Deuterium depletion results in several fold ...

Deuterium depletion results in several fold increases in the median survival time of cancer patients during oncotherapy

Gábor Somlyai, Mária Debrődi, Ildikó Somlyai, Orsolya Abonyi, László G. Boros

Concentration of deuterium and some other vital elements and glucose in human serum Deuterium

12–14 mmol/L (150 ppm)

Calcium

2.24–2.74 mmol/L

Magnesium

0.75–1.2 mmol/L

Potassium

3.5–5.1 mmol/L

Glucose

3.3–6.1 mmol/L

Pre-clinical studies In vitro

xCELLigence® System (Roche Applied Biosciences & ACEA Biosciences) was used to follow cell growth as a function of deuterium depletion

The xCELLigence System monitors cellular events in real time without the incorporation of labels. The System measures electrical impedance across micro-electrodes integrated on the bottom of tissue culture E-Plates. Reference: Methods Mol Biol. 2011;740:33-43. doi: 10.1007/978-1-61779-108-6_6

Avidin Ltd.- xCELLigence System

MCF-7

D-depletion started

Deuterium depletion inhibits the growth rate of MCF-7 breast cancer, A549 lung cancer and HT199 melanoma cell lines 150 ppm (physiological D-concentration)

A549

135 ppm

125 ppm 115 ppm 105 ppm

85 ppm 65 ppm 40 ppm

HT199

The results show a dose dependent inhibitory effect of DDW on the growth rate of transformed cell lines.

Unpublished data of L. Nagy et al., Avidin LLC. Hungary

Decreasing the D-concentration by small increments improves the sensitivity of A549 lung cancer cell line

0 ppm 1 ppm 2.5 ppm 5 ppm

D-concentration of the media was decreased from 150 ppm by 0 ppm, 1 ppm, 2.5 ppm and 5 ppm in every 8 hours. Even 1 ppm decrease resulted lower growth rate and the repeated decrease of D-concentration improved the sensitivity of the cells. Unpublished data of L. Nagy et. al., Avidin LLC. Hungary

DDW as a potential adjuvant treatment in combination with Doxorubicin − HT-199 melanoma cells

150 ppm 85 ppm 450 nM Dox 85 ppm + 450 nM Dox 900 nM Dox 85 ppm + 900 nM Dox

Unpublished data of L. Nagy et al., Avidin LLC. Hungary

Pre-clinical studies In vivo

DDW induced apoptosis and inhibited mitosis in mice xenotransplanted with human prostate tumor (PC-3) %5 4

3

3.6% 3.0%

2

1.5%

1 0

1.0% control group (14)

Average tumor volume: 3.7 cm3

treated group (13) 0.97 cm3

mitosis apoptosis No treatment for 18 days, followed by 12 days of DDW-treatment G. Somlyai, et al., (1998) Synthesis and Application of Isotopically Labelled Compounds 1997. Edited by J.R. Heys and D.G. Mellilo, 1998 John Wiley, Sons Ltd., 137-141

Per os DDW-treatment as single treatment and its combination with surgery resulted in complete remission in 50% of cases in breast and rectum tumors

Dogs

Cats

Length of treatment

Breast tumor

81

14

2-6 months

Rectum tumor

43

3

2-6 months

Complete response (CR)

DDW+ Surgery

DDW

40 (42%)

11 (11%)

4 (9%)

19 (41%)

Dr. med. vet. M. Szabó, Veterinary Clinic, Budapest, Hungary

DDW as single treatment resulted complete response in a 5-year-old dog with haemangionsarcoma on the neck

Before DDW treatment

After 10-week treatment

After 4-week treatment

After 14-week treatment Dr. med. vet. M. Szabó, Veterinary Clinic, Budapest, Hungary

Registered deuterium-depleted anticancer medicinal product for veterinary use ̶ Vetera-DDW-25® (084/1/2011 MgSzH ÁTI) • Main indication: is pre- and post treatment of surgical interventions or single treatment in the case of non-operable tumors. • Tumors showed a response rate higher than 70%; more than 50% of the animals achieved complete recovery. • The relapse rate can be halved with the application of Vetera-DDW-25. • Since the authorization (1999) of VeteraDDW-25 no pharmacovigilance report has been received.

