Noncovalent complexation of amphotericin-B with Poly(α-glutamic acid).

A noncovalent complex of amphotericin B (AmB) and poly(α-glutamic acid) (PGA) was prepared to develop a safe and stable formulation for the treatment of leishmaniasis. The loading of AmB in the complex was in the range of ∼20-50%. AmB was in a highly aggregated state with an aggregation ratio often above 2.0. This complex (AmB-PGA) was shown to be stable and to have reduced toxicity to human red blood cells and KB cells compared to the parent compound; cell viability was not affected at an AmB concentration as high as 50 and 200 μg/mL respectively. This AmB-PGA complex retained AmB activity against intracellular Leishmania major amastigotes in the differentiated THP-1 cells with an EC50 of 0.07 ± 0.03-0.08 ± 0.01 μg/mL, which is similar to Fungizone (EC50 of 0.06 ± 0.01 μg/mL). The in vitro antileishmanial activity of the complex against Leishmania donovani was retained after storage at 37 °C for 7 days in the form of a solution (EC50 of 0.27 ± 0.03 to 0.35 ± 0.04 μg/mL) and for 30 days as a solid (EC50 of 0.41 ± 0.07 to 0.63 ± 0.25 μg/mL). These encouraging results indicate that the AmB-PGA complex has the potential for further development

Development of targeted siRNA nanocomplexes to prevent fibrosis in experimental glaucoma filtration surgery

RNA interference induced by double-stranded, small interfering RNA (siRNA) molecules has attracted great attention as a naturally-occurring approach to silence gene expression with high specificity. The Myocardin-Related Transcription Factor/Serum Response Factor (MRTF/SRF) pathway is a master regulator of cytoskeletal gene expression and thus represents a promising target to prevent fibrosis. A major hurdle to implementing siRNA therapies is the method of delivery and we have thus optimised lipid-peptide-siRNA (LPR) nanoparticles containing MRTF-B siRNAs as a targeted approach to prevent conjunctival fibrosis. We tested fifteen LPR nanoparticle formulations with different lipid compositions, surface charges and targeting or non-targeting peptides in human conjunctival fibroblasts. In vitro, the LPR formulation of DOTMA/DOPE lipid with the targeting peptide Y (LYR) was the most efficient in MRTF-B gene silencing and non-cytotoxic compared to the non-targeting formulation. In vivo, subconjunctival administration of LYR nanoparticles containing MRTF-B siRNAs doubled bleb survival in a pre-clinical rabbit model of glaucoma filtration surgery. Furthermore, MRTF-B LYR nanoparticles reduced the MRTF-B mRNA by 29.6% in rabbit conjunctival tissues, which led to significantly decreased conjunctival scarring with no adverse side effects. LYR-mediated delivery of siRNA shows promising results to increase bleb survival and to prevent conjunctival fibrosis after glaucoma filtration surgery

Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.Leona M. and Harry B. Helmsley Charitable Trust (3-SRA-2014-285-M-R)United States. National Institutes of Health (EB000244)United States. National Institutes of Health (EB000351)United States. National Institutes of Health (DE013023)United States. National Institutes of Health (CA151884)United States. National Institutes of Health (P41EB015871-27)National Cancer Institute (U.S.) (P30-CA14051

Molecular yardsticks.

The synthesis and chemistry of a family of Equilibrium Transfer Alkylating Crosslink (ETAC) reagents with extended conjugation were developed. These cross-functionalized reagents were designed for protein cross-link studies. The exchange chemistry of the three carbon bridge 2,2-bis(4-tolylsulfonylmethyl)-m-nitroacetophenone with sulfur and nitrogen nucleophiles was studied where it was determined that: (1) the m-nitrobenzoyl group is a sufficient activating function; (2) the sulfonyl function is a suitable leaving group for undergoing an elimination necessary for the ETAC process. The extended reagents were then prepared. These substituted diene, triene, and tetraene-1-one ETAC reagents were synthesized by a titanium tetrachloride-diisopropylethylamine aldol/dehydration sequence. The exchange chemistry of the extended ETAC reagents with mercaptans was a function of the length and substitution of the extended reagents. Thus, 2-(2-hydroxyethylsulfonylmethyl)-m-nitro-2(Z),4-pentadienophenone undergoes reaction with two equivalents of ethanethiol to form the dithiolether, while 2-(2-hydroxyethylsulfonylmethyl)-5-(p-nitrophenyl)-m-nitro-2(Z),4(E)-pentadienophenone remained unchanged under the same reaction conditions. The exchange chemistry with dimercaptans is a more accurate model of a protein system. The reactions with dimercaptans gave macrocyclic structures. The isolation of crystalline macrocycles containing trans double bonds in 50-55% yields was possible with several of the extended ETAC reagents. The architecture of the adding bis-mercaptan was also important; the adding bis-mercaptan must be long enough to bridge the extended ETAC reagent. The ETAC reagents were successful in the cross-linking of proteins. The aldol/dehydration reaction mediated by titanium tetrachloride was extensively studied. It was possible to prepare: (1) a water soluble extended ETAC reagent, 2-(2-trimethylammoniumethylthiomethyl)-m-nitro-2(Z),4-pentadienophenone; (2) an aliphatic extended ETAC reagent, 3-(2-hydroxyethylsulfonylmethyl)hexa-3(Z),5-diene-2-one; and (3) an extended double-armed ETAC reagent, 2- (3-(2-hydroxyethylsulfonyl)-1-propenyl) -m-nitro-2(E),4-hexadienophenone. These ETAC reagents underwent the corresponding exchange reactions with mono and bis-mercaptans.Ph.D.BiochemistryHealth and Environmental SciencesImmunologyOrganic chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128490/2/9023523.pd

