Nové směry ve vývoji nanoterapeutik BlankaŘíhová Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic, (rihova@biomed.cas.cz)
Paul Ehrlich a jeho kouzelná střela Psal se rok 1906 Studies in Immunity, Plenum Press, New York, 1906
POŽADAVKY NA LÉČIVA NOVÉ GENERACE musí se akumulovat v cílové tj. nádorové tkáni - agresivnější léčba vedlejší účinky musí být minimální - podávání ambulantně cirkulace v krevním oběhu musí být prodloužená - podávání nemusí být tak časté léčivo nesmí být během cirkulace toxické - působení je omezeno jen na postiženou tkáň/orgán náklady nesmí být příliš vysoké
OD VÝZKUMU K LÉČBĚ 50 000 nové látky 5 000 testy in vitro 500 testy in vivo 5 klinické testy 1 použití v klinické praxi
LÉČIVA NOVÉ GENERACE SE SCHOPNOSTÍ PASIVNÍ NEBO AKTIVNÍ AKUMULACE a b c nerozpustné liposomy rozpustné imunotoxiny vodorozpustné polymerní systémy
SMĚROVANÁ POLYMERNÍ LÉČIVA
Free drug Targeted drug
KONFERENCE NIH/NCI-WASHINGTON ZÁŘÍ 2002 a aktivní směrování b pasivní směrování
Active targeting cell-surface receptors on target tumor cells
Free drug Targeted drug Target cell Target cell
Extracellular matrix Intracellular fluid Endosome Plasma membrane Lysosome
Passive targeting (accumulation) EPR effect
a) Normal tissue Intact endothelium Effective lymphatic drainage b) Tumor tissue Discontinuous endothelium Limited lymphatic drainage
EPR effect (Enhanced Permeability and Retention) effect
POLYMERNÍ NOSIČE Dextrany Polygalakturonová kyselina Polyglutamová kyselina PEG Poly-L-lysin Blokové kopolymery N-(2-hydroxypropyl)metakrylamid (HPMA)
N-(2-hydroxypropyl)methacrylamide HPMA
HPMA NOSIČ neimunogenní nemitogenní netoxický neaktivuje komplement neukládá se v organizmu
STRUCTURE OF PRODRUGS PRODRUGS with ACTIVE TARGETING PRODRUGS with PASSIVE TARGETING
STRUCTURE OF ANTIBODY-TARGETED HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN DOX
STRUCTURE OF (PK1) HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN DOX
in vivo veritas
Elimination (Cancer Immunosurveillance) Equilibrium γδ NK Genetic instability/ immune selection Protection
Je to závažné? Kčemu vlastně imunitní systém potřebujeme?
STATISTIKA 40 % žen mezi 40 50 lety má potenciální zhoubné nádory v prsu incidence je 1% 55% mužů mezi 50 60 lety má potenciální zhoubné nádory v prostatě incidence je 1% 90% populace ve věku 60 70 let má potenciální zhoubné nádory štítné žlázy incidence je 0.1%
Vznik nádoru může usnadnit dočasné nebo chronické selhání imunitního systému, jiná onemocnění, stres, špatná výživa, nedostatek odpočinku, drogy a podobně Imunitní systém
Organismus se nádoru brání, imunitní systém se ale postupně Imunitní systém vyčerpává, nádor roste
Jsou nasazena klasická cytostatika, která sice zničí rychle se dělící nádorové buňky, ale částečně také rychle se dělící buňky imunitního systému
Uplatní se částečně poškozený imunitní systém při odstranění Imunitní systém této zbytkové choroby?