Vetera-DDW-25® hypodermic injection for veterinary use

Prospective study – Prostate cancer

Phase II, double blind, randomized clinical trial for prostate cancer (Prospective study) 

44 evaluated patients (22 treated, 22 control)  Duration of treatment: 4 months  Treated group: conventional treatment + DDW 85 ppm 

Placebo group: conventional treatment + placebo 150 ppm

Inclusion criteria was histologically confirmed prostate cancer. Patients consumed DDW instead of their entire daily fluid intake.

A. Kovacs et al., (2011) Journal of Cancer Therapy 2, 548-556

Cumulative PSA decreased by 80% in treated vs. 47% in placebo group 521

600

PSA (ng/mL)

500

Before treatment

406.4

400

277.4

After treatment

300

200

80.3

100

0

treated group

control group A. Kovacs et al., (2011) Journal of Cancer Therapy 2, 548-556

DDW-treated group had a significant decrease in prostate volume from baseline to the last treatment visit compared to placebo Treated group

Control group

Before treatment

After treatment -160 cm3

-54 cm3 p= 0.0019 A. Kovacs et al., (2011) Journal of Cancer Therapy 2, 548-556

Survival was longer, death rate was lower in the DDW-treated group (one year survival)

Cumulated efficacy

AliveAlive

Death

Dead

Treated group

20 (91%)

2 (9%)

Control group

13 (59%)

9 (41%)

Death rate p=0.034 Survival p=0.029 A. Kovacs et al., (2011) Journal of Cancer Therapy 2, 548-556

Evaluation of the DDW population in retrospective study

Key parameters for retrospective data collection • • • • • • •

Date of diagnosis Type of tumor (location of primer tumor) Date of DDW start End date of DDW treatment End date of the follow-up period Type of response during DDW treatment Dead/alive status at the end of the follow-up

Retrospective data collection • Enrollment period: Oct. 1992 – Oct. 2014 • 1,827 cancer patients were treated with DDW for at least 1 day • 1,034 women (56.6%) 793 men (43.4%) • Age: average: 54±16 years, median: 57 years • Body weight: 69±17 kgs, median: 70 kgs

The prevalance of different tumor types in the US and in DDW population Tumor type genitals digestive respiratory breast urinary skin lymphoma endocrine leukemia oral cavity unspecified myeloma brain, nervous system bones, joints, soft tissue total

prevalence, US

prevalence, DDW

prevalence %, US

prevalence %, DDW

338450 289610 242550 235030 141610 81220 79990 65630 52380 42440 34160 24050

270 338 304 342 81 45 54 13 107 42 16 12

20,32 17,39 14,56 14,11 8,50 4,88 4,80 3,94 3,14 2,55 2,05 1,44

14,78 18,50 16,64 18,72 4,43 2,46 2,96 0,71 5,86 2,30 0,88 0,66

23380

146

1,40

7,99

15040

57

0,90

3,12

1665540

1827

100

100

http://www.cancer.org/acs/groups/content/@research/documents/webcontent/acspc-042151.pdf

Mortality in the US and in DDW population Tumor type

Deaths, US

Deaths, DDW

genital digestive respiratory breast urinary skin lymphoma endocrine leukemia oral cavity unspecified myeloma

58970 147260 163660 40430 30350 12980 20170 2820 24090 8390 44990 11090

58 105 109 83 19 11 8 1 19 12 1 4

brain, nervous system

14320

45

bones, joints, soft tissue

6200

12

585720 (35,1%)

487 (26,6%)

total

http://www.cancer.org/acs/groups/content/@research/documents/webcontent/acspc-042151.pdf

Main parameters in the statistical evaluation of the follow-up population (1,827 patients), using DDW for at least one day 2,265 years

1,512 years

3,777 years 3,104 years 6,881 years

date of diagnosis

end of follow-up start of DDW

end of DDW administration

Kaplan Meier survival curve in the retrospectively evaluated population of 1,827 cancer patients treated with DDW (all tumor types)

