European Core Consultancy and Global Advisory Meetings

European Core Consultancy and Global Advisory Meetings

Toxicidad de Efavirenz y Lopinavir/r sobre los adipocitos Pere Domingo Unidad de Enfermedades Enfermedades Infecciosas Infecciosas Hospital de la Santa creu creu ii Sant Sant Pau Pau Universitat Universitat Autnoma Autnoma de de Barcelona Barcelona Barcelona [email protected] Contenido Implicaciones,consecuencias y visibilidad de la lipoatrofia Frmacos y lipoatrofia (EFV vs. LPV/r) Mecanismos patognicos en la lipoatrofia El papel de la mitocondria EFV, LPV/r y adipocitos in vitro EFV, LPV/r y adipocitos ex vivo Implicaciones de la lipoatrofia Calidad de vida Efectos psicolgicos Baja autoestima Disminucin de la autoconfianza Ansiedad y depresin Efectos sociales Dao en la calidad de la relaciones sociales Alienacin social Dificultad para encontrar ropa adecuada Efectos fsicos (ej, dolor al sentarse) Disfuncin sexual

Descenso de la adherencia 3 ODonovan CA et al, 7th IWADRL Nov 2005, Dublin, #34; James J et al, Dermatol Surg 2002; 28:979-986 Santos CP et al, AIDS 2005;19(S4):S14-S21; Reynolds NR et al, AIDS Care Oct 2006; 18(7):663-73 La lipoatrofia se hace clnicamente evidente despus de perder el 40%-50% de la grasa en la extremides Grasa en las extremidades (kg) Estudios en VIH, Controles sanos voluntarios* 10 9 8 7 6 5 4 3 2 1 0 9.11 7.11 Adultos VIH+, Lipoatrofia clinicamente evidente al comienzo* 7.22 3 3 Mujer Hombre Hombre 3.1 4

3.55 3.66 2.77 2.68 Todos los estudios, principalmente hombres *Todos los estudios utilizaron DEXA para medir la grasa. 1. Engelson ES et al. Am J Clin Nutr. 1999;69:11621169; 2. Carr A et al. AIDS. 1998;12:F51F58; 3. Moyle G et al. CROI 2005. Abstract 44LB; 4. McComsey GA et al, Clin Infect Dis. 2004;38:263270; 5. Martin A et al. AIDS. 2004;18:10291036; 6. Milinkovic A et al. Antivir Ther. 2007;12:407415; 7. Carr A et al. Lancet. 2004;363:429438; 8. Slama L. Antivir Ther. 2007, in press. 4 Importancia del diagnstico temprano: La lipoatrofia es difcil de revertir Mnima ganancia en la grasa en las extremidades 48 semanas despus del cambio de los NRTI 9 8.1 Limb fat (kg) 8 7 6 5 4 2.95 3 3.41 2 1 0 Normal 1

Pacientes lipoatrficos Pacientes tras el 2 al comienzo cambio de anlogos 2 Otros estudios dieron resultados similares 5 1. Engelson ES et al. Am J Clin Nutr. 1999;69:11621169; 2. Moyle GJ et al. AIDS. 2006;20:20432050; 3. Carr A et al. AIDS. 1998;12:F51F58. Cambio % de la mediana desde la basal ACTG 5142: Efecto diferente de LPV/r y EFV sobre la grasa perifrica: LPV/r < EFV ACTG 5142 Grasa en las extremidades 20 +18 15 +9.8 10 5 +1.4 .013 <.001 .007 0 -5 -10 -15 EFV LPV/r

LPV/r + EFV 0 EFV LPV/r LPV/r + EFV 48 188 191 197 Haubrich R et al. 14th CROI 2007; Los Angeles. Abstract #38. 6 P Value 96 171 166 173 Weeks ACTG 5142: La prevalencia de lipoatrofia fue menor con Kaletra que con EFV % de pacientes con lipoatrrofia (>20% prdida de grasa perifrica) Evolucin de la lipoatrofia (> 20% de prdida de grasa en las extremidades) EFV+2 ITIAN 50% 40% LPV/r+EFV Valores de P a 96s LPV/EFV vs LPV: 0,023 LPV/EFV vs EFV: <0,001 LPV vs EFV: 0,003 32% 30%

21% 20% 10% 10% 17% 9% 7% 0% 0% 0% Basal EFV LPV/r LPV/r + EFV 7 LPV/r +2 ITIAN 48 semanas 188 191 197 96 semanas 171 166 173 Haubrich R et al. AIDS. 2009 Jun 1;23(9):1109-18 . ACTG 5142. La prevalencia de lipoatrofia fue menor con LPV/r que con EFV independientemente de los anlogos utilizados Porcentaje de pacientes con lipoatrofia por subgrupos, semana 96 60% 50% 40% 51% n=4 1 40%

n=63 33% n=43 EFV Kaletra 30% 16% n=73 20% 10% 12% n=67 6% n=50 0% d4T 8 AZT TDF Haubrich R et al. AIDS. 2009 Jun 1;23(9):1109-18 . ACTG 5142. La prevalencia de lipoatrofia fue menor con LPV/r que con EFV independientemente de la definicin de lipoatrofia elegida EFV Kaletra % pacientes 50 40 30 40%

