An Investigation into Multifaceted Mechanisms of Action of Allantoin in Wound Healing

Amy Paller1, Ron Nardi2, Hung Do3, Allen Reha3, Christopher Viereck3, Jeffrey Castelli3, Jay Barth3

1Northwestern University Feinberg Medical School, Chicago, IL, USA; 2Scioderm - An Amicus Therapeutics Company, Durham, NC, USA; 3Amicus Therapeutics, Inc., Cranbury, NJ, USA

Conflict of Interest: AP: Scioderm consultant, honoraria, investigator, grants. RN: Consultant to Amicus. HD, AR, CV, JC, JB: Amicus employee

NP-NN-US-0004 Background • Allantoin is a heterocyclic organic compound produced by animals, bacteria, and plants • While different allantoin-containing preparations have been used clinically to study its therapeutic effects in wound healing, its mechanisms of action are not known with certainty Methods • Systematic literature search for preclinical and clinical studies to further understand the mechanism of action (MOA) of allantoin in the context of wound healing • The following search terms were used: wound, burn, scar, pruritus, anti-inflammatory, antioxidant, fibroblast, collagen, antifungal, antimicrobial, necrotic, and keratolytic • ~100 preclinical studies (in vitro and animal models) and ~30 clinical studies were found focusing on MOA impacting the wound healing process Anti-Inflammatory Effects

• Allantoin 5% emulsion resulted in fewer inflammatory cells in wounds compared with no treatment at all time points (Wistar rat model). • Allantoin 1% treatment resulted in nearly complete inhibition of edema caused by chemical burn with phorbol myristate acetate (PMA) applied to mouse ear.

Number of Inflammatory Cells in a % Reduction of Edema Caused by PMA Rat Wound Model by Treatment

1000 Control (no treatment) 100 96.11 ± 5.41 92.15 ± 0.32 Emulsion 800 Emulsion with 5% allantoin 80 68.4 ± 7.04 600 60 400 a 200 40

0 20 Number of Inflammatory Cells Inflammatory of Number 0 7 14 21 28

Days From Wound Infliction PMA (%) Causedby of Edema Inhibition 0 Allantoin aP<0.05 for emulsion containing 5% allantoin compared with α-Bisabolol (0.1%) 18 β-glycyrrhetinic acid control group. (1%) (1%)

Araújo LU et al. Acta Cir Bras. 2010;25:460-466. Couteau C et al. Arch Dermatol Res. 2012;304:817-821. Anti-Inflammatory Effects Allantoin Reduced Expression of Pro-inflammatory Markers and Cytokines in Cell Culture Assays

Red Box: Control Blue Box: Allantoin (200 µM) Treatment • In cell cultures, allantoin CD40 Expression 15,000 CD83 Expression 8000 200 µM led to 10,000 downregulation of 6000 4000 maturation surface 5000

2000 Intensity (by FACS) (by Intensity

molecule CD40 and Fluorescence Mean Intensity (by FACS) (by Intensity Mean Fluorescence Fluorescence Mean 0 dendritic cell 0 maturation marker CD83, and a slight increase in the secretion IL-12 p70 Secretion PBL Proliferation 3000 b c c of tumor growth factor a 60 (TGF)-β 2000 40

1000 20

0 % Proliferation Concentration (pg/mL) Concentration 0 aP<0.05; bP<0.01; cP<0.001. TGF-β, tumor growth factor beta; FACS, fluorescence-activated cell sorting; DC, dendritic cells; DC + B-met, DCs incubated with β-methasone (50 ng/mL); DC + All, DCs incubated with allantoin (200 µM); DC + Enox, DCs incubated with enoxolon (50 µm); DC + Bisa, DCs incubated with bisabolol (0.01%); DC + BZ4, DCs incubated with benzophenone-4 (0.01%); PHA, phytohemagglutinin; ELISA, enzyme-lined immunosorbent assay. Frikeche J et al. Arch Dermatol Res. 2015;307:211-218. Keratolytic Activity

• In patients with psoriasis, 14-days open-label treatment with allantoin decreased disease manifestations including a reduction in hyperkeratosis

Effects of Allantoin (2% w/w) in Psoriasis Example Study Results: Effect of Allantoin Preparation With Lanacolic Vehicle on Hyperkeratosis1 Degreea of hyperkeratosis +++ ++ + 0 Patients prior to therapy, n 9 6 0 0 7 days post-treatment 3 7 5 0 14 days post-treatment 0 6 2 7 aDegree expressed as: + + + (very expressed), + + (moderately expressed degree), + (poorly expressed), 0 (not expressed).

