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Nanotoxikológia I: Nanogyógyszerek Immunológiai Mellékhatásai: pseudoallergia és immunogenitás

Szebeni J á nos Nanomedicine Research and Education Center Semmelweis University, Budapes t Hungary. Nanotoxikológia I: Nanogyógyszerek Immunológiai Mellékhatásai: pseudoallergia és immunogenitás. NANOMEDICINES: THE GOOD AND THE BAD. Unknown longterm toxicities Systemic Organ Immune

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Nanotoxikológia I: Nanogyógyszerek Immunológiai Mellékhatásai: pseudoallergia és immunogenitás

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  1. SzebeniJános Nanomedicine Research and Education Center Semmelweis University, Budapest Hungary Nanotoxikológia I:Nanogyógyszerek Immunológiai Mellékhatásai: pseudoallergia és immunogenitás

  2. NANOMEDICINES: THE GOOD AND THE BAD Unknown longterm toxicities Systemic Organ Immune Expensive • 38 products in market • 7 000 M USD/y • 157 products in R&D • 207 companies • 13000 publications • 59 journals • 422000 google hit

  3. „Problem” • Immune recognition & toxicity represent a barrier to the clinical use of many nanomedicines • Although clinicians handle most acute and longterm immune toxicities, current approaches of prediction, prevention and treatment are not sufficiently specific and effective

  4. Nanodrug carrier systems 10-6 liposzómák 10-7 polimer vezikulum Méret (m) micellum 10-8 Szén nanocsövek polimer konjugátum fullerén aptamer quantum dot dendrimer spio 10-9 10-10

  5. Drug Adverse Events, 2,2 M / year 5th cause of death 75-85% Systemic Organ Toxicity 15-25% immune toxicity Immune suppression Proinflammatory/cytokine storm hypersensitivity reactions 23% IgE-mediated (allergic) 77% Non-IgE-mediated (pseudoallergic, 420,000/year) Direct triggering Activation of „allergy” cells Mast cells, basophils, macrophages Receptor-mediated triggering (C3a, C5a receptors)

  6. Nanomedicine-immune system cross-talk Specific (adaptive) Nonspecific (innate) Humoral Natural antibodies Complement antibodies Cellular PMN, NK cells, macrophages T and B cells Mast cells Dendritic cells

  7. Adverse immuneeffects of nanomedicines

  8. Detection limits of the immune system 10000 1000 100 Liposomes (MLV) Particle size (nm) sMW drugs liposomes (SUV) 10 micelles carbon nanotubes (MWCNT) dendrimers 1 fullerenes 103 104 106 107 0 105 MW

  9. Morphological similarity between liposomes and pathogenic human viruses Doxil HIV-1 100 nm 100 nm paramixo Human viruses orthomixo pox bunya herpes papova 300 nm adeno reo

  10. Membrane proteins protecting cells against complement attack

  11. Complement activation IgE • Hypersensitivity reactions • Infusion, naphylactoid, anaphylactic, idiosyncratic... • pseudoallergic • Complement activation-related pseudoallergy, CARPA

  12. Clinical Symptoms of Pseudoallergy

  13. Unique features of pseudoallergicdrug reactions • first treatment (no prior exposure) • milder or absent upon re-exposure • spontaneous resolution • pulmonary infiltration • high reaction rate (2-10%) or higher

  14. Doxil package insert Hypersensitivity to Doxil C activation in human sera in vitro WARNING: Acute infusion-related reactions, sometimes reversible upon terminating or slowing infusion,occurred in up to 10% of patients. Serious and sometimes fatal allergic/anaphylactoid-like infusion reactions have been reported. Medications/emergency equipment to treat suchreactions should be available for immediate use ...” Source: www.doxil .com J. Liposome Res. 10:347-361, 2000

  15. C activation by Liposomes Classical Pathway Alternative Pathway Natural antibodies C1q IgG C3 C1q CRP C1q IgM C1q

  16. Drugs causing hypersensitivity reactions

  17. Mechanism of CARPA C activation Reactogenic nanoparticles Release of primary mediators Release of Secondary mediators Mast cells Basophil leukocytes

