Toxicidad de Efavirenz y Lopinavir/r sobre los adipocitos Pere Domingo Unidad de Enfermedades Infecciosas Hospital de la Santa creu i Sant Pau Universitat Autònoma de Barcelona Barcelona pdomingo@santpau.cat

Implicaciones,consecuencias y visibilidad de la lipoatrofia Contenido Implicaciones,consecuencias y visibilidad de la lipoatrofia Fármacos y lipoatrofia (EFV vs. LPV/r) Mecanismos patogénicos 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 psicológicos Baja autoestima Disminución de la autoconfianza Ansiedad y depresión Efectos sociales Daño en la calidad de la relaciones sociales Alienación social Dificultad para encontrar ropa adecuada Efectos físicos (ej, dolor al sentarse) Disfunción sexual Descenso de la adherencia Lipoatrophy, a potential side effect of HIV infection and HIV therapy, leads to other psychological, social, and physical consequences. These consequences are outlined in the proceeding slides. 3 O’Donovan 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 3

La lipoatrofia se hace clínicamente evidente después de perder el 40%-50% de la grasa en la extremides Estudios en VIH, Controles sanos voluntarios* Adultos VIH+, Lipoatrofia clinicamente evidente al comienzo* 10 9.11 9 8 7.11 7.22 7 6 Grasa en las extremidades (kg) 5 4 3.55 3.66 33 3.14 2.77 2.68 3 A review of the body fat composition of normal controls in HIV trials reveals that women have about 9 kgs of limb fat, vs. 7 kgs for men. In trials of HIV positive adults (of which 83% and 98% of the study subjects were men) presenting with clinically evident lipoatrophy, subjects had between 2.7 and 4.4 kgs of limb fat at inclusion. This suggests that 40%-50% fat loss is necessary for lipoatrophy to be clinically apparent. 2 1 Mujer Hombre Hombre Todos los estudios, principalmente hombres *Todos los estudios utilizaron DEXA para medir la grasa. 1. Engelson ES et al. Am J Clin Nutr. 1999;69:1162–1169; 2. Carr A et al. AIDS. 1998;12:F51–F58; 3. Moyle G et al. CROI 2005. Abstract 44LB; 4. McComsey GA et al, Clin Infect Dis. 2004;38:263–270; 5. Martin A et al. AIDS. 2004;18:1029–1036; 6. Milinkovic A et al. Antivir Ther. 2007;12:407–415; 7. Carr A et al. Lancet. 2004;363:429–438; 8. Slama L. Antivir Ther. 2007, in press. 4

Importancia del diagnóstico temprano: La lipoatrofia es difícil de revertir Mínima ganancia en la grasa en las extremidades 48 semanas después del cambio de los NRTI 9 8.1 8 7 6 Limb fat (kg) 5 3.41 4 2.95 3 2 The incentive for early diagnosis and possible prevention of developing lipoatrophy lies in the difficulty of reversing this condition once established. The RAVE study was a randomized, open-label, comparative study of switching from a thymidine nucleoside analogue to either tenofovir disoproxil fumarate (DF) or abacavir in 105 individuals on successful antiretroviral therapy with clinically evident moderate to severe lipoatrophy. Body and limb fat were measured by DEXA at baseline to 48 weeks. The results of this study were published by Moyle and colleagues, and showed that switching HIV-infected patients with lipoatrophy from an NRTI-containing (ZDV or d4T) regimen to abacavir or tenofovir DF (TDF) did significantly increase limb fat volume over 48 weeks, but not rapidly or to the normal values observed in uninfected controls. 1 Normal 1 Pacientes lipoatróficos al comienzo 2 Pacientes tras el cambio de análogos 2 Otros estudios dieron resultados similares 5 1. Engelson ES et al. Am J Clin Nutr. 1999;69:1162–1169; 2. Moyle GJ et al. AIDS. 2006;20:2043–2050; 3. Carr A et al. AIDS. 1998;12:F51–F58.

