Effect of atorvastatin on cyclosporine pharmacokinetics in liver transplant recipients

BACKGROUND: The development of hyperlipidemia after liver transplant is frequently treated with hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) such as atorvastatin. As atorvastatin and the primary immunosuppressant drug, cyclosporine, are metabolized by the same pathway, there is the potential for an interaction. OBJECTIVE: To determine the effect of atorvastatin on cyclosporine pharmacokinetics in liver transplant recipients. METHODS: Six stable, long-term adult liver transplant recipients from a single center who developed posttransplant dyslipidemia were recruited to participate in a 14-day, open-label study of atorvastatin 10 mg/d coadministered with standard posttransplant immunosuppression using constant oral doses-of cyclosporine and corticosteroids. A 10-point pharmacokinetic profile was performed prior to and on day 14 after commencement of atorvastatin therapy. Cyclosporine concentrations were measured by HPLC-electrospray-tandem mass spectrometry. The AUC was calculated by the linear trapezoidal rule, with other parameters determined by visual inspection. RESULTS: Atorvastatin coadministration increased the cyclosporine AUC by 9% (range 0-20.6%; 3018 vs 3290 ng(.)h/mL; p = 0.04). No significant change was evident for other cyclosporine pharmacokinetic parameters. Total cholesterol and low-density lipoprotein cholesterol levels were significantly lower on day 14 than at baseline (p < 0.02). One patient developed a twofold increase in transaminases after 2 weeks of atorvastatin therapy, but no other clinical or biochemical adverse events were recorded. CONCLUSIONS: Atorvastatin coadministration increases the cyclosporine AUC by approximately 10% in stable liver transplant recipients. This change in systemic exposure to cyclosporine is of questionable clinical significance. Atorvastatin is effective in reducing cholesterol levels in liver transplant recipients.

Inhibition of epidermal growth factor receptor expression by RNA interference in A549 cells

AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by RNA interference (RNAi). METHODS: A549 cells were transfected using short small interfering RNAs (siRNAs) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded RNA (dsRNA) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsRNA-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsRNA-EGFR reduced the cell number by 85.0 %, decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to cisplatin by four-fold. CONCLUSION: Sequence specific dsRNA-EGFR were capable of suppressing EGF receptor expression, hence significantly inhibiting cellular proliferation and motility, and enhancing chemosensitivity of A549 cells to cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGF receptor overexpressing cells can help extend the list of available therapeutic modalities in the treatment of non-small-cell lung carcinoma (NSCLC).

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices

Objectives: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. Methods: Blood samples (50 mu L) were prepared by protein precipitation followed by C-18 solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. Results: The assay was linear from 10 to 2000 mu g/L (r(2) > 0.996, n = 9). Inter-day,analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 mu g/L were 94.9-103.5% and

CEDIA (R) sirolimus assay compared with HPLC-MS/MS and HPLC-UV in transplant recipient specimens

The role of the therapeutic drug monitoring laboratory in support of immunosuppressant drug therapy is well established, and the introduction of sirolimus (SRL) is a new direction in this field. The lack of an immunoassay for several years has restricted the availability of SRL assay services. The recent availability of a CEDIA (R) SRL assay has the potential to improve this situation. The present communication has compared the CEDIA (R) SRL method with 2 established chromatographic methods, HPLC-UV and HPLC-MS/MS. The CEDIA (R) method, run on a Hitachi 917 analyzer, showed acceptable validation criteria with within-assay precision of 9.1% and 3.3%, and bias of 17.1% and 5.8%, at SRL concentrations of 5.0 mu g/L and 20 mu g/L, respectively. The corresponding between-run precision values were 11.5% and 3.3% and bias of 7.1% and 2.9% at 5.0 mu g/L and 20 mu g/L, respectively, The lower limit of quantification was found to be 3.0 mu g/L. A series of 96 EDTA whole-blood samples predominantly from renal transplant recipients were assayed by the 3 methods for comparison. It was found that the CEDIA (R) method showed a Deming regression line of CEDIA = 1.20 X HPLC-MS/MS - 0.07 (r = 0.934, SEE = 1.47), with a mean bias of 20.4%. Serial blood samples from 8 patients included in this evaluation showed that the CEDIA (R) method reflected the clinical fluctuations in the chromatographic methods, albeit with the variable bias noted. The CEDIA (R) method on the H917 analyzer is therefore a useful adjunct to SRL dosage individualization in renal transplant recipients.

