Core shell lipid-polymer hybrid nanoparticles with combined docetaxel and molecular targeted therapy for the treatment of metastatic prostate cancer.

Many prostate cancers relapse after initial chemotherapy treatment. Combining molecular and chemotherapy together with encapsulation of drugs in nanocarriers provides effective drug delivery and toxicity reduction. We developed core shell lipid-polymer hybrid nanoparticles (CSLPHNPs) with poly (lactic-co-glycolic acid) (PLGA) core and lipid layer containing docetaxel and clinically used inhibitor of sphingosine kinase 1 (SK1) FTY720 (fingolimod). We show for the first time that FTY720 (both free and in CSLPHNPs) re-sensitizes castrate resistant prostate cancer cells and tumors to docetaxel, allowing a four-fold reduction in effective dose. Our CSLPHNPs showed high serum stability and a long shelf life. CSLPHNPs demonstrated a steady uptake by tumor cells, sustained intracellular drug release and in vitro efficacy superior to free therapies. In a mouse model of human prostate cancer, CSLPHNPs showed excellent tumor targeting and significantly lower side effects compared to free drugs, importantly, reversing lymphopenia induced by FTY720. Overall, we demonstrate that nanoparticle encapsulation can improve targeting, provide low off-target toxicity and most importantly reduce FTY720-induced lymphopenia, suggesting its potential use in clinical cancer treatment.

Combination of RAD001 (everolimus) and docetaxel reduces prostate and breast cancer cell VEGF production and tumour vascularisation independently of sphingosine-kinase-1.

Resistance to docetaxel is a key problem in current prostate and breast cancer management. We have recently discovered a new molecular mechanism of prostate cancer docetaxel chemoresistance mediated by the mammalian target of rapamycin (mTOR)/sphingosine-kinase-1 (SK1) pathway. Here we investigated the influence of this pathway on vascular endothelial growth factor (VEGF) production and tumour vascularisation in hormone resistant prostate and breast cancer models. Immunofluorescent staining of tumour sections from human oestrogen receptor (ER)-negative breast cancer patients showed a strong correlation between phosphorylated P70S6 kinase (mTOR downstream target), VEGF and SK1 protein expression. In hormone-insensitive prostate (PC3) and breast (MDA-MB-231 and BT-549) cancer cell lines the mTOR inhibitor RAD001 (everolimus) has significantly inhibited SK1 and VEGF expression, while low dose (5 nM) docetaxel had no significant effect. In these cell lines, SK1 overexpression slightly increased the basal levels of VEGF, but did not block the inhibitory effect of RAD001 on VEGF. In a human prostate xenograft model established in nude mice, RAD001 alone or in combination with docetaxel has suppressed tumour growth, VEGF expression and decreased tumour vasculature. Overall, our data demonstrate a new mechanism of an independent regulation of SK1 and VEGF by mTOR in hormone-insensitive prostate and breast cancers.

Everolimus (RAD001) sensitizes prostate cancer cells to docetaxel by down-regulation of HIF-1α and sphingosine kinase 1.

Resistance to docetaxel is a key problem in current prostate cancer management. Sphingosine kinase 1 (SK1) and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways have been implicated in prostate cancer chemoresistance. Here we investigated whether their combined targeting may re-sensitize prostate cancer cells to docetaxel.In hormone-insensitive PC-3 and DU145 prostate cancer cells the mTOR inhibitor everolimus (RAD001) alone did not lead to significant cell death, however, it strongly sensitized cells to low levels (5 nM) of docetaxel. We show that mTOR inhibition has led to a decrease in hypoxia-inducible factor-1α (HIF-1α) protein levels and SK1 mRNA. HIF-1α accumulation induced by CoCl2 has led to a partial chemoresistance to RAD001/docetaxel combination. SK1 overexpression has completely protected prostate cancer cells from RAD001/docetaxel effects. Using gene knockdown and CoCl2 treatment we showed that SK1 mRNA expression is downstream of HIF-1α. In a human xenograft model in nude mice single RAD001 and docetaxel therapies induced 23% and 15% reduction in prostate tumor volume, respectively, while their combination led to a 58% reduction. RAD001 alone or in combination with docetaxel has suppressed intratumoral mTOR and SK1 signaling, however as evidenced by tumor size, it required docetaxel for clinical efficacy. Combination therapy was well tolerated and had similar levels of toxicity to docetaxel alone.Overall, our data demonstrate a new mechanism of docetaxel sensitization in prostate cancer. This provides a mechanistic basis for further clinical application of RAD001/docetaxel combination in prostate cancer therapy.