Median Survival Time (MST) 121 months = 10.1 years

Correlation between the length of DDW treatment and median survival time (MST)

MST correlates with the length of DDW treatment 180

160

MST (months)

145 148

140 120

121 120 115 115

156 158 158 158 156

130 132

100 80 60 40

1827 1793

1752

1689

1464

1312

1177

1060

963

889

820

152

135

117

97

74

69

55

34

41

63

225

1

31

61

91

765

697

Number of evaluated patients

68

20 0

121 151 181 211 241 271 301 331 361 Time (days)

Number of patients in the given time periods

Longer duration of DDW treatment resulted in longer MST

215

250

200

150

100

56

74

50

512

288

112

91-180

181-270

271-360

0

Time (days)

Kaplan Meier survival curve in the subgroups of patients with different durations of treatment 100 90 80 Duration of DDW (days) 91-180 181-270 271-360

70 60 50

DDW MST n 91-180 days 56 months (512 patients) 181-270 days 74 months (288 patients) 271-360 days 215 months (112 patients)

40 30 20 10 0

50

100

150 200 Time (months)

250

300

350

MST vs. time elapsed from the date of diagnosis till the start of DDW treatment

The earlier the DDW treatment started after diagnosis, the longer was the MST MST (months) 120

98

100

77

80

54

60

40

20

1,173

209

131

Number of patients

0

Within 1 year

Between 1 and 2 years

Over 2 years Start of DDW after diagnosis

Kaplan Maier survival curve in the subgroups of patients in correlation with the start of DDW treatment after diagnosis 100 90 80

DDW start: Within 1 year Between 1-2 years Over 2 years

70 60 50 40 30 20 0

50

100

150 Time (months)

200

DDW start from diagnosis within 1 year between 1-2 years over 2 years

250

300

MST (months) 98 months 77 months 54 months

Survival in subgroups of breast cancer patients (232) defined on the time elapsed from diagnosis to DDW start MST: L/E

I.: no applicable

100

(18 deaths out of 114 pts 80

399.8 y / 1 death / 22.2 y) II.: 49 months / 118 pts

60 time from DG_to_DDWstart I. (0-365 days) II. (> 365 days) 40

20

p < 0.0001

0 0

50


200

250

K. Krempels, et al. (2013) Journal of Cancer Research & Therapy, 1(8):194–200

Establishing optimal DDW dose

The expression of the DDW dose Deuterium depletion unit (DdU) Main variable factors:

- body weight (kg) - D-concentration of DDW (ppm) - daily volume of DDW (L)

Examples for the calculation of DdU Body weight: 60 kg Daily volume of DDW (L): 1.66 L D-concentration of DDW: 65 ppm Dosage: 2.36 DdU

Body weight : 80 kg Daily volume of DDW (L): 1.33 L D-concentration of DDW: 105 ppm Dosage: 0.75 DdU

Correlation between body weight and MST in the DDW population

Design for the retrospective evaluation of breast cancer patients • 232 breast cancer patients were involved from February 1993 to April 2011

• 74 patients were diagnosed in stage IV having distant metastasis in 135 organs • The possible correlation between the DdU and response were evaluated patient by patient Journal of Cancer Research & Therapy 2013, 1(8):194–200

Relation between DDW-dose and response to DDW-treatment in advanced cases (DdU> 1 or ≤1)

DdU

CR

PR

NC

PD

Complete response

Partial response

No change

Progression of disease

>1

12 (30%)

16 (40%)

3 (7%)

9 (23%)

≤1

3 (7%)

10 (24%)

10 (24%)

19 (45%)

p=0.0028 Journal of Cancer Research & Therapy 2013, 1(8):194–200

Relation between DDW-dose and the response to DDW-treatment in advanced cases (DdU> 0.6 or ≤0.6) DdU

CR

PR

NC

PD

Complete response

Partial response

No change

Progression of disease

> 0.6

13 (23%)

21 (37%)

6 (10%)

17 (30%)

≤ 0.6

2 (8%)

5 (20%)

7 (28%)

11 (44%)

p=0.046 Journal of Cancer Research & Therapy 2013, 1(8):194–200

Retrospective study – Prostate cancer

Sub-groups of patients with prostate tumor in the retrospective study 20 patients: the 46 patients had distant metastasis during the follow-up

33 patients: the metastasis was limited to the bone

32 patients had adenocarcinoma

1 patient had anaplastic adenocarcinoma

10 patients had metastasis in other organ (liver, lung, bladder, etc.)

