DIC/Oxyma-based accelerated synthesis and oxidative folding studies of centipede toxin RhTx.

Coupling reagents play crucial roles in the iterative construction of amide bonds for the synthesis of peptides and peptide-based derivatives. The novel DIC/Oxyma condensation system featured with the low risk of explosion displayed remarkable abilities to inhibit racemization, along with efficient coupling efficiency in both manual and automated syntheses. Nevertheless, an ideal reaction molar ratio in DIC/Oxyma condensation system and the moderate reaction temperature by manual synthesis remain to be further investigated. Herein, the synthetic efficiencies of different reaction ratios between DIC and Oxyma under moderate reaction temperature were systematically evaluated. The robustness and efficiency of DIC/Oxyma condensation system are validated by the rapid synthesis of linear centipede toxin RhTx. Different folding strategies were applied for the construction of disulfide bridges in RhTx, which was further confirmed in assays of circular dichroism and patch-clamp electrophysiology evaluation. This work establishes the DIC/Oxyma-based accelerated synthesis of peptides under moderate condensation conditions, which is especially useful for the manual synthesis of peptides. Besides, the strategy presented here provides robust technical supports for the large-scale synthesis and oxidative folding of RhTx.

Outcome and risk of ocular complications of managing children with chronic anterior uveitis with topical rimexolone 1.

PURPOSE: To investigate the efficacy and safety of 1% rimexolone ophthalmic suspension in children with chronic anterior uveitis under real-life conditions in a tertiary center. METHODS: This is a retrospective longitudinal study. Medical records were analyzed at baseline, 1, 3, 6 and 12 months before and after switching to rimexolone for best-corrected visual acuity (BCVA), oral steroid use, number of flares, IOP and anti-glaucoma management. RESULTS: Twenty-four patients (41 eyes) diagnosed with either anterior uveitis (n = 25, 60.0%) or panuveitis (n = 16, 40%) were enrolled. The mean age was 10.5 years (4-16 years). The number of patients requiring oral prednisolone reduced from 8 patients (32.0%) at baseline to 3 patients (20.0%) at 12 months (P < 0.001). Following baseline, the median number of uveitis flares reduced from 2.0 (inter-quartile range (IQR) 1.0-2.75) to 1.0 (IQR 0.0-1.0) compared to the 12 months before baseline (P < 0.001). The mean IOP reduced from baseline (22.0 ± 7.3 mmHg) to 1 month (18.8 ± 8.7 mmHg, P = 0.01) and remained stable up to 12 months (15.9 ± 5.0 mmHg, P < 0.001). Average BCVA, dose of oral prednisolone and anti-glaucoma treatments did not change compared to the baseline. The development for IOP ≥ 30 mmHg was associated with a known corticosteroid response [odds ratio (OR) 6.8, P = 0.003] and a dose > 7.5 mg/day oral prednisolone (OR 4.4, P = 0.033). CONCLUSIONS: Rimexolone 1% ophthalmic suspension is an effective and safe topical steroid for pediatric anterior uveitis.

PLGA/PLCA casting and PLGA/PDPA electrospinning bilayer film for prevention of postoperative adhesion.

Postoperative adhesion is a common complication and preventing adhesions during or immediately after operation is particularly important. The application of solid barrier materials represents the most successful clinical strategy to prevent postoperative adhesion. However, a simple physical barrier effect might be insufficient in preventing adhesion satisfactorily. Multilayered structures can be designed with an outer layer as the barrier and an inner layer to respond to relative drug release. In this article, bilayer film composed of a PLGA/PLCA casting layer as barrier and PLGA/PDPA electrospinning layer to respond to the release of anti-fibrosis drug l-Phe was designed and synthesized. The adhesion prevention effect of the above PLGA/PLCA/PDPA bilayer film was examined and compared with single PLGA/PLCA casting film and single PLGA/PDPA electrospinning film by applying rabbit sidewall defect-cecum abrasion model. As demonstrated by histological observation and immunohistochemical analysis, the bilayer film was the most effective of the three films in postoperative adhesion prevention in terms of both physical barrier effect and anti-fibrosis effect of the PDPA macromolecular prodrug. Besides anti-fibrosis effect, PDPA could also suppress excess proliferation of vascular endothelial cells and microvessel caused by long-term stimulation of implantation materials to the surrounding tissues. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2030-2039, 2019.

