Ramizol® encapsulation into extended release PLGA micro- and nanoparticle systems for subcutaneous and intramuscular administration: in vitro and in vivo evaluation

Objective: Novel antibiotic Ramizol® is advancing to clinical trials for the treatment of gastrointestinal Clostridium difficile associated disease. Despite this, previous studies have shown a rapid plasma clearance upon intravenous administration and low oral bioavailability indicating pure drug is unsuitable for systemic infection treatment following oral dosing. The current study aims to investigate the development of poly-lactic-(co-glycolic) acid (PLGA) particles to overcome this limitation and increase the systemic half-life following subcutaneous and intramuscular dosing.
The development of new antibiotic treatments will help in combatting the rising incidence of antimicrobial resistance.
Ramizol® was encapsulated into PLGA nano and microparticles using nanoprecipitation and emulsification solvent evaporation techniques. Formulations were analyzed for particle size, loading level and encapsulation efficiency as well as in vitro drug release profiles. Final formulation was advanced to in vivo pharmacokinetic studies in Sprague–Dawley rats.
Formulation technique showed major influence on particle size and loading levels with optimal loading of 9.4% and encapsulation efficiency of 92.06%, observed using emulsification solvent evaporation. Differences in formulation technique were also linked with subsequent differences in release profiles. Pharmacokinetic studies in Sprague–Dawley rats confirmed extended absorption and enhanced bioavailability following subcutaneous and intramuscular dosing with up to an 8-fold increase in Tmax and T1/2 when compared to the oral and IV routes.
Conclusions: Subcutaneous and intramuscular dosing of PLGA particles successfully increased systemic half-life and bioavailability of Ramizol®. This formulation will allow further development of Ramizol® for systemic infection eradication.

Preclinical development of Ramizol, an antibiotic belonging to a new class, for the treatment of Clostridium difficile colitis

Abstract: Antibiotic-resistant bacteria is a major threat to human health and is predicted to become the leading cause of death from disease by 2050. Despite the recent resurgence of research and development in the area, few antibiotics have reached the market, with most of the recently approved antibiotics corresponding to new uses for old antibiotics, or structurally similar derivatives thereof. We have recently reported an in silico approach that led to the design of an entirely new class of antibiotics for the bacteria-specific mechanosensitive ion channel of large conductance: MscL. Here, we present the preclinical development of one such antibiotic, Ramizol, a first generation antibiotic belonging to that class. We present the lack of interaction between Ramizol and other mammalian channels adding credibility to its MscL selectivity. We determine the pharmacokinetic profile in a rat model and show <0.1% of Ramizol is absorbed systemically. We show this non-systemic nature of the antibiotic translates to over 70% survival of hamsters in a Clostridium difficile colitis model. Lastly, initial in vitro data indicate that resistance to Ramizol occurs at a low frequency. In conclusion, we establish the potential of Ramizol as an effective new treatment for C. difficile associated disease.

Zolav®: a new antibiotic for the treatment of acne

Background: Acne is a prominent skin condition affecting >80% of teenagers and young adults and ~650 million people globally. Isotretinoin, a vitamin A derivative, is currently the standard of care for treatment. However, it has a well-established teratogenic activity, a reason for the development of novel and low-risk treatment options for acne.
Objective: To investigate the effectiveness of Zolav®, a novel antibiotic as a treatment for acne vulgaris.

Materials and methods: Minimum inhibitory concentration of Zolav® against Propionibacterium acnes was determined by following a standard protocol using Mueller-Hinton broth and serial dilutions in a 96-well plate. Cytotoxicity effects on human umbilical vein endothelial cells and lung cells in the presence of Zolav® were investigated by determining the growth inhibition (GI50) concentration, total growth inhibition concentration, and the lethal concentration of 50% (LC50). The tryptophan auxotrophic mutant of Escherichia coli strain, WP2 uvrA (ATCC 49979), was used for the AMES assay with the addition of Zolav® tested for its ability to reverse the mutation and induce bacterial growth. The in vivo effectiveness of Zolav® was tested in a P. acnes mouse intradermal model where the skin at the infection site was removed, homogenized, and subjected to colony-forming unit (CFU) counts.

