Designing α-helical peptides with enhanced synergism and selectivity against Mycobacterium smegmatis: Discerning the role of hydrophobicity and helicity.

Recently, we reported on a series of short amphipathic α-helical peptides, comprising the backbone sequence (LLKK)2, with the ability to kill susceptible and drug-resistant Mycobacterium tuberculosis. In this study, the effect of key physicochemical parameters including hydrophobicity and helicity of α-helical peptides on anti-mycobacterial activity and synergism with rifampicin was investigated. The most hydrophobic analogue, W(LLKK)2W, displayed low selectivity against mycobacteria while peptides with intermediate hydrophobicity were shown to be equally active, yet significantly less toxic. Furthermore, proline substitution impeded the formation of stable amphipathic structures, rendering P(LLKK)2P as one of the least active analogues. Terminal capping with isoleucine was found to promote α-helical folding and the resultant peptide demonstrated the highest selectivity and minimal cytotoxicity against mammalian macrophages. Flow cytometric analysis revealed that enhancements in hydrophobicity and α-helicity increased the rate and extent of peptide-mediated membrane permeabilization. This finding corroborated the hypothesis that synergism between the peptides and rifampicin was likely mediated via peptide-induced pore formation. The rapid, concentration-dependent membrane depolarization, leakage of intracellular ATP and calcein release from PE/PG LUVs supported the membrane-lytic mechanism of action of the peptides. Together, these findings suggest that hydrophobicity and α-helicity significantly impact anti-mycobacterial activity and optimization of both parameters is necessary to develop synthetic analogues with superior selectivity indices and enhanced synergistic potential with conventional antibiotics. STATEMENT OF SIGNIFICANCE: There is an urgent clinical need for the discovery of new antimicrobials, effective not just for drug susceptible, but also rapidly emerging drug-resistant TB. Recently, we reported on a series of short amphipathic α-helical peptides, comprising the backbone sequence (LLKK)2, with the ability to kill susceptible and drug-resistant M. tuberculosis. In this study, we evaluated a series of synthetic α-helical (LLKK)2 peptides over a range of hydrophobicities for their activity against mycobacteria and provide the first report on the modulating effect of hydrophobicity and α-helicity on the antimicrobial mechanisms of synthetic AMPs and their synergism with first-line antibiotics. These findings demonstrate the applicability of strategies employed here for the rational design of AMPs with the aim of improving cell selectivity and synergistic interactions when co-administered with first-line antibiotics in the fight against drug-resistant tuberculosis.

Synthetic modifications of the immunomodulating peptide thymopentin to confer anti-mycobacterial activity.

Effective global control of tuberculosis (TB) is increasingly threatened by the convergence of multidrug-resistant TB and the human immunodeficiency virus (HIV) infection. TB/HIV coinfections exert a tremendous burden on the host's immune system, and this has prompted the clinical use of immunomodulators to enhance host defences as an alternative therapeutic strategy. In this study, we modified the clinically used synthetic immunomodulatory pentapeptide, thymopentin (TP-5, RKDVY), with six arginine residues (RR-6, RRRRRR) at the N- and C-termini to obtain the cationic peptides, RR-11 (RKDVYRRRRRR-NH2) and RY-11 (RRRRRRRKDVY-NH2), respectively. The arginine residues conferred anti-mycobacterial activity to TP-5 in the peptides as shown by effective minimum inhibitory concentrations of 125 mg/L and killing efficiencies of >99.99% against both rifampicin-susceptible and -resistant Mycobacterium smegmatis. The immunomodulatory action of the peptides remained unaffected as shown by their ability to stimulate TNF-α production in RAW 264.7 mouse macrophage cells. A distinct change in surface morphology after peptide treatment was observed in scanning electron micrographs, while confocal microscopy and dye leakage studies suggested bacterial membrane disruption by the modified peptides. The modified peptides were non-toxic and did not cause hemolysis of rat red blood cells up to a concentration of 2000 mg/L. Moreover, RY-11 showed synergism with rifampicin and reduced the effective concentration of rifampicin, while preventing the induction of rifampicin resistance. The synthetic peptides may have a potential application in both immunocompetent and immunocompromised TB patients.

Co-delivery of thioridazine and doxorubicin using polymeric micelles for targeting both cancer cells and cancer stem cells.

