesting a broader therapeutic window for the combinations. Under the experimental conditions, the PLC decrease showed an interesting correlation with the anti-rejection effects in these two-drug regimens. Thus, in rats the PLC is helpful for monitoring the biological activity of FTY720 at low doses (<0.1 mg/kg per day), i.e. in the range of the steep part of its dose–response relationship.

Identification of a new metabolite of macrolide immunosuppressant, like rapamycin and SDZ RAD, using high performance liquid chromatography and electrospray tandem mass spectrometry

Previously unknown metabolites from the two macrolide immunosuppressants rapamycin (sirolimus) and SDZ RAD [40-O-(2-hydroxyethyl)rapamycin] obtained after in vitro incubation with human liver microsomes have been purified. Structure elucidation was performed by nanoelectrospray ionization tandem mass spectrometry applying low energy collision activated dissociation. This ionization method is, as shown here, a powerful tool to determine metabolic pathways by analysis of even low abundance products. Product ion spectra of the isolated metabolites indicate a new kind of biotransformation reaction for rapamycin and SDZ RAD. The proposed metabolic pathway starts with an ester hydrolysis which leads to a ring-opened structure. A dehydration on C33–C34 and a supplementary hydrogenation at C33–C34 result in a structure similar to the ring-opened isomer with an single bond at C33–C34.