Recent advances in development of amphotericin B formulations for the treatment of visceral leishmaniasis.

PURPOSE OF REVIEW: Amphotericin B (AmpB) is considered the first-line treatment for visceral leishmaniasis in areas in which resistance to antimony is prevalent. This review describes recent advances in clinically available and novel drug delivery systems of AmpB to treat visceral leishmaniasis. RECENT FINDINGS: Over the past two decades, lipid-based AmpB formulations developed to tackle the toxicity of AmpB have been used clinically for the treatment of visceral leishmaniasis. Liposomal AmpB (AmBisome) has been the most successful lipid formulation, and recent clinical studies on visceral leishmaniasis have shown the potential of single-dose AmBisome treatment as well as its use in short course combinations with other antileishmanial drugs. Current research is focussed on the development of more stable and affordable nonlipid formulations of AmpB. Although a diverse range of nonlipid-based AmpB formulations have been evaluated, none have yet reached the clinic. SUMMARY: Liposomal AmpB (AmBisome) has become a standard treatment, by intravenous infusion, for visceral leishmaniasis and the basis for new short course treatments. There have been extensive efforts to develop new AmpB formulations on the basis of polymers, lipids or physical aggregates of AmpB to replace the costly lipid-based formulations. However, no nonlipid-based AmpB delivery systems have yet reached the clinic

Noncovalent Complexation of Amphotericin‑B with Poly(α-glutamic acid)

A noncovalent complex of amphotericin B (AmB) and poly­(α-glutamic acid) (PGA) was prepared to develop a safe and stable formulation for the treatment of leishmaniasis. The loading of AmB in the complex was in the range of ∼20–50%. AmB was in a highly aggregated state with an aggregation ratio often above 2.0. This complex (AmB–PGA) was shown to be stable and to have reduced toxicity to human red blood cells and KB cells compared to the parent compound; cell viability was not affected at an AmB concentration as high as 50 and 200 μg/mL respectively. This AmB–PGA complex retained AmB activity against intracellular <i>Leishmania major</i> amastigotes in the differentiated THP-1 cells with an EC₅₀ of 0.07 ± 0.03–0.08 ± 0.01 μg/mL, which is similar to Fungizone (EC₅₀ of 0.06 ± 0.01 μg/mL). The <i>in vitro</i> antileishmanial activity of the complex against <i>Leishmania donovani</i> was retained after storage at 37 °C for 7 days in the form of a solution (EC₅₀ of 0.27 ± 0.03 to 0.35 ± 0.04 μg/mL) and for 30 days as a solid (EC₅₀ of 0.41 ± 0.07 to 0.63 ± 0.25 μg/mL). These encouraging results indicate that the AmB–PGA complex has the potential for further development

Antileishmanial activity, uptake, and biodistribution of an amphotericin B and poly(α-Glutamic Acid) complex.

A noncovalent, water-soluble complex of amphotericin B (AMB) and poly(α-glutamic acid) (PGA), with AMB loadings ranging from 25 to 55% (wt/wt) using PGA with a molecular weight range of 50,000 to 70,000, was prepared as a potential new treatment for visceral leishmaniasis (VL). The AMB-PGA complex was shown to be as active as Fungizone (AMB deoxycholate) against intracellular Leishmania donovani amastigotes in differentiated THP-1 cells. The in vitro uptake of the AMB-PGA complex by differentiated THP-1 cells was similar to that of Fungizone and higher than that of AmBisome (liposomal AMB). The AMB-PGA complex also displayed a dose-response profile similar to that of AmBisome in vivo in BALB/c mice against L. donovani, with 50% effective doses (ED50s) of 0.24 ± 0.03 mg/kg of body weight for the AMB-PGA complex and 0.24 ± 0.06 mg/kg for AmBisome. A biodistribution study with mice indicated that the AMB-PGA complex cleared more rapidly from plasma than AmBisome, with a comparable low level of distribution to the kidneys