Retrospective analysis of a single-center clinical experience toward development of curative treatment of 123 pemphigus patients with a long-term follow-up: efficacy and safety of the multidrug protocol combining intravenous immunoglobulin with the cytotoxic immunosuppressor and mitochondrion-protecting drugs.

BACKGROUND: Pemphigus vulgaris (PV) is a life-long IgG autoantibody-mediated blistering disease affecting the mucosal surfaces lined by the stratified epithelium (oral, nasal, genital) and sometimes also the skin. While corticosteroid treatment is life saving, the high dose and prolonged courses required for disease control are associated with significant adverse effects, including death. Although introduction of rituximab (RTX) provided for a favorable outcome, the high relapse rate, that is, up to 80%, precludes successful use of RTX as a monotherapy. Intravenous immunoglobulin (IVIg) is being increasingly utilized as off-label therapy for a variety of autoimmune and inflammatory diseases, including PV and pemphigus foliaceus (PF). AIMS: The goal of pemphigus research is to develop an effective treatment modality that would allow patients to achieve and maintain a stable clinical remission without the need for additional treatments, or cure. MATERIALS AND METHODS: This article summarizes clinical outcome of 123 pemphigus patients treated with a combination of IVIg, an immunosuppressive cytotoxic drug (ICD) and mitochondrion-protecting drugs in the Blistering Disease Clinic at the University of California, Irvine from 2007 to 2017. RESULTS: The mean time to disease control was 0.2 months and time to complete remission - 1.7 months. Duration of complete remission on drugs until relapse or end of treatment was 19.3 months. The mean duration of complete remission off drugs until relapse was 15.8 months. That until end of follow up was 48.4 months, with a minimum of 14 and a maximum of 91 months. The overall complete remission rate off all drugs was 100%, with 12% overall relapse rate. Most relapses, 8.1 vs. 3.3%, occurred during the time of treatment, compared to posttreatment. No patients had more than a single relapse. The duration of the posttreatment follow-up ranged from 9 to 97 months with a mean of 64.8 months, or 5.4 years. The total number of IVIg cycles ranged from 26 in patients without a relapse to 37 in patients with a relapse. The clinical outcome in patients that received IVIg with RTX or another ICD were found to be very similar. DISCUSSION: Thus, the multidrug IVIg regimen allowed to achieve three principal treatment objectives: (i) rapid control of pemphigus symptoms; (ii) stable disease remission; and (iii) overall safety of treatment. CONCLUSIONS: While the individualized therapeutic approaches to eradicate the autoreactive B cell clones causing disease in each particular PV or PF patient are being developed, all pemphigus patients can benefit from the treatment protocol described in this study.

Biomolecular study of human thymidylate synthase conformer-selective inhibitors: New chemotherapeutic approach.

Thymidylate synthase (TS) is a well-validated target for the therapy of adult cancers. Propane-1,3-diphosphonic acid (PDPA) has significant inhibitory properties against human thymidylate synthase (hTS) relative to mouse TS which is not predicted to adopt an inactive conformer. The current research aims to identify novel, lead inhibitors of hTS and examine the prediction that they bind selectively to hTS enzymes existing in different conformational equilibria. Conformer-selectivity was evaluated through performing activity inhibition studies, as well as intrinsic fluorescence (IF) studies in comparison to the known orthosteric inhibitor raltitrexed (RTX). Human TS was isolated from recombinant bacteria expressing either native hTS, capable of conformational switching, or an actively stabilized mutant (R163K-hTS). The examined test compounds were rationally or virtually predicted to have inhibitory activity against hTS. Among these compounds, glutarate, N-(4-carboxyphenyl) succinamic acid, and diglycolic anhydride showed higher selectivity towards native hTS as compared to R163K-hTS. The active site inhibitor RTX showed significantly higher inhibition of R163K-hTS relative to hTS. Targeting hTS via conformational selectivity represents a future approach for overcoming reported resistance towards active-state TS analogs.

