Dr Alessandro Perra , Guna S,p.a.Italy

Future Health Summit

LOW DOSE CYTOKINES IN SUPPORTIVE THERAPY FOR CANCER

From research: Up to date pre-clinical evidence

*****

DUBLINMay 26th, 2016

Alessandro Perra – Scientific Director of GUNA S.p.a.

Low Dose Medicine into the Universities

MASTER BIENNALE DI II LIVELLOLOW DOSE MEDICINE IN

PSICO-NERUO-ENDOCRINO-IMMUNOLOGIA

Applicazioni in Medicina Generale, nelle Disfunzioni

Neuro-Muscolo-Scheletriche e nell’ Healthy Aging

© Dipartimento Scientifico Guna S.p.a.

LOW DOSE MEDICINE

Low Dose Medicine integrates state of the art scientific advances in:

P.N.E.I.Molecular

Biology

Nano-Pharmacology

linked toQuantum

Physics


BIO-REGULATING PHYSIOLOGY

LOW DOSE CYTOKINES

LOW DOSE NEURO

PEPTIDES

LOW DOSE HORMONES

LOW DOSE GROWTH FACTORS

LOW DOSE MEDICINEA NOVEL, INTEGRATED, SYSTEMIC APPROACH TO DISEASES


CENTRAL NERVOUS SYSTEM&

AUTONOMIC SYSTEM

IMMUNE SYSTEM

ENDOCRINE SYSTEM

HOMEOSTATIC Control Systems and bi-directional cross-talk

Ader, R., Psychoneuroimmunology, IV edition, vol. 1 e 2, Academic Press, Amsterdam 2007. It is the classical text on the matter, pubblished for the first time in 1981.

PNEI


STRESS AXIS

Th1

Th2

Reduction of

IFN-γ

X

CD8+

Tc

Not correctstimulation of

Ts


•Cohen M, et al. Increased emotional distress in daughters of breast cancer patients is associated with decreased natural cytotoxic activity, elevatedlevels of stress hormones and decreased secretion of Th1 cytokines. Int J Cancer. 2002;100(3):347-54.•Montoro J, et al. Stress and allergy. J Investig Allergol Clin Immunol. 2009;19 Suppl 1:40-7.•He Y, et al. Psychological stress exerts effects on pathogenesis of hepatitis B via type-1/type-2 cytokines shift toward type-2 cytokine response. PLoSOne. 2014;9(8):e105530.•Radice E, et al. Low-doses of sequential-kinetic-activated interferon-gamma enhance the ex vivo cytotoxicity of peripheral blood natural killer cellsfrom patients with early-stage colorectal cancer. A preliminary study Intern. Immunopharm 2014;19:66-73.

DOWN-REGULATION OF Th1

AN ALTERATED CROSS-TALK DUE TO AN

UNBALANCE* BETWEEN SIGNALING MOLECULES

PLAYS A CRUCIAL ROLE IN THE ONSET OF

INFLAMMATORY, ALLERGIC AND AUTO-IMMUNE

DISEASES

*ALTERATED CONCENTRATIONS

P.N.E.I. NETWORK AND SIGNALING MOLECULES


HEALTH

PHYSIOLOGICAL CONCENTRATION

DISEASE

DISEASEHYPO-CONCENTRATION

HYPER-CONCENTRATION10-6

10-15

C O P E Cytokines & Cells Online Pathfinder Encyclopedia

Version 26.7 (Spring 2011 Edition)


g (gram)= 1

10 -1 = 0.1

10 -2 = 0.01

mg (milligram)= 10 -3 = 0.001

μg (microgram)= 10 -6 = 0.000001

ng (nanogram)= 10 -9 = 0.000000001

pcg (picogram)= 10 -12 = 0.000000000001

fg (fentogram)= 10 -15 = 0.000000000000001

DEFINITIONS


The membrane receptor plays a KEY role.

ONLY physiological concentrations are able toactivate or reactivate the membrane receptors and consequently, stimulate the physiological function

of a target cell.

14

UP- AND DOWN-REGULATION

- Akahoshi T et al. Interleukin 1 stimulates its own receptor expression on human fibroblasts through the endogenousproduction of prostaglandin(s). J Clin Invest. 1988 Oct;82(4):1219-24

- Samanta AK et al. Interleukin 8 (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own receptorexpression on human neutrophils. J Biol Chem. 1990 Jan 5;265(1):183-9.


