Tacrolimus-loaded lecithin-based nanostructured lipid carrier and nanoemulsion with propylene glycol monocaprylate as a liquid lipid: Formulation characterization and assessment of dermal delivery compared to referent ointment.

Nanostructured lipid carriers (NLC) and nanoemulsions (NE) are colloid carriers which could improve dermal delivery of tacrolimus. The aims of this study were to evaluate effects of different formulation and process parameters on physicochemical characteristics and stability of lecithin-based NLC with glyceryl palmitostearate as solid and propylene glycol monocaprylate as liquid lipid and to compare the influence of different inner structure of tacrolimus-loaded NLC and corresponding NE on physicochemical characteristics, stability, entrapment efficiency, in vitro drug release and overall skin performance. Solid/liquid lipid ratio, total amount of lipids, homogenization pressure and cooling after the preparation were identified as critical variables in NLC development. Moreover, tacrolimus-loaded NLC emerged as more stabile carrier than NE. Differential stripping performed on porcine ear skin revealed significantly higher tacrolimus amount in stratum corneum from nanocarriers compared to referent ointment (Protopic®). Similarly the highest amount of tacrolimus in hair follicles was obtained using NLC (268.54 ±â€¯92.38 ng/cm2), followed by NE (128.17 ±â€¯48.87 ng/cm2) and Protopic® (77.61 ±â€¯43.25 ng/cm2). Contrary, the highest permeation rate through full-thickness porcine ear skin was observed for Protopic®, implying that the selection of experimental setup is critical for reliable skin performance assessment. Overall, developed NLC could be suggested as promising carrier in a form of lotion for tacrolimus dermal delivery.

Oral fingolimod reduces glutamate-mediated intracortical excitability in relapsing-remitting multiple sclerosis.

OBJECTIVE: Fingolimod is an effective disease modifying therapy for multiple sclerosis (MS). Beyond its main action on peripheral lymphocytes, several noteworthy side effects have been demonstrated in vitro, among which modulation of neural excitability. Our aim was to explore cortical excitability in vivo in patients treated with fingolimod 0.5mg/day. METHODS: Paired-pulse TMS was applied on the left primary motor cortex in 13 patients affected by relapsing-remitting MS, the day before the first dose of fingolimod (T0) and 60days later (T1). Resting motor threshold, baseline motor evoked potentials, short interval intracortical inhibition (at 1, 3, 5ms) and intracortical facilitation (at 7, 9, 11 and 13ms) were estimated at T0 and T1. RESULTS: Intracortical facilitation was reduced at T1, without any changes in short interval intracortical inhibition. CONCLUSIONS: Fingolimod selectively reduced intracortical facilitation, which is mainly mediated by glutamate. SIGNIFICANCE: This is the first in vivo confirmation of the effects of fingolimod on glutamatergic drive in treated humans. Our results suggest a novel neuromodulatory activity of fingolimod with potential effect on glutamate-mediated excitotoxicity in vivo, as already seen in animal models.

A sphingosine-1 phosphate agonist (FTY720) limits trauma/hemorrhagic shock-induced multiple organ dysfunction syndrome.

