Colloidal systems for the delivery of cyclosporin A to the anterior segment of the eye.

Due to the eye's specific anatomical and physiological conformation, the treatment of eye diseases is a real challenge for pharmaceutical therapy. The presence of efficient protective barriers (i.e., the conjunctival and corneal membranes) and protective mechanisms (i.e., blinking and nasolachrymal drainage) makes this organ particularly impervious to local drug therapy. To overcome these issues, numerous strategies have been envisioned using pharmaceutical technology. Many formulations currently on the market or still under development are emulsions or colloidal systems intended to enhance precorneal residence time and corneal penetration, causing a consequent increase in drug bioavailability after instillation. After a review of some recent developments in the field of cyclosporin A formulations for the eye, a novel micellar formulation of cyclosporine A based on a diblock methoxy-poly(ethylene glycol)-hexysubstituted poly(lactides) (MPEG-hexPLA) is described.

Novel micelle carriers for cyclosporin A topical ocular delivery: in vivo cornea penetration, ocular distribution and efficacy studies.

Cornea transplantation is one of the most performed graft procedures worldwide with an impressive success rate of 90%. However, for "high-risk" patients with particular ocular diseases in addition to the required surgery, the success rate is drastically reduced to 50%. In these cases, cyclosporin A (CsA) is frequently used to prevent the cornea rejection by a systemic treatment with possible systemic side effects for the patients. To overcome these problems, it is a challenge to prepare well-tolerated topical CsA formulations. Normally high amounts of oils or surfactants are needed for the solubilization of the very hydrophobic CsA. Furthermore, it is in general difficult to obtain ocular therapeutic drug levels with topical instillations due to the corneal barriers that efficiently protect the intraocular structures from foreign substances thus also from drugs. The aim of this study was to investigate in vivo the effects of a novel CsA topical aqueous formulation. This formulation was based on nanosized polymeric micelles as drug carriers. An established rat model for the prevention of cornea graft rejection after a keratoplasty procedure was used. After instillation of the novel formulation with fluorescent labeled micelles, confocal analysis of flat-mounted corneas clearly showed that the nanosized carriers were able to penetrate into all corneal layers. The efficacy of a 0.5% CsA micelle formulation was tested and compared to a physiological saline solution and to a systemic administration of CsA. In our studies, the topical CsA treatment was carried out for 14 days, and the three parameters (a) cornea transparency, (b) edema, and (c) neovascularization were evaluated by clinical observation and scoring. Compared to the control group, the treated group showed a significant higher cornea transparency and significant lower edema after 7 and 13 days of the surgery. At the end point of the study, the neovascularization was reduced by 50% in the CsA-micelle treated animals. The success rate of cornea graft transplantation was 73% in treated animals against 25% for the control group. This result was as good as observed for a systemic CsA treatment in the same animal model. This new formulation has the same efficacy like a systemic treatment but without the serious CsA systemic side effects. Ocular drug levels of transplanted and healthy rat eyes were dosed by UPLC/MS and showed a high CsA value in the cornea (11710 ± 7530 ng(CsA)/g(tissue) and 6470 ± 1730 ng(CsA)/g(tissue), respectively). In conclusion, the applied formulation has the capacity to overcome the ocular surface barriers, the micelles formed a drug reservoir in the cornea from, where a sustained release of CsA can take place. This novel formulation for topical application of CsA is clearly an effective and well-tolerated alternative to the systemic treatment for the prevention of corneal graft rejection.

Upregulation of vasopressin V1A receptor mRNA and protein in vascular smooth muscle cells following cyclosporin A treatment.

