Discovery of ASP5286: A novel non-immunosuppressive cyclophilin inhibitor for the treatment of HCV.

Evidence indicates that hepatitis C virus (HCV) utilizes cellular cyclophilin proteins in its replication, and cyclophilin inhibitors represent a new class of anti-HCV agents. We have established an efficient synthetic methodology to generate FR901459 derivatives via N, O-acyl migration reaction while avoiding total synthesis. Through a detailed structure-activity relationship study, we improved anti-HCV activity while decreasing immunosuppressive activity. Additionally, we discovered the importance of substitution at the 3 position for not only improving anti-HCV activity but also pharmacokinetic profile. Finally, by striking an appropriate balance between potency, solubility, and permeability, we discovered ASP5286 (13) as a potential clinical candidate for anti-HCV therapy.

Preparation and comparison of tacrolimus-loaded solid dispersion and self-microemulsifying drug delivery system by in vitro/in vivo evaluation.

This study aimed to compare the dissolution and the intestinal absorption of tacrolimus in self-microemulsifying drug delivery system (SMEDDS) and solid dispersion (SD). Poloxamer 188 SD was prepared by the combination of the solvent evaporation method and the freeze drying method. Hydroxypropyl methylcellulose (HPMC) SD was prepared by the solvent evaporation method combined with the vacuum drying method. The formation of SD was confirmed by SEM images which showed new solid phases. The SMEDDS was composed of oil (Labrafil M1944 CS 28%), surfactant (Cremophor EL 48%) and co-surfactant (Transcutol P 24%). The self microemulsion formed by the SMEDDS upon aqueous media had spherical droplets with a hydrodynamic size of 46.0±3.2nm. The dissolution of tacrolimus from SD and SMEDDS was performed in sink and non-sink conditions with various pH. As revealed by the DSC and FT-IR, the tacrolimus was molecularly or amorphously dispersed in the SMEDDS and SD. The in vivo intestinal absorption study in rats showed that both SMEDDS and SD improved the absorption of tacrolimus over the raw tacrolimus while the SMEDDS exhibited lower absorption rate constant (Ka) and apparent permeability coefficients (Papp) than the SD. The self-prepared SD with poloxamer 188 or HPMC had comparable intestinal absorption as compared with Prograf®. The tacrolimus-loaded SMEDDS and SD would be further compared by in vivo pharmacokinetic study.

Discovery, Total Synthesis and Key Structural Elements for the Immunosuppressive Activity of Cocosolide, a Symmetrical Glycosylated Macrolide Dimer from Marine Cyanobacteria.

A new dimeric macrolide xylopyranoside, cocosolide (1), was isolated from the marine cyanobacterium preliminarily identified as Symploca sp. from Guam. The structure was determined by a combination of NMR spectroscopy, HRMS, X-ray diffraction studies and Mosher's analysis of the base hydrolysis product. Its carbon skeleton closely resembles that of clavosolides A-D isolated from the sponge Myriastra clavosa, for which no bioactivity is known. We performed the first total synthesis of cocosolide (1) along with its [α,α]-anomer (26) and macrocyclic core (28), thus leading to the confirmation of the structure of natural 1. The convergent synthesis featured Wadsworth-Emmons cyclopropanation, Sakurai annulation, Yamaguchi macrocyclization/dimerization reaction, α-selective glycosidation and ß-selective glycosidation. Compounds 1 and 26 potently inhibited IL-2 production in both T-cell receptor dependent and independent manners. Full activity requires the presence of the sugar moiety as well as the intact dimeric structure. Cocosolide also suppressed the proliferation of anti-CD3-stimulated T-cells in a dose-dependent manner.

Total synthesis of periploside A, a unique pregnane hexasaccharide with potent immunosuppressive effects.