BACKGROUND: Trauma/hemorrhagic shock (T/HS) is one of the major consequences of battlefield injury as well as civilian trauma. FTY720 (sphingosine-1-phosphate agonist) has the capability to decrease the activity of the innate and adaptive immune systems and, at the same time, maintain endothelial cell barrier function and vascular homeostasis during stress. For this reason, we hypothesize that FTY720, as part of resuscitation therapy, would limit T/HS-induced multiple organ dysfunction syndrome in a rodent T/HS model. METHODS: Rats subjected to trauma/sham shock (T/SS) or T/HS (30 mm Hg × 90 min) were administered FTY720 (1 mg/kg) post-T/HS during volume resuscitation. Lung injury (permeability to Evans blue dye), polymorphonuclear leukocyte (PMN) priming (respiratory burst activity), and red blood cell (RBC) rigidity were measured. In addition, lymph duct-cannulated rats were used to quantify the effect of FTY720 on gut injury (permeability and morphology) and the biologic activity of T/HS versus T/SS lymph on PMN-RBC and RBC deformability. RESULTS: Trauma/hemorrhagic shock-induced increased lung permeability, PMN priming, and RBC rigidity were all abrogated by FTY720. The systemic protective effect of FTY720 was only partially at the gut level, because FTY720 did not prevent T/HS-induced gut injury (morphology or permeability); however, it did abrogate T/HS lymph-induced increased respiratory burst and RBC rigidity. CONCLUSIONS: FTY720 limited T/HS-induced multiple organ dysfunction syndrome (lung injury, red cell injury, and neutrophil priming) as well as T/HS lymph bioactivity, although it did not limit gut injury.

PLA/PEG-PPG-PEG/dexamethasone implant prepared by hot-melt extrusion for controlled release of immunosuppressive drug to implantable medical devices, Part 2: in vivo evaluation.

Hot-melt extrusion (HME) plays an important role in preparing implants as local drug delivery systems in pharmaceutical fields. Here, a new PLA/PEG-PPG-PEG/Dexamethasone (PLA/F68/Dex) implant prepared by HME has been developed. Importantly, the implant was successfully achieved to control release of immunosuppressive drug to an implanted device. In particular, this implant has not been reported previously in pharmaceutical fields. FTIR and XRD were adopted to investigate the properties of the samples. The in vivo release study showed that the maximum value of Dex release from the implants was approximately 50% at 1 month. The in vivo degradation behavior was determined by UV spectrophotometer and scanning electron microscopy studies, and the weight loss rate of the implants were up to 25% at 1 month. Furthermore, complete blood count (CBC) test, serum chemistry and major organs were performed, and there is no significant lesion and side effects observed in these results. Therefore, the results elucidated that the new PLA/F68/Dex implant prepared by HME could deliver an immunosuppressive drug to control the inflammatory reaction at the implant site.

Evaluation of the lymphocyte trafficking drug FTY720 in vaginal tissues.

BACKGROUND: FTY720 is an immunomodulatory agent that reduces lymphocytes in peripheral tissues and circulation. Such agents may be effective as vaginal microbicides for HIV prevention. Systemic or vaginal application of FTY720 may reduce lymphocyte concentrations in genital tissues, reducing HIV target cell numbers. METHODS: Five female pigtail macaques received topical vaginal gel FTY720 (n = 2), intravenous (i.v.) FTY720 (n = 2), or placebo gel (n = 1) in this pilot study. Circulating and mucosal lymphocytes and genital mucosa, cytokines, and tissue histology were analyzed to document topical and i.v. FTY720 effects. RESULTS: Topical and i.v. FTY720 appeared to decrease the levels of cervicovaginal IL-8, IL-1ra, and genital inflammatory cells. Small sample size precluded statistical analysis. Topical administration had no overt adverse effects. CONCLUSIONS: This study introduces FTY720 as an immunomodulatory agent for the vaginal mucosa, compares topical effects to those of i.v. administration, and provides the basis for future studies involving FTY720 for HIV prevention.

Therapeutic approach to steroid-resistant dermatitis using novel immunomodulator FTY720 (Fingolimod) in combination with betamethasone ointment in NC/Nga mice.