Novel diaroylhydrazine ligands as iron chelators: coordination chemistry and biological activity

The search for orally effective drugs for the treatment of iron overload disorders is an important goal in improving the health of patients suffering diseases such as beta-thalassemia major. Herein, we report the syntheses and characterization of some new members of a series of N-aroyl-N'-picolinoyl hydrazine chelators (the H2IPH analogs). Both 1:1 and 1:2 Fe-III:L complexes were isolated and the crystal structures of Fe(HPPH)Cl-2, Fe(4BBPH)Cl-2, Fe(HAPH)(APH) and Fe(H3BBPH)(3BBPH) were determined (H2PPH=N,N'-bis-picolinoyl hydrazine; H(2)APH=N-4-aminobenzoyl-N'-picolinoyl hydrazine, H(2)3BBPH=N-3-bromobenzoyl-N'-picolinoylhydrazine and H(2)4BBPH=N-(4-bromobenzoyl)-N'-(picolinoyl)hydrazine). In each case, a tridentate N,N,O coordination mode of each chelator with Fe was observed. The Fe-III complexes of these ligands have been synthesized and their structural, spectroscopic and electrochemical characterization are reported. Five of these new chelators, namely H2BPH (N-(benzoyl)-N'-(picolinoyl)hydrazine), H2TPH (N-(2-thienyl)-N'-(picolinoyl)-hydrazine), H2PPH, H(2)3BBPH and H(2)4BBPH, showed high efficacy at mobilizing Fe-59 from cells and inhibiting Fe-59 uptake from the serum Fe transport protein, transferrin (Tf). Indeed, their activity was much greater than that found for the chelator in current clinical use, desferrioxamine (DFO), and similar to that observed for the orally active chelator, pyridoxal isonicotinoyl hydrazone (H2PIH). The ability of the chelators to inhibit Fe-59 uptake could not be accounted for by direct chelation of Fe-59-Tf. The most effective chelators also showed low antiproliferative activity which was similar to or less than that observed with DFO, which is important in terms of their potential use as agents to treat Fe-overload disease.

Pharmacokinetic considerations relating to tacrolimus dosing in the elderly

Relaxation of the upper age limits for solid organ transplantation coupled with improvements in post-transplant survival have resulted in greater numbers of elderly patients receiving immunosuppressant drugs such as tacrolimus. Tacrolimus is a potent agent with a narrow therapeutic window and large inter- and intraindividual pharmacokinetic variability. Numerous physiological changes occur with aging that could potentially affect the pharmacokinetics of tacrolimus and, hence, patient dosage requirements. Tacrolimus is primarily metabolised by cytochrome P450 (CYP) 3A enzymes in the gut wall and liver. It is also a substrate for P-glycoprotein, which counter-transports diffused tacrolimus out of intestinal cells and back into the gut lumen. Age-associated alterations in CYP3A and P-glycoprotein expression and/or activity, along with liver mass and body composition changes, would be expected to affect the pharmacokinetics of tacrolimus in the elderly. However, interindividual variation in these processes may mask any changes caused by aging. More investigation is needed into the impact aging has on CYP and P-glycoprotein activity and expression. No single-dose, intense blood-sampling study has specifically compared the pharmacokinetics of tacrolimus across different patient age groups. However, five population pharmacokinetic studies, one in kidney, one in bone marrow and three in liver transplant recipients, have investigated age as a co-variate. None found a significant influence for age on tacrolimus bioavailability, volume of distribution or clearance. The number of elderly patients included in each study, however, was not documented and may have been only small. It is likely that inter- and intraindividual pharmacokinetic variability associated with tacrolimus increase in elderly populations. In addition to pharmacokinetic differences, donor organ viability, multiple co-morbidity, polypharmacy and immunological changes need to be considered when using tacrolimus in the elderly. Aging is associated with decreased immunoresponsiveness, a slower body repair process and increased drug adverse effects. Elderly liver and kidney transplant recipients are more likely to develop new-onset diabetes mellitus than younger patients. Elderly transplant recipients exhibit higher mortality from infectious and cardiovascular causes than younger patients but may be less likely to develop acute rejection. Elderly kidney recipients have a higher potential for chronic allograft nephropathy, and a single rejection episode can be more devastating. There is a paucity of information on optimal tacrolimus dosage and target trough concentration in the elderly. The therapeutic window for tacrolimus concentrations may be narrower. Further integrated pharmacokinetic-pharmaco-dynamic studies of tacrolimus are required. It would appear reasonable, based on current knowledge, to commence tacrolimus at similar doses as those used in younger patients. Maintenance dose requirements over the longer term may be lower in the elderly, but the increased variability in kinetics and the variety of factors that impact on dosage suggest that patient care needs to be based around more frequent monitoring in this age group.