STRUCTURE OF PRODRUGS PRODRUGS with ACTIVE TARGETING PRODRUGS with PASSIVE TARGETING
A B Survival of mice PRIMARY TREATMENT Dox-HPMA HYD Survival of mice RE-TRANSPLANTATION Dox-HPMA HYD controls 1x75 mg/kg Dox 1x25 mg/kg
Re-transplantation
A B Survival of mice PRIMARY TREATMENT Dox-HPMA HYD Survival of mice RE-TRANSPLANTATION Dox-HPMA HYD controls 1x75 mg/kg Dox 1x25 mg/kg
CYTOTOXIC AND IMMUNOMOBILIZING ACTIVITY (CIA) OF POLYMER-BOUND DRUGS Cancer-FREE cancer cells with target Ags 3 days? MDR cells cancer cells without target Ags
Immunomobilization
Cytotoxicity and immunostimulation double attack on cancer cells with polymeric therapeutics
Polymeric drugs based on HPMA cytotoxic immunomodulation component
Protection against the second cancer attack
HPMA polymer Polymer drugs based on HPMA Tumor cells sensitive or resistant to the treatment with polymeric drugs drug (doxorubicin) bound through spacer Immunogenic cancer cell death And release of tumor antigenes Induction of anti-tumor immunity (activation of dendritic cells, natural killer cells, T and B lymphocytes)
Happy tumor cells hidden and growing in defenceless environment Treatment with HPMA-based polymeric conjugates......has not only a direct cytotoxic effect on cancer cells but also causes their unmasking and exposing to effector mechanisms of immune system
Systemic antitumor resistance initiated by the treatment with polymeric drugs depends on Dose Time The immune system of the host
Dose
A B Survival of mice PRIMARY TREATMENT Dox-HPMA HYD Survival of mice RE-TRANSPLANTATION Dox-HPMA HYD controls 1x75 mg/kg Dox 1x25 mg/kg
More aggressive treatment lower resistance
Time
RESISTANCE AGAINST BCL1 LEUKEMIA DEPENDS ON THE INTERVAL BETWEEN TUMOR CELL INOCULATION AND APPLICATION OF THE ANTIBODY-TARGETED CONJUGATE (DOX-HPMA-B1MAB) Conjugate survival of survival of experimental mice re-transplanted mice (day) (n = 10) (1 x 10 4 BCL1 cells i.p.) 3 100% 0% 7 100% 30% 11 100% 20% 15 60% 0% Control (PBS) 0% 0%
vaccination window optimal time frame treatment given too early or too late after transplantation of cancer cells induces only a limited resistance
Too early the immune system is not supplied with a sufficient amount of antigens (cancer cells) for effector-cell activation Too late the effector mechanisms of the cancer-bearing host s immune system are already exhausted and unable to be activated, resulting in only very limited cancer resistance
The immune system of the host
The effect of Dox-HPMA AM /DOX-HPMA HYD mixture (1:2) on the growth of EL4 T cell lymphoma: conventional mice 100 Survival of mice % of survival 80 60 40 20 0 0 10 20 30 40 50 60 days DOX-HPMA AM /Dox-HPMA HYD mixture (1:2) controls
The effect of Dox-HPMA AM /DOX-HPMA HYD mixture (1:2) on the growth of EL4 T cell lymphoma: nu/nu mice 100 Survival of mice % of survival 80 60 40 20 0 0 10 20 30 40 50 60 days DOX-HPMA AM /Dox-HPMA HYD mixture (1:2) controls
Conventional mice full resistance Immunocompromized (nu/nu) mice no resistance
Anti-cancer drug testing conventional immunocompromised mice mice
Patient Immune system is not exhausted immune system is exhausted (nu/nu mice model) efficient treatment both components of the polymeric drugs are involved limited efficacy of the treatment only cytotoxic component of the polymeric drug is involved