The therapeutic efficacy of the novel immunomodulator FTY720 (Fingolimod), alone and in combination with betamethasone ointment, was examined in the NC/Nga mouse model of spontaneous steroid-resistant dermatitis. Male NC/Nga mice which had developed severe dermatitis were divided into six groups: 1) a biweekly betamethasone group (betamethasone ointment, twice a week), 2) a daily betamethasone group (betamethasone ointment, six times a week), 3) an FTY720 group (FTY720, orally, three times a week), 4) a biweekly combination group (oral FTY720 plus betamethasone ointment, twice a week), 5) a daily combination group (oral FTY720 plus betamethasone ointment, six times a week) and 6) a placebo group (vehicle alone). The therapeutic efficacy was evaluated in terms of severity of dermatitis, epidermal hypertrophy, accumulation and degranulation of mast cells and infiltrated CD3+ T cells into the dermis after 4 weeks of treatment. Biweekly and daily betamethasone treatments had little effect, confirming that the dermatitis was steroid-resistant. In the FTY720 and biweekly combination groups, the dermatitis showed no marked improvement. In the daily combination group, the dermatitis was significantly (p<0.05, Mann-Whitney U-test) improved as compared with the FTY720 group, biweekly and daily betamethasone groups and placebo group. Further, epidermal hypertrophy and accumulation of mast cells were suppressed. Therefore, combination therapy with FTY720 and daily betamethasone ointment is a promising candidate for treatment of steroid-resistant atopic dermatitis.

Novel Th1-biased adjuvant, SPO1, enhances mucosal and systemic immunogenicity of vaccines administered intranasally in mice.

Oil-in-water emulsions are potent human adjuvants commonly used in effective pandemic influenza vaccines; however, such emulsions that can induce both Th1-biased systemic immune responses and strong mucosal immune responses via an easy method of administration are lacking. To address this need for new adjuvants, we developed a novel oil/water emulsion, SPO1, which allows convenient mucosal immunization via an intranasal spray as well as by parenteral routes. Our report shows that SPO1 was able to boost up immunological resistance by inducing effective mucosal and serum antibodies, and the immune response was polarized to a Th1 pattern, as demonstrated by high IgG2α antibody levels and interferon-gamma production by splenocytes from intranasally (i.n.) immunized mice. Up-regulation of co-stimulatory and antigen-presenting molecules on dendritic cells was also observed in vivo after i.n. immunization, suggesting a possible mechanism for the adjuvant effects of SPO1. Another explanation may simply be a depot of antigen at the immunization site, as evidenced by in vivo imaging of i.n. immunized mice. In conclusion, our results demonstrate that a novel oil/water emulsion, SPO1, is a potent Th1 adjuvant for use in influenza and other vaccines, as it induces strong mucosal and systemic immune responses.

FTY720 improves functional recovery after spinal cord injury by primarily nonimmunomodulatory mechanisms.

Spinal cord injury (SCI) is an incapacitating injury that can result in limited functional recovery. We have previously shown increases in the lysophospholipid mediator, sphingosine-1-phosphate (S1P), in the spinal cord after contusion injury. To apply S1P receptor modulation to the treatment of SCI, we examined the therapeutic effects of FTY720, an S1P receptor agonist, on locomotor recovery after SCI in mice. Oral administration of FTY720 shortly after contusion SCI significantly improved motor function recovery, as assessed by both Basso Mouse Scale scores and Rotarod Performance test results. FTY720 induced lymphopenia and reduced T-cell infiltration in the spinal cord after SCI but did not affect the early infiltration of neutrophils and the activation of microglia. In addition, plasma levels and mRNA expression of inflammatory cytokines in the spinal cord after SCI were not attenuated by FTY720. Vascular permeability and astrocyte accumulation were both decreased by FTY720 in the injured spinal cord. The therapeutic effects of FTY720 were not solely dependent on immune modulation, as confirmed by the demonstration that FTY720 also ameliorated motor function after SCI in mice with severe combined immunodeficiency. Finally, the S1P(1) receptor agonist, SEW2871, partly mimicked the therapeutic effect of FTY720. Our data highlight the importance of immune-independent functions of FTY720 in decreasing vascular permeability and astrogliosis in the injured spinal cord and promoting locomotor function recovery after SCI.

Poly(ß-cyclodextrin)/curcumin self-assembly: a novel approach to improve curcumin delivery and its therapeutic efficacy in prostate cancer cells.