Cyclosporine A induced changes to plasma and erythrocyte antioxidant defences

Organ transplant recipients develop pronounced cardiovascular disease, and decreased antioxidant capacity in plasma and erythrocytes is associated with the pathogenesis of this disease. These experiments tested the hypothesis that the immunosuppressant cyclosporine A (CsA) alters erythrocyte redox balance and reduces plasma antioxidant capacity. Female Sprague-Dawley rats were randomly assigned to a control or CsA treated group. Treatment animals received 25 mg/kg/day of CsA via intraperitoneal injection for 18 days. Control rats were injected with the same volume of the vehicle. Three hours after the final CsA injection, rats were exsanguinated and plasma analysed for total antioxidant status (TAS), alpha-tocopherol, malondialdehyde (MDA), and creatinine. Erythrocytes were analysed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities, alpha-tocopherol, and MDA. CsA administration resulted in a significant (P < 0.05) decrease in plasma TAS and significant increases (P < 0.05) in plasma creatinine and MDA. Erythrocyte CAT was significantly (P < 0.05) increased in CsA treated rats compared to controls. There were no significant differences (P > 0.05) in erythrocyte SOD, GPX, G6PD, alpha-tocopherol or MDA between groups. In summary, CsA alters erythrocyte antioxidant defence and decreases plasma total antioxidant capacity.

Pioglitazone inhibits cell growth and reduces matrix production in human kidney fibroblasts

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists are increasingly used in patients with diabetes, and small studies have suggested a beneficial effect on renal function, but their effects on. extracellular matrix (ECM) turnover are unknown. The aims of this study were to investigate the effects of the PPAR-gamma agonist pioglitazone on growth and matrix production in human cortical fibroblasts (CF). Cell growth and ECM production and turnover were measured in human CF in the presence and absence of 1 and 3 muM pioglitazone. Exposure of CF to pioglitazone caused an antiproliferative (P < 0.0001) and hypertrophic (P < 0.0001) effect; reduced type IV collagen secretion (P < 0.01), fibronectin secretion (P < 0.0001), and proline incorporation (P < 0.0001); decreased MMP-9 activity (P < 0.05); and reduced tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 secretion (P < 0.001 and P < 0.0001, respectively). These effects were independent of TGF-beta1. A reduction in ECM production was similarly observed when CF were exposed to a selective PPAR-gamma agonist (L-805645) in concentrations that caused no toxicity, confirming the antifibrotic effects of pioglitazone were mediated through a PPAR-gamma-dependent mechanism. Exposure of CF to high glucose conditions induced an increase in the expression of collagen IV (P < 0.05), which was reversed both in the presence of pioglitazone (1 and 3 muM) and by L-805645. In summary, exposure of human CIF to pioglitazone causes an antiproliferative effect and reduces ECM production through mechanisms that include reducing TIMP activity, independent of TGF-beta1. These studies suggest that the PPAR-gamma agonists may have a specific role in ameliorating the course of progressive tubulointerstitial fibrosis under both normoglycemic and hyperglycemic states.

