Download Evaluation of Carbohydrate Derived Fulvic Acid (CHD-FA)

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
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•
•
•
•
•
•
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