A novel PCD/CUR self-assembly approach for improved curcumin delivery to prostate cancer cells is described. The formation of PCD/CUR was confirmed using FTIR, DSC, TGA, and SEM/TEM, and their stability and solubility under physiological conditions was demonstrated. A mechanism for self-assembly is proposed. Intracellular uptake of the self-assemblies was studied by flow cytometry and immunofluorescence microscopy. The therapeutic efficacy was determined by cell proliferation and colony formation assays using C4-2, DU145 and PC3 prostate cancer cells. The results suggest that the PCD/CUR formulation could be a useful system for improving curcumin delivery and its therapeutic efficacy in prostate cancer.

Oral gene therapy for hypoparathyroidism: a rat model.

The use of nonionic polymeric micelles orally to protect and deliver plasmid DNA in vivo was investigated. Parathyroid hormone (PTH)(1-34) gene (179 bp) was inserted into a human cytomegalovirus promoter (PCMV) and E. coli competent cells were used to amplify the cDNA. Polymeric micelle formations (100 microl) formed from PCMV-PTH(1-34) cDNA (7.2 microg/microl) and 6% (w/v) polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) was administered at 8-hr intervals for 48 hr and then at 8-hr intervals for 24 hr weekly for 3 weeks. Parathyroidectomized rats receiving 150 microl of EDTA (10 mM) before each dose of formation served as the study group; rats receiving drinking water, EDTA (10 mM), PCMV-PTH(1-34) cDNA and PCMV-PTH(1-34) cDNA plus EDTA at the same amount and time intervals served as the control groups. Serum levels of calcium and PTH(1-34) were measured weekly for 4 weeks. Immunohistochemical stain for PTH(1-34), reverse transcriptase polymerase chain reaction for PTH(1-34) mRNA and the relative density of PTH(1-34) mRNA were performed at 2 and 4 weeks after oral gene therapy in different organs. One third to three of five rats in the control groups died after parathyroidectomy. Serum levels of calcium and PTH(1-34) were higher in the study than in the control groups. In the study group, positive stain of PTH(1-34) and PTH(1-34) mRNA could be found in those organs. Relative densities of PTH(1-34) mRNA were higher in the study than in the drinking water group in different organs. Oral gene therapy can maintain calcium and PTH(1-34) levels in parathyroidectomized rats.

[Study on self-microemulsifying drug delivery system of Jiaotai Pill active components].

OBJECTIVE: To develop self-microemulsifying drug delivery system (SMEDDS) of Jiaotai Pill active components and its quality control method. METHODS: The solubility of berberine hydrochloride in different auxiliary-materials were investigated, and the self-microemulsion formula was optimized by studying the self-microemulsifying efficiency. The content of berberine hydrochloride and cinnamaldehyde in the preparation were determined by HPLC. RESULTS: The optimal self-microemulsion formula was composed of OP, propanediol, cinnamon oil, and total alkaloid from Rhizoma Coptidis with the ratio of 4:8:3:6. The average size of the microemulsion particle was 15.8 nm; the average content of berberine hydrochloride and cinnamaldehyde in the preparation was above 20.0% and 10.0%, respectively. CONCLUSION: The acquired microemulsion with small particle size is stable. The determination method of berberine hydrochloride and cinnamaldehyde in the preparation is accurate and reliable.

Vaginal distribution of Replens and K-Y Jelly using three imaging techniques.