Polymorphisms in glutathione S-transferases and non-melanoma skin cancer risk in Australian renal transplant recipients

Caucasian renal transplant recipients from Queensland, Australia have the highest non-melanoma skin cancer (NMSC) risk worldwide. Although ultraviolet light (UVR) exposure is critical, genetic factors also appear important. We and others have shown that polymorphism in the glutathione S-transferases (GST) is associated with NMSC in UK recipients. However, the effect of high UVR exposure and differences in immunosuppressive regimen on these associations is unknown. In this study, we examined allelism in GSTM1, GSTM3, GSTT1 and GSTP1 in 361 Queensland renal transplant recipients. Data on squamous (SCC) and basal cell carcinoma (BCC), UVR/tobacco exposure and genotype were obtained. Associations with both NMSC risk and numbers were examined using logistic and negative binomial regression, respectively. In the total group, GSTM1 AB [P = 0.049, rate ratio (RR) = 0.23] and GSTM3 AA (P = 0.015, RR = 0.50) were associated with fewer SCC. Recipients were then stratified by prednisolone dose (less than or equal to7 versus >7 mg/day). In the low-dose group, GSTT1 null (P = 0.006, RR = 0.20) and GSTP1 Val/Val (P = 0.021, RR = 0.20) were associated with SCC numbers. In contrast, in the high-dose group, GSTM1 AB (P = 0.009, RR = 0.05), GSTM3 AB (P = 0.042, RR = 2.29) and BB (P = 0.014, RR = 5.31) and GSTP1 Val/Val (P = 0.036, RR = 2.98) were associated with SCC numbers. GSTM1 AB (P = 0.016) and GSTP1 Val/Val (P = 0.046) were also associated with fewer BCC in this group. GSTP1 associations were strongest in recipients with lower UVR/tobacco exposure. The data confirm our UK findings, suggesting that protection against UVR-induced oxidative stress is important in NMSC development in recipients, but that this effect depends on the immunosuppressant regimen.

Generalized, pruritic, ulcerating maculopapular rash necessitating cessation of sirolimus in a liver transplantation patient

The use of sirolimus as an alternative to calcineurin antagonists has enabled the continuation of immunosuppression in patients with renal impairment with preservation of kidney function. Sirolimus is generally well tolerated, with the main causes of cessation of therapy related to its effect on blood lipid profile as well as leukopenia and thrombocytopenia. We report a case of a debilitating ulcerating maculopapular rash necessitating cessation of the drug in a liver transplantation patient. A 56-year-old Caucasian liver transplantation patient presented with a diffuse, debilitating rash attributed to sirolimus use. This ultimately necessitated cessation of the immunosuppressant with subsequent resolution of her symptoms. From a review of the current literature, this is a highly unusual adverse reaction to sirolimus.

Complexes of cytotoxic chelators from the dipyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

In an effort to better understand the antiproliferative effects of the tridentate hydrazone chelators di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and di-2-pyridyl ketone benzoyl hydrazone (HPKBH), we report the coordination chemistry of these ligands with the divalent metal ions, Mn, Co, Ni, Cu, and Zn. These complexes are compared with their Fe-II analogues which were reported previously. The crystal structures of Co(PKIH)(2), Ni(PKIH)(2), Cu(PKIH)(2), Mn(PKBH)(2), Ni(PKBH)(2), Cu(PKBH)(2), and Zn(PKBH)(2) are reported where similar bis-tridenate coordination modes of the ligands are defined. In pure DMF, all complexes except the Zn-II compounds exhibit metal-centered M-III/II (Mn, Fe, Co, Ni) or M-II/I (Cu) redox processes. All complexes show ligand-centered reductions at low potential. Electrochemistry in a mixed water/DMF solvent only elicited metal-centered responses from the Co and Fe complexes. Remarkably, all complexes show antiproliferative activity against the SK-N-MC neuroepithelioma cell line similar to (HPKIH) or significantly greater than that of the (HPKBH) ligand which suggests a mechanism that does not only involve the redox activity of these complexes. In fact, we suggest that the complexes act as lipophilic transport shuttles that allow entrance to the cell and enable the delivery of both the ligand and metal which act in concert to inhibit proliferation.