Results: Susceptibility testing of Zolav® against P. acnes showed a minimum inhibitory concentration of 2 µg/mL against three strains with no cytotoxicity and no mutagenicity observed at the highest concentrations tested, 30 µM and 1,500 µg/plate, respectively. The use of Zolav® at a concentration of 50 µg/mL (q8h) elicited a two-log difference in CFU/g between the treatment group and the control.

 This study demonstrates the potential of Zolav® as a novel treatment for acne vulgaris.

A new antibiotic with potent activity targets MscL

Abstract: The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of non-toxic antimicrobials for the bacteria-sepcific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modelling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant Staphylococcus aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections. 

Aqueous based synthesis of antimicrobial-decorated graphene

Abstract: Ramizol® (1,3,5-tris[(1E)-2’-(4’-benzoic acid)vinyl]benzene) is a potent amphiphilic anti-microbial agent. It is essentially a planar molecule and can interact with the surface of graphene via extended π-π interactions. Herein we demonstrate the utility of Ramizol® in potentially acting as a molecular ‘wedge’ to exfoliate graphene and stabilize it in water. The non-covalent attachment of Ramizol® on the graphene surface enables release of Ramizol® by altering the pH of the solution. Furthermore, the stabilized composite material demonstrates antibacterial activity against Staphylococcus aureus which leads to potential in biomedical applications with graphene acting as a drug carrier as well as enhancing the structural strength of the composite material.

Inspiration from old dyes: New tris-stilbene compounds as potent Gram-positive anti-bacterial agents

Abstract: Herein we describe the preparation and structure-activity relationship studies on range of stilbene based compounds and their antibacterial activity. Two related compounds, each bearing carboxylic acid moieties, exhibit good activity against several bacterial strains, including methicillin-resistant Staphylococcus aureus MRSA (ATCC 33592 and NCTC 10442). Compound 10 was most active against Moraxella catarrhalis with minimum inhibitory concentrations (MICs) of 0.12-0.25 μg/mL and against Staphylococcus spp. with MICs ranging from 2-4 μg/mL. The derivative 17 showed increased activity with MICs of 0.06-0.25 μg/mL against M. catarrhalis and 0.12 μg/mL against Staphylococcus spp. This level of activity is similar to that reported for S. aureus for antibiotics, such as vancomycin, with MICs of ≤2.0 μg/mL and clindamycin with MICs of ≤0.5 μg/mL. As an indicator of toxicity, 17 was tested for its ability to lyse sheep erythrocytes, and showed low haemolytic activity. Such results highlight the value of tris(stilbene) compounds as antibacterial agents providing suitable properties for further development.


A novel antimicrobial agent reduces oxidative stress in cells

Abstract: 1,3,5-Tris[(1E)-2′-(4′′-benzoic acid)vinyl]benzene] (Ramizol™) is a potent and non-toxic synthetic antimicrobial agent, and we now establish that it is also a potent inhibitor of reactive oxygen species (ROS) generation, with similar antioxidant activity to α-tocopherol (Vitamin E), which is a standard antioxidant drug. The results show that Ramizol™ decreases ROS to basal levels in PC12 cells and mouse and guinea pig myocytes, and demonstrate its protective nature from oxidative damage to the cells. The continuing challenge to discover antibiotics with no or few side effects makes Ramizol™ a very attractive candidate for the potential treatment of infections.


The synthesis of fluorescent DNA intercalator precursors through efficient multiple heck reactions

Abstract: A highly efficient synthesis of p-carboethoxy-tristyryl and carboethoxy-terastyrenyl benzene derivatives through a multiple Heck cross coupling reaction is reported. This reaction provides an efficient route to DNA intercalator precursors containing a benzene core.


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