Steroid-induced ocular hypertension in the pediatric age group.

PURPOSE: Comparing the effects of topical Rimexolone versus Dexamethasone and Rimexolone versus Fluorometholone on the intraocular pressure in children <13 years. METHODS: A total of 40 patients (80 eyes) undergoing bilateral recession strabismus surgery were divided into two groups. Group A included 20 children (40 eyes); for each, one eye was randomized to receive 1% Rimexolone and the fellow eye received 0.1% Dexamethasone. Group B included 20 children (40 eyes); for each, one eye was randomized to receive 1% Rimexolone and the fellow eye received 0.1% Fluorometholone. Patients received eye drops for two consecutive weeks. Preoperative and postoperative intraocular pressure values for weeks 1, 2, 3, 4, and 6 were measured. The ocular-hypertensive response of all patients was categorized as either high, intermediate or low (Armaly-Becker Classification). RESULTS: After a 2-week treatment for both groups, peak and maximal intraocular pressure changes were reached. Changes were significantly higher in the Dexamethasone-treated eyes than in the Rimexolone- and Fluorometholone-treated eyes, which had a comparable change. (Week 2 intraocular pressure Group A: 14.15 ± 3.23 mmHg vs 17.95 ± 4.27 mmHg; Group B: 15.1 ± 2.27 mmHg vs 15.2 ± 2.73 mmHg). In both groups, the increase was statistically significant compared to the baseline intraocular pressure (preoperative intraocular pressure Group A: 13.2 ± 3.53 mmHg vs 13.1 ± 3.43 mmHg; Group B: 12.55 ± 2.98 mmHg vs 12.15 ± 3.31 mmHg). Intraocular pressure returned to near preoperative values over the following four consecutive weeks (Week 6 intraocular pressure Group A: 12.25 ± 2.67 mmHg vs 12.55 ± 2.95 mmHg; Group B: 12.15 ± 2.8 mmHg vs 12.00 ± 2.75 mmHg). None of the patients were high responders. CONCLUSION: Dexamethasone caused a higher elevation in intraocular pressure than Rimexolone and Fluorometholone in children. The ocular-hypertensive response was transient after the 2-week course.

Automatic procedures for the synthesis of difficult peptides using oxyma as activating reagent: A comparative study on the use of bases and on different deprotection and agitation conditions.

Solid-Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthesis of peptides and small proteins. However, the assembly of peptide sequences classified as "difficult" poses severe synthetic problems in SPPS for the occurrence of extensive aggregation of growing peptide chains which often leads to synthesis failure. In this framework, we have investigated the impact of different synthetic procedures on the yield and final purity of three well-known "difficult peptides" prepared using oxyma as additive for the coupling steps. In particular, we have comparatively investigated the use of piperidine and morpholine/DBU as deprotection reagents, the addition of DIPEA, collidine and N-methylmorpholine as bases to the coupling reagent. Moreover, the effect of different agitation modalities during the acylation reactions has been investigated. Data obtained represent a step forward in optimizing strategies for the synthesis of "difficult peptides".

Synthesis of novel 4'-acylamino modified 21E-benzylidene steroidal derivatives and their cytotoxic activities.

A series of 4'-acylamino modified Δ1,4-pregnadien-21E-benzylidene-3,20-dione derivatives (6a-v) was synthesized from the commercially available progesterone (1). These title compounds were evaluated for their toxicity against brine shrimp (Artemia salina) and cytotoxic activities against two human cancer cell lines (HeLa and MCF-7). The results revealed that compound 6f exhibited promising in vitro cytotoxic activity to the two cancer cell lines and the nature of acylamino functional group in the benzylidene moiety had a significant influence on cytotoxicity.