91 patients

45 patients were free of metastasis

bone metastasis appeared within 1 year from the diagnosis (DDW started after the diagnosis of the bone metastasis)

12 patients: the bone metastasis appeared later than 1 year from the diagnosis

3 patients had secondary tumor


Kaplan Meier survival curve in the sub-group of DDW-treated patients (20), who developed bone metastasis within 1 year from the diagnosis of prostate cancer

DDW MST: 64.8 months

Historical control MST: 15-20 months A. Kovacs et al., (2011) Journal of Cancer Therapy 2, 548-556

Sub-groups of patients with prostate tumor in the retrospective study 20 patients: the 46 patients had distant metastasis during the follow-up





91 patients


bone metastasis appeared within 1 year from the diagnosis (DDW started after the diagnosis of the bone metastasis)




Kaplan Meier survival curve in the sub-group of DDWtreated patients (12), who developed bone metastasis later than one year from the diagnosis of prostate cancer

MST: not calculable Cumulative follow-up period was 103 years, two patients died out of 12 (1 death case/51.5 years) Historical control: 5 years survival less than 1% A. Kovacs et al., (2011) Journal of Cancer Therapy 2, 548-556

Sub-groups of patients with prostate tumor in the retrospective study

46 patients had distant metastasis during the follow-up





91 patients


20 patients: the bone metastasis appeared within 1 year from the diagnosis (DDW started after the diagnosis of the bone metastasis)




Kaplan Meier survival curve in the subgroup of DDW-treated patients (45), having no metastasis during the follow-up period

MST: not calculable Cumulative follow-up was 157 years, 4 patients died out of 45 (1 death case/39.2 years) Historical control: 5 years survival 56%


Retrospective study – Lung cancer

Kaplan Meier survival curve in DDW-treated lung cancer patients (304) 100

80

60

MST 48 months 40

20

0 0

50

100 Time (months)

150

200

Kaplan Meier survival curve in the DDW-treated lung cancer patients (304) for males (157) and females (147) 100

80

60

male female

40

MST Males 40 months Females 87 months

20

0 0

50

100

150

200

Time (monts)

Z. Gyöngyi, et al. (2013) Nutrition and Cancer, 65:2, 240-246

Evaluation of lung cancer patients 129 patients, enrolled from 1992, evaluated in 2010

304 patients, enrolled from 1992, evaluated in 2015

Historical control

MST of male patients

25.9 months

40 months

7.5 months

MST of female patients

74.1 months

87 months

10.3 months

MR scan of a 54-year-old female patient with brain metastasis from SCLC at the time of the diagnosis (07.29.2001) (Size of brain metastasis: 20x30x40 mm)

K. Krempels, et al. (2008) Integrative Cancer Therapies 7(3):172-81.

MR scan of a 54-year-old female patient with brain metastasis from SCLC after three months from the diagnosis (10.22.2001) (Size of brain metastasis: 15x20x23 mm)


MR scan of the patient with brain metastasis from SCLC, 23 months after the diagnosis (06.30.2003), complete remission with residual tissue


MR scan of the patient with brain metastasis from SCLC, 43 months after the diagnosis (02.26.2005), complete remission with residual tissue


CT scan of the 54-year-old female patient with 30x50x32 mm lung tumor at the time of the diagnosis (07.31.2001) and 23 months later (06.30.2003)


Retrospective study – Breast cancer

Kaplan Meier survival curve of 232 DDW-treated breast cancer patients L/E 100 90 80

MST: 12.3 years

70 60 50 40 30 20 10 0

100

200 Time (months)

300

400


Study arms in the retrospective evaluation of breast cancer patients

According to staging at the diagnosis: group A: advanced breast cancer, n=74 patients group P: primary breast cancer, n=158 patients