In this study, thioridazine (THZ), which was reported to kill cancer stem cells, was used in a combination therapy with doxorubicin (DOX) to eradicate both cancer cells and DOX-resistant cancer stem cells to mitigate the reoccurrence of the disease. Both THZ and DOX were loaded into micelles with sizes below 100 nm, narrow size distribution and high drug content. The micelles were self-assembled from a mixture of acid-functionalized poly(carbonate) and poly(ethylene glycol) diblock copolymer (PEG-PAC) and urea-functionalized poly(carbonate) (PUC) and PEG diblock copolymer (PEG-PUC). The drug-loaded mixed micelles (MM) were used to target both cancer cells and stem cells via co-delivery. Cancer stem cells were sorted by a side population assay from BT-474 and MCF-7 human breast cancer cell lines, and identified by CD44+/CD24- phenotype. The cytotoxicity of various formulations was evaluated on the sorted cancer stem cells (side population SP cells), sorted non-stem-like cancer cells (non-side population NSP cells) and unsorted cancer cells. Antitumor activity was also evaluated on BT-474 xenografts in nude mice. As compared with NSP cells, DOX suppressed SP cell growth less effectively, while THZ and THZ-MM were more effective in the inhibition of SP cells. A stronger inhibitory effect was observed on SP cells with the co-delivery of free DOX and THZ or DOX-MM and THZ-MM as compared to free DOX or DOX-MM. THZ and THZ-MM were capable of lowering the population of SP cells in unsorted cells. In the BT-474 xenografts, the co-delivery of DOX-MM and THZ-MM produced the strongest antitumor efficacy, and both THZ and THZ-MM showed strong activity against cancer stem cells. This combination therapy may provide a promising strategy for breast cancer treatment by targeting both cancer cells and cancer stem cells.

Anti-mycobacterial activities of synthetic cationic α-helical peptides and their synergism with rifampicin.

The rapid emergence of multi-drug resistant tuberculosis (TB) and the lack of effective therapies have prompted the development of compounds with novel mechanisms of action to tackle this growing public health concern. In this study, a series of synthetic cationic α-helical antimicrobial peptides (AMPs) modified with different hydrophobic amino acids was investigated for their anti-mycobacterial activity, both alone and in synergistic combinations with the frontline anti-tuberculosis drug rifampicin. The addition of thiol groups by incorporating cysteine residues in the AMPs did not improve anti-mycobacterial activity against drug-susceptible and drug-resistant Mycobacterium tuberculosis, while the enhancement of peptide hydrophobicity by adding methionine residues increased the efficacy of the primary peptide against all strains tested, including clinically isolated multidrug-resistant mycobacteria. The peptide with the optimal composition M(LLKK)2M was bactericidal, and eradicated mycobacteria via a membrane-lytic mechanism as demonstrated by confocal microscopic studies. Mycobacteria did not develop resistance after multiple exposures to sub-lethal doses of the peptide. In addition, the peptide displayed synergism with rifampicin against both Mycobacterium smegmatis and Mycobacterium bovis BCG and additivity against M. tuberculosis. Moreover, such combination therapy is effective in delaying the emergence of rifampicin resistance. The ability to potentiate anti-TB drug activity, kill drug-resistant bacteria and prevent drug resistance highlights the potential utility of the peptide in combating multidrug-resistant TB.

Galactose-functionalized cationic polycarbonate diblock copolymer for targeted gene delivery to hepatocytes.

To mediate selective gene delivery to hepatocytes via the asialoglycoprotein receptors (ASGP-Rs), we designed and synthesized well-defined and narrowly dispersed galactose- and glucose-functionalized cationic polycarbonate diblock copolymers (designated as Gal-APC and Glu-APC, respectively) using organocatalytic ring-opening polymerization of functionalized carbonate monomers, with a subsequent quaternization step using bis-tertiary amines to confer quaternary and tertiary amines for DNA binding and endosomal buffering, respectively. The sugar-functionalized diblock copolymers effectively bound and condensed DNA to form positively charged nanoparticles (<100 nm in diameter and ≈30 mV zeta-potential) that were stable under high physiological salt conditions. In comparison to the control Glu-APC/DNA complexes, Gal-APC/DNA complexes mediated significantly higher gene expression in ASGP-R positive HepG2 cells with no significant difference observed in ASGP-R negative HeLa cells. The co-incubation of Gal-APC/DNA complexes with a natural ASGP-R ligand effectively led to a decrease in gene expression, hence providing evidence for the ASGP-R mediated endocytosis of the polyplexes. Importantly, the Gal-APC/DNA complexes induced minimal cytotoxicities in HepG2 cells at the N/P ratios tested. Taken together, the galactose-functionalized cationic polycarbonate diblock copolymer has potential for use as a non-viral gene vector for the targeted delivery of therapeutic genes to hepatocytes in the treatment of liver diseases.