ACTG 5142: Efecto diferente de LPV/r y EFV sobre la grasa periférica: LPV/r < EFV ACTG 5142 Grasa en las extremidades P Value +18 20 15 .013 +9.8 10 <.001 5 .007 Cambio % de la mediana desde la basal +1.4 -5 EFV LPV/r -10 LPV/r + EFV -15 Weeks 48 96 EFV 188 171 LPV/r 191 166 LPV/r + EFV 197 173 Haubrich R et al. 14th CROI 2007; Los Angeles. Abstract #38.

ACTG 5142: La prevalencia de lipoatrofia fue menor con Kaletra que con EFV Evolución de la lipoatrofia (> 20% de pérdida de grasa en las extremidades) Valores de P a 96s LPV/EFV vs LPV: 0,023 LPV/EFV vs EFV: <0,001 LPV vs EFV: 0,003 EFV 188 171 LPV/r 191 166 LPV/r + EFV 197 173 Haubrich R et al. AIDS. 2009 Jun 1;23(9):1109-18 .

Porcentaje de pacientes con lipoatrofia por subgrupos, semana 96 ACTG 5142. La prevalencia de lipoatrofia fue menor con LPV/r que con EFV independientemente de los análogos utilizados Porcentaje de pacientes con lipoatrofia por subgrupos, semana 96 51% n=41 40% n=63 33% n=43 16% n=73 12%n=67 6% n=50 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 definición de lipoatrofia elegida 40% 32% 28% % pacientes 20% 17% 13% 10% 4% p=0,0027 p=0,0004 p=0,0016 p=0,0008 Haubrich R et al. AIDS. 2009 Jun 1;23(9):1109-18 .

Estudio transversal alemán Estudio transversal alemán. Resultados: Características de los pacientes LPV/r EFV Nº de pacientes 139 165 Edad en años 44.7 45.6 Duración media del tratamiento [años] 3.4 3.6 Adeherencia autoreferedia [%] > 95 El 45% estabán tomando TDF, 28% AZT/3TC y 12% ABC/3TC Alta concordancia entre las opiniones de los pacientes y de los médicos Van Lunzen J et al. IAS Ciudad del Cabo Jul 2009 #CDB111

% pacientes que reportan pérdida de gras en las piernas Estudio transversal alemán. El porcentaje de pacientes que reportan haber pérdido grasa en las piernas fue menor con Kaletra que con EFV p < 0,05 % pacientes que reportan pérdida de gras en las piernas El 79 % de los pacientes tratados con Kaletra no reportó signos de pérdida de grasa en las piernas frente a sólo 68% entre los tratados con EFV. Van Lunzen J et al. IAS Ciudad del Cabo Jul 2009 #CDB111

% pacientes que reportan pérdida de gras en las piernas Estudio transversal alemán. Este resultado se repitió en los tratados con AZT/3TC p < 0,05 % pacientes que reportan pérdida de gras en las piernas Van Lunzen J et al. IAS Ciudad del Cabo Jul 2009 #CDB111

% pacientes que reportan pérdida de gras en las piernas Estudio transversal alemán. Entre los tratados con TDF/3TC o FTC también hubo menos pacientes que notaron pérdida de grasa en las piernas en el grupo de Kaletra que en el de EFV p < 0,05 % pacientes que reportan pérdida de gras en las piernas Van Lunzen J et al. IAS Ciudad del Cabo Jul 2009 #CDB111

Causas de la lipoatrofia Virus VIH Paciente Genética/Ambiente HAART This slide highlights the major factors influencing the development of HIV-associated lipoatrophy: host, virus and therapy. Lipoatrofia 14 14

Mecanismos fisiopatológicos de la lipodistrofia a nivel celular X 400 Control1 X 200 Patient Aumento del número de adipocitos pequeños1 Disfunción mitocondrial Proliferación mitocondrial y morfología alterada2 Ciclo celular y diferenciación adipocitaria alterada Descenso del tamaño de los adipocitos1-3 Apoptosis aumentada4 Sustitución de los adipocitos por tejido fibrosos3 Mediadores proinflamatorios Infiltración de macrófagos2,3 Expresión de IL-6 y TNF - aumentada3 Patient X 400 Incremento del número de macrófagos3 On a cellular level lipoatrophy results from deleterious effects on adipose tissue. This micrographs show hemalyotoxin and eosin (H&E) staining of adipocytes. The cells (adipocytes and macrophages), the mitochondria, and inflamatory mediators underlying lipoatrophy are highlighted. 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