28% 20 32% 17% 20% 10% 10 13% 4% 0 >10% p=0,0027 9 >20% >30% >40% p=0,0004 p=0,0016 p=0,0008 Haubrich R et al. AIDS. 2009 Jun 1;23(9):1109-18 . Estudio transversal alemn. Resultados: Caractersticas de los pacientes LPV/r EFV N de pacientes Edad en aos

Duracin media del tratamiento [aos] 139 44.7 3.4 165 45.6 3.6 Adeherencia autoreferedia [%] > 95 > 95 El 45% estabn tomando TDF, 28% AZT/3TC y 12% ABC/3TC Alta concordancia entre las opiniones de los pacientes y de los mdicos 10 Van Lunzen J et al. IAS Ciudad del Cabo Jul 2009 #CDB111 Estudio transversal alemn. El porcentaje de pacientes que reportan haber prdido grasa en las piernas fue menor con Kaletra que con EFV % pacientes que reportan prdida de gras en las piernas 50 p < 0,05 40 32 30 21 20 10 0 Kaletra EFV

El 79 % de los pacientes tratados con Kaletra no report signos de prdida de grasa en las piernas frente a slo 68% entre los tratados con EFV. 11 Van Lunzen J et al. IAS Ciudad del Cabo Jul 2009 #CDB111 Estudio transversal alemn. Entre los tratados con TDF/3TC o FTC tambin hubo menos pacientes que notaron prdida de grasa en las piernas en el grupo de Kaletra que en el de EFV % pacientes que reportan prdida de gras en las piernas 50 p < 0,05 40 30 22 20 10 10 0 Kaletra 13 EFV Van Lunzen J et al. IAS Ciudad del Cabo Jul 2009 #CDB111 Causas de la lipoatrofia Virus VIH Paciente Gentica/Ambiente HAART Lipoatrofia 14

Mecanismos fisiopatolgicos de la lipodistrofia a nivel celular Disfuncin mitocondrial Control1 Patient Aumento del nmero de adipocitos pequeos1 X 400 X 200 Proliferacin mitocondrial y morfologa alterada2 Ciclo celular y diferenciacin adipocitaria alterada Descenso del tamao de los adipocitos1-3 Apoptosis aumentada4 Sustitucin de los adipocitos por tejido fibrosos3 Mediadores proinflamatorios Patient Incremento del nmero de macrfagos3 Infiltracin de macrfagos2,3 Expresin de IL-6 y TNF - aumentada3 X 400 1. 2. 3. 4. 15

Bastard JP, et al. Lancet 2002: 359: 1028-31 Nolan D, et al. AIDS 2003; 17: 121-3 Jan V, et al. Antivir Ther 2004; 9: 555-64 Domingo P, et al. AIDS 1999; 13: 2261-7 Investigacin para esclarecer el papel de Kaletra en la lipoatrofia Mitocondria Adipocito T. Adiposo Dr. Esplugues Dr. Domingo Dr. A. Carr Dr. Domingo Individuo Estudio 613 ACTG 5142 Dr. van Lunzen 16 Estructura mitocondrial AND mitocondrial (mtDNA) Mitocondria. Estructura interna Membrana interna 16 kb, 2-10 copias/mitocondria en la matriz La replicacin est controlada por genes mitocondriales La DNA polymerasa- mitocondrial es la enzima requerida para la replicacin del mtDNA La inhibicin de la mtDNA polymerasa- conduce a la deplecin del mtDNA Transcripcin protenas mitocondriales

Polimerasa del RNA (mtRNA) y factores de transcripcin La apoptosis es parte del proceso natural celular y se incrementa durante la disfuncin mitocondrial En el contexto de la disfuncin mitocondrial Se incrementa la apoptosis celular Puede aparecer hiperlactatemia Chan DC, Cell June 2006; 125;7:1241-1252 McBride HM, Current Biology July 2006;16:R551-R560 17 HIV Matriz Cristae Membrana externa Adipocito TEM Toxicida inducida por los anlogos: Inhibicin de la mtDNA Polymerasa- Los ensayos enzimticos con NRTIs demuestran la inhibicin de la mtDNA polymerasa- 1,2: zalcitabina (ddC) > didanosina (ddI) > stavudina > zidovudina (ZDV) > lamivudina = abacavir = tenofovir Resultados Metodos2 Clulas humanas tratadas con NRTIs Southern blot para cuantificar mtDNA El grado de inhibicin de la DNA polymerase- inhibition varia con los NRTI Algunos NRTIs inhiben la mtDNA polymerasa- 1. Kakuda TN. Clin Ther. 2000;22(6):685708; 2. Birkus G et al. Antimicrob Agents Chemother. 2002;46:716723. 18