Dipyridyl thiosemicarbazone chelators with potent and selective antitumor activity form iron complexes with redox activity

There has been much interest in the development of iron (Fe) chelators for the treatment of cancer. We developed a series of di-2-pyridyl ketone thiosemicarbazone (HDpT) ligands which show marked and selective antitumor activity in vitro and in vivo. In this study, we assessed chemical and biological properties of these ligands and their Fe complexes in order to understand their marked activity. This included examination of their solution chemistry, electrochemistry, ability to mediate redox reactions, and antiproliferative activity against tumor cells. The higher antiproliferative efficacy of the HDpT series of chelators relative to the related di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues can be ascribed, in part, to the redox potentials of their Fe complexes which lead to the generation of reactive oxygen species. The most effective HDpT ligands as antiproliferative agents possess considerable lipophilicity and were shown to be charge neutral at physiological pH, allowing access to intracellular Fe pools.

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 mu l) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3 -> 255.3). The assay was linear from 0.2 to 25 mu g/l (r(2) > 0.997, n = 5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 mu g/l) were 95.8-103.2 and < 5.5%, respectively. At the lower limit of quantification (0.2 mu g/l) the interand intra-day analytical recovery was 99.0-102.8% with imprecision of < 7.6% (n = 5). The assay had a mean relative recovery of 100.5 +/- 5.8% (n = 15). Extracted samples were stable for 16 h. IFTY720 quality control samples were stable at room temperature for 16 h at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans. (c) 2006 Elsevier B.V. All rights reserved.

Comparison of the reintroduced MEIA (R) assay with HPLC-MS/MS for the determination of whole-blood sirolimus from transplant recipients

Therapeutic monitoring with dosage individualization of sirolimus drug therapy is standard clinical practice for organ transplant recipients. For several years sirolimus monitoring has been restricted as a result of lack of an immunoassay. The recent reintroduction of the microparticle enzyme immunoassay (MEIA (R)) for sirolimus on the IMx (R) analyser has the potential to address this situation. This Study, using patient samples, has compared the MEIA (R) sirolimus method with an established HPLC-tandem mass spectrometry method (HPLC-MS/MS). An established HPLC-UV assay was used for independent cross-validation. For quality control materials (5, 11, 22 mu g/L), the MEIA (R) showed acceptable validation criteria based on intra-and inter-run precision (CV) and accuracy (bias) of < 8% and < 13%, respectively. The lower limit of quantitation was found to be approximately 3 mu g/L. The performance of the immunoassay was compared with HPLC-MS/MS using EDTA whole-blood samples obtained from various types of organ transplant recipients (n = 116). The resultant Deming regression line was: MEIA = 1.3 x HPLC-MS/MS+ 1.3 (r = 0.967, s(y/x) = 1) with a mean bias of 49.2% +/- 23.1 % (range, -2.4% to 128%; P < 0.001). The reason for the large and variable bias was not explored in this study, but the sirolimus-metabolite cross-reactivity with the MEIA (R) antibody could be a substantive contributing factor. Whereas the MEIA (R) sirolimus method may be an adjunct to sirolimus dosage individualization in transplant recipients, users must consider the implications of the substantial and variable bias when interpreting results. In selected patients where difficult clinical issues arise, reference to a specific chromatographic method may be required.

Role of lipid-mobilising factor (LMF) in protecting tumour cells from oxidative damage

Lipid-mobilising factor (LMF) is produced by cachexia-inducing tumours and is involved in the degradation of adipose tissue, with increased oxidation of the released fatty acids through an induction of uncoupling protein (UCP) expression. Since UCP-2 is thought to be involved in the detoxification of free radicals if LMF induced UCP-2 expression in tumour cells, it might attenuate free radical toxicity. As a model system we have used MAC13 tumour cells, which do not produce LMF. Addition of LMF caused a concentration-dependent increase in UCP-2 expression, as determined by immunoblotting. This effect was attenuated by the β3 antagonist SR59230A, suggesting that it was mediated through a β3 adrenoreceptor. Co-incubation of LMF with MAC13 cells reduced the growth-inhibitory effects of bleomycin, paraquat and hydrogen peroxide, known to be free radical generators, but not chlorambucil, an alkylating agent. There was no effect of LMF alone on cellular proliferation. These results indicate that LMF antagonises the antiproliferative effect of agents working through a free radical mechanism, and may partly explain the unresponsiveness to the chemotherapy of cachexia-inducing tumours. © 2004 Cancer Research UK.