1. The major side effects of the immunosuppressive drug cyclosporin A (CsA) are hypertension and nephrotoxicity. It is likely that both are caused by local vasoconstriction. 2. We have shown previously that 20 h treatment of rat vascular smooth muscle cells (VSMC) with therapeutically relevant CsA concentrations increased the cellular response to [Arg8]vasopressin (AVP) by increasing about 2 fold the number of vasopressin receptors. 3. Displacement experiments using a specific antagonist of the vasopressin V1A receptor (V1AR) showed that the vasopressin binding sites present in VSMC were exclusively receptors of the V1A subtype. 4. Receptor internalization studies revealed that CsA (10(-6) M) did not significantly alter AVP receptor trafficking. 5. V1AR mRNA was increased by CsA, as measured by quantitative polymerase chain reaction. Time-course studies indicated that the increase in mRNA preceded cell surface expression of the receptor, as measured by hormone binding. 6. A direct effect of CsA on the V1AR promoter was investigated using VSMC transfected with a V1AR promoter-luciferase reporter construct. Surprisingly, CsA did not increase, but rather slightly reduced V1AR promoter activity. This effect was independent of the cyclophilin-calcineurin pathway. 7. Measurement of V1AR mRNA decay in the presence of the transcription inhibitor actinomycin D revealed that CsA increased the half-life of V1AR mRNA about 2 fold. 8. In conclusion, CsA increased the response of VSMC to AVP by upregulating V1AR expression through stabilization of its mRNA. This could be a key mechanism in enhanced vascular responsiveness induced by CsA, causing both hypertension and, via renal vasoconstriction, reduced glomerular filtration.

PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

A novel cyclosporin a aqueous formulation for dry eye treatment: in vitro and in vivo evaluation.

PURPOSE: The aim of the present study was the in vitro and in vivo evaluation of a novel aqueous formulation based on polymeric micelles for the topical delivery of cyclosporine A for dry eye treatment. METHODS: In vitro experiments were carried out on primary rabbit corneal cells, which were characterized by immunocytochemistry using fluorescein-labeled lectin I/isolectin B4 for the endothelial cells and mouse monoclonal antibody to cytokeratin 3+12 for the epithelial ones. Living cells were incubated for 1 hour or 24 hours with a fluorescently labeled micelle formulation and analyzed by fluorescence microscopy. In vivo evaluations were done by Schirmer test, osmolarity measurement, CyA kinetics in tears, and CyA ocular distribution after topical instillation. A 0.05% CyA micelle formulation was compared to a marketed emulsion (Restasis). RESULTS: The in vitro experiments showed the internalization of micelles in the living cells. The Schirmer test and osmolarity measurements demonstrated that micelles did not alter the ocular surface properties. The evaluation of the tear fluid gave similar CyA kinetics values: AUC = 2339 ± 1032 min*μg/mL and 2321 ± 881.63; Cmax = 478 ± 111 μg/mL and 451 ± 74; half-life = 36 ± 9 min and 28 ± 9 for the micelle formulation and Restasis, respectively. The ocular distribution investigation revealed that the novel formulation delivered 1540 ± 400 ng CyA/g tissue to the cornea. CONCLUSIONS: The micelle formulation delivered active CyA into the cornea without evident negative influence on the ocular surface properties. This formulation could be applied for immune-related ocular surface diseases.

The direct effects of tacrolimus and cyclosporin A on isolated human islets: A functional, survival and gene expression study.

The use of immunosuppressive drugs in transplanted patients is associated with the development of diabetes, possibly due to β-cell toxicity. To better understand the mechanisms leading to post-transplant diabetes, we investigated the actions of prolonged exposure of isolated human islets to therapeutical levels of tacrolimus (Tac) or cyclosporin A (CsA). Islets were isolated from the pancreas of multiorgan donors by enzymatic digestion and density gradient centrifugation. Functional, survival and molecular studies were then performed after 4 days of incubation with therapeutical concentrations of Tac or  CsA. Glucose-induced insulin secretion was significantly decreased in Tac, but not in CsA exposed islets, which was associated with a reduction of the amount of insulin granules as shown by electron microscopy. The percentage of apoptotic β-cells was higher in Tac than CsA exposed islets. Microarray experiments followed by Gene Set Enrichment Analysis revealed that gene expression was more markedly affected upon Tac treatment. In conclusion, Tac and CsA affect features of beta-cell differently, with several changes occurring at the molecular level.