High cut-off dialysis in chronic haemodialysis patients.

BACKGROUND: Haemodialysis patients suffer from chronic systemic inflammation and high incidence of cardiovascular disease. One cause for this may be the failure of diseased kidneys to eliminate immune mediators. Current haemodialysis treatment achieves insufficient elimination of proteins in the molecular weight range 15-45 kD. Thus, high cut-off dialysis might improve the inflammatory state. DESIGN: In this randomized crossover trial, 43 haemodialysis patients were treated for 3 weeks with high cut-off or high-flux dialysis. Inflammatory plasma mediators, monocyte subpopulation distribution and leucocyte gene expression were quantified. RESULTS: High cut-off dialysis supplemented by a low-flux filter did not influence the primary end-point, expression density of CD162 on monocytes. Nevertheless, treatment reduced multiple immune mediators in plasma. Such reduction proved - at least for some markers - to be a sustained effect over the interdialytic interval. Thus, for example, soluble TNF-receptor 1 concentration predialysis was reduced from median 13·3 (IQR 8·9-17·2) to 9·7 (IQR 7·5-13·2) ng/mL with high cut-off while remaining constant with high-flux treatment. The expression profile of multiple proinflammatory genes in leucocytes was significantly dampened. Treatment was well tolerated although albumin losses in high cut-off dialysis would be prohibitive against long-term use. CONCLUSIONS: The study shows for the first time that a dampening effect of high cut-off dialysis on systemic inflammation is achievable. Earlier studies had failed due to short study duration or insufficient dialysis efficacy. Removal of soluble mediators from the circulation influences cellular activation levels in leucocytes. Continued development of less albumin leaky membranes with similar cytokine elimination is justified.

Therapeutic potential of targeting SK1 in human cancers.

Sphingosine kinase 1 (SK1) is a lipid enzyme with oncogenic properties that converts the proapoptotic lipids ceramide and sphingosine into the antiapoptotic lipid sphingosine-1-phosphate and activates the signal transduction pathways that lead to cell proliferation, migration, the activation of the inflammatory response, and the impairment of apoptosis. There is compelling evidence that SK1 activation contributes to cancer progression leading to increased oncogenic transformation, tumor growth, resistance to therapies, tumor neovascularization, and metastatic spread. High levels of SK1 expression or activity have been associated with a poor prognosis in several human cancers. Recent studies using cancer cell and mouse models demonstrate a significant potential for SK1-targeting therapies to synergize with the effects of chemotherapy and radiotherapy; however, until recently the absence of clinically applicable SK1 inhibitors has limited the translation of these findings into patients. With the recent discovery of SK1 inhibiting properties of a clinically approved drug FTY720 (Fingolimod), SK1 has gained significant attention from both clinicians and the pharmaceutical industry and it is hoped that trials of newly developed SK1 inhibitors may follow soon. This review provides an overview of the SK1 signaling, its relevance to cancer progression, and the potential clinical significance of targeting SK1 for improved local or systemic control of human cancers.

Randomized controlled pilot study of 2 weeks' treatment with high cutoff membrane for hemodialysis patients with elevated C-reactive protein.