Disfuncin mitocondrial y apoptosis Deplecin DNAmt, ... Disfuncin cadena respiratoria Reactive oxygen species MPTP 19 (M) Concentracin de oxgeno Oxygen concentration Inhibicin aguda de la respiracin mitocondrial por EFV 100 Control EFV 25uM 50 0 6 8 10 12 14 16 Tiempo

(min) Time (min) 100 120,00 Efects of EFV (M) on respiration rate *** ** 80 *** % *** 40 (M)) Data not shown 80,00 60,00 40,00 20,00 20 0 21 ** P< 0.01 *** P< 0.001 *** 60 20 100,00

% % of control % of control 100 18 5 10 15 25 50 Blas-Garca A, et al. 15th CROI. Boston, 2008 #984 100 M 0,00 ATV 50 RTV 25 LPV 16 MetOH M La inhibicin de la funcin mitocondrial resulta en una reduccin dosis dependiente del ATP intracelular Fig.3 Intracellular ATP levels 16

ATP(nmol/mg protein) 14 12 * 10 8 6 4 ** 2 0 Control 22 EFV 10 Blas-Garca A, et al. 15th CROI. Boston, 2008 #984 EFV 25 EFV 50 M Adipocyte differentiation: a highly regulated process Adipoblast Stem cell mitosis Preadipocyte mitosis Immature adipocyte

Organelle reconstitution Mature adipocyte TG storage E2F, pRb p130/p107 TNF- TNF- PPAR C/EBP/ SREBP1/ADD1 PPAR C/EBP FA ligand activated transcription factors Gregoire FM, Physiological Reviews July 1998; 78(3):783-809 23 Lipogenic gene induction LPL, HSL, aP2, perilipin DGAT, GLUT4 Mechanisms of NRTI-Induced Mitochondrial Toxicity PPAR- gene expression relative to ACTB Adipocyte differentiation factors (nuclear transcription factor PPAR-) Transcription of mitochondrial chain respiratory subunits (COX-1, Cox-2, and Cyt b) Negative regulatory effects impede adipogenesis and respiratory chain complexes (eg. Complex IV via COX1 inhibition) NRTI effects on peripheral

mononuclear blood cells suggest a possible role in other tissues 0.06 PPAR- : ACTB NRTI therapy for 6 weeks (ZDV/3TC or d4T/3TC ) In the absence of mtDNA depletion, thymidine analogue NRTIs downregulate: 0.05 0.04 P = 0.003 0.03 0.02 0.01 0.0 Baseline Week 2 COX-1 gene expression relative to ACTB housekeeping gene at 6 weeks of ZDV/3TC or d4T/3TC twice-daily in 1.8 monocytes COX1 : ACTB 20 HIV- volunteers received dual housekeeping gene at baseline and at 2 weeks of ZDV/3TC or d4T/3TC twicedaily in adipocytes 1.6 1.4 1.2 1 0.8 0.6

P = 0.01 P = 0.005 0.4 0.2 0 Baseline Week 6 Week 12 * Median = 66 weeks Mallon PW et al. J Infect Dis. 2005;191:16861696. 24 Follow up* Effect of EFV and LPV/ r on differentiating adipocytes Effect of EFV and LPV/r on mature adipocytes Control (No drug) Control (No drug) EFAVIRENZ 0.5 MM EFAVIRENZ 2 MM 4 MM 10 MM 2 MM

4 MM 20 MM Massive cell death - no image available KALETRA 0.5 MM 25 KALETRA 2 MM 4 MM 10 MM Diaz-Delfn J, et al. 10th IWADRL. London, 2008 #P-01 2 MM 4 MM 20 MM Effect of EFV and LPV/r on gene expression Log mtDNA copy number Adipogenesis (PPAR) 1.2 # 1 0.8 # * * 0.6

* * * 0.4 0.2 0 Control 0.5 * * * 2 4 EFV (Mmol/l) 10 0.5 4 20 LPV/r (Mmol/l) * 40 *P<0.05, **P<0.01, ***P< 0.001 vs. control, # EFV vs. LPV/r Mitochondrial DNA Log mtDNA copy number 1.4 26 1.2 1