BACKGROUND: Determination of vaginal distribution is important to the development of potential vaginal microbicidal or spermicidal products. STUDY DESIGN: This was a descriptive study of three imaging techniques with a randomized crossover assignment of two gels and activity status within each technique. METHOD: Each of three sites utilized one technique. Three nulligravid women and three parous women were to be enrolled at each site. We studied the effects of time, ambulation, parity and body mass index on vaginal spreading of two commonly used gels, K-Y Jelly and Replens. Imaging by magnetic resonance imaging and gamma scintigraphy was performed at 5, 20, 35 and 50 min after insertion of 3.5 mL of gel. Imaging with a fiberoptic probe was performed at 5 and 20 min after insertion. RESULTS: Initial application of the gel resulted in approximately two thirds of maximum coverage possible, both in linear extent along the vaginal axis and in surface area covered. Over the next 45 min, spreading increased to about three quarters of the maximum possible. Ambulation generally increased linear spreading and the proportions of women with gel at the introitus and os. Effects of parity and body mass index (BMI) were similar on most measures of gel spreading, with nulligravid women tending toward greater spread than parous women and women of high BMI usually showing somewhat greater spread than women of normal weight. Differences between the two gels were not seen when all conditions of application were considered together. CONCLUSION: In vivo imaging of gel distribution demonstrated that ambulation, parity and BMI affect vaginal gel spreading. The three imaging techniques have advantages and disadvantages and provide complementary information for microbicide development.

Efficacy and safety of percutaneous transhepatic portal embolization before right liver resection using an ethibloc/lipiodol mixture: a single-center experience.

AIM: The purpose of this study was to evaluate the safety and efficacy of percutaneous transhepatic portal vein embolization of the right portal vein with an Ethibloc/Lipiodol mixture to induce hypertrophy of the left liver lobe in patients with primarily unresectable liver tumor. METHODS: 15 patients (8 primary liver tumors, 7 liver metastases) underwent portal vein embolization. Liver volumetry, duration of hospitalization, complication rates, relevant laboratory values were documented. RESULTS: In 13/15 patients (84.6%) embolization could be performed with a median of 8.8 ml (range 1.5-28 ml) Ethibloc/Lipiodol. One minor procedure-related complication (subcapsular hematoma) occurred, which did not affect the two-step liver resection. No patient developed acute liver failure after embolization or liver resection. The volume of the left liver lobe increased significantly (p = 0.0015) by 25% from a median of 750 ml (587-1,114 ml) to 967 ml (597-1,249 ml). 11/13 (81.8%) of the embolized patients underwent liver resection at a median of 49 days after embolization. Median hospitalization time was 4 days after embolization and 7 days after liver resection. Median overall survival of the 11 operated patients was 376 days. CONCLUSION: Percutaneous transhepatic portal vein embolization using an Ethibloc/Lipiodol mixture is a safe, feasible, and efficient interventional procedure.

Warm water or oil-assisted colonoscopy: toward simpler examinations?

OBJECTIVES: Completion rates, pain, and difficulties during the exam are still problems in colonoscopy. New methods of lubrication, rarely considered a matter of study, may help in this respect. Our aim was to compare an oil-assisted technique with a modified warm water method applied during colonoscopy. METHODS: A prospective, randomized, and controlled study was planned in which three groups of patients were submitted to colonoscopy: a standard lubricating method (water-soluble jelly: group A, 170 patients) was adopted in a control group, whereas the standard method plus injection into the colon of corn seed oil (group B, 170 patients) or warm water (group C, 170 patients) were employed in the other groups. The main variables evaluated were: the success rate for total intubation, the time required to reach the cecum and the time needed to examine the colon at withdrawal, and the level of pain and degree of difficulty associated with the examination. RESULTS: Successful intubation to the cecum was significantly more frequent (P < 0.01 and P < 0.001, respectively) in the oil group (group B, 155/166) and in the warm water group (group C, 156/163) than in the control group (group A, 138/164), and less time was needed (P < 0.001); no significant difference was found between group B and C. Furthermore, no significant differences were found with regard to time for examination at withdrawal among the three groups. Level of pain and degree of difficulty during colonoscopy were significantly lower in the oil (P < 0.001) and in the warm water (P < 0.001) groups than in the control group, but no significant difference was found between group B and C. Neither side effects were observed for patients nor damage to the instrument. CONCLUSIONS: Warm water and oil-assisted colonoscopy could be simple, safe, and inexpensive methods for easier and less painful examinations.