Periploside A is a pregnane hexasaccharide identified from the Chinese medicinal plant Periploca sepium, which features a unique seven-membered formyl acetal bridged orthoester (FABO) motif and potent immunosuppressive activities. Here, we show the synthesis of this molecule in a total of 76 steps with the longest linear sequence of 29 steps and 9.2% overall yield. The FABO motif is constructed via a combination of Sinaÿ's and Crich's protocol for the formation of orthoester and acetal glycosides, respectively. The 2-deoxy-ß-glycosidic linkages are assembled stereoselectively with judicious choice of the glycosylation methods. The epimer at the spiro-quaternary carbon in the FABO motif has also been elaborated in a stereo-controlled manner. This epimer, as well as the synthetic analogues bearing the FABO motif, retain largely the inhibitory activities of periploside A against the proliferation of T-lymphocyte, indicating the importance of the chemical connection of the FABO motif to their immunosuppressive activity.

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.

Synthesis and biological evaluation of α,ß-unsaturated lactones as potent immunosuppressive agents.

Compounds having α,ß-unsaturated lactones display a variety of biological activities. Many research groups have tested both natural and unnatural α,ß-unsaturated lactones for as-yet undiscovered biological properties. We synthesized α,ß-unsaturated lactones with various substituents at the δ-position and studied their immunosuppressive effects, that is, the inhibition of Interleukin-2 (IL-2) production. Among the compounds synthesized, the benzofuran-substituted α,ß-unsaturated lactone 4h showed the best inhibitory activity toward IL-2 production in Jurkat e6-1 T lymphocytes (IC(50)=66.9 nM) without cytotoxicity at 10 µM. The results indicated that 4h may be useful as a potent immunosuppressive agent, as well as in IL-2-related studies.

Discovery and characterization of potent and selective 4-oxo-4-(5-(5-phenyl-1,2,4-oxadiazol-3-yl)indolin-1-yl)butanoic acids as S1P1 agonists.

S1P(1) receptor driven lymphopenia has proven utility in the treatment of an array of autoimmune disease states. As a part of our efforts to develop potent and selective S1P(1) receptor agonists, we have identified a novel chemical series of 4-oxo-4-(5-(5-phenyl-1,2,4-oxadiazol-3-yl)indolin-1-yl)butanoic acid S1P(1) receptor agonists.

Oxadiazole derivatives containing 1,4-benzodioxan as potential immunosuppressive agents against RAW264.7 cells.

A series of oxadiazole derivatives containing 1,4-benzodioxan (4a-4s) have been first synthesized for their potential immunosuppressive activity. Among the compounds, compound 4i showed the most potent biological activity against RAW264.7 cells (inhibition=37.66±2.34% for NO overproduction and IC(50)=0.05µM for iNOS). Docking simulation was performed to position compound 4i into the iNOS structure active site to determine the probable binding model. RT-PCR experiment results demonstrated that some of these compounds possessed good immunosuppressive activity against iNOS, especially for compound 4i. Therefore, compound 4i with potent inhibitory activity may be a potential agent.

Synthesis, biological evaluation of chrysin derivatives as potential immunosuppressive agents.

A series of novel chrysin derivatives was firstly synthesized and evaluated on their immunosuppressive activity in the search for potential immunosuppressive agents. Among them, compounds 5c displayed the most potent immunosuppressive inhibitory activity with IC(50) of 0.78 µM, which was comparable to that of cyclosporin A (IC(50) = 0.06 µM). The preliminary mechanism of compound 5c inhibition effects was also detected by flow cytometry (FCM), and the compound exerted immunosuppressive activity via inducing the apoptosis of activated lymph node cells in a dose dependent manner. Furthermore, the estimated LD(50) (in mg/kg) in vivo of compound 5c is 738.2, which indicated that compound 5c was low toxic.

Synthesis and in vitro evaluation of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)-pteridines as dual immunosuppressive and anti-inflammatory agents.

Screening of a pteridine-based compound library led to the identification of compounds exhibiting immunosuppressive as well as anti-inflammatory activity. Optimization afforded a series of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)pteridine analogues. The most potent congeners in this series displayed low nM IC(50) values in the Mixed Lymphocyte Reaction (MLR) assay. In addition, these compounds also have potent anti-inflammatory activity as measured in the Tumor Necrosis Factor (TNF) assay.

Synergistic effects of Isatis tinctoria L. and tacrolimus in the prevention of acute heart rejection in mice.

