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Evaluation of Carbohydrate Derived Fulvic Acid (CHD-FA) as a Topical Broad-Spectrum Antimicrobial for Drug Resistant Wound Infections David S. Perlin, PhD Executive Director and Professor Public Health Research Institute Rutgers-New Jersey Medical School Project Dates: 2012-2016 Funding Amount: 1,530,400 Pilot Study funded by: October 2012 CDMRP Joint Program Committee-2/Military Infectious Diseases Research Program Review Militarily Relevant Issue to be Solved • Increase in battlefield wound infections due to growing drug resistant bacteria and molds • Infectious complications cause enhanced morbidity and mortality Aspergillus Klebsiella Drug Resistance Acinetobacter Solution Carbohydrate Derived Fulvic Acid (CHD-FA) • • • A stable topical agent that is effective against a wide range of drug resistant bacteria and fungi that can be deployed in theatre to limit or eliminate wound infections CHD-FA is a broad spectrum anti-microbial/anti-inflammatory compound active on drug resistant Gram positive, Gram negative, and mold pathogens that can be applied safely topically. Fulvic acid is a humic substance formed during the decay of plant and animal residues in the environment but which is often contaminated. Carbo-Hydrate-Derived Fulvic Acid (CHD-FA) is a pure form of fulvic acid manufactured from a carbohydrate source and is contaminant-free Fulvic Acid Project Description • Assess in vitro susceptibility of CHD-FA against MDR bacteria and fungal pathogens • Evaluate efficacy of CHD-FA in a rat wound infection model (open, burn) with MDR bacterial and fungal pathogens • • • • • • • Rat Wound Model Anesthetize SD rats Cut two 0.9 cm sym excision wounds Glue cylindrical chamber to wound Add pathogen and seal with Tegaderm Tx BID w/ saturated dressing 30 min postinoculation and on days 1-6 Obtain daily digital images, evaluate wound health Assess histopathology and wound-related genes Study Timeline Successes To Date • Microbial burden reduction – 5.2, 4.5, 6.3 log reduction on day 1,3,6 in P. aeruginosa infection model compared to the untreated – 2, 1.8, 5.4 log reduction on day 1,3,6 in MRSA infection model compared to the untreated • Decreased molecular inflammation – Dampened IL-6 level in CHD-FA treated group at days 3 and 6 compared to the baseline in P. aeruginosa infection model – In comparison, 3500 fold increase in expression of IL-6 at day 6 in the untreated group compared to the baseline • Enhanced wound healing by histopathology – Lower neutrophils as early as day 3 in CHD-FA treated group compared to the untreated in P. aeruginosa infection model Potent in vitro properties against target bacterial and fungal pathogens CHD-FA promotes wound closure and wound healing Wounds infected with 1×108 CFU MRSA, treated with CHD-FA as indicated for 10 days CHD-FA effectively reduces microbial burdens in wounds infected by MDR P. aeruginosa relative to colistin Pseudomonas infected Day 3 Wounds CHD-FA Treated Untreated Bacterial burdens of wounds infected with P. aeruginosa at Day 1, 3 and 6 endpoints Day 1 Day 3 Log Fold Avg. Log CFU Changes Range Day 6 Log Fold Avg. Log CFU Changes Range Log Fold Changes 7.2 NA NA 4.5 0.9 0.0-1.7 6.3 0.4 7.6 NA -0.4 Treatment Avg. Log CFU Range No Tx Control 5.2 NA NA 7.5 NA NA CHD-FA Sterilized NA 5.2 3.0 1.7-4.2 AB Control Sterilized NA 5.2 7.1 NA CHD-FA gel (4.6%) dressing twice a day applied to the wound site resulted in a rapid wound sterilization. Study P. aeruginosa 500 CFU 0.5h Tx P. aeruginosa 500 CFU Histopathology confirms wound healing and decreased inflammation •Less neutrophils present in the highest CHD-FA dose relative to the Colistin (antibiotic control) sample. •The histopathologic analysis from day 6, clearly demonstrates improved wound healing in the treated groups. The sham treated group still had an increased presence of neutrophils and no cellular