CENTRAL NERVOUS SYSTEM&

AUTONOMIC SYSTEM

IMMUNE SYSTEM

ENDOCRINE SYSTEM

HOMEOSTATIC Control Systems and bi-directional cross-talk

Ader, R., Psychoneuroimmunology, IV edition, vol. 1 e 2, Academic Press, Amsterdam 2007. It is the classical text on the matter, pubblished for the first time in 1981.

PNEI


16

Th-1/Th-2 BALANCE

Th2

Crohn’s DiseasePsoriasisVitiligo

…

AsthmaAtopy

…

Th1TReg

Th1 UP-REGULATION

Th2 DOWN-REGULATION Th1 DOWN-REGULATION

Th2 UP-REGULATION

Th2

Th1Th17


HEALTH

HYPER

HYPO

Cytokines UP• IL-1• IL-6• TNF-α• IL-17• INF-γ• IL-2• IL-8

Cytokines DOWN• TGF-β • IL-4• IL-10

Cytokines DOWN• IL-1• IL-6• TNF-α• IL-17• INF-γ• IL-2• IL-8

Cytokines UP• TGF-β • IL-4• IL-10

Physiological concentration• IL-1• IL-6• TNF-α• IL-17• INF-γ• IL-2• IL-8

Physiological concentration• TGF-β • IL-4• IL-10


Raphael I et al. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine (2014), http://dx.doi.org/10.1016/j.cyto.2014.09.011

Neither good nor bad in Nature

PROBLEMIs it possible to modulate the action of cytokines and other

signaling molecules?

IF DISEASES ARE EXPRESSIONS, CONSEQUENCES OF

CHANGED CONCENTRATION OF MESSENGER MOLECULES…


Antagonistic cytokines are utilized in orderto slow down a biological effect; Same

cytokines in order to enhance a biologicalfunction.

The concept of BALANCE and the use of SKA low dose cytokines


Th0

Th2Th1

IL-12, INF-γ IL-4

INF-γ DOWN-REGULATES

IL-12 UP-REGULATESIL-4 UP-REGULATES

IL-4 DOWN-REGULATES

Th subsets cross-regulate expansion and functions each other.

- Cooke , A. Th17 in Inflammatory Conditions. 2006, Rev Diabetic Stud 3: 72-7- Bettelli E. et al. Th17: the third member of the effector T cell trilogy. Current Opinion in Immunology 2007, 19: 652-657

Inflammatory diseases Allergy

THE CONCEPT OF BALANCE – RECIPROCITY of TH CELLS


© D

ipa

rtim

en

to S

cie

nti

fico

Gu

na

S.p

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IL-10 4C

A novel, systemic, integrated,

synergistic approach for supportive therapy in

Cancer© Dipartimento Scientifico Guna S.p.a.


D’Amico L, Ruffini E, Ferracini R, Roato I (2012) Low Dose of IL-12 stimulates T Cell response in cultures of PBMCs derived from Non Small Cell Lung Cancer Patients. Journal of Cancer Therapy 3: 337-342.

Th0

Th2Th1

TReg

TGF-βDOWN-REGULATES DOWN-REGULATES

Plasticity and Rigidity of

Relationship among Th1-Th2-Th3



1.7 1.7

0.9

4.5

0.8

0.6

0.4

1.8

0.2

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

basale CH3 CH4 placebo IL12 10ng/ml

CD

4/T

reg

day 5

day 10

healthy ctrl 1pg/ml 0.01pg/ml SKA basal 10 ng/ml

basale CH3 CH4 placebo IL12 10 ng/ml

Series1 30.7 19 48.4 48.3 4.8

0

10

20

30

40

50

60

%C

D4

/IFN

gam

ma

Day 5

CH3 CH4 placebo IL12 10 ng/ml

Series1 32.8 56 46.2 4.2

0

10

20

30

40

50

60

%C

D4

/IFN

gam

ma

Day 10


IL-12 0.01pg/ml SKAIL-12 1pg/ml SKA

Legenda:

•Left column =not-treated control group

•Central column = patients treated withconventional dosage of IFN-gamma (1 ng/ml)

•Right column = patientstreated with IFN-gammaGUNA (0.25 fg/ml)

Healthysubjects

Non-metastaticcancerpatients

MetastaticcancerpatientsHealthy subjects

Non metastatic

cancer patients

Metastatic cancer

patients


Radice E, Bellone G, Miranda V.

