VETERINARYELECTROCHEMOTHERAPY

DEVICES

by Loïck RoyantChief Executive Officer of LEROY Biotech

November 28th, 2016

Presentation agendaINTRO

Q&A

01 Principles of ECT*

02 ECT* session

03 Case studies

04 Electroporators

05 Clinics equiped with ELECTROvet

ECT*= ElectroChemoTherapy

01 electrochemotherapy

‣ Since 2006 on human patients Success rate : 84.1% (on 1894 tumours) (Miklavcic, 2013)

‣ Response rate at 4 years on sarcoids: 97.9% (Tamzali, 2012)

‣ Recovery rate squamous cell carcinoma on cats: 87.5% (Tozon, 2014)

‣ Tumour cells selective (more sensitive to cytotoxic agent and electric pulses because of shape and size of tumour cells)

‣ Used alone to cure small-sized tumours

‣ Before, during or after surgery or radiation therapy

Key features

01 Principles of electrochemotherapy

1 2 3Injection of anticancer drug

(bleomycin or cisplatin)Application of electrical pulses (electroporation)

Consequence : Drugs remain trapped in the cells

cell membranedrug

Pulsed Electric Field

drug

Electroporation allows molecules to either permeate into the cell orleave the cell. ce

ll m

embr

ane

Rapid and significant increase of drugs into the cell,Cytotoxicity increased (bleomycin >1000-fold, cisplatin > 80-fold)Vascular lock within tumour vessels,Immune system activation

4

02.1 Electrochemotherapy session

General anesthesia Injection of cytotoxic

Plan a follow-up visit after 2 weeks :If tumour is entirely necrotic, no more sessions needed.If tumour still shows signs of evolution, plan a new session.

Follow-up

Surgical scrub Electroporation

1 2 3 4 52 weeks1

2nd session

2-5 days

1 session = 20 mins

(approx.)

10 mins max

Never more than 5 sessions

Side effects: oedema, necrosis.

02.2 What volume of drug inject ?‣ The objective is to saturate the tumour with cytotoxic drugs.

‣ Calculate the volume of the tumour :

‣ The formula for calculating the volume of the tumor: V= ab²π/6 (cm3)

‣ Concentration : Cisplatin 1mg/ml - Bleomycin 1000IU/ml

a

b

‣ a = 1st length of the tumour (cm)‣ b = 2nd length of the tumor (cm)

Greatest length of the tumor (=

a) - cm

Length perpendicular to

a (= b) - cm

For a circular tumor, volume of tumor (V=

ab²π/6) cm3

cisplatin dose to be injected intra tumor

- (0,3 mg / cm3)

Volume of cisplatin to be injected intra tumor (ml)concentration 1mg/ml

Bleomycin dose to be injected intra tumor -

(0,5 mg / cm3)

Volume of bleomycin to be injected intra tumor (ml)concentration 1000IU/ml

0,5 cm 0,5 cm 0,07 cm3 0 mg 0,02 ml 0 mg 0,03 ml1 cm 1 cm 0,52 cm3 0,2 mg 0,16 ml 0,3 mg 0,26 ml

1,5 cm 1,5 cm 1,77 cm3 0,5 mg 0,53 ml 0,9 mg 0,88 ml2 cm 2 cm 4,19 cm3 1,3 mg 1,26 ml 2,1 mg 2,09 ml

2,5 cm 2,5 cm 8,18 cm3 2,5 mg 2,45 ml 4,1 mg 4,09 ml3 cm 3 cm 14,13 cm3 4,2 mg 4,24 ml 7,1 mg 7,07 ml

3,5 cm 3,5 cm 22,44 cm3 6,7 mg 6,73 ml 11,2 mg 11,22 ml4 cm 4 cm 33,49 cm3 10 mg 10,05 ml 16,7 mg 16,75 ml5 cm 5 cm 65,42 cm3 19,6 mg 19,63 ml 32,7 mg 32,71 ml

02.3 How to inject cytotoxic ?

‣ Use 3ml or 5ml syringes lockable cap (like Luer-Lock)‣ Use hypodermic needles gauge 18 or 20‣ Make injection of cytotoxic spaced 6mm apart (approx.) in one plane‣ 1 cm margin all around the tumour