evidence of healing or remodeling. •In both the CHD-FA and Colistin treated groups, remodeling is present and in the highest CHD-FA dose, epithelialization is evident as seen with the Colistin (antibiotic) treated group. •Compared to untreated control, the treated groups are in the advanced stages of wound healing by their high fibroblast, angiogenesis and epithelialization scores Wound healing gene expression profiling • 84 key genes central to the wound healing response • Wound healing progresses via three overlapping phases: inflammation, granulation and tissue remodeling. Wound Healing RT2 Profiler PCR Array Gene Table Symbol Acta2 Description Smooth muscle alpha-actin Symbol Cxcl1 Actc1 Actin, alpha, cardiac muscle 1 Angpt1 Symbol Itga2 Description Integrin, alpha 2 Symbol Ptgs2 Cxcl11 Description Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating Chemokine (C-X-C motif) ligand 11 Itga3 Integrin, alpha 3 Rac1 Angiopoietin 1 Cxcl3 Chemokine (C-X-C motif) ligand 3 Itga4 Integrin, alpha 4 Rhoa Ccl12 Chemokine (C-C motif) ligand 12 Cxcl5 Chemokine (C-X-C motif) ligand 5 Itga5 Chemokine (C-C motif) ligand 7 Egf Epidermal growth factor Itga6 Integrin, alpha 5 (fibronectin polypeptide) alphaalpha receptor, 6 Integrin, Serpine1 Ccl7 Cd40lg CD40 ligand Egfr Epidermal growth factor receptor Itgav Integrin, alpha V Tagln Cdh1 Cadherin 1 F13a1 Itgb1 Integrin, beta 1 Tgfa Transforming growth factor alpha Col14a1 Collagen, type XIV, alpha 1 F3 Coagulation factor XIII, A1 polypeptide Coagulation factor III Itgb3 Integrin, beta 3 Tgfb1 Transforming growth factor, beta 1 Col1a1 Collagen, type I, alpha 1 Fga (thromboplastin, tissue factor) Fibrinogen alpha chain Itgb5 Integrin, beta 5 Tgfbr3 Col1a2 Collagen, type I, alpha 2 Fgf10 Fibroblast growth factor 10 Itgb6 Integrin, beta 6 Timp1 Transforming growth factor, beta receptor III inhibitor 1 TIMP metallopeptidase Col3a1 Collagen, type III, alpha 1 Fgf2 Fibroblast growth factor 2 Mapk1 Mitogen activated protein kinase 1 Tnf Col4a1 Collagen, type IV, alpha 1 Fgf7 Fibroblast growth factor 7 Mapk3 Mitogen activated protein kinase 3 Vegfa Col4a3 Collagen, type IV, alpha 3 Hbegf Vitronectin Hgf Mmp1 Macrophage migration inhibitory factor 1a Matrix metallopeptidase Vtn Collagen, type V, alpha 1 Heparin-binding EGF-like growth factor Hepatocyte growth factor Mif Col5a1 Wisp1 Col5a2 Collagen, type V, alpha 2 Ifng Interferon gamma Mmp2 collagenase)2 (interstitial metallopeptidase Matrix Wnt5a WNT1 inducible signaling pathway protein 1 integration Wingless-type MMTV Col5a3 Collagen, type V, alpha 3 Igf1 Insulin-like growth factor 1 Mmp7 Matrix metallopeptidase 7 Actb site family, Actin,member beta 5A Csf2 Colony stimulating factor 2 (granulocyte-macrophage) Colony stimulating factor 3 Il10 Interleukin 10 Mmp9 Matrix metallopeptidase 9 B2m Beta-2 microglobulin Csf3 Il1b Interleukin 1 beta Pdgfa Hprt1 Ctgf (granulocyte) Connective tissue growth factor Il2 Interleukin 2 Plat Platelet-derived growth factor alpha polypeptide activator, tissue Plasminogen Ldha Hypoxanthine phosphoribosyltransferase Lactate dehydrogenase A1 Ctnnb1 Il4 Interleukin 4 Plau Plasminogen activator, urokinase Rplp1 Ribosomal protein, large, P1 Ctsg Catenin (cadherin associated protein), beta Cathepsin G1 Il6 Interleukin 6 Plaur Ctsk Cathepsin K Il6st Interleukin 6 signal transducer Plg Plasminogen activator, urokinase receptor Plasminogen Ctsl1 Cathepsin L1 Itga1 Integrin, alpha 1 Pten Phosphatase and tensin homolog Itga2 Integrin, alpha 2 Ptgs2 Itga3 Integrin, alpha 3 Rac1 Prostaglandin-endoperoxide 2 synthase toxin C3 botulinum Ras-related 1 member substrate gene family, Ras homolog Rhoa Serpine1 Stat3 Serpin peptidaseAinhibitor, clade E activator of plasminogen (nexin, and activator transducer Signal Stat3 Description Prostaglandin-endoperoxide synthase 2 Ras-related C3 botulinum toxin substrate 1 member Ras homolog gene family, A Serpin