0.8 0.6 0.4 0.2 0 Control 0.5 2 4 EFV (Mmol/l) Diaz-Delfn J, et al. 10th IWADRL. London, 2008 #P-01 10 0.5 2 4 LPV/r (Mmol/l) 10 EFV increases pro-inflammatory cytokines and reduces adiponectin c/w LPV/r in human adipose cells EFV: 4 M - increase in levels of MCP-1 (5.3-fold), IL-5 (4.2-fold), IL-8 (18.7-fold), PAI-1 (5.7-fold) and HGF (5.5-fold) Significant reduction of adiponectin and leptin (17% and 19% of levels vs controls respectively) LPV/r: No induction of MCP-1, IL-8 and PAI-I Minor but significant reduction in release of adiponectin and leptin (71% and 76% of levels vs controls, respectively) HGF and IL-6 were induced to a similar extent in response to LPV/r and to EFV mRNA: Adiponectin mRNA significantly decreased with [EFV] 2 M and [LPV/r] 4 M (EFV inhibition of mRNA significantly worse). Leptin mRNA significantly decreased with [EFV] 2 M and [LPV/r] 10 M Conclusions:

In human adipocytes in culture, EFV causes a higher release of pro-inflammatory cytokines and a more profound impairment in the release of adiponectin compared to LPV/r. 27 Domingo P et al, 5th IAS 2009, #TUPEB167 Efectos ex vivo de los frmacos antiretrovirales Adiponectina P = NS 140 P = NS 120 120 100 100 80 80 60 60 40 40 20 20 0 0 Naive

TRU+EFV Naive MCP-1 30 P = NS 25 -20 TRU-KAL 40 35 30 20 25 15 20 15 10 10 5 5 0 28 0 Naive TRU+EFV Naive TRU+KAL

Efectos ex vivo de los frmacos antiretrovirales PPAR 8 9 P = NS 7 8 P = NS 7 6 6 5 5 4 4 3 3 2 1 2 0 1 -1 0 -2 Naive

Naive TRU+EFV TNF 12 P = NS 10 TRU+KAL P = NS 5 4,5 4 8 3,5 6 3 2,5 4 2 2 1,5 1 0 ,5 -2 0 Naive

29 TRU+EFV Naive TRU+KAL Efectos ex vivo de los frmacos antiretrovirales 40000 Cyt B P P == 0,04 0,04 35000 35000 P P == 0,008 0,008 30000 30000 25000 25000 20000 20000 15000 15000 10000 10000 5000

5000 0 0 Naive TRU+EFV 3000 ADNmt Naive TRU+KAL 4500 2800 4000 2600 3500 2400 3000 2200 2000 2500 1800 2000 1600 1500 1400

1000 1200 Naive 30 TRU+EFV 500 Naive TRU+KAL HIV Infection Contributes to Lipodystrophy Through Alteration of Gene Expression in Adipose Tissue Compared to HIV-negative controls, ARV-nave HIV-positive patients had significant Decreases in mRNA of COX-2, COX-4, UCP2, C/EBP-, PPAR-, GLUT4, LPL, leptin, and adiponectin Increases in PGC-1, TNF, and -2 microglobulin PPAR- mRNA/18S rRNA (x10-4) TNF- mRNA/18S rRNA (x10-6) COX-2 mRNA/18S rRNA (x10-2) 1.5 1.0 P=0.0002 P<0.0001 P=0.001 P=0.001 P<0.0001 2 1

0 0 Control Nave Non-LD LD Giralt M et al. Antivir Ther. 2006;11:729740. 31 P=0.051 P=0.0004 3 P=0.046 P=0.018 0.5 4 P=0.023 P=0.045 P=0.0001 5 P=0.027 Control Nave Non-LD LD Control Nave Non-LD LD

Cambio % de la mediana desde la basal ACTG 5142: Efecto diferente de LPV/r y EFV sobre la grasa perifrica: LPV/r < EFV ACTG 5142 Grasa en las extremidades 20 +18 15 +9.8 10 5 +1.4 .013 <.001 .007 0 -5 -10 -15 EFV LPV/r LPV/r + EFV 0 EFV LPV/r LPV/r + EFV 48 188 191 197 Haubrich R et al. 14th CROI 2007; Los Angeles. Abstract #38.

32 P Value 96 171 166 173 Weeks Grasa subcutnea La toxicidad grasa es bifsica TARc Tiempo Tiempo 33 Conclusiones Tres factores implicados en la LD Virus Husped Frmacos La patogenia de la lipodistrofia es multifactorial Mitocondria Diferenciacin Inflamacin EFV es ms txico que LPV/r in vitro. A 48 semanas dicha toxicidad no es apreciable ex vivo. El curso de la LD es bifsico 34 Agradecimientos Hospital de la Santa Creu i Sant Pau M del Mar Gutierrez M Gracia Mateo

M Antonia Sambeat Angels Fontanet Jessica Muoz Jessica Martnez 35 Biologia U.B. Francesc Villarroya Marta Giralt Joan C. Domingo Julieta Daz

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