Evaluation of combined use of Oxyma and HATU in aggregating peptide sequences.

Polypeptides are finding increasing applications as therapeutics because of their specificity that often translates into excellent safety, tolerability, and efficacy profiles in humans. New synthetic methodologies for their preparation are thereby continuously sought to reduce the costs associated to chain assembly and purification. Although solid-phase peptide synthesis has become one of the most advanced synthetic procedures at both laboratory and industrial scale, the process is often complicated by aggregation phenomena originating from the combined occurrence of intermolecular and intramolecular hydrogen bonding, hydrophobic interactions, or other effects. Altogether, these effects cause accumulation of many side products and synthetic mixtures extremely hard to separate and purify, strongly affecting the costs of the final material. In the attempt to optimize the coupling steps of some well-known aggregating or otherwise difficult to obtain peptides, we have comparatively investigated the use of Oxyma/DIC and HATU/Sym-collidine as second coupling reagents in double coupling settings for the preparation of some model peptides. Comparative analytical data obtained on the unpurified products with the two different protocols clearly show that the use of Oxyma/DIC largely improves the content of the target molecules in the final crude materials, making the synthesis more convenient and cost-effective. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Structure Based Docking and Molecular Dynamic Studies of Plasmodial Cysteine Proteases against a South African Natural Compound and its Analogs.

Identification of potential drug targets as well as development of novel antimalarial chemotherapies with unique mode of actions due to drug resistance by Plasmodium parasites are inevitable. Falcipains (falcipain-2 and falcipain-3) of Plasmodium falciparum, which catalyse the haemoglobin degradation process, are validated drug targets. Previous attempts to develop peptide based drugs against these enzymes have been futile due to the poor pharmacological profiles and susceptibility to degradation by host enzymes. This study aimed to identify potential non-peptide inhibitors against falcipains and their homologs from other Plasmodium species. Structure based virtual docking approach was used to screen a small non-peptidic library of natural compounds from South Africa against 11 proteins. A potential hit, 5α-Pregna-1,20-dien-3-one (5PGA), with inhibitory activity against plasmodial proteases and selectivity on human cathepsins was identified. A 3D similarity search on the ZINC database using 5PGA identified five potential hits based on their docking energies. The key interacting residues of proteins with compounds were identified via molecular dynamics and free binding energy calculations. Overall, this study provides a basis for further chemical design for more effective derivatives of these compounds. Interestingly, as these compounds have cholesterol-like nuclei, they and their derivatives might be well tolerated in humans.

Synthesis and biological activity of two pregnane derivatives with a triazole or imidazole ring at C-21.

Pregnane derivatives are studied as agents for the treatment of different hormone-dependent diseases. The biological importance of these steroids is based on their potential use against cancer. In this study, we report the synthesis, characterization and biological activity of two pregnane derivatives with a triazole (3ß-hydroxy-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-20-one; T-OH) or imidazole (3ß-hydroxy-21-(1H-imidazol-1-yl)pregna-5,16-dien-20-one; I-OH) moieties at C-21. These derivatives were synthesized from 16-dehydropregnenolone acetate. The activity on cell proliferation of the compounds was measured on three human cancer cells lines: prostate cancer (PC-3), breast cancer (MCF7) and lung cancer (SK-LU-1). The cytotoxic and antiproliferative effects of T-OH and I-OH were assessed by using SBR and XTT methods, respectively. The gene expressions were evaluated by real time PCR. In addition, results were complemented by docking studies and transactivation assays using an expression vector to progesterone and androgen receptor. Results show that the two compounds inhibited the three cell lines proliferation in a dose-dependent manner. Compound I-OH downregulated the gene expression of the cyclins D1 and E1 in PC-3 and MFC7 cells; however, effect upon Ki-67, EAG1, BIM or survivin genes was not observed. Docking studies show poor interaction with the steroid receptors. Nevertheless, the transactivation assays show a weak antagonist effect of I-OH on progesterone receptor but not androgenic or antiandrogenic actions. In conclusion, the synthesized compounds inhibited cell proliferation as well as genes key to cell cycle of PC-3 and MCF7 cell lines. Therefore, these compounds could be considered a good starting point for the development of novel therapeutic alternatives to treat cancer.