New immunosuppresor strategies in the treatment of murine lupus nephritis.

Renal involvement in systemic lupus erythematosus is a common complication that significantly worsens morbidity and mortality. Although treatment with corticosteroids and cytotoxic drugs may be useful in many cases, morbidity associated with these drugs and the relapsing nature of the disease make it necessary to develop new treatment strategies. Five-month old female NZB/W F1 mice were divided into the following groups: CYP group (n = 10), cyclophosphamide (CYP) 50 mg/kg intraperitoneally every 10 days; RAPA 1 group (n = 10) oral daily sirolimus (SRL), 1 mg/kg; RAPA 12 group (n = 13), oral daily SRL, 12mg/kg; FTY group (n = 10), oral fingolimod (FTY720), 2 mg/kg three times per week. An additional group of 13 non-treated mice were used as a control (control group). Follow-up was performed over four months. Animal survival, body weight, anti-DNA antibodies and proteinuria were determined. Kidneys were processed for conventional histology and immunofluorescence for IgG and complement. Total histological score (HS) was the sum of mesangial expansion, endocapillary proliferation glomerular deposits, extracapillary proliferation, interstitial infiltrates, tubular atrophy and interstitial fibrosis. All treated groups had lower proteinuria at the end of the follow-up with respect to the control group (P < 0.0001). Serum anti-DNA antibodies were appropriately controlled in RAPA 1 and CYP groups, but not in FTY or RAPA 12 groups. SRL and CYP arrested, and perhaps reversed almost all histological lesions. FTY720 ameliorated histological lesions but did not control mesangial expansion or interstitial infiltrates. SRL produces great improvement in murine lupus nephritis, while FTY720 seems a promising alternative if used in appropriate doses.

Fingolimod. Mitsubishi Pharma/Novartis.

Mitsubishi Pharma Corp and Novartis AG are developing fingolimod, an orally active immunosuppressant affecting lymphocyte re-circulation, for the potential prevention of transplant rejection and the treatment of autoimmune diseases, including multiple sclerosis. Fingolimodis a synthetic sphingosine analog that becomes phosphorylated in vivo and acts as a sphingosine-1-phosphate receptor agonist.

Percutaneous portal vein embolisation in preparation for extended hepatic resection of primary nonresectable liver tumours.

BACKGROUND: In patients with malignant primary and secondary liver tumours or proximal bile duct carcinoma radical surgery is superior to all other therapeutic modalities in terms of survival and quality of life. Radical resection, however, often requires the removal of a large amount of liver parenchyma, resulting in a marked reduction of functional liver tissue with the risk of liver failure. AIM: Preoperative partial portal vein embolisation induces hypertrophy of the controlateral liver and thereby increases the safety of extended liver resections. PATIENTS AND METHODS: Between January 1997 and February 2001 we applied this strategy in 19 patients with primary and secondary nonresectable hepatobiliary malignancies, in whom the estimated amount of the remnant liver was < or =25% of the liver volume. RESULTS: The increase in volume ranged between 7 and 245%. Radical extended liver resection was performed in 13 patients (68%) without mortality. After a mean observation time of 22 months patient survival was 19 months with six tumour-related deaths during the second year after surgery. The remaining seven patients are alive and well with tumour recurrence in one. CONCLUSION: Preoperative partial portal vein embolisation allows more patients with previously unresectable liver tumours to benefit from a potentially curative resection.

Intralesional Ethibloc injections in primary aneurysmal bone cysts: an efficient and safe treatment.