Survival from the diagnosis in the subgroups defined on the staging at the diagnosis L/E 100

80

60 staging at the diagnosis A P 40

MST

A: 52 months (4.3 years)/ 74 pts

20

Historical control: 20-24 months (1.6-2 years)

0 0

50


200

250

P: 217 months (18.1 years)/ 158 pts

P < 0.0001


Retrospective study – Pancreas cancer

Kaplan-Meier estimator curve of survival probability of pancreatic cancer patients MST A: 39 months (3.2 years)/ 18 pts /within 60 days after diagnosis

B: 16 months (1.3 years)/14 pts/over 60 days after diagnosis Historical control: 6-7 months / 30 pts All groups underwent standard cytostatic treament due to an unresectable disease

Retrospective study – Glioblastoma multiforme (GBM)

Inoperable glioblastoma multiforme, radiotherapy combined with DDW treatment

34 patients MST: 26 months

Historical control MST: 4-7 months

May Deuterium Depletion lower the risk of developing cancer?

Main phases of tumor development Changes in genome and metabolism

Healthy cell

Pre-tumorous stage

Additional changes in genome and metabolism

Tumorous cells 1 mm

4-5 years

Vessels grow into the tumor

Fast growth of tumor start

Detectable tumor 1 cm

Effect of D-concentration on the migration of MCF-7 (breast tumor) cells 150ppm D 135ppm D 120ppm D 105ppm D

RTCA (Real Time Cell Analyzer) DP system that is a migration and invasion assay system uses the Boyden Chamber principle but does not involve any fixation or labelling of the cells. Unpublished data of Avidin Ltd.

Effect of D-concentration on the migration of HT199 (melanoma) cells 150ppm D 135ppm D 120ppm D 105ppm D

RTCA (Real Time Cell Analyzer) DP system that is a migration and invasion assay system uses the Boyden Chamber principle but does not involve any fixation or labelling of the cells. Unpublished data of Avidin Ltd.

Relative ratio of glycogen, deoxiribose, ribose synthesis in MIA-PaCa pancreatic carcinoma cells 120 100 80 60 40 20 0

25 ppm

50 ppm

100 ppm

150 ppm

(D-conc.)

In healthy cells the glycogen:deoxiribose ratio is tipically close to 1:1. British Journal of Cancer (2004) 91, 2094–2100. doi:10.1038/sj.bjc.6602243

Scheme of study to investigate the preventive role of deuterium depletion in carcinogen-treated (DMBA) mice

Chemical carcinogen

Myc Hras Kras

Bcl2 Chemical carcinogen

+

p53

DDW Gene expression 24 hours after induction

Deuterium-depleted water (DDW; 25 ppm D) supresses early gene expression having key role in cancer development after carcinogen treatment (DMBA) in mice 120

Gene expression%

100

80 Myc

Hras

60

Tp53 BCL2 40

Kras

20

0 Tap water 1

2

3

Tap w + DMBA 1

2

3

DDW 1

2

3

DDW + DMBA 1

2

3

Experimental Protocol to Verify the Preventive Effect of D-depletion in Carcinogen-treated Mice

24 (control group) normal water tumor induction 48 mice

One-year follow-up

with chemical agent (DMBA)

24 (treated group) DDW

One-Year Survival of Carcinogen-treated Mice

cell proliferation (% of 150 ppm treatment)

Effect of DDW on the proliferation of HT-29 cell line 100 80 60 40

20

100

1000

Deuterium concentration (ppm) M. Molnár, Semmelweis University, Budapest,Hungary

Effect of D2O on COX-2 expression in HT-29 cells

COX-2 (DENSITOMETRIC UNITS)

20

80 150 200 500 1000 D2O (ppm) COX-2

1200 1000

800 600 400 200 20

80

150

200

500

1000

D2O (ppm) M. Molnár, Semmelweis Univ. Medical School, Budapest, Hungary

Effect of DDW on the prostaglandin production in HT-29 cells 12

Prostaglandin E2 (ng/mg protein)

10 8 6 4 2

20

80 150 500 1000 Deuterium concentration (ppm) M. Molnár, Semmelweis Medical School, Budapest, Hungary

Conclusions • DDW reduces the migration of tumor cells on dose dependent manner • DDW has an impact on the metabolism of cancer cells • DDW inhibits the expression of genes having role in tumor development • DDW may become the very first drug which is dedicated to prevent cancer without causing toxic effect

May Integration of Deuterium Depletion into Oncotherapies Help Us to Cure Cancer?