Antitumor efficacy of a novel CLA-PTX microemulsion against brain tumors: in vitro and in vivo findings.

BACKGROUND: Considering the observations that linoleic acid conjugated with paclitaxel (CLA-PTX) possesses antitumor activity against brain tumors, is able to cross the blood-brain barrier, but has poor water solubility, the purpose of this study was to prepare a novel CLA-PTX microemulsion and evaluate its activity against brain tumors in vitro and in vivo. METHODS: The in vitro cytotoxicity of a CLA-PTX microemulsion was investigated in C6 glioma cells. The in vivo antitumor activity of the CLA-PTX microemulsion was evaluated in tumor-bearing nude mice and rats. The pharmacokinetics of the CLA-PTX microemulsion were investigated in rats, and its safety was also evaluated in mice. RESULTS: The average droplet size of the CLA-PTX microemulsion was approximately 176.3 ± 0.8 nm and the polydispersity index was 0.294 ± 0.024. In vitro cytotoxicity results showed that the IC(50) of the CLA-PTX microemulsion was 1.61 ± 0.83 µM for a C6 glioma cell line, which was similar to that of free paclitaxel and CLA-PTX solution (P > 0.05). The antitumor activity of the CLA-PTX microemulsion against brain tumors was confirmed in our in vivo C6 glioma tumor-bearing nude mice as well as in a rat model. In contrast, Taxol(®) had almost no significant antitumor effect in C6 glioma tumor-bearing rats, but could markedly inhibit growth of C6 tumors in C6 glioma tumor-bearing nude mice. The pharmacokinetic results indicated that CLA-PTX in solution has a much longer circulation time and produces higher drug plasma concentrations compared with the CLA-PTX microemulsion. The results of the acute toxicity study showed that the LD(50) of CLA-PTX solution was 103.9 mg/kg. In contrast, the CLA-PTX microemulsion was well tolerated in mice when administered at doses up to 200 mg/kg. CONCLUSION: CLA-PTX microemulsion is a novel formulation with significant antitumor efficacy in the treatment of brain tumors, and is safer than CLA-PTX solution.

A novel domperidone hydrogel: preparation, characterization, pharmacokinetic, and pharmacodynamic properties.

The purpose of the present study was to prepare a novel domperidone hydrogel. The domperidone dispersion was prepared by the solvent evaporation method. The characteristics of domperidone dispersion were measured by dynamic light scattering (DLS), scanning electronic microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffractometry, and solubility test, respectively. Domperidone hydrogel was prepared by directly incorporating the domperidone dispersion in Carbopol hydrogel to increase its mucoadhesive properties to gastrointestinal tract (GIT). The in vivo pharmacokinetic and pharmacodynamic studies were investigated to evaluate the relative oral bioavailability and the propulsion efficacy of domperidone hydrogel as compared with market domperidone tablet (Motilium tablet). The particle size of domperidone dispersion in distilled water was 454.0 nm. The results of DSC and X-ray indicated that domperidone in dispersion was in amorphous state. The solubility of domperidone in the dispersion in distilled water, pH of 1, 5, and 7 buffer solution was 45.7-, 63.9-, 13.1-, and 3.7-fold higher than that of raw domperidone, respectively. The area under the plasma concentration curve (AUC(0-24)) in domperidone hydrogel was 2.2-fold higher than that of tablet. The prolonged propulsion efficacy in the domperidone hydrogel group compared to that in tablet group was observed in the pharmacodynamic test.

The antiangiogenic efficacy of NGR-modified PEG-DSPE micelles containing paclitaxel (NGR-M-PTX) for the treatment of glioma in rats.