Chronic inflammation in hemodialysis (HD) patients is associated with cardiovascular complications and mortality. Circulating immune active proteins in the molecular range 15-45 kD that cannot be efficiently cleared by high-flux (HF) dialysis may be causally involved. We intended to test the feasibility of using a high cutoff (HCO) dialyzer in chronic HD patients and its influence on inflammation and monocyte activation. The Gambro HCO1100 dialyzer was compared to a conventional HF membrane in a randomized double-blind crossover trial in 19 chronic HD patients selected for the presence of elevated serum C-reactive protein levels. Patients were treated for six consecutive dialysis sessions (2 weeks) with each membrane. Safety analysis recorded adverse events and albumin losses through the protein-leaking membranes. Efficacy analysis observed reductions in the number of proinflammatory (CD14+CD16+) monocyte subpopulations in circulating blood. Treatment with the HCO membrane was well tolerated, although the number of adverse events was slightly higher. Despite significant serum albumin loss (from 34.1 ± 2.7 to 29.6 ± 3.0 g/L; P < 0.01), there was no need to supplement albumin, and rising activity of cholinesterase during HCO treatment indicated compensation by enhanced hepatic synthesis. The HCO membrane cleared high amounts of proinflammatory cytokines, but did not reduce predialysis inflammatory monocytes and markers. Although the time of HD session was extended, the study was hampered by a lower Kt/V in the HCO compared to the HF period. Treatment of chronic HD patients with this HCO dialyzer for 2 weeks is tolerable in terms of albumin loss and able to clear proinflammatory cytokines; however, this was not sufficient to decrease monocyte activation. Therefore, a more selective, less albumin-leaking membrane is desirable to allow prolonged high-efficient dialysis with more effective cytokine clearance.

Therapeutic potential of targeting sphingosine kinase 1 in prostate cancer.

Sphingosine kinase 1 (SK1) is a lipid enzyme with oncogenic properties that converts the proapoptotic lipid sphingosine into the antiapoptotic lipid sphingosine-1-phosphate, which activates the signal transduction pathways that lead to cell proliferation, migration, activation of the inflammatory response and impairment of apoptosis. Compelling evidence suggests that SK1 activation contributes to cancer progression leading to increased oncogenic transformation, tumor growth, resistance to therapies, tumor neovascularization and metastatic spread. High levels of SK1 expression or activity have been associated with poor prognosis in several cancers, including those of the prostate. Recent studies using prostate cancer cell and mouse models demonstrate a significant potential for SK1-targeting therapies to synergize with the effects of docetaxel chemotherapy and radiotherapy. However, until recently the absence of clinically applicable SK1 inhibitors has limited the translation of these findings into patients. With the recent discovery that clinically approved drug fingolimod has SK1-inhibiting properties, SK1 has gained significant attention from both clinicians and the pharmaceutical industry and it is hoped that trials of newly developed SK1 inhibitors might follow soon.

FTY720 (fingolimod) sensitizes prostate cancer cells to radiotherapy by inhibition of sphingosine kinase-1.

Radiotherapy is widely used as a radical treatment for prostate cancer, but curative treatments are elusive for poorly differentiated tumors where survival is just 15% at 15 years. Dose escalation improves local response rates but is limited by tolerance in normal tissues. A sphingosine analogue, FTY720 (fingolimod), a drug currently in phase III studies for treatment of multiple sclerosis, has been found to be a potent apoptosis inducer in prostate cancer cells. Using in vitro and in vivo approaches, we analyzed the impact of FTY720 on sphingolipid metabolism in hormone-refractory metastatic prostate cancer cells and evaluated its potential as a radiosensitizer on cell lines and prostate tumor xenografts. In prostate cancer cell lines, FTY720 acted as a sphingosine kinase 1 (SphK1) inhibitor that induced prostate cancer cell apoptosis in a manner independent of sphingosine-1-phosphate receptors. In contrast, γ irradiation did not affect SphK1 activity in prostate cancer cells yet synergized with FTY720 to inhibit SphK1. In mice bearing orthotopic or s.c. prostate cancer tumors, we show that FTY720 dramatically increased radiotherapeutic sensitivity, reducing tumor growth and metastasis without toxic side effects. Our findings suggest that low, well-tolerated doses of FTY720 could offer significant improvement to the clinical treatment of prostate cancer.