  18. Facts about liposomal CARPA • All types of liposomes can cause CARPA in animals and man, with neutral SUV being the least reactogenic. • CARPA can be screened for in vitro, by measuring C activation in serum, and in vivo, by injecting liposomes iv. in animals. • The sensitivity of different species to liposomal CARPA decreases in the following order: pig> dog> rabbit> sheep > rat> mouse. • Pigs are extremely sensitive to liposome reactions and can be used therefore to model the reaction in hypersensitive humans. • Both in pigs and dogs the cardiopulmonary changes can decrease or entirely disappear after the second or third dosing, a reflection of tachyphylaxis (tolerance induction). • The latter phenomenon allows the development of desensitization protocols using empty (placebo) liposomes.

  19. Significance of CARPA • Rare, but serious –occasionally deadly- anaphylactic reactions may surface only in phase III-IV postmarket surveillance; • can be fatal (in cardiac patients) • cannot be predicted by standard allergy tests • may lead to drug withdrawal • May contribute to immunogenicity • change pharmacokinetics, compromise efficacy • cause toxicity, including HSRs • Regulatory authorities increasingly demand experimental verification of short- and longterm immune tolerance.

  20. Why CARPA tests are recommended? • Regulatory authorities increasingly demand experimental verification of short- and longterm immune tolerance. „It is essential to adopt an appropriate strategy for the development of adequate screening and confirmatory assays to measure an immune response against a therapeutic protein.” „All new human pharmaceuticals should be evaluated for the potential to produce immunotoxicity.” „Methods include standard toxicity studies and additional immunotoxicity studies conducted as appropriate…”

  21. Drug Withdrawals *Source: US FDA http://www.fda.gov/CDER/drug/advisory/technetium99.htm 2005 - „…Palatin Technologies, the manufacturer of NeutroSpec (Technetium (99m Tc) fanolesomab) is voluntarily suspending marketing of NeutroSpec effective immediately due to serious safety concerns” „… FDA received reports from PalatinTechnologies of 2 deaths and 15 additional life-threatening adverse events in patients receiving NeutroSpec.”*

  22. Manifestations of porcine CARPA • Hemodynamic alterations • rise of PAP • rise or decline of SAP • declince of CO and pCO2 • Cardiac abnormalities • tachycardia, bradycardia, arrhythmias • ventricular fibrillation, arrest • Skin reaction • erythema, • rash • Blood abnormalities • Leukocytosis • leukopenia • thrombocytosis • thrombopenia ~1/100 of human total dose

  23. 2002 - US FDAGUIDANCE FOR INDUSTRY:IMMUNOTOXICOLOGY EVALUATION OFINVESTIGATIONAL NEW DRUGS „Immunotoxicology is a rapidly advancing field ... New endpoints are needed for such adverse effects as systemic hypersensitivity, autoimmunity, immunogenicity and photoallergy.” Source: US FDA www.fda.gov

  24. Ambisome reaction in pigs

  25. The reactogenicity of Doxil in pigs is tachyphylactic 0.5 mg/kg Zymosan 0.01 mg/kg Doxil 0.01 mg/kg Doxil 0 27 67 Minutes after start of injections

  26. Prediction of CARPA • Prediction of the immune reactivity of the drug • Incubation in large number of human sera with the drug and measurement of C activation in vitro • SC5b-9, C3a, C5a, CH50 (poster by Z. Rozsnyay) • Testing of physiologic changes in sensitive animal models • Pigs , dogs, rats (poster by R. Urbanics) • Prediction of individual hypersensitivity to a certain drug • Measurement in individual sera of • C activation by the drug in vitro (poster by Z. Rozsnyay) • Factor H levels (BioSystems International plasma scan technology) • ADA levels in blood (poster by G. Kozma) • Sensitivity to zymosan activation (poster by G. Kozma)