1. Cajkovac M et al. Pharmazie. 1992;47:39-43; Anti-microbial Effects

• Allantoin exhibited antibacterial activity against many bacterial strains1 and exhibited antifungal and antiviral activity against several strains of microorganisms in vitro2 • Allantoin in a solid lipid nanoparticle containing copaiba oil resulted in potent antifungal activity3 Antibacterial Activity Tested Against Standard and Isolated Strains of Microorganisms1 Escherichia coli Pseudomonas aeruginosa Proteus mirabilis Klebsiella pneumoniae MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) Compound ATCC 35218 Isolated ATCC 10145 Isolated ATCC 7002 Isolated RSKK 574 Isolated strain ESβL+ strain strain ESβL+ strain ESβL+ Allantoin 8 128 4 32 8 128 8 128 Ampicillin 2 >128 - - 2 >128 2 >128 (reference) Ofloxacin 0.12 0.5 1 64 <0.12 1 <0.12 0.5 (reference)

Acinetobacter baumannii Staphylococcus aureus Enterococcus faecalis Bacillus subtilis MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) MIC (µg/mL) Compound RSKK 02026 Isolated ATCC 25923 Isolated ATCC 29212 Isolated ATCC 6633 Isolated strain strain MRSA strain strain Allantoin 2 64 16 >128 8 128 8 16 Ampicillin 2 >128 <0.12 >128 0.5 >128 0.12 0.5 (reference) Ofloxacin 0.12 64 0.25 64 1 32 - - (reference)

MIC, minimum inhibitory concentration. 1. Özçelik B et al. Pharm Biol. 2011;49:396-402. 2. Özçelik B et al. Pharm Biol. 2011;49:396-402. 3. Svetlichny G et al. Pharmazie. 2015;70:155-164. Fibroblasts • Allantoin and crude extracts of allantoin-containing Arrabidaea chica added to human fibroblast cell cultures demonstrated a concentration-dependent increase in fibroblast proliferation • Allantoin and A. chica extract significantly improved surgically induced wound healing in Wistar rats by days 5 and 10 compared with saline control Concentration-Dependent Fibroblast Growth Wound Healing Observed as Percent of Percentage Induced by Allantoin and A. chica Extract Contraction for Each Treatment Group

100 a* Saline a* 200 Allantoin Allantoin 80 A. chica A. chica 150 60

a* 40 a* 100 20 a*b†

Fibroblast Growth Percentage Growth Fibroblast 0 50

10-1 0.25 100 0.5 101 25 102 250 Percent of Contraction Wounds of of Contraction Percent Concentration (µg/mL) 2nd Day 5th Day 10th Day

Jorge MP et al. J Ethnopharmacol. 2008;118:361-366. aDifferent from saline; bdifferent from allantoin (100 mg/mL). *P<0.001 †P<0.01. Jorge MP et al. J Ethnopharmacol. 2008;118:361-366. Collagen Synthesis

• Allantoin and A. chica extract improved collagen synthesis in the wounds of Wistar rats analyzed 11 days after injury • Allantoin 5% emulsion and the emulsion alone increased collagen stimulation compared with control (without treatment) at all times

Effects of Allantoin and A. chica Extract on Effects of Allantoin on Collagen Synthesis Collagen Synthesis in Wistar Rat Wounds in Wistar Rat Wounds a 0.30 a 400,000 Control (without treatment)

0.25 Emulsion 300,000 Emulsion with 5% allantoin 0.20 b

0.15 200,000

0.10 100,000

Hydroxyproline (µg/mL) Hydroxyproline 0.05 Collagen Staining Intensity Intensity Staining Collagen

0.00 0 0 7 14 21 28 Saline Allantoin A. chica (Control) Days From Wound Initiation

aP<0.001 compared with fibroblast control group. Araújo LU et al. Acta Cir Bras. 2010;25:460-466. Jorge MP et al. J Ethnopharmacol. 2008;118:361-366. Summary Fibroblast Anti-inflammatory Effects Keratolytic Antimicrobial Collagen Synthesis Proliferation . Allantoin reduced . In patients with . Allantoin showed in . Allantoin . Allantoin chemotaxis of psoriasis, allantoin vitro antiviral, demonstrated demonstrated inflammatory cells decreased disease antibacterial, and increased fibroblast increased collagen into rat wounds manifestations antifungal activity proliferation in cell synthesis in . Allantoin application . In vitro, allantoin based on 2 culture assays cultured fibroblast prevented the edema dispersed psoriatic separate studies . Allantoin increased . Histology of associated with scales into solution tissue regeneration rat wound models chemical burns in and epithelialization demonstrated mice in rat wound models increased collagen . In cell culture, synthesis allantoin decreased immune activity and inhibited cytokine secretion

• Studies used different formulations of allantoin and there was a lack of information on the stability or dermal penetration of allantoin • Additional research is warranted to further characterize clinically relevant mechanisms of action in human skin and to determine the relative role of these various mechanisms in wound healing