Although immunosuppressive treatments are available for acute cardiac rejection no viable treatment exists for long-term cardiac graft failure. Moreover, the extended use of calcineurin inhibitor immunosuppressants, the mainstay of current treatment for cardiac transplantation, leads to significant side effects such as nephrotoxicity and an increased risk of cardiac disease. Because some agents used in Traditional Chinese Medicine (TCM) have strong immunosuppressive effects coupled with low toxicity, we investigated the effect of Compound K (K), the synthesized analogue of highly unsaturated fatty acids from Isatis tinctoria L., either as a single treatment or combined with tacrolimus (FK-506) on acute cardiac allograft rejection. We compared the ability of K alone, or in combination with FK-506, to inhibit acute heart transplant rejection both in vitro and in vivo. We found that the inhibition of lymphocyte proliferation was positively correlated with K concentration. K significantly reduced IL-2 and IFN-gamma expression levels and significantly inhibited lymphocyte proliferation in both a lymphocyte transformation test and a mixed lymphocyte reaction (MLR). We also found that the inhibitory effect of a combination of K and a sub-therapeutic dose of FK-506 (SubFK-506) was stronger than that of full-dose FK-506 alone. Oral administration of K reduced acute cardiac allograft rejection in mice and had no apparent toxicity. In vivo, the immunosuppressive effect of K combined with a half-dose of FK-506 was equivalent to that of a full-dose of FK-506 alone. K combined with a half-dose of FK-506 reduced the expression levels of IL-2 and IFN-gamma (both within the graft and in the recipients' serum) more effectively than a full-dose of FK-506. These results show that K has significant immunosuppressive effects both in vitro and in vivo. When used as a combination therapy with FK-506 we see a powerful inhibition of rejection with no obvious toxic side effects. The mechanism of action is postulated to involve the inhibition of IL-2 and IFN-gamma expressions by lymphocytes, rather than the activation of Tr1 cells via the production of IL-10.

Amelioration of adjuvant induced arthritis by apocynin.

The premise of the study was to investigate the antiarthritic potential of apocynin (APO) in Balb/c mice (in vivo). The experiment showed a dose-dependent decrease in oedema and showed a suppression of proinflammatory cytokines such as TNF-alpha and IL-1beta and mediators such as prostaglandin E(2) (PGE(2)) and LTB(4). At oral doses of 0.5, 1.0, 2.0 and 4.0 mg/kg once daily during the course of the experiment, APO induced an inhibition of T cell mediated immune response causing suppression of CD4+ and CD8+ T cells and of intracellular interferon-gamma (IFN-gamma) by flow cytometry in arthritic mice. In parallel there was a dose-dependent inhibition in vascular permeability causing an inhibition in the migration of leucocytes and exudate volume at the site of the inflammatory reaction. These observations validate the immunoregulatory potential of apocynin.

Etanercept en la psoriasis. Experiencia clínica / Clinical experience with etanercept in the treatment of psoriasis