Investigación para esclarecer el papel de Kaletra en la lipoatrofia Mitocondria Adipocito T. Adiposo Individuo Dr. Esplugues Dr. Domingo Dr. A. Carr Dr. Domingo Estudio 613 ACTG 5142 Dr. van Lunzen

Estructura mitocondrial AND mitocondrial (mtDNA) 16 kb, 2-10 copias/mitocondria en la matriz La replicación está controlada por genes mitocondriales La DNA polymerasa-γ mitocondrial es la enzima requerida para la replicación del mtDNA La inhibición de la mtDNA polymerasa-γ conduce a la depleción del mtDNA Transcripción – proteínas mitocondriales Polimerasa del RNA (mtRNA) y factores de transcripción La apoptosis es parte del proceso natural celular y se incrementa durante la disfunción mitocondrial En el contexto de la disfunción mitocondrial Se incrementa la apoptosis celular Puede aparecer hiperlactatemia Mitocondria. Estructura interna HIV Membrana interna Membrana externa Cristae Matriz Adipocito TEM Overview of the mitochondria structure and its primary cellular functions. Mitochondria serve to make ATP, however other mitochondrial functions include: Inducing apoptosis (a normal cellular process). Uncoupling proteins that can uncouple the membrane potential and generate heat. Fe and Ca binding and storage. It should be noted that an increase in the measurement of lactate is the hallmark of mitochondrial complications. The spectrum of disease ranges from mild to moderate asymptomatic (or sub-clinical) hyperlactatemia to fulminate and life threatening lactic acidosis. Responsible for 90% of the reactive oxygen species leading to oxidative stress 17 Chan DC, Cell June 2006; 125;7:1241-1252 McBride HM, Current Biology July 2006;16:R551-R560 17

Toxicida inducida por los análogos: Inhibición de la mtDNA Polymerasa-γ Los ensayos enzimáticos con NRTIs demuestran la inhibición de la mtDNA polymerasa- 1,2: zalcitabina (ddC) > didanosina (ddI) > stavudina > zidovudina (ZDV) > lamivudina = abacavir = tenofovir Metodos2 Células humanas tratadas con NRTIs Southern blot para cuantificar mtDNA Resultados El grado de inhibición de la DNA polymerase- inhibition varia con los NRTI This slide shows the main results of two in vitro experiments, which test NRTI on cultured cells. The Birkus study (Birkus G, et al. Antimicrob Agents Chemother. 2002;46:716-723.) was performed in HepG2 human hepatoblastoma cells (and also on SkMC skeletal muscle cells), for only 9 days (not long enough biologically for mitochondrial to turnover) with substantially high doses (upto 100x more than the physiological dose in plasma). Human hepatoblastoma cells (HepG2) incubated in the presence of each agent for 9 days. Relative mtDNA content was determined as the ratio of hybridization signals from mtDNA- and chromosomal DNA-specific probes. Additionally, these NRTIs have different toxicity profiles, which include: skeletal myopathy; lactic acidosis; hepatic steatosis; cardiomyopathy; peripheral neuropathy; pancreatitis; CNS degenerative disease; and rhabdomyolysis. Walker performed a study similar to Birkus, but in transformed cells for 25 days. Zalcitabine (ddC) Didanosine (ddI) Stavudine (d4T) Zidovudine (ZDV) Lamivudine (3TC) Abacavir (ABC) Tenofavir (TDF) Algunos NRTIs inhiben la mtDNA polymerasa- 18 1. Kakuda TN. Clin Ther. 2000;22(6):685–708; 2. Birkus G et al. Antimicrob Agents Chemother. 2002;46:716–723. 18