Investigation of benzoyloximes as benzoylating reagents: benzoyl-Oxyma as a selective benzoylating reagent.

Hydroxybenzotriazole (HOBt) and HOBt-derived reagents have been classified as Class I explosives, with restrictions on their transportation and storage. We explored a range of benzoylated oxime-based reagents as alternatives to benzoyloxybenzotriazole (BBTZ) for the selective benzoylation of carbohydrate polyols. Benzoylated oximes derived from 2-hydroximino-malononitrile, ethyl 2-hydroximino-2-cyanoacetate (Oxyma), and tert-butyl 2-hydroximino-2-cyanoacetate were most effective for benzoylation of a simple primary alcohol, with yields approaching that obtained for BBTZ. When applied to carbohydrate diols, the most effective reagent was identified as benzoyl-Oxyma. Benzoyl-Oxyma is a highly crystalline, readily prepared alternative to BBTZ, useful in the selective benzoylation of carbohydrate polyols.

Systemic adalimumab induces peripheral corneal infiltrates: a case report.

BACKGROUND: Tumor necrosis factor-alpha inhibitors are widely used agents in the treatment of immune disorders such as rheumatoid arthritis and inflammatory bowel disease. Despite their anti-inflammatory action, paradoxical drug-induced inflammatory events have been occasionally associated with the use of infliximab, etanercept, and in a lesser extent adalimumab. However, eye involvement is uncommon and anterior uveitis is the only reported ocular adverse manifestation. It can be induced by etanercept, but has also been described during adalimumab therapy. We present here the first report of recurrent peripheral corneal infiltrates following subcutaneous injections of adalimumab. CASE PRESENTATION: A 34 year-old Caucasian woman with Crohn's disease presented to the emergency department with bilateral red eyes and discomfort 36 hours after she received her bimonthly dose of subcutaneous adalimumab. Examination revealed bilateral peripheral corneal infiltrates with characteristic features of immune infiltrates. Symptoms and infiltrates regressed after topical corticosteroid therapy, but recurred after each adalimumab injection over the following weeks. CONCLUSION: Paradoxical immune reactions associated with tumor necrosis factor-alpha inhibitors may result either from hypersensitivity mechanisms, or from immune-complex deposition via anti-adalimumab antibodies. Both mechanisms could explain this newly described manifestation. Care should be taken to search for corneal infiltrates in the event of red eye symptoms during adalimumab therapy since they respond to topical corticosteroids and do not necessarily prompt the discontinuation of the immunosuppressive therapy.

Effect of new hybrids based on 5,16-pregnadiene scaffold linked to an anti-inflammatory drug on the growth of a human astrocytoma cell line (U373).

In spite of the fact that anaplastic astrocytoma is an uncommon disease, very often the pathology of this disease is associated with lethal effects due to the late diagnosis and unspecific treatments. This paper reports the synthesis and the biological effect on the growth of U373 cell line (human anaplastic astrocytoma) of new hybrid compounds based on 5,16-pregnadiene scaffold linked to an anti-inflammatory drug (6a-e). Moreover, we also determined the cell growth effect of five non-steroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, indomethacin and sulindac) as well as the free steroidal alcohol 5. The results from this study indicated that sulindac as well as compound 5 decreased the number of U373 cells at different concentrations. However, when an anti-inflammatory drug was bound to the steroidal structure (5), the resulting compounds (6a-e) showed an enhanced biological effect with exception of hybrid 6c. Furthermore, derivative 6e (sulindac hybrid) did not allow cell growth during six days of experiment at a concentration of 10 µM. The overall data indicated that these molecules showed an anti-proliferative activity on anaplastic astrocytoma cell line.