Actualités en hépatologie [Highlights in hepatology]

This review highlights recent advances in hepatology, including new insights into the clinical penetrance of hereditary hemochromatosis, the development of non-immunosuppressive cyclosporin A analogs for the treatment of chronic hepatitis C, thrombopoietin receptor agonists for thrombocytopenia in cirrhosis, the development of vasopressin V2 receptor antagonists (vaptans) for the management of ascites and hyponatremia in portal hypertension, the description of chronic hepatitis E in immunosuppressed patients, and the development of sorafenib as the first molecularly targeted therapy with a demonstrated benefit in the treatment of advanced hepatocellular carcinoma. These new developments will be summarized and discussed critically, with a particular emphasis on their potential implications for current and future clinical practice.

Les hépatopathies auto-immunes et leurs traitements [Auto-immune liver diseases and their treatment]

There are three main types of auto-immune liver disease, auto-immune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis. In the case of auto-immune hepatitis, prednisone therapy, with or without azathioprine, can improve quality of life and halt progression to cirrhosis. If there is no response or if the therapy is poorly tolerated, mycophenolate mofetil or cyclosporin should be considered. Ursodeoxycholic acid (UDCA), at a dosage of 13 to 15 mg/kg/day slows the progression of fibrosis in patients with primary biliary cirrhosis. Pruritus may be treated with cholestyramine, rifampicin or opiate antagonists. Ursodeoxycholic acid at a dosage of 20 to 30 mg/kg/day will slow the evolution of fibrosis.

Suivi du patient après transplantation cardiaque: monitoring et adaptation de l'immunosuppression [Monitoring and adjustment of immunosuppression after heart transplantation].

Heart transplantation remains the best therapeutic option for the treatment of end-stage heart failure. However, good survival rates can be obtained only if patients are closely monitored, particularly for their immunosuppressive regimens. Currently, a triple-drug regimen usually based on calcineurin-inhibitors (cyclosporin A or tacrolimus), anti-proliferative agents and steroids is used in most recipients. New agents such as the mTOR inhibitors, a more recently developed class of immunosuppressive drugs, can also be used in some patients. The aim of this article is to review currently used immunosuppressive regimens after heart transplantation, and to propose some individualized options depending on specific patient characteristics and recent pharmacological developments in the field.

Assessing multiple sclerosis activity: is the in vitro production of tumor necrosis factor-alpha, interleukins 2, 6, 4, and 10, and immunoglobulin G of value?

Tumor necrosis factor (TNF) alpha, interleukins (IL) 2, 4, 6, and 10, and IgG oligoclonal bands (IgG OB) in vitro production was assessed, after whole-blood stimulation with lipopolysaccharide or concanavalin A, in 61 patients presenting with relapsing-remitting, relapsing-progressive, or chronic progressive multiple sclerosis. Multiple sclerosis patients were receiving no treatment or azathioprine (AZA), cyclosporin, cyclophosphamide, subcutaneous interferon (IFN) beta 1 a, or corticosteroids (CST). Statistical correlations significantly showed that: (a) AZA lowers TNF-alpha (P = 0.002) and increases IL-4 production (P = 0.0024), and IFN-beta 1 a increases TNF-alpha and decreases IL-4 levels; (b) CST has a negative effect on TNF-alpha, IL-6, and IL-4 synthesis; and (c) AZA, IFN-beta 1 a, and CST diminish IgG OB synthesis (P = 0.001). Although our study of the dynamics of TNF-alpha, IL-2, IL-4, IL-6, and IL-10 in vitro production generally found no statistically significant correlations (partly explained by the limited number of values in the various groups), IL-6 was shown to drop during the periods surrounding relapse (P = 0.05) in the absence of treatment, while TNF-alpha (P = 0.04) and IL-6 (P < 0.05) dropped before exacerbation in the presence of AZA. In vitro production of TNF-alpha was closely and positively correlated with that of IL-6, independently of clinical features. The enhanced production of IL-10 detected before or at relapse with AZA and IFN-beta 1 a (trends) may interfere with initiation of the immune reaction and with the development of new CNS lesions. Some discrepancies with previously published results stress the difficulties in studying the state of stimulation of different populations of leukocytes by using a variety of in vitro stimuli and in establishing a correlation between mRNA studies and the amount of final or active protein produced.

