1. 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

2. 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 Drug Resistance Acinetobacter Aspergillus Klebsiella

3. 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

4. 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 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 post- inoculation and on days 1-6 Obtain daily digital images, evaluate wound health Assess histopathology and wound-related genes Project Description Rat Wound Model

5. Study Timeline

6. 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

7. Potent in vitro properties against target bacterial and fungal pathogens

8. CHD-FA promotes wound closure and wound healing Wounds infected with 1×10 8 CFU MRSA, treated with CHD-FA as indicated for 10 days

9. CHD-FA gel (4.6%) dressing twice a day applied to the wound site resulted in a rapid wound sterilization. Bacterial burdens of wounds infected with P. aeruginosa at Day 1, 3 and 6 endpoints Pseudomonas infected Day 3 Wounds CHD-FA Treated Untreated CHD-FA effectively reduces microbial burdens in wounds infected by MDR P. aeruginosa relative to colistin

10. 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

11. Wound Healing RT 2 Profiler PCR Array Gene Table 84 key genes central to the wound healing response Wound healing progresses via three overlapping phases: inflammation, granulation and tissue remodeling. W ound healing gene expression profiling

12. Temporal changes of wound healing gene expression Day 3 Day 6 Day 10 No animal available. All rats in untreated group died before day 10.

13. 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

14. Wound healing gene expression profiling revealed strong anti-inflammatory properties of CHD-FA

15. CHD-FA modulates key inflammatory mediators in vitro Sherry et al. BMC Oral Health (2013)13:47.

16.  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 Genetic profiling summary

17. 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

18. Challenges To ascertain MOA of CHD-FA To assess value for preventing burn infections To confirm efficacy in humans beyond anecdotal reports

19. Competing Solutions Wound management standard of care – Antibiotic treatment – Anti-inflammatory agents: steroids

20. Microbial burden Inflammation in burns Inflammation Standard of care Steroids for burns Antibiotics Resistance developed Burden rebound CHD-FA application Δ inflammation Δ burden Progression and factors influencing wound infections

21. 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

22. 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 carbohydrate- derived fulvic acid is as a topical broad- spectrum antimicrobial for drug resistant wound infections. MHSRS 2014.

23. 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 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. Human lower leg ulcers These are established contaminated wounds with mixed organism infections, i.e. Gram+ plus Gram- plus fungi. Comparative study with best alternative care.

24. Plans to Apply for Additional Funding CDMRP clinical trial funding NIAID Pharmaceutical company sponsor