Introducción. Etanercept es uno de los nuevos fármacos biológicos surgidos para el tratamiento de la psoriasis. Ha demostrado ser una opción eficaz en un alto porcentaje de pacientes, provocando mejoras en el PASI (Psoriasis Assessment and Severity Index) que se mantienen en el tiempo. Además, resulta igualmente adecuado para el control de la artritis psoriásica. Por otra parte, el perfil de seguridad es excelente, con una toxicidad órgano específica mucho menor que los fármacos clásicos y la aparición de escasos efectos secundarios. Muchos de los datos publicados hasta el momento proceden de los distintos estudios clínicos que se han realizado con esta medicación, pero se necesitan trabajos que reflejen la experiencia en la práctica clínica diaria con el manejo de esta terapia en condiciones normales. Métodos. Estudio observacional, retrospectivo, en el que se recogen los 36 pacientes con psoriasis a los que administramos etanercept durante el período de tiempo comprendido entre marzo de 2004 y marzo de 2006. Exponemos la experiencia de nuestro centro en la utilización de este fármaco, con la evolución clínica y los problemas a los que nos hemos enfrentado. Resultados. El PASI se evaluó antes de comenzar el tratamiento y a los tres y seis meses de seguimiento de los pacientes. A los tres meses de tratamiento 13 de los pacientes (36,11 %) habían alcanzado el PASI 50, y 16 pacientes (44,44 %) habían alcanzado el PASI 75. Dos de los pacientes (5,56 %) experimentaron una mejoría de su psoriasis, sin alcanzar el PASI 50, y sólo 4 pacientes (11,11 %) no mostraron mejoría clínica o incluso empeoraron. A los 6 meses se observó un aumento de la eficacia, con 27 pacientes (75 %) que alcanzaron el PASI75 y 6 pacientes (16,67 %) que llegaron a obtener el PASI 50, 2 pacientes (5,56 %) no mostraron ningún beneficio tras la terapia. A los 6 meses 13 de los pacientes (36,1 %) habían alcanzado el PASI 90. En ninguno de los casos se presentaron acontecimientos adversos de importancia que obligaran a suspender el tratamiento. Once de los pacientes siguen en tratamiento con etanercept en el momento actual, ya que se ha mantenido la eficacia y no han presentado efectos adversos importantes. Conclusiones. Exponemos nuestra experiencia clínica con la utilización de etanercept para el tratamiento de la psoriasis en placas, con un perfil muy favorable de eficacia y seguridad. Proponemos la estandarización de la visita clínica al paciente con psoriasis, con recogida exhaustiva de datos en cada visita y la creación de un sistema nacional de registro de datos de pacientes con tratamientos biológicos (AU)

Background. Etanercept is one of the new biologic agents available for treating psoriasis. It has proved an effective option in a high percentage of patients, leading to sustained improvements in the psoriasis are aseverity index (PASI). Likewise, it is effective at controlling psoriatic arthritis, and its safety profile is excellent, with a much lower specific organ toxicity than traditional drugs and few side effects. Many of the data published to date are derived from clinical trials with this medication, but further studies are needed on the use of this therapy to manage patients in daily clinical practice. Methods. This was a retrospective observational study of 36 patients with psoriasis who received etanercept between March 2004 and March 2006. We describe the experience of using this agent at our hospital, with the clinical outcomes and the problems we have faced. Results. The PASI score was assessed before treatment and at 3 and 6 months of patient follow-up. After 3 months of treatment, 13 patients (36.11 %) had achieved a 50 % improvement in PASI score (PASI50), and 16 patients (44 %) had achieved a 75 % improvement (PASI75). Two of the patients (5.56 %) experienced an improvement in their disease without reaching PASI50 and only 4 patients (11.11 %) did not show clinical improvement or deteriorated. After 6 months, efficacy improved, with 27 patients (75 %) achieving PASI75, 6 patients (16.67 %) achieving PASI50, and 2 patients (5.56 %) showing no benefit from treatment. After 6 months, 13 of the patients (36.1 %) had achieved a 90% improvement in PASI score. No adverse events of sufficient significance to warrant discontinuation of treatment were reported. At present, 11 of the patients remain on etanercept treatment as efficacy has been sustained and they have not experienced any adverse events of note. Conclusions. Our clinical experience with the use of etanercept for treating plaque psoriasis shows a favorable efficacy and safety profile. We propose a standardized procedure for consultations with psoriasis patients involving extensive data collection on each visit and the creation of a national surveillance system for patients under treatment with biologic agents (AU)

Discovery and synthesis of new immunosuppressive alkaloids from the stem of Fissistigma oldhamii (Hemsl.) Merr.

Three new alkaloids (1-3) and twenty-one known compounds were isolated from the stem of Fissistigma oldhamii (Hemsl.) Merr. which was the ruler herb in an approved Traditional Chinese herbal formula used for treatment of rheumatoid arthritis in China and synthesis of one new immunosuppressive alkaloid was achieved. These compounds, including the crude extracts of this herb, exhibited strong activities in the inhibition of T and B cell proliferation.

Chemistry and biology of cyclic depsipeptides of medicinal and biological interest.