  27. Treatment of CARPA • Current methods • Empirical • slow infusion, break or termination of infusion • Parmacological • steroids, NSAIDs, antihistamines, • Emergency measures • CPR, epinephrine, oxygen, fluids • Prediction of individual hypersensitivity to a certain drug • Measurement in individual blood/sera of • C activation by the drug in vitro • Factor H levels • Sensitivity to zymosan activation

  28. Prevention of CARPA via desensitization • Theoretical basis • tachyphylactic nature of HSRs • Weak, subclinical reactions also can lead to tachyphylaxis • Realization • Slow infusion of low dose of placebo (empty) liposomes

  29. Slow infusion of empty (placebo) Doxil leads to tachyphylactic response to subsequent Doxil injections B C A D E Passive TOLERANCE PAP (mm Hg, % of baseline) 0 0 0 0 5 5 5 10 10 10 15 5 10 15 0 5 10 15 Doxebo (0,002) 1. Doxil (0,005) 3. Doxil (0,1) 2. Doxil (0,05) Zymosan (0,5) Minutes

  30. Limitations of the CARPA hypothesis • C activation does not fully account for HSR • The correlation betwen C activation and clinical HS reactions is not absolute • The rate of C activation is higher than that of HSRs • There are clinical reactions without measurable C activation • C-independent, direct activation of cells of innate immunity (e.g., macrophages), may also explain HSRs

  31. Need for more models • To understand the variability of human reaction (< 10%) • Pigs, dogs 100 % react • Rats react to 100-1000X higher dose • Mice do not react • To understand the lack of absolute correlation with C activation • C activation was present in more patients than reactors • is not rate limiting • It may be a necessary but not sufficient precondition

  32. Immunogenicity of PEG-L:The accelerated blood clearance (ABC) phenomenon • Dams, et al, (2000). Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes. J Pharmacol Exp Ther 292, pp. 1071-9. • Ishida, et al, 2004-2009, J Control Release95, 105, 112, 115, Int J Pharm. 255, pp. 167-74, Pharm Res 10, pp. 2270-9

  33. Mechanism of ABC

  34. Evidence of immunosuppression by Doxil • Doxil in mice interferes with the clearance of bacteria from blood. Storm, et al.,1998, Clin Cancer Res 4, 111 • Escalation of Doxil dose (from 2.5 to 20 mg/kg)saturates clearance in mice. with disproportional accumulation of doxorubicin in tumorGabizon et al., 2002, J. Drug Targeting 10,539 • Prolongation of the circulation T1/2 of Doxil at repeated administrations in manGabizon, et al., 2007, Cancer Chemother. Pharmacol. 61, 695 • Inhibition of HSRs to carboplatin by co-administered Doxil Alberts, et al., 2008, Gynecol. Oncol. 108, 90

  35. CARPA assays in vitro • Complement activation assays in vitro • C3a, SC5b-9, C4d, Bb ELISAs, • CH50 • Complement activation-related reaction tests ex vivo • ELISA assays for SC5/9, histamine, TXB2, • CH50 • Basophil leukocyte activation in whole blood • FACS analysis of CD203c upregulation

  36. Pharmacological Prevention of CARPA • Commonly applied • anti-inflammatory agents • Steroids • NSAID • Ibuprofen • acetaminophenol • Antihistamines • H1,H2 • Potential • IVIG • C1INH • Anti C5 mAb (Soliris, Alexion) • Macrophage / RES / mast cell blockers

  37. Complement activationby Doxil in cancerpatientsin vivo 10 min after start of infusion • Tumor: gynaecological and other solid (head/neck, pancreas, esophagus, thyroid, neuroendocrine, melanoma) carcinomas • Treatment: 40-100 mg Doxil in infusion, no antiallergic premedication • Conclusions: • Doxil activates C in • 50-85% of patients. • C activation is more expressed and frequent in the reactor group. • C activation does not necessarily lead to clinical reaction Ann Oncol. 2003 Sep;14(9):1430-7.

  38. Complement activation in vitro by reactogenic drugs

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