Nové směry ve vývoji nanoterapeutik

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1 Nové směry ve vývoji nanoterapeutik BlankaŘíhová Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic,

2 Paul Ehrlich a jeho kouzelná střela Psal se rok 1906 Studies in Immunity, Plenum Press, New York, 1906

3 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

4 OD VÝZKUMU K LÉČBĚ nové látky testy in vitro 500 testy in vivo 5 klinické testy 1 použití v klinické praxi

5 LÉČIVA NOVÉ GENERACE SE SCHOPNOSTÍ PASIVNÍ NEBO AKTIVNÍ AKUMULACE a b c nerozpustné liposomy rozpustné imunotoxiny vodorozpustné polymerní systémy

6 SMĚROVANÁ POLYMERNÍ LÉČIVA

7 Free drug Targeted drug

8

9 KONFERENCE NIH/NCI-WASHINGTON ZÁŘÍ 2002 a aktivní směrování b pasivní směrování

10 Active targeting cell-surface receptors on target tumor cells

11 Free drug Targeted drug Target cell Target cell

12 Extracellular matrix Intracellular fluid Endosome Plasma membrane Lysosome

13 Passive targeting (accumulation) EPR effect

14 a) Normal tissue Intact endothelium Effective lymphatic drainage b) Tumor tissue Discontinuous endothelium Limited lymphatic drainage

15 EPR effect (Enhanced Permeability and Retention) effect

16 POLYMERNÍ NOSIČE Dextrany Polygalakturonová kyselina Polyglutamová kyselina PEG Poly-L-lysin Blokové kopolymery N-(2-hydroxypropyl)metakrylamid (HPMA)

17 N-(2-hydroxypropyl)methacrylamide HPMA

18 HPMA NOSIČ neimunogenní nemitogenní netoxický neaktivuje komplement neukládá se v organizmu

19 STRUCTURE OF PRODRUGS PRODRUGS with ACTIVE TARGETING PRODRUGS with PASSIVE TARGETING

20 STRUCTURE OF ANTIBODY-TARGETED HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN DOX

21 STRUCTURE OF (PK1) HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN DOX

22

23 in vivo veritas

24 Elimination (Cancer Immunosurveillance) Equilibrium γδ NK Genetic instability/ immune selection Protection

25 Je to závažné? Kčemu vlastně imunitní systém potřebujeme?

26 STATISTIKA 40 % žen mezi lety má potenciální zhoubné nádory v prsu incidence je 1% 55% mužů mezi lety má potenciální zhoubné nádory v prostatě incidence je 1% 90% populace ve věku let má potenciální zhoubné nádory štítné žlázy incidence je 0.1%

27 Vznik nádoru může usnadnit dočasné nebo chronické selhání imunitního systému, jiná onemocnění, stres, špatná výživa, nedostatek odpočinku, drogy a podobně Imunitní systém

28 Organismus se nádoru brání, imunitní systém se ale postupně Imunitní systém vyčerpává, nádor roste

29 Jsou nasazena klasická cytostatika, která sice zničí rychle se dělící nádorové buňky, ale částečně také rychle se dělící buňky imunitního systému

30 Uplatní se částečně poškozený imunitní systém při odstranění Imunitní systém této zbytkové choroby?

31 STRUCTURE OF PRODRUGS PRODRUGS with ACTIVE TARGETING PRODRUGS with PASSIVE TARGETING

32 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

33 Re-transplantation

34 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

35 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

36 Immunomobilization

37 Cytotoxicity and immunostimulation double attack on cancer cells with polymeric therapeutics

38 Polymeric drugs based on HPMA cytotoxic immunomodulation component

39 Protection against the second cancer attack

40 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)

41 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

42 Systemic antitumor resistance initiated by the treatment with polymeric drugs depends on Dose Time The immune system of the host

43 Dose

44 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

45 More aggressive treatment lower resistance

46 Time

47 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% % 20% 15 60% 0% Control (PBS) 0% 0%

48 vaccination window optimal time frame treatment given too early or too late after transplantation of cancer cells induces only a limited resistance

49 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

50 The immune system of the host

51 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 days DOX-HPMA AM /Dox-HPMA HYD mixture (1:2) controls

52 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 days DOX-HPMA AM /Dox-HPMA HYD mixture (1:2) controls

53 Conventional mice full resistance Immunocompromized (nu/nu) mice no resistance

54 Anti-cancer drug testing conventional immunocompromised mice mice

55 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

56 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

57 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

58 makrofág (APC) dendritická buňka NK buňka (LAK buňka) CD4 Th lymfocyt CD8 CTL plasmatická buňka sekretující protilátky protilátka

59 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 survival (%) days spleen cells 100:1 resistant donors CD8+ 20:1 CD8+ 10:1 control (EL-4 only)

60 The treatment generates a tumorspecific long-lasting memory, which is based mainly on effector cell of specific immunity, the CD8 + CTLs

61 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

62 Immunonormalization

63

64 Dox-HPMA copolymer, PK1 (FCE 28068) Paclitaxel-HPMA copolymer (PNU ) Cisplatin-HPMA copolymer (AP 5280) Dox-HPMA copolymer-gal, PK2 (FCE 28069) Dox-HPMA copolymer-huig No polymer-related toxicity was observed

65 STRUCTURE OF ANTIBODY-TARGETED HPMA COPOLYMER CARRIER-BOUND DOXORUBICIN DOX

66 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)

67 METASTATIC LUNG TUMORS % of regression Treatment D-P-HuIg

68 40 LEUKOCYTES x 10 9 cells/l free drug D-P-HuIg

69 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)

70 TAA cytokeratin cytokeratin

71 ACTIVITY OF NK CELLS IN HUMAN PATIENTS Appli- day E.G. J.K. K.R. K.H. cation I st II nd III rd healthy donor NK % of K562 killing

72 ACTIVITY OF LAK CELLS IN HUMAN PATIENTS Appli- day E.G. J.K. K.R. K.H. cation Ist 0 N.D N.D IInd IIInd healthy donor LAK % of Daudi killing

73 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

74 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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