antimicrobial photodynamic therapy with vulnofast products by molteni Therapeutics

1. Antimicrobial efficacy of
VULNOFAST®
plus

2. Presentation Outline
2
• 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

3. 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.
N
N
NN
N N
N
N
Zn
O
N
+
O
N
+
O
N
+
O
N
+
Cl
-
Cl
-
Cl
- Cl
-
C68H64Cl4N12O4Zn
F.W. = 1320.5
3

4. Presentation Outline
4
• 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

5. VULNOFAST®
: pioneering the PDT
treatment 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:
5

6. 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
6
* 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.

7. 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).
7
• 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).

8. 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)
8

9. 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).
9

10. Presentation Outline
10
• 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

11. Aim of the study
11
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.

12. Presentation Outline
12
• 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

13. Experiment protocol
13
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

14. 14
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

15. 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)
15
Experiment protocol

16. 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
16
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

17. Presentation Outline
17
• VULNOFAST®
(RLP068/Cl):
• VULNOFAST®
products
• VULNOFAST®
plus antimicrobial efficacy in APDT vs
Staphylococcus aureus ATCC 43300:
• Aim of the study
• Experiment description
• Data analysis
• Results
• Conclusion

18. 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.
18

19. 19
Statistical analysis
LOG10 CFU
Soggetto Trattamento Exp1 Exp2 Exp3 Exp4
1 A 7.083 6.315 6.514 6.602
2 A 6.444 6.160 6.590 6.985
3 A 6.933 6.342 6.763 NaN
4 A 6.869 6.330 7.017 6.404
5 A 6.549 6.109 6.576 6.031
6 A 6.273 5.872 6.860 6.833
7 A 6.519 6.455 6.362 6.114
8 A 6.717 6.096 7.233 7.183
1 B 5.820 5.944 6.685 NaN
2 B 5.283 6.061 6.796 6.057
3 B 7.374 5.613 6.458 6.498
4 B 5.703 5.580 6.352 6.881
5 B 6.690 6.011 6.732 6.109
6 B NaN 5.810 6.583 6.260
7 B 6.078 6.214 6.607 6.505
8 B 6.724 5.914 NaN 7.127
1 C 2.000 3.936 4.182 3.972
2 C 3.591 3.811 5.205 NaN
3 C 2.845 4.109 4.863 4.775
4 C 3.470 4.332 5.199 4.716
5 C 3.724 4.380 5.342 4.161
6 C 3.255 4.130 4.063 6.905
7 C 3.568 3.380 5.288 7.117
8 C 3.519 5.296 6.088 6.510
1 D 3.760 4.057 4.740 4.562
2 D 4.397 5.053 5.477 5.336
3 D 3.512 4.556 4.869 4.949
4 D 3.789 NaN 3.900 5.537
5 D 3.556 4.927 6.229 5.444
6 D 3.398 4.580 4.908 6.035
7 D 3.398 4.898 5.613 5.162
8 D 2.778 4.623 5.145 5.157
Experimental data used for the statistical
analysis

20. Presentation Outline
20
• VULNOFAST®
(RLP068/Cl):
• VULNOFAST®
products
• VULNOFAST®
plus antimicrobial efficacy in APDT vs
Staphylococcus aureus ATCC 43300:
• Aim of the study
• Experiment description
• Data analysis
• Results
• Conclusion

21. APDT treatment after 60 min incubation time
21
VULNOFAST®
plus antimicrobial efficacy in
APDT vs Staphylococcus aureus ATCC 43300
60 minutes
A B C D
2
4
6
8
* *
CFU(log10)
60 minutes
Mean SEM Log10 diff
A 6.7064 0.1295
B 6.4204 0.1384 0.29
C 4.1377 0.1295 2.57
D 4.3419 0.1295 2.36
* p values << 0.001

22. APDT treatment after 30 min incubation time
22
30 minutes
A B C D
2
4
6
8
* *
CFU(log10)
30 minutes
Mean SEM Log10 diff
A 6.4014 0.1508
B 6.1922 0.1508 0.21
C 4.8114 0.1508 1.59
D 4.9717 0.1508 1.43
VULNOFAST®
plus antimicrobial efficacy in
APDT vs Staphylococcus aureus ATCC 43300
* P values << 0.001

23. Presentation Outline
23
• VULNOFAST®
(RLP068/Cl):
• VULNOFAST®
products
• VULNOFAST®
plus antimicrobial efficacy in APDT vs
Staphylococcus aureus ATCC 43300:
• Aim of the study
• Experiment description
• Results
• Data analysis
• Conclusion

24. 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
24

25. Thanks
for your attention
Molteni Therapeutics s.r.l.
Via I. Barontini, 8 – Loc. Granatieri, 50018 Scandicci (FI)
Tel. +39 055 7361285
www.moltenitherapeutics.it
info@moltenitharapeutics.it