Biomarkers as a tool for management of immunosuppression in transplant patients.

Therapeutic drug monitoring is a well-established approach in transplantation medicine to guide immunosuppressive therapy. However, it cannot always predict the effects of immunosuppressive drugs on immune cells, because it does not reflect any aspect of an individual patient's immune system. Pharmacodynamic monitoring is a more recent strategy to provide information about the biologic effect of a specific drug or drug combination on the individual transplant patient. Currently, there is a large number of different biomarkers that either directly (specific markers) or indirectly (global markers) relate to the pharmacodynamic effects of immunosuppressive drugs and are under investigation as potential candidates to be introduced in clinical practice. Such biomarkers may be useful to identify patients at risk of developing acute rejection, infection, or cancer as well as patients who are suitable for minimization of immunosuppressant therapy and may be helpful to manage the timing and rate of immunosuppressant weaning. Serial longitudinal monitoring may allow maintenance of an individualized immunosuppressive regimen. Thus, biomarker monitoring is a potential complementary tool to therapeutic drug monitoring. This review summarizes the current state of knowledge about the use of a number of global or drug-specific pharmacodynamic biomarkers. It is not a comprehensive overview of the literature available, but rather an evidence-based reflection by experts who are intensively involved in scientific work in this field.

Polyoma BK virus-associated nephropathy in kidney-transplant patients: Effects of leflunomide on T-cell functions and disease outcome.

BACKGROUND: In kidney-transplant recipients, leflunomide has been shown to be efficient for treating polyomavirus BK virus-associated-nephropathy (PVAN). However, it is unknown whether the beneficial effect of leflunomide is related to it having a lower immunosuppressive effect than mycophenolate mofetil (MMF), or to its anti-viral activity. The aim of this study was to assess i) T-cell functions before and after conversion from MMF to leflunomide in kidney-transplant patients with PVAN, and ii) effects of leflunomide on PVAN outcome. PATIENTS AND METHODS: Twelve patients were enrolled in this study. At PVAN diagnosis, MMF was replaced by leflunomide. Other immunosuppressive drug doses and levels were maintained unchanged. T-cell functions, i.e., intralymphocyte cytokine expression (IL-2 and TNF-alpha), T-cell activation [i.e., transferrin receptor (CD71) and interleukin (IL)-2 alpha-chain (CD25) expression], and T-cell proliferation were measured using a flow-cytometry whole-blood assay before and at one month after conversion. RESULTS: Despite a slight decrease in tacrolimus trough levels, no significant change in T-cell-function biomarkers was observed after conversion. After a follow-up of 6 (4-30) months, five patients were cleared of the virus, and decreased viral load was observed in four patients. Only one patient suffered a graft loss. No difference in immunological parameters was observed between patients who were cleared or not of BKV. CONCLUSION: Results of this pilot study suggest that the potential benefits of leflunomide to treat PVAN in kidney-transplant patients is not related to reduced immunosuppression induced by replacing MMF by leflunomide. Virological studies are required to determine the anti-BKV effect of leflunomide.

Pharmacodynamic evaluation of the first dose of mycophenolate mofetil before kidney transplantation.