OBJECTIVE: Ethibloc is a fibrogenic and thrombogenic agent recently proposed for the treatment of bone cysts. The purpose of this study is to report the results of direct Ethibloc injection in primary aneurysmal bone cyst (ABC) in children. DESIGN AND PATIENTS: Seventeen patients, aged from 2 to 18 years (mean 8 years), were treated with either a single injection (14 patients) or supplementary injections (3 patients) of Ethibloc. The histological diagnosis was assessed following surgical biopsy and was retrospectively reviewed. The mean follow-up was 5 years (range 18 months to 11 years). RESULTS: At 5 year follow-up, 14 of 17 patients demonstrated complete healing manifest by increased cortical and septal thickening. Surgical excision was required in three patients, in two of whom the ABC increased rapidly in size despite the injection, and in one of whom the healing was incomplete. We observed inflammatory reactions in 16 of 17 patients with local pain and fever. Three patients developed a small cutaneous fistula which resolved spontaneously in a few weeks. No major complications such as deep infection, pulmonary embolism, epiphyseal necrosis or malignant degeneration were observed. CONCLUSION: Percutaneous direct Ethibloc injection is a safe, efficient and noninvasive treatment for ABC. The authors highlight the frequent local reactions.

Combined FTY720/cyclosporine A treatment promotes graft survival and lowers the peripheral lymphocyte count in DA to lewis heart and skin transplantation models.

OBJECTIVE: The immunomodulator, FTY720, lowers the peripheral lymphocyte count (PLC) by inducing migration of circulating lymphocytes to secondary lymphoid organs. We investigated the efficacy of mono- vs. combined-FTY720/CsA therapy on graft survival (GS) and on lowering the PLC in a solid organ and a skin graft model, using strains with strong MHC disparity. METHODS: Heterotopic cardiac or tail skin grafting was performed using the DA (RT1a) to Lewis (RT1(1)) rat strain combination. FTY720 was administered as a single daily dose by gavage alone or in combination with subcutaneously delivered CsA. PLC, body weight and drug concentrations were determined on day 7, 28, or the day of rejection. MAIN FINDINGS: In placebo-treated animals the heart and skin allografts rejected after 6 and 8 days. FTY720 delayed rejection of both the solid organ and skin grafts. The maximal effect was achieved at 1 mg x kg(-l) x day(-1) FTY720, resulting in a median survival time (MST) of 14 days for both allotransplants comparable to the effect achieved by 1 mg x kg x day(-1) CsA in both models. In the cardiac graft experiment with CsA co-administration, doses of 0.3 and 1 mg/kg were used. Under these conditions very small doses of FTY720 were effective in maintaining grafts throughout the treatment period. Adding higher FTY720 doses to the 1 mg x kg(-1) x day(-1) CsA was needed to effectively extend the skin GS, e.g. 0.3 mg x kg(-l) x day(-1) FTY720 prolonged GS from 13 to 47.5 days MST, i.e. well beyond the 28 day-treatment period. CsA did not influence the PLC at clinically relevant doses. FTY720 lowered the PLC significantly and dose-dependently, at doses lower than those needed for the prolongation of both cardiac and skin GS with FTY720 monotherapy. In rats with skin grafts the PLC was markedly lowered up to 1 mg x kg(-1) x day(-1) FTY720, whereas, in the heart model, it was lowered up to 0.1 mg x kg(-1) x day(-1). Independently of the graft type, within the combination regimens 0.3 mg x kg(-1) x day(-1) FTY720 achieved a maximal PLC depletion. CONCLUSIONS: Combining FTY720 and CsA was very well tolerated with respect to weight gain and lack of any clinically detectable infections. In the strain combination used FTY720 monotherapy was less effective than previously reported in maintaining grafts. The two-drug regimens extended strikingly the GS for both models. However, the prolongation of the heart GS was smoothly dose-related with FTY720 doses ranging from 0.01 to 1 mg x kg(-1) x day(-1) , whereas, the skin graft prolongation was modest at doses up to 0.1 mg x kg(-1) x day(-1) and remarkably enhanced at 0.3 and 1 mg x kg(-1) x day(-1) FTY720.