Methadone maintenance treatment and St. John's Wort - a case report.

St. John's wort, a popular over-the-counter drug for treatment of depression, might reduce concentrations of drugs such as cyclosporin and indinavir and lead to drug resistance and treatment failure. No studies as yet have examined its influence on methadone plasma levels. The trough methadone plasma levels were measured in four patients (2 males, median age: 31 years; range 19 - 40 years) in methadone maintenance treatment just before the introduction of St. John's wort (900 mg/d) and after a median period of 31-day treatment (range 14 - 47). The study was proposed to addict patients about to start an antidepressant therapy. Introduction of St. John's wort resulted in a strong reduction of (R,S)-methadone concentration-to-dose ratios in the four median patients included, with a median decrease to 47 % of the original concentration (range: 19 % - 60 % of the original concentration). Two patients reported symptoms that suggested a withdrawal syndrome. Thus, prescription of St. John's wort might decrease methadone blood levels and induce withdrawal symptoms which, if not correctly identified and handled (by changing the antidepressant or by increasing the methadone dose), might cause unnecessary discomfort to the patient, lead to resumption of illicit drug uses, or be a risk factor for discontinuation of the methadone or antidepressant treatment.

Loco-regional administration of anticancer agents : isolated lung perfusion with doxorubicin hypoxic abdominal stop-flow perfusion with gemcitabine