BACKGROUND AND OBJECTIVES: The effect of mycophenolate mofetil (MMF) on T cell function has not been evaluated in patients undergoing kidney transplantation. The aim of this study was to assess the effect of 1g of MMF on T cell function, that is, intralymphocyte cytokine expression, T cell activation (CD25 and CD71), and T cell proliferation, as well as inosine monophosphate dehydrogenase (IMPDH) activity, to better understand the relationship between pharmacokinetic and pharmacodynamic markers in patients receiving the first dose of MMF before kidney transplantation. PATIENTS: Twenty-four patients undergoing a kidney transplantation from a living donor were enrolled in this study. RESULTS: Compared with baseline (before MMF intake), T cell proliferation (93%), IMPDH activity (74%), CD25 (46%), and CD71 (50%) expression significantly decreased during the first hour after MMF intake, in parallel to the rise in MPA concentration. Thereafter, all pharmacodynamic markers, except IMPDH activity, returned back to baseline level. There was a complex inverse relationship between pharmacokinetic and pharmacodynamic markers. The inhibition of T cell proliferation was highly correlated to IMPDH activity, but also to T cell activation markers. CONCLUSION: The administration of MMF to patients is associated not only with a dramatic decrease in both T cell proliferation and IMPDH activity, but also with in a decrease in CD25 and CD71 expression.

Cytokines correlate with age in healthy volunteers, dialysis patients and kidney-transplant patients.

T-cell functions are currently used as biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs or as disease biomarkers of inflammation/sepsis and organ rejection. In order to evaluate co-factors potentially influencing the expression of the immunological biomarkers, we explored T-cell proliferation, T-cell activation (CD25 and CD71 expressions) and intra-lymphocyte cytokine production (interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha) in healthy volunteers, dialysis patients and stable kidney-transplant patients treated with standard immunosuppressive therapy, i.e. tacrolimus, mycophenolic acid with or without steroids. Age was positively correlated with TNF-alpha expression in all three patient populations, and with IL-2 expression in healthy volunteers and kidney-transplant patients. Further age was correlated with inhibition of lymphocyte proliferation in healthy volunteers and with the T-cell activation marker CD25 in kidney-transplant patients. In healthy volunteers lymphocyte proliferation was higher in woman as compared to men. Other biomarkers of T-cell function were independent of the gender. In the kidney-transplant patient group a significantly lower expression of all biomarkers of T-cell functions compared to healthy volunteers and dialysis patients. In dialysis patients we found significant increased IL-2 expression compared to healthy volunteers, while the other T-cell functions were not significantly different. Further time on dialysis had no effect on the level of biomarker expression. In conclusion we found decreased T-cell functions in kidney-transplant patients compared to healthy volunteers and dialysis patients, increased IL-2 expression in dialysis patients compared to healthy volunteers and in all three populations we found a correlation of age and intra-T-lymphocyte TNF-alpha expression.

In vitro mitogen-stimulated T-cell from hepatitis C virus-positive liver transplantation candidates, increases T-cell activation markers and T-cell proliferation.

The incidence of acute rejection is significantly higher in hepatitis C virus (HCV) liver-transplant patients than in patients who have received a graft for other liver diseases, i.e., mainly alcoholic cirrhosis. The aim of this study was to assess T-cell function, i.e., intralymphocyte cytokine expression (IL-2 and TNF-alpha), T-cell activation [i.e., transferrin receptor (CD71) and interleukin (IL)-2 alpha-chain (CD25) expression], and T-cell proliferation using a flow-cytometry whole-blood assay in patients waiting for a liver transplantation (n=49). Our data suggest that, in mitogen-stimulated T-cells, (i) intra-lymphocyte cytokine expression is significantly higher in patients with liver disease than in healthy volunteers (n=25); (ii) the expression of T-cell activation markers is decreased in patients with liver cirrhosis compared to healthy volunteers, and (iii) the expression of T-cell activation markers and T-cell proliferation are increased in patients with HCV infection (n=15) compared to those without HCV infection (n=34), particularly compared to patients with alcoholic liver disease (n=19). Circulating CD19-positive cells count was also significantly higher in HCV-positive patients. In conclusion, in vitro, mitogen-stimulated T-cell seem to induce a higher immune response in the blood from patients waiting for a liver transplant for HCV-related liver disease than those without HCV infection, and particularly those with alcoholic liver disease.