STRUCTURE OF PRODRUGS ENZYMATICALLY ACTIVATED PRODRUGS ph-sensitive PRODRUGS 3 H 2 C C H 2 C C CH CH 3 C O NH CH 2 HC OH CH 3 x C H 3 HO C O Gly Phe Leu Gly NH y H C H 2 CH 3 C C NH O CH 2 HC OH CH 3 x O OH H C H 2 CH 3 C C O X NH N y C CH 2 OH O HO O OH O OMe OMe O OH H O O CH 3 OH CH 2 OH CH 2 OH O OH NH 2.HCl
A B Survival of mice PRIMARY TREATMENT Dox-HPMA AM or Dox-HPMA AM -HuIg Survival of mice RE-TRANSPLANTATION Dox-HPMA AM or Dox-HPMA AM -HuIg controls Dox-HPMA AM -HuIg Dox-HPMA AM
makrofág (APC) dendritická buňka NK buňka (LAK buňka) CD4 Th lymfocyt CD8 CTL plasmatická buňka sekretující protilátky protilátka
The anti-tumour immunity could be transferred to naive mice: Winn s assay tumour neutralization in vivo: 1x10 5 EL-4 cells were transplanted s.c. together with cells of the Dox-PHPMA-HuIg-cured animals to naïve recipients 100 80 survival (%) 60 40 20 0 0 10 20 30 40 50 60 days spleen cells 100:1 resistant donors CD8+ 20:1 CD8+ 10:1 control (EL-4 only)
The treatment generates a tumorspecific long-lasting memory, which is based mainly on effector cell of specific immunity, the CD8 + CTLs
Specificity The same tumor used for the primary transplantation and re-transplantation full resistance Different tumors used for the primary transplantation and re-transplantation limited resistance
Immunonormalization
Dox-HPMA copolymer, PK1 (FCE 28068) Paclitaxel-HPMA copolymer (PNU 166945) Cisplatin-HPMA copolymer (AP 5280) Dox-HPMA copolymer-gal, PK2 (FCE 28069) Dox-HPMA copolymer-huig No polymer-related toxicity was observed
STRUCTURE OF ANTIBODY-TARGETED HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN DOX
Effects of Hulg - it contains a mixture of natural antibodies, some of them might react with tumor markers - it stimulates production of IL-12 and other anti-tumor and anti-angiogenic cytokines - it enhances NK cell activity - it increases MW of the conjugate (EPR effect)
METASTATIC LUNG TUMORS 60 50 % of regression 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Treatment D-P-HuIg
40 LEUKOCYTES 35 30 x 10 9 cells/l 25 20 15 10 5 0 1 2 3 4 11 12 13 14 15 16 17 18 25 26 27 28 29 30 31 32 33 34 free drug D-P-HuIg
Nine tumor markers β -microglobulin 2 α-fetoprotein (AFP) CA 72-4 CA 125 CA 15-3 CA 19-9 CEA Ferritin Neuron-specific enolase (NSE)
TAA cytokeratin cytokeratin
ACTIVITY OF NK CELLS IN HUMAN PATIENTS Appli- day E.G. J.K. K.R. K.H. cation I st 0 24 23 9 11 3 45 21 19 33 II nd 0 22 10 10 11 3 27 25 16 12 III rd 0 23 10 8 13 3 42 12 6 18 healthy donor 24 24 NK % of K562 killing
ACTIVITY OF LAK CELLS IN HUMAN PATIENTS Appli- day E.G. J.K. K.R. K.H. cation Ist 0 N.D. 25 11 4 3 N.D. 17 15 14 IInd 0 20 14 17 18 3 40 18 16 15 IIInd 0 17 16 13 8 3 50 20 5 20 healthy donor 24 24 LAK % of Daudi killing
Acknowledgement Institute of Macromolecular Chemistry AS CR, v.v.i. Karel Ulbrich Tomáš Etrych Martin Hrubý Petr Chytil Hana Krakovičová Daniela Plocová Robert Pola Jiří Strohalm Martin Studenovský Vladimír Šubr
Acknowledgement Institute of Microbiology, AS CR, v.v.i. Jaroslav Betka Jan Bouček Ondřej Hovorka Helena Chmelová Markéta Ibrahimová Martina Kabešová Jiřina Kovářová Marek Kovář Lubomír Kovář Veronika Pakanová Milada Šírová Jakub Tomala David Větvička
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