171 out of 1,827 patients (all tumor types) started DDW treatment in tumor-free status 1,656 tumorous patients

171 tumor-free patients

Cumulative time from diagnosis to the end of follow-up

6,079 year

801 year

MST

106 months (9.1 year) Not calculable

Average time between diagnosis and start of DDW

646 days

374 days

Average length of DDW consumption

440 days

571 days

Number of patients died

476 (28.7%)

11 (6.4%)

Deaths/Cumulative follow-up period

12.7 years

72.8 years

Survival from the start of DDW treatment in the subgroups of breast cancer patients defined on restaging at the involvement MST:

L/E 100

A:

P:

80

R: 1 death / 48 pts / 221.1 year 60 restaging at the DDWstart A P R 40

20

P < 0.0001

0 0

50


200

250


Protocol for the integration of deuterium depletion into the currently applied oncotherapies Break in DDW administration

Start of DDW again

Start of DDW

Start of DDW again Break again

R Diagnosis Patient is in remission, but still keep drinking DDW Start of treatment (surgery, radiotherapy, chemotherapy)

Survival from the diagnosis in subgroups of breast cancer patients undergoing single and continuous or repeated DDW treatments L/E 100

MST: Y: 293 months/24.4 years / 53 pts

80

N: 108 months/9 years / 179 pts 60 method of the DDW treatment Y (repeated) N (single and continuous) 40

20

P < 0.0001

0 0

100

200 Time (months)

300

400


Main rules and conclusions of the application of DDW • Start DDW treatment as close to diagnosis as possible • Administer DDW longer than 4 months • Administer DDW for at least 9 months to achieve the best result • DdU must be or exceed 1 • In remission do not stop repeating cures for 3-4 months within 6-8 years • DDW in healthy population may reduce the incidence of cancer

Mechanism of cell cycle regulation

Albert Szent-Györgyi Nobel laureate (1893–1986)

„To regulate the cell growth you need a red and a green light” Albert Szent-Györgyi

Metabolic water with low deuterium content as a natural deuterium depleting mechanism

Malate

http://high-fat-nutrition.blogspot.com/2012/07/protons-wheres-bias.html

h

Increase of intracellular pH is necessary to proliferation

Na+ H+

cell division

decreasing H+concentration

Pouyssegur et al., PNAS, 81 4833-4837, 1984

H+

decreasing H+concentration

cell division

tumor

R. Perona et al., Nature, 334 438-440, 1988

The H+ transport system will prefer to eliminate H+ resulting an increase in D/H ratio Applying DDW increasing D+concentration

increasing D+concentration

H+

decreasing H+-

concentration

H+

Signal is a + higher D/H ratio decreasing H concentration

cell division

no signal

no cell division

Effect of D depletion on the rate of recovery from acid load in A4 cells 505/440 fluorescence excitation ratio

3.75

3.68

3.57

3.48 150 ppm 90 ppm 3.39

3.30

0

200

400

600

800

1000

time (s)

Laskay, G., Universitiy of Szeged

Changes in serum Na+ concentration Day

N

Mean

SD

Min. value

Max. value

mmol/L

0 90

30 30

141.1 139.0

2.4298 2.0095

136.8 135.4

147.3 143.0 p=0.00002

Serum Na+ was the only measured parameter, which changed into the same direction in 90% of the patients. Serum Na+ concentration significantly decreased between day 0 and day 90.