Pharmacodynamic effects of everolimus on anti-CD3 antibody-stimulated T-lymphocyte proliferation and interleukin-10 synthesis in stable kidney-transplant patients.

Everolimus (rapamycin derivative, RAD) is a new immunosuppressive drug that prevents allograft rejection. Herein, the pharmacodynamics of everolimus in human renal-allograft recipients is evaluated. Single doses of everolimus (0.75-10mg), combined with a maintenance immunosuppressive therapy based on CyA, decreased lymphocyte proliferation. In addition, the effect of multiple doses of everolimus (0.75-10mg) given daily for 21 days, to stable renal-allograft patients (n=11), was investigated. Everolimus treatment resulted in immediate inhibition (25-55%) of lymphocyte proliferation in renal-allograft recipients; values returning to baseline by 14 days after cessation of everolimus treatment. Placebo-treated patients showed no decrease in lymphocyte proliferation. Interestingly, everolimus reduced IL-10 synthesis by 20-60% in renal-allograft recipients. Phagocytosis rates were not changed by everolimus. In vitro, everolimus inhibited lymphocyte proliferation and IL-10 synthesis dose dependently in anti-CD3 mAb and LPS stimulated peripheral blood mononuclear cell cultures derived from human volunteers.

Pharmacodynamic monitoring of the conversion from mycophenolate mofetil to enteric-coated mycophenolate sodium in stable kidney-allograft recipients.

BACKGROUND: The formulations of mycophenolic acid, i.e., mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS), seem to have different pharmacokinetic profiles. The aim of this study was to compare the effects MMF and EC-MPS on T-cell proliferation, T-cell activation, T-cell function, and lymphocyte subsets. CLINICAL STUDY AND METHODS: Ten stable kidney-transplant patients on standard maintenance therapy of tacrolimus and MMF (1 g/d), with or without steroids, were converted from MMF to EC-MPS at equivalent dose (720 mg/d). Tacrolimus and steroid doses remained unchanged before, and at 1, 2, 3, and 6 months (M) after conversion. Intra T-lymphocyte cytokines IL-2 and TNF-alpha, lymphocyte-activation surface markers (CD25 and CD71), T-cell proliferation (PCNA+ PI(high)), total lymphocyte count, as well as lymphocytes subsets (CD2, CD3, CD4, CD8, CD19, NK cells) were measured by flow cytometry before conversion and at M1, M2, M3, and M6. RESULTS: We found no significant differences of MMF versus EC-MPS on lymphocyte function. T-cell proliferation and T-cell activation (CD25 and CD71 expression), but not cytokine expression (TNF-alpha and IL-2), showed a trend to increase after conversion from MMF to EC-MPS. Total lymphocyte, CD2, CD3, CD4, CD8, and NK cells counts were not significantly modified. CONCLUSION: This study revealed a trend to a lower immunosuppression with EC-MPS as compared to MMF in stable renal transplant patients.

Reconstitution of immunodeficient SCID/beige mice with human cells: applications in preclinical studies.

Experimental studies of the in vivo behaviour of human cells and tissues have become possible with the development of immunodeficient mice strains. Such mice accept readily allogeneic or xenogeneic grafts, including grafts of human cells or tissues, without rejection. In this review we describe different immunodeficient mouse strains that have been used for reconstitution by human immune cells. We subsequently go through the experience that we and others have had with reconstitution, and mention the adverse effects, in particular xenogeneic graft versus host reactions. The use of haematopoietic stem cells avoids such reactions but the immunological reconstitution may take several months. We then report the use of immunodeficient mice for the study of chronic vascular rejection of human mesenteric arteries due to cellular or humoral alloreaction. We have shown that SCID/beige mice grafted with a human artery at the place of the aorta developed a thickening of the intima of the human artery after 5-6 weeks, when they were reconstituted with spleen cells from another human donor. The thickening is mainly due to a proliferation of smooth muscle cells. The same type of lesion developed if they received injection of antibodies towards HLA class I antigens. The arteries of the mouse did not develop any lesion. The arterial lesions closely resembled those seen after clinical organ transplantation. Mice that received spleen cells from the same human donor developed little or no lesions. An important aspect of this experimental transplantation model is the possibility to test drugs that may be used in clinical transplantation. In recent experiments we have shown that novel immunosuppressive drugs can inhibit the hyperproliferation of smooth muscle cells in vitro. Preclinical testing in reconstituted SCID/beige mice grafted with human arteries will permit the evaluation of the potential use of these drugs to prevent chronic vascular rejection. The model also allows pharmacodynamic studies that give information on the biological impact of different drugs that may be used in experimental or clinical transplantation.