Disfunción mitocondrial y apoptosis Depleción DNAmt, ... Disfunción cadena respiratoria Y D De forma tentativa, la deplección de ADNmt inducida por los ITIAN puede provocar una disfunción de la CRM con la generación de productos intemediarios del metabolismo del oxígeno como son los radicales libres de oxígeno que entre otras acciones tóxicas provoca la apertura del poro de la transición en la permeabilidad mitocondrial, permitiendo el escape de proteínas mitocondriales con capacidad para iniciar la secuencia apoptótica. Adicionalmente, la disfunción de la CRM y la generación de radicales libres de oxígeno puede en última instancia provocar el colapso del potencial de membrana interna mitocondrial lo que conduce a permeabilización por ruptura mecánica de la membrana mitocondrial externa Reactive oxygen species MPTP

Inhibidores metabólicos Disfunción mitocondrial Consecuencias potenciales de la disfunción mitocondrial: Disfunciones celulares: - DNA polymerase-g1-3 - Desacopladoras1-3 - Transportadoras3 - Stress Oxidativo1-4 - Apoptosis2,3 - Fosforilación2,3 - Procesamiento proteolítico3 Glicosilación3 Complicaciones agudas: Neuropatia1-3 Esteatosis hepática1-3 Miopatía1-3 Pancreatitis1-3 Acidosis Láctica1-3 Complicaciones a largo plazo: Lipodystrophy1-4 Hypoxia Privación de glucosa Inhibidores metabólicos Glucose Fatty acids LDH Lactate Pyruvate NAD+ NADH Malonyl CoA CPT-1 PDH -oxidation ADP/ATP AK translocator ATP NAD+ NADH Acetyl CoA AAC ATP IV Cito c NADH Electron transport chain III Krebs cycle CoQ -KDH NAD+ II NAD+ NADH I NADH Mitochondrion matrix H+ Mitochondria are considered the engine of the cells. The primary role of the mitochondrion is to provide energy to the cell in the form of ATP. Gluocose is the main row material for energy production but mitochondria can also use fatty acid and even amino acids. AAC: acetyl CoA carboxylase CPT-1: carnitine palmitoyl transferase-1 PDH: pyruvate dehydrogenase CoQ: coenzime Q Cito c: cytochrome c NAD: nicotinamide adenine dinucleotide a-KDH: alpha ketoglutarate dehydrogenase Inner membrane AMPk Inner membrane space Outer membrane Cellular cytosol Stress metabólico Adapted from Kakuda TN. Clin Ther 2000: 22: 685-708; Dobrzyn P, et al. PNAS 2004; 101: 6409-14; Dagan T. Mitochondrion. 2002;1:397–412; 2. Day L et al. Mitochondrion. 2004;4:95–109; 3. Gerschenson M. Mitochondrion. 2004;4(5-6):763–777; 4. McComsey GA. J Acquir Immune Defic Syndr. 2003;34:45–49.

Inhibición aguda de la respiración mitocondrial por EFV Time (min) Oxygen concentration (μM) 100 MetOH ATV RTV LPV 50 25 16 M Data not shown Efects of EFV (M) on respiration rate This slide shows the acute and dose dependent inhibitory effect of EFV on the rate of oxygen consumption following its addition to the gas-tight chamber. Protease inhibitors have no effect on respiration rate. *** ** P< 0.01 *** P< 0.001 ** *** *** *** % of control % of control M 5 10 15 25 50 100 Blas-García A, et al. 15th CROI. Boston, 2008 #984

La inhibición de la función mitocondrial resulta en una reducción dosis dependiente del ATP intracelular Fig.3 Intracellular ATP levels Control EFV 10 25 * ** 50 µM Blas-García A, et al. 15th CROI. Boston, 2008 #984