Effect of D-concentration on the growth rate of HT-29 (colon) cell line HT29_56hours

500ppm 200ppm 300ppm 800ppm 150ppm

HT-29; 56 hours (150-800 ppm D) 1,6

Effect ratio (%)

1,4 1,2 1 0,8 0,6 0,4 0,2 0 150ppm

200ppm

300ppm

500ppm

800ppm

Optical density of XTT (OD450) after incubation with L929 vs. deuterium concentration of medium

Somlyai, G., et al. (1993) FEBS Lett. 317, 1-4

The mitochondria communicate with the cytosol • The probability of the cell division may depend on the D/H ratio in the mitochondria and the cytosol • When a growth hormon binds to the membrane it stimulates the Na+/H+ antiport, which prefer to eliminate the lighter isotope of H will increase the D/H ratio within the cytoplasm • If there is no properly working mitochondria in the cells, it makes easier to increase the D/H to threshold level

The road to human anticancer drug registration

Production of DDW as active ingredient according to GMP with franctional distillation (2015)

Storage tanks for purified water and deuteriumdepleted water

Grade D Clean areas

Production of Depletin® for clinical use

Registration of DDW as active substance at the European Medicine Agency (EMA)

CLINICAL STUDY PROTOCOL A MULTICENTER, SINGLE ARM, OPEN LABEL, EXPLORATORY PROSPECTIVE PHASE II STUDY TO EXPLORE THE EFFICACY, SAFETY AND TOLERABILITY OF DEUTERIUM DEPLETED WATER IN PREVIOUSLY UNTREATED PATIENTS WITH ASYMPTOMATIC CHRONIC LYMPHOCYTIC LEUKEMIA BUT WHO ARE AT HIGH RISK OF PROGRESSION. STUDY NUMBER HYD-HD85/45-CLL-01 PRODUCT Depletin, supplied as water with deuterium concentration 45, 65 or 85 ppm EUDRACT NUMBER 2014-002060-32 INDICATION CHRONIC LYMPHOCYTIC LEUKEMIA CLINICAL PHASE II PROTOCOL VERSION 3.0 DATE 23 March 2015 SPONSOR HYD Pharma Inc. Branch office: Fürj u. 2. H-1124 Budapest, Hungary Phone: +36 1 365 1660 Registered seat: Alsó kikötő sor 11. H-6726 Szeged, Hungary MEDICAL SAFETY MONITOR Edwin Klumper, M.D., Ph.D., M.B.A. OVERALL STUDY DIRECTOR Gábor Somlyai, Ph.D. THIS CONFIDENTIAL DOCUMENT IS THE PROPERTY OF HYD PHARMA INC. FOR CANCER RESEARCH AND DRUG DEVELOPMENT. NO UNPUBLISHED INFORMATION CONTAINED IN THIS DOCUMENT MAY BE DISCLOSED WITHOUT PRIOR WRITTEN APPROVAL OF HYD PHARMA INC. FOR CANCER RESEARCH AND DRUG DEVELOPMENT.

Special thanks to • • • • •

• • • •

Gábor Jancsó and György Jákli, KFKI Atomic Energy Research Institute, Budapest, Hungary Miklós Molnár, Semmelweis University Medical School, Budapest, Hungary András Kovács, Imre Guller, Saint John’s Hospital, Budapest, Hungary Ildikó Somlyai, Krisztina Krempels, Krisztina Balog, Orsolya Abonyi, HYD LLC. for Cancer Research and Drug Development, Budapest, Hungary Boros G. László, Kalifornia Egyetem, Los Angeles (UCLA), Gyermekgyógyászat (Pediatrics), Los Angeles Orvosbiológiai Kutató Intézet, Los Angeles Biomedical Research Insititute at the Harbor-UCLA Medical Center (LABIOMED) Zoltán Gyöngyi, Department of Public Health, Medical School, University of Pécs, Pécs, Hungary Mariann Szabó, Veterinary Clinic, Budapest, Hungary Tamás Berkényi, Alpha-Vet Veterinary Hospital, Székesfehérvár, Hungary Lilian Z. Fehér, László G. Puskás, Lajos Nagy, Avidin Ltd, Szeged, Hungary, Biological Research Center of the Hungarian Academy of Sciences, Laboratory of Functional Genomics, Szeged, Hungary † Gábor Laskay, Department of Plant Biology, University of Szeged, Szeged, Hungary

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