Aminopeptidase N (APN), recognized by Asn-Gly-Arg (NGR) peptides, is expressed in the pericytes associated with the BBB, and the main objective of this study is to confirm the hypothesis that NGR-modified DSPE-PEG micelles containing paclitaxel (NGR-M-PTX) can bind to and kill brain tumor angiogenic blood vessels and penetrate into the brain tumor interstitial space, resulting in direct cell death. NGR-M-PTX is prepared by a thin-film hydration method. The in vitro targeting characteristics of NGR-modified micelles on BMEC (murine brain microvascular endothelial cells) were investigated. The effect of NGR-M-PTX on BMEC proliferation and the cytotoxicity of NGR-M-PTX in C6 glioma cells were also tested. The antitumor activity NGR-M-PTX was evaluated in C6 glioma tumor-bearing rats in vivo. The particle size of NGR-M-PTX was approximately 54.2 nm. The drug encapsulation efficiency of NGR-M-PTX was 82.11 ± 2.82%. The cellular coumarin-6 level of NGR-M-coumarin-6 in the BMEC was about 2.2-fold higher than that of M-coumarin-6. BMEC proliferation was significantly inhibited by NGR-M-PTX. NGR-M-PTX had a much lower IC(50) value than M-PTX and free drug. The growth of C6 glioma tumor was markedly inhibited by NGR-M-PTX compared with Taxol. In conclusion, our results show that antiangiogenic therapy using NGR-M-PTX exhibits potent in vivo antitumor activity in a C6 glioma-bearing animal model.

Antiangiogenic activity of sterically stabilized liposomes containing paclitaxel (SSL-PTX): in vitro and in vivo.

The purpose of this present study was to evaluate the antiangiogenic activity of sterically stabilized liposomes containing paclitaxel (SSL-PTX). The SSL-PTX was prepared by the thin-film method. The release of paclitaxel from SSL-PTX was analyzed using a dialysis method. The effect of SSL-PTX on endothelial cell proliferation and migration was investigated in vitro. The antitumor and antiangiogenic activity of SSL-PTX was evaluated in MDA-MB-231 tumor xenograft growth in BALB/c nude mice. The release of paclitaxel from SSL-PTX was 22% within 24 h. Our in vitro results indicated that SSL-PTX could effectively inhibit the endothelial cell proliferation and migration at a concentration-dependent manner. We also observed that metronomic SSL-PTX induced marked tumor growth inhibition in MDA-MB-231 xenograft model via the antiangiogenic mechanism, unlike that in paclitaxel injection (Taxol) formulated in Cremophor EL (CrEL). Overall, our results suggested that metronomic chemotherapy with low-dose, CrEL-free SSL-PTX should be feasible and effective.

The therapeutic efficacy of conjugated linoleic acid - paclitaxel on glioma in the rat.

Considering the effects of conjugated linoleic acid (CLA) on anti-tumor and anti-angiogenic in brain tumor, synergistic anti-tumor activity with taxane as well as potential activity for transporting chemotherapeutic agents across the blood-brain barrier (BBB), the purpose of this study was to synthesize CLA-paclitaxel (CLA-PTX) conjugate which could reach to the brain tissue and target brain tumor. The CLA was covalently linked to PTX. The conjugate was stable in PBS and rat plasma in vitro and had no microtubule assembly activity in solution and slight effect of arresting cell cycle progression at the G(2)-M phase. The in vitro cytotoxicity of conjugate was lower than that of PTX (p < 0.05). The conjugate showed higher cellular uptake efficiency on C6 glioma cells. The entire pharmacokinetic index revealed the significant enhancement of the conjugate pharmacokinetics compared with that in PTX (p < 0.01). The conjugate, unlike PTX, could distribute in brain tissue and retained higher concentrations throughout 360 h. The anti-tumor efficacy in brain tumor-bearing rats after administering conjugate was significantly higher than that after giving Taxol (p < 0.01). In conclusion, this CLA-PTX conjugate showed great potential to become a new prodrug of PTX and the methodology can be applied to other anticancer drugs.

In vitro and in vivo studies on plasma-to-blood ratio of paclitaxel in human, rabbit and rat blood fractions.

Many pharmacokinetic studies of paclitaxel formulations with or without Cremophor (CrEL) have been performed on experimental animals. However, limited studies describe the different pharmacokinetic behaviors of paclitaxel in animals. The different distribution of drug in blood fractions may have great effect on its pharmacokinetic behaviors. Our present study was designed to study the characteristics of paclitaxel distribution in human, rabbit and rat blood, by measuring plasma-to-blood ratio (PBR) of paclitaxel in vitro and in vivo, and analyzing the results of equilibrium dialysis of paclitaxel with erythrocyte, plasma and hemoglobin. It was demonstrated that the paclitaxel PBR values in rat, unlike those in rabbit, are most significantly different from those in human, which may be due to distinct affinity of paclitaxel to blood fractions among different species. The effect of CrEL on increasing paclitaxel plasma concentration and in vitro & in vivo correlation in animal PBR values were observed. The findings in this study are of significance in the evaluation of the newly developed formulations of paclitaxel.