Adipocyte differentiation: a highly regulated process Immature adipocyte Lipogenic gene induction LPL, HSL, aP2, perilipin DGAT, GLUT4 TG storage FA ligand activated transcription factors Preadipocyte PPARγ C/EBPα Organelle reconstitution Adipoblast mitosis E2F, pRb p130/p107 Mature adipocyte Stem cell mitosis PPARδ C/EBPβ/δ SREBP1/ADD1 TNF-α TNF-α The process of adipocyte differentiation leads from un undifferentiated mesenchimal cell to a highly specilaized cell whose mission is to store energy. This process is finely tuned by multiple trasncription factors, but the master reglatory factor is PPARg not only becuase of its intervention from the very beginning ot the differentitation process but also because it induces the acyivation of multiple genes which are going to induce to promote tg storage within he adipocyte. But in this process, as in many others, there are not only factors gavoring the process but also factors which mayinduce adipocyte dedifferentiation the main of them being TNF alpha. La diferenciación adipocitaria es un proceso finamente controlado por la activación secuencia de diversos factores de transcripción que van a promover el paso desde una celula mesenquimal indiferenciada hasta una célula altamente especializada en la reserva de energía. Gregoire FM, Physiological Reviews July 1998; 78(3):783-809

Mechanisms of NRTI-Induced Mitochondrial Toxicity PPAR- gene expression relative to ACTB housekeeping gene at baseline and at 2 weeks of ZDV/3TC or d4T/3TC twice-daily in adipocytes COX-1 gene expression relative to ACTB housekeeping gene at 6 weeks of ZDV/3TC or d4T/3TC twice-daily in monocytes 20 HIV- volunteers received dual NRTI therapy for 6 weeks (ZDV/3TC or d4T/3TC ) In the absence of mtDNA depletion, thymidine analogue NRTIs down-regulate: 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 0.05 0.04 PPAR-γ : ACTB P = 0.003 0.03 0.02 0.01 0.0 Baseline Week 2 1.8 In this study, Mallon found a decrease in transcription of mitochondrial RNA and oxidative phosphorylation changes, but not a decrease in DNA. Independent of HIV, NRTIs decrease transcription of mtRNA in vivo. The absence of mtDNA depletion suggests that NRTIs may cause mitochondrial dysfunction by means other than through inhibition of DNA polymerase-. Disruption of the lipid metabolism gene expression offers an explanation for NRTI-induced lipoatrophy. 1.6 1.4 1.2 COX1 : ACTB 1 P = 0.005 P = 0.01 0.8 0.6 0.4 0.2 Baseline Week 6 Week 12 Follow up* * Median = 66 weeks 24 Mallon PW et al. J Infect Dis. 2005;191:1686–1696. 24

Effect of EFV and LPV/r on differentiating adipocytes Effect of EFV and LPV/r on mature adipocytes Effect of EFV and LPV/r on differentiating adipocytes Control (No drug) Control (No drug) EFAVIRENZ EFAVIRENZ 0.5 μM 2 μM 4 μM 10 μM 2 μM 4 μM 20 μM Massive cell death - no image available KALETRA Efavirenz does not affect morphological adipocyte differentiation at 0.5 M . It severely impairs morphological adipocyte differentiation at 2 M and at 4 M and causes cell death on human adipogenic cells at 10 M. Kaletra does not affect morphological differentiation at 0.5 M and lead to a progressive dose-dependent impairment of differentiation leading to very low levels of differentiation at 10 M, the highest dose tested The major differences observed in the comparison of Efavirenz and Kaletra effects on differentiation may be summarized as: at 10 M, Efavirenz is toxic for adipogenic cells whereas Kaletra is not. at equivalent concentrations of Efavirenz and Kaletra (2 M and 4 M) Efavirenz causes always a more profound effect impairing morphological adipogenic differentiation than Kaletra. KALETRA 0.5 μM 2 μM 4 μM 10 μM 2 μM 4 μM 20 μM Diaz-Delfín J, et al. 10th IWADRL. London, 2008 #P-01