Push the plungerand pull out in the same time

02.4 How to apply the electrical field?

Place electrodes on the tumour

Push on left pedal « CHARGE »

Push on right pedal « PULSE »

« bip - bip » = OK

« biiiiiiiiip » = NOK

02.5 Where to apply the electrical field?

1st application 3rd application

And so on until treated the whole surface of the tumour and 1 cm margin all around is treated.

etc …

2nd application 4th application

1 2 3 4Rotate +90° move of 1 cm

rotate -90°Rotate +90°

(!) muscular contractions (!)

(!) muscular contractions (!)LL

LL

LL

LL

02.6 What does the electric field look like?« L » shape electrodes « needle » electrodes

Distance between electrodes : 1cmElectric Field Strength : 1300 V.cm-1 Average permeabilized volume : 1cm3

1 cm

0,5 cm

Distance between electrodes : 0,5 cmElectric Field Strength : 1000 V.cm-1 Average permeabilized volume : 0,9 cm3

03 Case studiesTumour type Cases Observation time Results

sarcoid 48 4 years 97.9% complete response

squamous cellcarcinoma 11 2 months to 3

years 87.5% objective response

fibrosarcoma 72 over 2 years

time to recurrence:12 months - intraoperative19 months - postoperative

(4 months for surgery alone)

mast cell tumor 25 over 3 years 62.5% complete response(comparison to surgery)

03 Case studies Horse sarcoid

Surgery + 3 sessions

3 sessions(no surgery)

(12 months later)

Y. Tamzali - ELECTROvet S13E.N.V. de Toulouse (France)

Y. Tamzali - ELECTROvet S13E.N.V. de Toulouse (France)

03 Case studies Horse sarcoid4 sessions, 2 weeks apart (no surgery) (8 months later)

Surgery + 3 sessions, 2-3 weeks apart

Y. Tamzali - ELECTROvet S13

(5 months later)

Y. Tamzali - ELECTROvet S13E.N.V. de Toulouse (France)

03 Case studies Horse carcinoma

3 sessions, 2-3 weeks apart (no surgery)

F. RELAVE - ELECTROvet S13Clinique de Conques (France)

03 Case studies Cat squamous cell carcinoma

2 sessions, 3 weeks apart (no surgery)

C. TRIPP — ELECTROvet S13Veterinary Cancer Specialty Care (USA)

(6 months later)

03 Case studies Dog fibrosarcoma

Radiation therapy + 3 sessions, 3 weeks apart

A. Alaphilippe— ELECTROvet S13Clinique des Cèdres (France)

(3 months later)

04 Electroporators

Recommended for equine clinics, veterinary schools and Universities.

✓ LCD touch screen to set up parameters✓ Showing curves of output electrical current ✓ Clinical trial mode to set up special pulses✓ Delivers standard ECT protocol✓ History of the treatment✓ 5 years warranty

Recommended for pet clinics

✓ Delivers standard ECT protocol✓ Easily transportable (5kg)✓ 3 years warranty

€ 4,850.-€ 12,950.-

€ 139.-

L shape contact electrodesP 2 «L» shape electrodesP 15 mm longP distance of 10 mmP Autoclavable

€ 139.-

Needle electrodesP 2 rows of 4 needlesP 15 mm longP distance of 5 mmP Autoclavable * *

05 Clinics equiped with ELECTROvet

UniversitiesUniversité de Montréal (Canada)Virginia-Maryland College of Veterinary Medicine (USA)University of Tennessee - College of Veterinary Medicine (USA)Auburn University - College of Veterinary Medicine (USA)Oregon State University - College of Veterinary Medicine (USA)Cornell University - College of Veterinary Medicine (USA)Universität Bern – Swiss Institute of Equine Medecine (Switzerland)Massey University Veterinary School (New Zeland)Faculté de médecine vétérinaire, Université de Liège (Belgium)Delft University (Netherland)Ecole Nationale Vétérinaire de Toulouse (France)University of Ljubljana (Slovenia)Universidad de Buenos Aires (Argentina)Madras Veterinary College (India)Universität Berlin, Klinik für Pferde (Germany)

