A water-soluble prodrug of cyclosporine A for ocular application: a stability study.

UNIL088 is a water-soluble prodrug of cyclosporine A (CsA) developed for topical eye delivery. Such a prodrug has to fulfil two paradoxical requirements as it must be rapidly hydrolysed under physiological conditions but also retain a long shelf-life in aqueous media. This study has been conducted to explore the stability of UNIL088 formulated as an eyedrop solution. The stability study of the prodrug was performed over a pH range of 5-7 at 20 degrees C and at various ionic strengths. The molecule was more stable at pH 5 than at pH 7 with conversion rate constant of 3.2 x 10(-3) and 26.0 x 10(-3)days(-1), respectively. The effect of temperature was studied at four different temperatures and activation energy was determined. Conversion of UNIL088 followed a pseudo-first-order kinetic with an activation energy of 79.4 kJ mol(-1). Due to its low solubility, CsA generated precipitated in the solution. The average size of CsA precipitates, determined by photon spectroscopy, was 0.22 and 1.08 microm at 7 and 14 days, respectively. The hydrolysis mechanism was partially elucidated by identification of the intermediate pSer-Sar-CsA.

Importance of single or blended polymer types for controlled in vitro release and plasma levels of a somatostatin analogue entrapped in PLA/PLGA microspheres.

The aim of the work was to develop biodegradable microspheres for controlled delivery of the somatostatin analogue vapreotide and maintenance of sustained plasma levels over 2-4 weeks after a single injection in rats. Vapreotide was microencapsulated into end-group capped and uncapped low molecular weight poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) by spray-drying and coacervation. Microspheres were prepared from single and blended (1:1) polymer types. The microparticles were characterized for peptide loading, in vitro release and pharmocokinetics in rats. Spray-drying and coacervation produced microspheres in the size range of 1-15 and 10-70 microm, respectively, and with encapsulation efficiencies varying between 46% and 87%. In vitro release of vapreotide followed a regular pattern and lasted more than 4 weeks, time at which 40-80% of the total dose were released. Microspheres made of 14-kDa end-group uncapped PLGA50:50 or 1:1 blends of this polymer with 35 kDa end-group uncapped PLGA50:50 gave the best release profiles and yielded the most sustained plasma levels above a pre-defined 1 ng/ml over approximately 14 days. In vitro/in vivo correlation analyses showed for several microsphere formulations a linear correlation between the mean residence time in vivo and the mean dissolution time (r=0.958) and also between the amount released between 6 h and 14 days and the AUC(6h-14d) (r=0.932). For several other parameters or time periods, no in vitro/in vivo correlation was found. This study demonstrates that controlled release of the vapreotide is possible in vivo for a duration of a least 2 weeks when administered i.m. to rats. These results constitute a step forward towards a twice-a-month or once-a-month microsphere-formulation for the treatment of acromegaly and neuroendocrine tumors.

Inhibition of human immunodeficiency virus type 1 replication in human cells by Debio-025, a novel cyclophilin binding agent.

Debio-025 is a synthetic cyclosporine with no immunosuppressive capacity but a high inhibitory potency against cyclophilin A (CypA)-associated cis-trans prolyl isomerase (PPIase) activity. A lack of immunosuppressive effects compared to that of cyclosporine was demonstrated both in vitro and in vivo. For three cyclosporines, the inhibitory potential against PPIase activity was quantitatively correlated with that against human immunodeficiency virus type 1 (HIV-1) replication. Debio-025 selectively inhibited the replication of HIV-1 in a CD4+ cell line and in peripheral blood mononuclear cells: potent activity was demonstrated against clinical isolates of various HIV-1 subtypes, including isolates with multidrug resistance to reverse transcriptase and protease inhibitors. Simian immunodeficiency virus and HIV-2 strains were generally resistant to inhibition by Debio-025; however, some notable exceptions of sensitive HIV-2 clinical isolates were detected. In two-drug combination studies, additive inhibitory effects were found between Debio-025 and 19 clinically used drugs of different classes. Clinical HIV-1 isolates that are naturally resistant to Debio-025 and that do not depend on CypA for infection were identified. Comparison of the amino acid sequences of the CypA binding domain of the capsid (CA) protein from Debio-025-sensitive and -resistant HIV-1 isolates indicated that resistance was mostly associated with an H87Q/P exchange. Mechanistically, cyclosporines competitively inhibit the binding of CypA to the HIV-1 CA protein, which is an essential interaction required for early steps in HIV-1 replication. By real-time PCR we demonstrated that early reverse transcription is reduced in the presence of Debio-025 and that late reverse transcription is almost completely blocked. Thus, Debio-025 seems to interfere with the function of CypA during the progression/completion of HIV-1 reverse transcription.

Naturally occurring capsid substitutions render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in Owl monkey cells.

In this study, we asked if a naturally occurring HIV-1 variant exists that circumvents CypA dependence in human cells. To address this issue, we sought viruses for CypA independence using Debio-025, a cyclosporine A (CsA) analog that disrupts CypA-capsid interaction. Surprisingly, viral variants from the Main group replicate even in the presence of the drug. Sequencing analyses revealed that these viruses encode capsid substitutions within the CypA-binding site (V86P/H87Q/I91V/M96I). When we introduced these substitutions into viruses that normally rely on CypA for replication, these mutants no longer depended on CypA, suggesting that naturally occurring capsid substitutions obviate the need for CypA. This is the first demonstration that isolates from the Main group naturally develop CypA-independent strategies to replicate in human cells. Surprisingly, we found that these capsid substitutions render HIV-1 capable of infecting Owl monkey (OMK) cells that highly restrict HIV-1. OMK cell resistance to HIV-1 is mediated via TRIM-Cyp, which arose from a retrotransposition of CypA into the TRIM5 alpha gene. Interestingly, saturation experiments suggest that the Pro86/Gln87/Val91/Ile96 capsid core is "invisible" to TRIM-Cyp. This study demonstrates that specific capsid substitutions can release HIV-1 from both CypA dependence in human cells and TRIM-Cyp restriction in monkey cells.

The nonimmunosuppressive cyclosporin analogs NIM811 and UNIL025 display nanomolar potencies on permeability transition in brain-derived mitochondria.

Cyclosporin A (CsA) is highly neuroprotective in several animal models of acute neurological damage and neurodegenerative disease with inhibition of the mitochondrial permeability transition (mPT) having emerged as a possible mechanism for the observed neuroprotection. In the present study, we have evaluated two new nonimmunosuppressive cyclosporin analogs NIM811 (Novartis) and UNIL025 (Debiopharm) for their ability to inhibit mPT in rat brain-derived mitochondria. Both NIM811 and UNIL025 were found to be powerful inhibitors of calcium-induced mitochondrial swelling under energized and deenergized conditions, and the maximal effects were identical to those of native CsA. The potencies of mPT inhibition by NIM811 and UNIL025 were stronger, with almost one order of magnitude higher potency for UNIL025 compared to CsA, correlating to their respective inhibitory action of cyclophilin activity. These compounds will be instrumental in the evaluation of mPT as a central target for neuroprotection in vivo.