peptidase inhibitor, clade E (nexin, plasminogen activator of Signal transducer and activator transcription Transgelin 3 Tumor necrosis factor (TNF superfamily, member Vascular endothelial growth 2) factor A Temporal changes of wound healing gene expression No animal available. All rats in untreated group died before day 10. Day 3 Day 6 Day 10 Temporal changes of individual gene targets Csf2 (GM-CSF), granulocyte-macrophage colony stimulating factor, a pleiotropic cytokine shown to be mitogenic for keratinocytes and to stimulate migration and proliferation of endothelial cells CXCL1 (growth-related oncogene-α, GRO-α) is potent regulator of neutrophil chemotaxis. In acute human excisional wounds, its expression profile correlated with keratinocyte migration and with neovascularization. IL10 is a proinflammation cytokine. It inhibits inflammation and scar formation Wound healing gene expression profiling revealed strong anti-inflammatory properties of CHD-FA CHD-FA modulates key inflammatory mediators in vitro Sherry et al. BMC Oral Health (2013)13:47. Genetic profiling summary Expression of genes required for wound healing were increased earlier and more pronounced in CHD-FA treated rats compared to untreated controls. For a majority of gene targets, expression levels return to baseline at day 6 in the CHD-FA treated rats, while remain stable or increase at day 6 post injury in the untreated animals. CHD-FA treatment accelerates wound healing process Overall Conclusions • CHD-FA is highly stable and safe. • It has potent antimicrobial behavior against a wide range of multidrug resistant Gram negative, Gram positive and fungal pathogens • Anti-infective and anti-inflammatory properties promote wound management and healing Challenges • To ascertain MOA of CHD-FA • To assess value for preventing burn infections • To confirm efficacy in humans beyond anecdotal reports Competing Solutions • Wound management standard of care – Antibiotic treatment – Anti-inflammatory agents: steroids Progression and factors influencing wound infections Standard of care Steroids for burns Antibiotics CHD-FA application Inflammation in burns Δ inflammation Resistance developed Burden rebound Δ burden Inflammation Microbial burden Limitations of standard of care: • Microbial rebound • Drug-resistant pathogens • Does not control inflammatory response Advantage of CHD-FA: • Extremely stable field use with long half-life • Active against MDR gram- strains with negligible resistance occurrence • Non-toxic • Has clinical history for other indications, esp. topical application Reportable Outcomes Zhao Y, Paderu P, Delmas G, Dolgov E, Lee MH, Senter M, Park S, Leivers S, Perlin DS (2015) Carbohydrate-derived fulvic acid is a highly promising topical agent to enhance healing of wounds infected with drug-resistant pathogens. J Trauma Acute Care Surgery 79: S121-129. Paderu P, Zhao Y, Delmas G, Park S, Leivers S, Perlin DS Carbohydrate-derived fulvic acid is a highly effective topical broad-spectrum antimicrobial for drug-resistant wound infections. ICAAC 2014. Perlin DS. Evaluation of carbohydratederived fulvic acid is as a topical broadspectrum antimicrobial for drug resistant wound infections. MHSRS 2014. Next Steps • Further in vivo evaluation of CHD-FA (open and burn model) with dressing pad against other key drug resistant bacteria and fungi • Progress into human clinical trials Human superficial burns Human lower leg ulcers To demonstrate enhanced healing and prevention and cure infection in human. Normal skin contaminations wound be determined and monitored on wound for duration of study. Comparator is sulfadiazine cream. These are established contaminated wounds with mixed organism infections, i.e. Gram+ plus Gram- plus fungi. Comparative study with best alternative care. Plans to Apply for Additional Funding • CDMRP clinical trial funding • NIAID • Pharmaceutical company sponsor