Effect of EFV and LPV/r on gene expression Adipogenesis (PPARγ) 1.2 # 1 # 0.8 * * Log mtDNA copy number 0.6 * * * 0.4 0.2 Control 0.5 2 4 10 0.5 4 20 40 EFV (μmol/l) LPV/r (μmol/l) *P<0.05, **P<0.01, ***P< 0.001 vs. control, # EFV vs. LPV/r 0.5 2 4 10 Log mtDNA copy number 0.2 0.4 0.6 0.8 1 1.2 1.4 Control LPV/r (μmol/l) EFV (μmol/l) Mitochondrial DNA Concerning marker genes of adipocyte differentiation, treatment of human adipocytes with: Kaletra causes a moderate but significant reduction in the expression of PPAR. A significant reduction in these mRNA was observed at concentrations of 4 M or higher. Efavirenz causes a progressive impairment in PPAR. First effects were observed at 0.5 M (significant reduction in PPAR mRNA) and a dramatic impairment occurred at 2 M and 4 M. The effect on the mitochondria caused by Kaletra or efavirenz has nothing to do with the mitochondrial DNA. Diaz-Delfín J, et al. 10th IWADRL. London, 2008 #P-01

EFV increases pro-inflammatory cytokines and reduces adiponectin c/w LPV/r in human adipose cells 4 µM - 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 µM and [LPV/r] ≥ 4 µM (EFV inhibition of mRNA significantly worse). Leptin mRNA significantly decreased with [EFV] ≥2 µM and [LPV/r] ≥ 10 µM 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. Background: Lopinavir/ritonavir (4:1) (L/r) and efavirenz are drugs of choice for initial antiretroviral therapy. These drugs may be involved in altered fat deposition and metabolism in patients. Local and systemic alterations mediated by adipose tissue in response to pathogenic insults may occur through altered release of pro-inflammatory cytokines and adipokines. Here we compared the effects of efavirenz respect to the L/r combination on the release of cytokines and adipokines by human adipocytes in culture. Methods: Human adipocytes were differentiated in culture from precursor cells obtained from liposuction from healthy individuals. Cells were treated with equivalent concentrations of L/r or efavirenz during the 10-day differentiation into adipocytes. Serum-free medium from the 4 last days of culture was used for the multiplex quantification of cytokines and adipokines (Linco, Luminex100IS), and lactate (Roche). Results: Efavirenz (4 m M) caused a dramatic increase in the levels of MCP-1 (5.3-fold), interleukin-5 (4.2-fold), interleukin-8 (18.7-fold), PAI-1 (5.7-fold) and HGF (5.5-fold) in the human adipocyte culture medium when compared with untreated adipocytes. NGF levels were unaltered. Moreover, efavirenz caused dramatic reduction of adiponectin and leptin levels (17% and 19% versus controls, respectively) in the medium. The induction of MCP-1, interleukin-8 and PAI-I observed for efavirenz did not occur with L/r (4 m M) treatment. HGF and IL-6 were induced to a similar extent in response to L/r and to efavirenz. L/r caused minor but significant reduction in the release of adiponectin and leptin (71% and 76% versus controls, respectively). Thus, adiponectin and leptin levels were significantly higher in medium from L/r-treated adipocytes than in medium from efavirenz-treated adipocytes (P < 0.05). Drug treatments did not alter lactate release. Conclusions: In human adipocytes in culture, efavirenz causes a higher release of pro-inflammatory cytokines and a more profound impairment in the release of adiponectin, an insulin sensitizing adipokine, respect to L/r treatment. Definitions: Cytokine: Signalling molecules used in cellular communication. Critical in development and functioning of immune response. Adipokine: A cytokine secreted by adipose tissue. Adiponectin: A protein hormone that regulates a number of metabolic processes, incl glucose regulation and fatty acid catabolism. Exclusively released by adipose tissue and blood levels are inversely correlated with body fat percentage. Leptin: Adipose derived hormone that regulate energy intake and energy expenditure, incl appetite and metabolism PAI-I: Plasminogen activator inhibitor (thus an inhibitor of fibrinolysis) MCP-1: Monocyte chemotactic protein-1. A small cytokine also known as chemokine ligand-2 HGF: Hepatocyte Growth Factor NGF: Nerve Growth Factor Domingo P et al, 5th IAS 2009, #TUPEB167

Efectos ex vivo de los fármacos antiretrovirales Adiponectina 20 40 60 80 100 120 140 Naive TRU+EFV P = NS -20 20 40 60 80 100 120 Naive TRU-KAL P = NS MCP-1 5 10 15 20 25 30 Naive TRU+EFV P = NS 5 10 15 20 25 30 35 40 Naive TRU+KAL