Enhancement of the immunomodulatory functions of ex vivo-generated dendritic cells from early-

stage colon cancer patients by consecutive exposure to low doses of sequential kinetic activated IL-4

and IL-12.

A preliminary study.

Translational Oncology 2015;8:327-38© Dipartimento Scientifico Guna S.p.a.

The aim of this study is to assess in vitro the ability of single or

sequentially combined exposure to very low doses of SKA IL-4

and IL-12 (10-8 µg/ml) to enhance DCs’ antigen presentation

capacity compared to the normally administered doses of

recombinant human (rh) IL-4 and IL-12.

Ex vivo study on dendritic cells obtained from the peripheral

blood of patients with early stage colorectal carcinoma (CRC) - in

the presence or absence of metastasis - and from healthy donors.

STUDY TYPE

AIM OF THE STUDY


EX VIVO STUDY

16 subjects

(10 males, 6 females; mean age 73; range 57-83)

With colon carcinoma

16

9 primary colon

carcinoma

2 Dukes A

7 Dukes B 1-2

7 metastatic colon

carcinoma

Dukes C 1-2

with lymph

node

metastasis

12 subjects

(7 males, 5 females; mean age 64; range 37-85)

Healthy donors

EX VIVO STUDY

INCLUSION CRITERIA

• Subjects affected by primary colon carcinoma needing surgery without

pre-surgery evidence of distance metatstasis a

PREPARATIONS

• SKA-IL-4 (10 fg/ml) GUNA Laboratories

• SKA-IL-12 (10 fg/ml) GUNA Laboratories

• rhIL-4 (1ng/ml) Pepro Tech Inc.

• rhIL-12 (1ng/ml) Pepro Tech Inc.

Translational Oncology 2015 8, 327-338DOI: (10.1016/j.tranon.2015.06.005)

(A) MoDC (monocyte derived denditic cells) allostimulatoryactivity of colon carcinoma patients (n = 16) versushealthy donors (n = 12). (B) MoDC allostimulatoryactivity of nonmetastatic (n = 9) versus metastatic (n= 7) colon carcinoma patients. MoDCs, generated byculturing PB CD14+ cells from tumor and normalsubjects in the presence of rhGM-CSF and rhIL-4 for6 days, were incubated with 1 × 105 allogeneic naïveCD4+ T cells at 1:40, 1:20, and 1:10 ratios for 5 daysfollowed by a 6-hour pulse of 3H-TdR. Results areexpressed as mean ± SE cpm of triplicate co-cultures. Statistical significance was determinedusing one-way ANOVA.

RESULTS

Translational Oncology 2015 8, 327-338DOI: (10.1016/j.tranon.2015.06.005)

.

Effects of standard-dose rhIL-4 and/or rhIL-12 and low-dose SKA-IL-4 and/or SKA-IL-12 on APC activity in MLR ofMoDCs from nonmetastatic colon carcinoma patients (n =6) and from metastatic colon carcinoma patients (n = 7).MoDCs were untreated or pretreated with SKA-IL-4 (48hours) and SKA-IL-12 (24 hours) as single agents orsequentially in parallel to the rh cytokines, and subjectedto MLR with allogeneic naïve T cells in different MoDC-to-T cell ratios. The figure shows the mean percentages ± SEof 3H-TdR incorporation in cpm. Statistical significance wasdetermined using one-way ANOVA.Rh/SKA cytokine pretreated nonmetastatic/metastaticcolon carcinoma MoDCs versus untreatednonmetastatic/metastatic colon carcinoma MoDCs: *P <.05 and **P < .01.Nonmetastatic colon carcinoma MoDCs versus metastaticcolon carcinoma MoDCs: ●●P < .01 and ●●●P < .0001.

RESULTS*

RECOMBINANT HIGH DOSE

LOW DOSE SKA

IL-4 (10 fg/ml SKA) IL-12 (10 fg/ml SKA)

IFN-g (10 fg/ml SKA)

IL-12 (10 fg/ml SKA)

GUNA IMMUNITY SYSTEM ACTIVATION IN CANCER

Biomedicowww.biomedico.ie

[email protected]

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FOR FURTHER INFORMATION


http://www.biomedico.ie/

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Dr Alessandro Perra , Guna S,p.a.Italy

Healthcare

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