antimicrobial activity of vulnafast plus

Antimicrobial efficacy of VULNOFAST® plus

Presentation Outline

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• VULNOFAST® (RLP068/Cl):• VULNOFAST® products

• VULNOFAST® plus antimicrobial efficacy in APDT vs Staphylococcus aureus ATCC 43300:

• Aim of the study

• Experiment protocol

• Data analysis

• Results

• Conclusion

RLP068/Cl: the main component of VULNOFAST®

• RLP068/Cl is the photosensitizer present in the VULNOFAST® products.

• RLP068/Cl is a tetrasubstituted Zinc-phthalocyanine derivative bearing quaternary ammonium groups, manufactured under cGMPs.

• RLP068/Cl is a patented compound and belongs to the original research carried out at Molteni Therapeutics.

NN

NNN N

NN

Zn

O

N+

O

N+

O

N+

O

N+

Cl-Cl

-

Cl- Cl

-

C68H64Cl4N12O4ZnF.W. = 1320.5

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• Data analysis

• Results

• Conclusion

VULNOFAST®: pioneering the PDTtreatment of skin lesions and ulcers

• VULNOFAST®: topically delivered sterile formulations comprising a red light photosensitiser (i.e. RLP068/Cl).

Two products (VULNOFAST® gel and VULNOFAST® plus) with different formulations.

• VULNOLIGHT®: a photodynamic light source, based on L.E.D. technology, specifically designed for the activation of VULNOFAST® products (light energy delivered - 60 J/cm2 in approximately 8 minutes).

Molteni has developed a therapeutic approach for the local treatment of skin lesions and ulcers by

means of photodynamic therapy, based on its proprietary products:

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VULNOFAST®: a new tool for ulcers and wounds topical treatment

• VULNOFAST® products are topically administered onto skin lesions or ulcers; after a suitable waiting time the lesion is illuminated with therapeutic red light.

• VULNOFAST® rapidly achieves a reduction of the pathogenic bacterial flora after the treatment without generating bacteria resistance*.

• VULNOFAST® can be used in combination with systemic antibiotic regimens. VULNOFAST® has been designed as a flexible and cost-effective tool to be

introduced in the ulcers’ management protocols.

• VULNOFAST® gel has been certified in the EU as a class IIb Medical Device in 2013; VULNOFAST® plus has been certified in the EU as a class IIb Medical Device in 2014

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* F. Giuliani, Martinelli M., Cocchi A., Arbia D., Fantetti L., and Roncucci G. (2010): In vitro resistance selection studies of RLP068/Cl, a new Zn(II) phthalocyanine suitable for antimicrobial photodynamic therapy. Antimicrob Agents Chemother 54(2): 637-642.

What is VULNOFAST® gel?

• VULNOFAST® gel is a sterile and single use medical device according to Directive 93/43/EEC. The product is preservative free.

• VULNOFAST® gel contains the proprietary photosensitizer RLP068/Cl

(0.3% w/w). VULNOFAST® gel has a non aqueous basis optimised to enhance the stability of RLP068/Cl.

• VULNOFAST® gel had been fully developed by Molteni Therapeutics (with the name G.68.γ/EtOH). In March 2013 the EU certification (CE mark) was granted by “Istituto Superiore di Sanità” (controlled by the Italian Ministry of Health; Notified Body identifier: 0373).

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• VULNOFAST® gel is presented in an amber glass vial (fill volume 2.5 mL) and it is repackaged into a

needleless sterile 1 mL polypropylene syringe for the administration to the patient ulcer / lesion.

• VULNOFAST® gel is manufactured under GMP by the qualified CMO SCM Pharma (Newcastle, UK), using the isolator technology (aseptic compounding, ca 500-600 units).

What is VULNOFAST® plus?

• VULNOFAST® plus is an improved formulation to facilitate use, increase stability and reduce costs of VULNOFAST® gel, mantaining the top-level quality standards of the original product.

• VULNOFAST® plus shares with the original VULNOFAST® gel the non aqueous formulation (propylene glycol basis) and the 0.3% content of RLP068/Cl.