Film/contact loading method improves the encapsulated amount of triazene anticancer compounds in polymeric micelles.

The development of organic solvent-free methods for the encapsulation of hydrophobic molecules is necessary for advances in micelle-mediated drug delivery. In this study we investigated the film/contact approach in which the use of organic solvents is limited to the preparation of a dry film before encapsulation. Unloaded micelles of five structurally related block copolymers were placed in contact with thin homogeneous films of two hydrophobic triazene anticancer compounds (1-(4-amidophenyl)-3-(4-acetylphenyl)triazene (1) and corresponding triazenido complex with triphenylphosphanegold(I) fragment (2)). The micelle surface becomes saturated with the drug, which eventually penetrates as a front into the core. Because the drug interacts with both the shell and the core microenvironments of micelle during the process, the maximum loading capacities were very sensitive to block copolymer micelle composition, ranging from 2.2 to 20.4% (wt./wt. of polymer). We conclude that micelles with poly[2-(diisopropylamino)ethyl methacrylate] (PDPA) cores are the best option for the encapsulation of triazene compounds because i) they are prepared in absence of organic phase; ii) the drug concentration in the particles is high enough for a therapeutic effect and iii) the responsiveness properties of PDPA is appropriate for practical applications in pH-triggered drug release systems.

Pregna-5,17(20)-dien-21-oyl amides affecting sterol and triglyceride biosynthesis in Hep G2 cells.

Synthesis of series [17(20)Z]- and [17(20)E]-pregna-5,17(20)-dien-21-oyl amides, containing polar substituents in amide moiety, based on rearrangement of 17α-bromo-21-iodo-3ß-acetoxypregn-5-en-20-one caused by amines, is presented. The titled compounds were evaluated for their potency to regulate sterol and triglyceride biosynthesis in human hepatoma Hep G2 cells in comparison with 25-hydroxycholesterol. Three [17(20)E]-pregna-5,17(20)-dien-21-oyl amides at a concentrations of 5 µM inhibited sterol biosynthesis and stimulated triglyceride biosynthesis; their regulatory potency was dependent on the structure of amide moiety; the isomeric [17(20)Z]-pregna-5,17(20)-dien-21-oyl amides were inactive.

Effect of combined topical heparin and steroid on corneal neovascularization in children.

BACKGROUND AND OBJECTIVE: To demonstrate the effect of topical heparin combined with topical steroid on corneal neovascularization (CN) in children. PATIENTS AND METHODS: Four children (5 eyes) with new-onset progressive CN in at least one eye received topical rimexolone or dexamethasone in combination with heparin until complete regression of CN was obtained. The regression of CN was documented by slit-lamp or anterior segment photography. RESULTS: All 5 eyes showed complete regression of CN within 5 months. An anti-angiogenic effect was found as early as 1 week after starting topical combination treatment. No ocular and systemic side effects were detected and treatment was well tolerated by all children. In the 3 eyes with involvement of the optical axis, symmetrical visual acuity was obtained by amblyopia treatment. Recurrence of the CN was detectable in 2 eyes at 1 and 6 months, respectively, after ending combination therapy. Both eyes responded favorably to re-treatment. CONCLUSION: Combination of topical heparin and steroid leads to rapid regression and complete inactivity of CN. This therapeutic approach is promising, especially in children with limited therapeutic alternatives and a high risk for amblyopia.

A new Oxyma derivative for nonracemizable amide-forming reactions in water.

An Oxyma derivative, (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-cyano-2-(hydroxyimino)acetate (2), displayed remarkable physicochemical properties as a peptide-coupling additive for peptide-forming reactions in water. Short peptides to oligopeptides could be synthesized by using 2, EDCI, and NaHCO(3) in water without measurable racemization. Significantly, a simple basic and acidic aqueous workup procedure can remove all reagents utilized in the reactions to afford only coupling products in consistently excellent yields.