Equine ClinicsBallarat Veterinary Practice (Australia)Mid-Atlantic Equine Medical Center (USA)EQUITOM - Dr. Tom Mariën (Belgium)Tierärztliche Klinik für Pferde Wolfesing (Germany)San Luis Equine Hospital (USA)Texas Equine Hospital (USA)Clinique de Groisbois (France)Clinique de l’Argonay (France)Clinique de Conques (France)Clinique vétérinaire du Dr. Pénide (France)Dierenkliniek de Morette (Belgium)Clinica veterinaria ippodromo di Merano (Italy)Chaparral Veterinary Medical Center (USA)Equine Services, PSC (USA)

Pet ClinicsPet Cancer Group, PLLC ( USA )Clinique des Cèdres (France)Pets stop vet Poliklinigi (Turkey)Veterinary Cancer Specialty Care (USA)Iowa Veterinary Specialties - Pet clinic (USA)Affiliated veterinary specialist Maitland (USA)Pawspice Vet Clinic of Dr. Villalobos (USA)Inland Valley Emergency Pet Clinic (USA)Hospital Veterinario Restelo (Portugal)Veterinary Emergency of Hawaii (USA)Dr. Alberto BAJO - Pet Clinic (Italy)Dr. Enrico SPONZA - Pet Clinic (Italy)Centro Veterinario 4 patas (Brasil)Paulo Roberto Martin (Brasil)Evidensia Tammisto (Finland)

Present in 18 Countries

November 2016

Annexe - References๏ Cemazar M, Miklavcic D, Mir L, Belehradek J Jr, Bonnay M, Fourcault D, Sersa G. Electrochemotherapy of tumours resistant to cisplatin: a study in a murine tumour model. Eur J Cancer.

2001. 37:1166-1172.๏ Cemazar M, Tamzali Y, Sersa G, Tozon N, Mir LM, Miklavcic D, Lowe R, Teissie J. Electrochemotherapy in veterinary oncology. J Vet Intern Med. 2008 Jul-Aug. 22(4) : 826-31.๏ Jarm T, Cemazar M, Miklavcic D, Sersa G. Antivascular effects of electrochemotherapy: implications in treatment of bleeding metastases. Expert Rev Anticancer Ther. 2010. 10:585-586.๏ Kodre V, Cemazar M, Pecar J, Sersa G, Cor A, Tozon N. Electrochemotherapy Compared to Surgery for Treatment of Canine Mast Cell Tumours. In Vivo. 2009. 23: 55-62.๏ Miklavcic D, Mali B, Bor K, Heller R, Sersa G. Electrochemotherapy: from the drawing board into medical practice. BioMedical Engineering Online. 2014. 13,29.๏ Mir L-M. Bases and rationale of the electrochemotherapy. EJC supplements 4. 2006. 38-44.๏ Mali B, Jarm T, Snoj M, Sersa G, Miklavcic D. Antitumor effectiveness of electrochemotherapy: A systematic review and meta-analysis. EJSO. 2013. 4-13.๏ Orlowski S, Belehradek J Jr, Paoletti C, Mir LM. Transient electropermeabilization of cells in culture. Increase of cytotoxicity of anticancer drugs. Biochem Pharmmacol. 1988. 37(24),

4727-4733.๏ Sersa G, Miklavcic D, Cemazar M, Rudolf Z, Pucihar G, Snoj M. Electrochemotherapy in treatment of tumours. EJSO 34. 2008. 232e240.๏ Spugnini EP, Vincenzi B, Baldi F, Citro G, Baldi A. Adjuvant Electrochemotherapy for the Treatment of Incompletely Resected Canine Mast Cell Tumors. Anticancer research. 2006. 26:

4585-4590.๏ Spugnini EP, Vincenzi B, Citro G, Santini D, Dotsinsky I, Mudrov N, Montesarchio V, Laieta MT, Esposito V, Baldi A. Adjuvant Electrochemotherapy for the treatment of incompletely excised

spontaneous canine sarcomas. In vivo. 2007. 21: 819-822.๏ Spugnini EP, Citro G, Mellone P, Dotsinsky I, Mudrov N, Baldi A. Electrochemotherapy for localized lymphoma: a preliminary study in companion animals. J Exp Clin Cancer Res. 2007.