SUMMARY Regional drug delivery is an approach designed to improve the selectivity of anticancer chemotherapy. The advantage of regional treatments lies in increasing the drug concentration in the affected organ, while the rest of the organism is spared, thus improving efficacy and limiting treatment toxicity. The goal of this thesis was to assess the distribution throughout the body and the disposition (pharmacokinetics) of two anticancer agents, doxorubicin and gemcitabine, administered by two different regional administration modalities: isolated lung perfusion (ILP) for pulmonary metastases from soft tissue sarcomas and abdominal stop-flow hypoxic perfusion for advanced pancreatic cancers, respectively. For this purpose, two high-performance liquid chromatography methods were developed and validated. The first enabled the determination of doxorubicin in four different biological matrices: serum, reconstituted effluent, tissues with low levels of doxorubicin and tissues with high levels of doxorubicin. The second allows the analysis of gemcitabine and its principal metabolite dFdU in plasma. The administration of doxorubicin by ILP was studied in three preclinical studies (one on pigs and two on rats). It was first shown that, regardless of the administration mode, doxorubicin was not homogeneously distributed throughout the lung and that some regions remained out of reach. Secondly, it was demonstrated that doxorubicin did not adequately reach the tumours despite very high levels found in the lung. Finally, an attempt to enhance the doxorubicin tumoural uptake by pharmacologic modulation using two P-glycoprotein inhibitors, cyclosporin and valspodar, was unsuccessful. The last part of this work involves the administration of gemcitabine by abdominal stop-flow as a part of a phase I clinical trial in patients with advanced pancreatic disease or resistant malignant ascites. The study has demonstrated that the regional exposure to gemcitabine was increased while the exposure of the entire organism was similar to standard intravenous administrations. From a toxicological perspective, the procedure was rather well tolerated. However, even if no clinical response is expected from a phase I study, no hints of clinical responses were unfortunately observed. In conclusion, even if loco-regional therapies may afford the pharmacological advantage of increasing anticancer drug levels at the tumour site, further studies of these investigational treatment modalities are warranted to ascertain whether they can provide a significant improvement of the cancer therapy for patients, in terms of treatment tolerability, improved responses and survival rates. RÉSUMÉ L'administration locorégionale d'agents anticancéreux est une approche destinée à augmenter la sélectivité du traitement. L'avantage des traitements régionaux repose sur le fait que la concentration du médicament cytostatique est augmentée dans l'organe où est localisée la tumeur, alors que le reste de l'organisme est épargné, améliorant ainsi en théorie l'efficacité du traitement et en limitant sa toxicité. Le but de ce travail de thèse avait pour objectif de préciser, la pharmacocinétique au sein de l'organisme de deux agents anticancéreux, la doxorubicine et la gemcitabine, administrés par deux types de perfusions loco-régionales: la perfusion isolée du poumon (ILP) pour les métastases pulmonaires de sarcomes des tissus mous, et la perfusion hypoxique (stop-flow) abdominale pour les cancers avancés du pancréas. Dans cette optique, deux méthodes de chromatographie liquide à haute performance ont été développées et validées. La première permet le dosage de la doxorubicine dans quatre milieux biologiques: le sérum, l'effluent reconstitué, ainsi que des tissus contenant des concentrations faibles et élevées en doxorubicine. La seconde méthode permet le dosage dans le plasma de la gemcitabine et de son principal métabolite, le dFdU. L'administration de doxorubicine par ILP a été étudiée dans trois études précliniques (une chez le porc et deux chez le rat). Il a été montré, dans un premier temps, que la doxorubicine n'était pas distribuée de façon homogène au sein du poumon, quel que soit son mode d'administration. Dans un deuxième temps, il a été démontré que le médicament n'atteignait pas les tumeurs de façon adéquate, malgré des concentrations très élevées au sein du tissu pulmonaire. Finalement, une tentative d'augmenter la pénétration tumorale de la doxorubicine par une modulation pharmacologique de la P-glycoprotéine en utilisant la cyclosporine et le valspodar n'a pas abouti. La dernière partie de ce travail concernait l'administration de gemcitabine par stop-flow abdominal dans le cadre d'une étude clinique de phase I menée auprès de patients atteints de cancers avancés du pancréas ou d'ascites malignes réfractaires. Cette étude a démontré que l'exposition régionale à la gemcitabine était augmentée, alors que l'exposition de l'organisme était similaire à une administration de dose standard par voie intraveineuse. D'un point de vue toxicologique la procédure fut relativement bien tolérée. Cependant, même s'il n'est pas attendu de réponses cliniques dans une étude de phase I, aucun signe de réponse au traitement n'a pu être malheureusement observé. En conclusion, même si les thérapies loco-régionales présentent -en théorie- l'avantage pharmacologique d'augmenter les taux du médicaments anticancéreux sur le site de la tumeur, d'autres études précliniques et cliniques sont nécessaires pour démontrer que ces nouvelles modalités de traitement, de nature investigationelle à présent, apportent une réelle amélioration pour la prise en charge des patients cancéreux, en terme de tolérance au traitement et de l'augmentation des taux de réponses et de survie.

Administration intra-vitréenne de liposomes pour la vectorisation de principes actifs [The use of liposomes as intravitreal drug delivery system].

Liposomes are vesicular lipidic systems allowing encapsulation of drugs. This article reviews the relevant issues in liposome structure (composition and size), and their influence on intravitreal pharmacokinetics. Liposome-mediated drug delivery to the posterior segment of the eye via intravitreal administration has been addressed by several authors and remains experimental. Liposomes have been used for intravitreal delivery of antibiotics, antivirals, antifungal drugs, antimetabolites, and cyclosporin. Encapsulation of these drugs within liposomes markedly increased their intravitreal half-life, and reduced their retinal toxicity. Liposomes have also shown an attractive potential for retinal gene transfer by intravitreal delivery of plasmids or oligonucleotides.