Efectos ex vivo de los fármacos antiretrovirales PPARg 8 9 P = NS 7 8 P = NS 7 6 6 5 5 4 4 3 3 2 2 1 1 -1 -2 Naive TRU+EFV Naive TRU+KAL TNFa -2 2 4 6 8 10 12 Naive TRU+EFV P = NS P = NS ,5 1 1,5 2 2,5 3 3,5 4 4,5 5 Naive TRU+KAL

Efectos ex vivo de los fármacos antiretrovirales Cyt B 5000 10000 15000 20000 25000 30000 35000 40000 Naive TRU+EFV P = 0,04 5000 10000 15000 20000 25000 30000 35000 Naive TRU+KAL P = 0,008 ADNmt 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 Naive TRU+EFV 500 1000 1500 2000 2500 3000 3500 4000 4500 Naive TRU+KAL

PPAR- mRNA/18S rRNA (x10-4) HIV Infection Contributes to Lipodystrophy Through Alteration of Gene Expression in Adipose Tissue Compared to HIV-negative controls, ARV-naïve HIV-positive patients had significant Decreases in mRNA of COX-2, COX-4, UCP2, C/EBP-a, PPAR-γ, GLUT4, LPL, leptin, and adiponectin Increases in PGC-1a, TNFa, and -2 microglobulin PPAR- mRNA/18S rRNA (x10-4) COX-2 mRNA/18S rRNA (x10-2) TNF-a mRNA/18S rRNA (x10-6) P<0.0001 P=0.0002 P=0.018 P=0.045 1.5 1.0 0.5 P=0.0004 P=0.0001 5 2 1 3 4 P=0.051 P=0.027 In this work from our group comparing gene expression of selected genes in naïve, non-lipodystropic and lipodystrophic patients , we can see how HIV-1 by itself causes underexpression of PPARg, and many other genes including …., and upregulation of inflammatory genes such as TNF alfa. This is an rt-PCT analysis measuring the expression of marker genes corresponding to mitochondrial function, adipocyte differentiation and metabolism in subcutaneous adipose tissue from healthy controls, untreated HIV-1-infected patients, and HIV-1-infected patients treated with HAART with or without HALS (HAART-associated Lipoatrophy). HIV+ patients (n=30, 10 per arm) Controls (n=10) Disturbances in adipose tissue gene expression are already present in untreated HIV-1-infected patients, thus indicating a role of HIV-1 infection itself in eliciting adipose tissue alterations that are worsened by HAART, which ultimately leads to HALS. P=0.023 P=0.046 P=0.001 P=0.001 Control Naïve LD Non-LD Control Naïve Non-LD LD Control Naïve LD Non-LD Giralt M et al. Antivir Ther. 2006;11:729–740.

ACTG 5142: Efecto diferente de LPV/r y EFV sobre la grasa periférica: LPV/r < EFV ACTG 5142 Grasa en las extremidades P Value +18 20 15 .013 +9.8 10 <.001 5 .007 Cambio % de la mediana desde la basal +1.4 -5 EFV LPV/r -10 LPV/r + EFV -15 Weeks 48 96 EFV 188 171 LPV/r 191 166 LPV/r + EFV 197 173 Haubrich R et al. 14th CROI 2007; Los Angeles. Abstract #38.

La toxicidad grasa es bifásica Grasa subcutánea TARc Tiempo

Conclusiones Tres factores implicados en la LD Virus Huésped Fármacos La patogenia de la lipodistrofia es multifactorial Mitocondria Diferenciación Inflamación EFV es más tóxico que LPV/r in vitro. A 48 semanas dicha toxicidad no es apreciable ex vivo. El curso de la LD es bifásico

Agradecimientos Biologia U.B. Hospital de la Santa Creu i Sant Pau Francesc Villarroya Marta Giralt Joan C. Domingo Julieta Díaz Hospital de la Santa Creu i Sant Pau Mª del Mar Gutierrez Mª Gracia Mateo Mª Antonia Sambeat Angels Fontanet Jessica Muñoz Jessica Martínez