26,3.๏ Spugnini EP, Baldi A, Vincenzi B, Bongiorni F, Bellelli C, Citro G, Porrello A. Intraoperative versus postoperative electrochemotherapy in high grade soft tissue sarcomas: a preliminary study

in a spontaneous feline model. Cancer Chemother Pharmacol. 2007. 59:375–381.๏ Spugnini EP, Filipponi M, Romani L, Dotsinsky I, Mudrov N, Baroni A, Ruocco E, Laieta MT, Montesarchio V, Cassandro R, Citro G, Baldi A. Local Control and Distant Metastasis after๏ Electrochemotherapy of a Canine Anal Melanoma. In Vivo. 2007. 21: 897-900.๏ Spugnini E, Dotsinsky I, Mudrov N, Bufalini M, Giannini G, Citro G, Feroce F, Baldi A. Adjuvant Electrochemotherapy for Incompletely Excised Anal Sac Carcinoma in a Dog. In Vivo. 2008.

47-50.๏ Spugnini EP, Dotsinsky I, Mudrov N, Citro G, D'Avino A, Baldi A. Biphasic pulses enhance bleomycin efficacy in a spontaneous canine genital tumor model of chemoresistance: Sticker

sarcoma. Journal of Experimental & Clinical Cancer Research. 2008. 27:58.๏ Spugnini EP, Citro G, Dotsinsky I, Mudrov N, Mellone P, Baldi A. Ganglioneuroblastoma in a cat: A rare neoplasm treated with electrochemotherapy. The Veterinary Journal. 2008. 298-291.๏ Spugnini EP, Dotsinsky I, Mudrov N, De Luca A, Codini C, Citro G, D’Avino A, Baldi A. Successful Rescue of an Apocrine Gland Carcinoma Metastatic to the Cervical Lymph Nodes by

Mitoxantrone Coupled with Trains of Permeabilizing Electrical Pulses (electrochemotherapy). In vivo. 2008. 22:51-54.๏ Spugnini EP, Renaud S M, Buglioni S, Carocci F, Dragonetti E, Murace R, Cardelli P, Vincenzi B, Baldi A, Citro G. Electrochemotherapy with cisplatin enhances local control after surgical

ablation of fibrosarcoma in cats: an approach to improve the therapeutic index of highly toxic chemotherapy drugs. Journal of Translational Medicine. 2011. 9:152.๏ Spugnini EP, Di Tosto G, Salemme S, Pecchia L, Fanciulli M, Baldi A. Electrochemotherapy for the treatment of recurring aponeurotic fibromatosis in a dog. Can Vet J. 2013. 54:606–609.๏ Tamzali Y, Borde L, Rols MP, Golzio M, Lyazrhi F, Teissie J. Successful treatment of equine sarcoids with cisplatin electrochemotherapy: A retrospective study of 48 cases. Equine Vet J.

2012 March. 44(2) : 214-220.๏ Teissié J, Escoffre JM, Paganin A, Chabot S, Bellard E, Wasungu L, Rols MP, Golzio M. Drug delivery by electropulsation: Recent developments in oncology. Int J Pharm. 2012 Feb. 423(1) :

3-6.๏ Tozon N, Kodre V, Sersa G, Cemazar M. Effective Treatment of Perianal Tumors in Dogs with Electrochemotherapy. Anticancer research. 2005. 25: 839-846.๏ Tozon N, Kodre V, Polona J, Sersa G, Cemazar M. Electrochemotherapy is highly effective for the treatment of canine perianal hepatoid adenoma and epithelioma. Acta Veterinaria. 2010.

Vol. 60, No. 2-3, 285-302.๏ Tozon N, Pavlin D, Sersa G, Dolinsek T, Cemazar M. Electrochemotherapy with intravenous bleomycin injection: an observational study in superficial squamous cell carcinoma in cats.๏ Journal of Feline Medicine and Surgery. 2014. Vol. 16(4) 291– 299.