• The product is sterile, single-use and with a low content of bacterial endotoxins. It is manufactured under non sterile conditions, then filter sterilized and aseptically filled.

• VULNOFAST® plus has the same intended use and the same classification (class IIb medical device) of the original product. GMP pilot batches for the CE certification (ca 6’000 units) had been manufactured and placed in a ICH stability programme by the qualified CMO COC Farmaceutici SpA (Bologna, Italy).

• In December 2014 the EU certification (CE mark) was granted by “Istituto Superiore di Sanità” (controlled by the Italian Ministry of Health; Notified Body identifier: 0373)

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Differences from VULNOFAST® gel: Product Packaging/Presentation

• VULNOFAST® plus is presented in easy-to-use plastic

monodose containers.

• Each market unit contains a strip of 5 monodose containers (LDPE, 2 mL each), protected from light by an aluminum bag.

• The product is administered on the patient lesion by

squeezing the monodose container (no need of accessories or repackaging into syringe).

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• Data analysis

• Results

• Conclusion

Aim of the study

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To demonstrate the efficacy equivalence of a single treatment of antimicrobial

photodynamic therapy (APDT) with VULNOFAST® plus formulation in a

mouse model of ulcera-like wound infection induced with a methicillin-

resistant (MRSA) strain of Staphylococcus aureus (ATCC 43300) respect

VULNOFAST® gel formulation used as the reference.


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• Data analysis

• Results

• Conclusion

Experiment protocol

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Bacterial suspension• MRSA ATCC 43300 was grown in brain-heart infusion

broth for 16-18 hours at 37°C• Cell harvested by centrifugation (1500 rpm – 15 min),

wash twice in sterile saline ad suspended in the same buffer

• Dilution of the bacteria solution in sterile saline to obtain the experimental suspension with an OD600 0.5 corresponding to a cell concentration of 5.0 × 108 cells/ml

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Full thickness wound mouse model*

• Anesthesia of CD1 female mice (28-30g; 4 weeks old)• Shaving of the back• One full-thickness wound (diameter 0,8 cm) was

established through the panniculus carnosus of each animal

• Wound was infected dropping 100 µl (5,0 × 107 cells/ml.) of bacterial suspension on a gauze placed over each wound.

• The lesion was closed by means of skin clips

This procedure results in a local infection after 2 days

* Kugelberg E, Norstrom T, Petersen TK et al. Establishment of a superficial skin infection model in mice by using Staphylococcus aureus and Streptococcus pyogenes. Antimicrob Agents Chemother 2005; 49:3435–41.

Experiment protocol

APDT treatment

• Anesthesia of the mice

• Mice were randomized into four groups (8 animal each)• Group A: infection control • Group B: Light control (no photosensitezer)• Group C: APDT treatment with VULNOFAST® gel• Group D: APDT treatment with VULNOFAST® plus

• Wound was opened and the gauze removed

• Gel was applied to the wound (25 µl), spread uniformly and left to act for 60 (Exp1 and Exp3) or 30 min. (Exp2 and Exp4)

• Illumination (8 min at 120 mW/cm2 corresponding to 60 J/cm2 of delivered light dose)

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Experiment protocol

Quantification of antimicrobial efficacy

• Animals were sacrificed

• Aseptically excision of a standard area of infected wound

• Homogenization in 2 ml of sterile saline

• Culturing serial dilution of bacterial suspension on Mannitol Salt Agar Plate

1616

10-6

900900 µl 900 µl 900 µl 900 µl 900 µl 900 µl

100 µl 100 µl 100 µl 100 µl 100 µl 100 µl

sample 10-1 10-2 10-3 10-4 10-5

Experiment protocol


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• Experiment description

• Data analysis

• Results

• Conclusion

Statistical analysis

• The statistical analysis was performed after a base-ten logarithmic transformation of the data obtained to improve data normality conditions

• The parametric analysis, performed for each experiment independently, is a One-Way ANOVA considering the treatment as the independent variable. The level of significance considered for the ANOVA, Tukey post test is p< 0.05

• The non-parametric analysis performed is the Kruskal Wallis test (Tukey test as post ANOVA test)

• The final analysis that analyzes together the two experiments at 60 minutes and 30 minutes is a Two-Way ANOVA with interaction where the considered variables are the treatment and the experiment.