Cyclodepsipeptides comprise a wide variety of cyclic peptides of natural origin and are characterized by the occurrence of at least one ester linkage. The great interest that this class of natural products has elicited in scientific community is explained by their wide range of biological activities, intriguing mechanisms of action and attractive molecular architecture. For example, they display a variety of biological effects, such as immunosuppressant, antibiotic, antifungi, antiinflammatory or antitumoral activities. In addition, many of these cyclic depsipeptides represent useful tools for the research of biological processes involved in cellular regulation. The present review deals with the most interesting aspects of the biology and the chemistry of some of these compounds.

Synthesis and evaluation of disubstituted N1- and N3-imidazolidin-2-ones acting as potential immunosuppressive agents.

New N1-mono and N1, N2-disubstituted imidazolidin-2-one with a significant immunosuppressive activity have been discovered. Among the 17 synthesized and tested compounds, five of them showed maximal inhibition of proliferation of concanavallin A (Con A)- stimulated splenocytes at 90 microM, identical to that obtained with cyclosporin A (CsA) at 5 microM, an optimal concentration.

Synthesis and immunosuppressive activity of 2-substituted 2-aminopropane-1,3-diols and 2-aminoethanols.

A series of 2-substituted 2-aminopropane-1,3-diols was synthesized and evaluated for their lymphocyte-decreasing effect and immunosuppressive effect on rat skin allograft. A phenyl ring was introduced into the alkyl chain of the lead compound 3, which is an immunosuppressive agent structurally simplified from myriocin (1, ISP-I) via compound 2. The potency of the various compounds was dependent upon the position of the phenyl ring within the alkyl side chain. The most suitable length between the quaternary carbon atom and the phenyl ring was two carbon atoms. 2-Substituted 2-aminoethanols were successively synthesized and evaluated for their T-cell-decreasing effect and immunosuppressive effect using a popliteal lymph node gain assay in rats. The absolute configuration at the quaternary carbon affected the activity, and the (pro-S)-hydroxymethyl group of compound 6 was essential for potent immunosuppressive activity. Favorable substituents for the (pro-R)-hydroxymethyl group of 6 were hydroxyalkyl (hydroxyethyl and hydroxypropyl) or lower alkyl (methyl and ethyl) groups. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (6, FTY720) was found to possess considerable activity and is expected to be useful as an immunosuppressive drug for organ transplantation.

Structure-immunosuppressive activity relationships of new analogues of 15-deoxyspergualin. 2. Structural modifications of the spermidine moiety.

A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent commercialized in Japan, was synthesized and tested in a graft-versus-host disease (GVHD) model in mice. Various substitutions of the spermidine "D" region were made in order to determine its optimum structure in terms of in vivo immunosuppressive activity. Various positions of methylation were first investigated leading to the discovery of the monomethylated malonic derivative 56h in which the pro-R hydrogen of the methylene alpha to the primary amine of the spermidine moiety has been replaced by a methyl group. Synthesis of the similarly methylated analogue of the previously reported glycolic derivative LF 08-0299 afforded 60e which demonstrated a powerful activity at a dose as low as 0.3 mg/kg in the GVHD model and was much more potent than DSG in the demanding heart allotransplantation model in rats. The improvement of in vivo activity was supposed to be related to an increase of the metabolic stability of the methylated analogues compared to the parent molecules. Due to its very low active dose, compatible with a subcutaneous administration in humans, and its favorable pharmacological and toxicological profile, 60e was selected as a candidate for clinical evaluation.

C32-O-phenalkyl ether derivatives of the immunosuppressant ascomycin: a tether length study.

A tether length study of C32-O-phenalkyl ether derivatives of ascomycin was conducted wherein it was determined that a 2-carbon tether provides optimum in vitro immunosuppressive activity. Oxygen-bearing substituents along the 2-carbon tether can further increase the potency of this design.

Potent immunosuppressive C32-O-arylethyl ether derivatives of ascomycin with reduced toxicity.

The synthesis of C32-O-arylethyl ether derivatives of ascomycin that possess equivalent immunosuppressant activity but reduced toxicity, compared to FK-506, is described.