Tabebuia avellanedae extracts inhibit IL-2-independent T-lymphocyte activation and proliferation.

In order to identify new, immune modulating compounds, aqueous extracts of plants pre-selected on ethno-pharmacological knowledge were screened for inhibitory effects in an anti-CD3 driven lymphocyte proliferation assay (MTT-assay). We found for the extract of the inner bark of Tabebuia avellanedae (Tabebuia) dose dependent and reproducible inhibitory effects on lymphocyte proliferation. We further analyzed Tabebuia in flow cytometry based whole blood T-cell function assays. We found that Tabebuia inhibited dose dependent ConA stimulated T-cell proliferation. Decreased T-lymphocyte proliferation was associated with dose dependent reduction of CD25 and CD71 expression on T-lymphocytes. In contrast Tabebuia exerted no effects on cytokine expression (Il-2 and TNF-alpha) by PMA/Ionomycin stimulated T-lymphocytes. Concentrations of Tabebuia used were not toxic for lymphocytes as verified by trypan blue exclusion assay. Further experiments showed that the immune inhibitory effects by Tabebuia were not mediated by its pharmacological lead compound beta-lapachone and only observed in aqueous but not in ethanol plant extracts.

Current perspectives on FTY720.

The search for effective immunosuppressants with fewer side effects continues not only for transplantation, but also for autoimmune diseases. With a novel mechanism of action (sphingosine-1 receptor modulation), oral FTY720 (fingolimod) has the potential to address this need. FTY720 has been preclinically tested with promising results in transplantation and autoimmune disease models. Phase I studies explored the pharmacokinetics and pharmacodynamics of this novel therapeutic concept. Recently, the surprising results of two sister Phase III studies in de novo renal transplant patients, as well as a Phase II study in patients with relapsing multiple sclerosis, were published. This review discusses these findings as well as their implications for the future of sphingosine-1 receptor modulation.

Validation of immunological biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs in humans.

Pharmacodynamic monitoring (PD) can evaluate the efficacy of immunosuppressive drug therapies. In this study, the expressions of PD biomarkers [lymphocyte proliferation, CD25 and CD71 expression, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-alpha) synthesis] were determined in whole-blood assays and were validated for their application in PD of immune modulators in future clinical trials. Initially, the assay conditions were re-evaluated. The measurement of T-lymphocyte proliferation and activation marker expression in whole-blood cultures resulted in optimized stimulation for 72 hours with 7.5 microg/mL concavalin A. Intracellular cytokine expression of CD3+ T-cells received optimized stimulation for 4 hours with 15 ng/mL phorbol 12-myristate 13-acetate and 0.75 microg/mL ionomycin. Statistical assay parameters (intra-assay, intra-individual, and interindividual variabilities) were determined. It was found that blood storage for up to 24 hours is possible without any change in biomarker expression. Dosage effects of immunosuppressive drugs (tacrolimus, cyclosporin A, sirolimus, mycophenolic acid, and methylprednisolone) were evaluated in vitro and the assay was applied successfully to dialysis, renal transplant, and liver transplant patients. We conclude that these biomarkers used in whole-blood assays are suitable for PD of immune modulators in clinical trials.