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Statistical analysisLOG10 CFU

Soggetto Trattamento Exp1 Exp2 Exp3 Exp41 A 7.083 6.315 6.514 6.6022 A 6.444 6.160 6.590 6.9853 A 6.933 6.342 6.763 NaN4 A 6.869 6.330 7.017 6.4045 A 6.549 6.109 6.576 6.0316 A 6.273 5.872 6.860 6.8337 A 6.519 6.455 6.362 6.1148 A 6.717 6.096 7.233 7.1831 B 5.820 5.944 6.685 NaN2 B 5.283 6.061 6.796 6.0573 B 7.374 5.613 6.458 6.4984 B 5.703 5.580 6.352 6.8815 B 6.690 6.011 6.732 6.1096 B NaN 5.810 6.583 6.2607 B 6.078 6.214 6.607 6.5058 B 6.724 5.914 NaN 7.1271 C 2.000 3.936 4.182 3.9722 C 3.591 3.811 5.205 NaN3 C 2.845 4.109 4.863 4.7754 C 3.470 4.332 5.199 4.7165 C 3.724 4.380 5.342 4.1616 C 3.255 4.130 4.063 6.9057 C 3.568 3.380 5.288 7.1178 C 3.519 5.296 6.088 6.5101 D 3.760 4.057 4.740 4.5622 D 4.397 5.053 5.477 5.3363 D 3.512 4.556 4.869 4.9494 D 3.789 NaN 3.900 5.5375 D 3.556 4.927 6.229 5.4446 D 3.398 4.580 4.908 6.0357 D 3.398 4.898 5.613 5.1628 D 2.778 4.623 5.145 5.157

Experimental data used for the statistical analysis


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• Data analysis

• Results

• Conclusion

APDT treatment after 60 min incubation time

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VULNOFAST® plus antimicrobial efficacy in APDT vs Staphylococcus aureus ATCC 43300

60 minutes

A B C D2

4

6

8

* *

CF

U (

log

10)

60 minutes Mean SEM Log10 diff

A 6.7064 0.1295 B 6.4204 0.1384 0.29C 4.1377 0.1295 2.57D 4.3419 0.1295 2.36

* p values << 0.001

APDT treatment after 30 min incubation time

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30 minutes

A B C D2

4

6

8

* *

CF

U (

log

10)

30 minutes Mean SEM Log10 diff

A 6.4014 0.1508 B 6.1922 0.1508 0.21C 4.8114 0.1508 1.59D 4.9717 0.1508 1.43

VULNOFAST® plus antimicrobial efficacy in APDT vs Staphylococcus aureus ATCC 43300

* P values << 0.001


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• Results

• Data analysis

• Conclusion

Conclusions

• A significant bacterial load reduction (p< 0.001) is obtained with a single APDT treatment with VULNOFAST® plus by using an incubation time of 60 min

• There is no difference in the antimicrobial efficacy of VULNOFAST® plus versus VULNOFAST® gel APDT treatment by using an incubation time of 60 min

• There is no statistical difference in bacterial load reduction of light controls versus infection controls

• A significant bacterial load reduction (p< 0.001) is obtained with a single APDT treatment with VULNOFAST® plus by using an incubation time of 30 min

• Even in this case there is no difference in the antimicrobial activity of the two compositions in a single APDT treatment, though data are affected by greater variability

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Thanks

for your attention

Molteni Therapeutics s.r.l.Via I. Barontini, 8 – Loc. Granatieri, 50018 Scandicci (FI) Tel. +39 055 [email protected]

http://www.moltenitherapeutics.it/

Health & Medicine

antimicrobial activity of vulnafast plus