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Resistência aos anti-retrovirais

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Apresentação em tema: "Resistência aos anti-retrovirais"— Transcrição da apresentação:

1 Resistência aos anti-retrovirais
Ricardo Sobhie Diaz Professor Adjunto e Livre Docente Chefe do Laboratório de Retrovirologia Escola Paulista de Medicina Universidade Federal de São Paulo

2 A resistência aos anti-retrovirais deverá ser o grande limitador da sobrevida dos pacientes em médio e longo prazo.

3 Tempo para o desenvolvimento de uma infecção oportunista integrante da categoria clínica C
Log Rank = 52,23 P<0,0001

4 Mutantes com resistência preexistem em indivíduos não tratados
Genoma do HIV-1 tem pb Índices de mutação de 1 erro/ pb 1010 virions gerados diariamente Portanto, todas as mutações possíveis são geradas diariamente

5 Zone of potential replication
Stopping drugs with different half-lives may lead to periods of monotherapy 24 48 36 12 Time (hours) Drug concentration Last dose Day 1 Day 2 Zone of potential replication MONOTHERAPY Stopping drugs in a treatment regimen with differing half lives may lead to periods of monotherapy with a long half-life drug, as well a s an extended period of time in the zone of HSP. Thus, pharmacokinetics are critical for ensuring effective treatment and preventing resistance development if a dose is missed. Taylor S, et al., 11th CROI, San Francisco, February 2004, #131

6 Clinical consequences of stopping ARVs with long half-lives
Nevirapine (NVP) and efavirenz (EFV) have extended time in the zone of highest selective pressure Steady-state half-life: NVP: 25–32 h EFV: 40–55 h Understanding the emergence of resistance requires characterization of pharmacokinetic/pharmacodynamic (PK/PD) relationships during intermittent adherence. Recent results by Taylor et al illustrate the clinical consequences of the long half-lives of NNRTIs. Taylor S, et al. 11th CROI, San Francisco 2004, #131

7 Incidence and persistence of resistance after single-dose NVP
NVP resistance in mothers and infants HIV+ women (n=157) and infants (n=21) with NVP resistance (K103N, V106A/M, Y181C, or G190A) after single-dose NVP for prevention of MTCT1 K103N persisted in mothers Y181C persisted in infants Mothers with persistent resistance had higher HIV RNA and lower CD4+ counts than those where resistance no longer detected at 6 months NVP drug levels after single dose persisted for up to 21 days2 Resistance (%) Mothers Infants 10 20 30 40 50 Baseline 6 weeks 6 months Treatment with NVP is effective and inexpensive; however, the data raise issues of use of this therapy in patients who may then receive combination therapy in the future. Morris L, et al: NVP has a low genetic barrier to resistance and a long plasma half-life of up to 21 days. NVP resistance has been reported in women and infants following single-dose NVP. NVP resistance in mothers receiving single-dose NVP may impact virologic response to treatment. NVP drug resistance among mothers at 4–8 weeks varies from 16.8% (in subtype A- HIVNET 012 trail) to 39% (in subtype C by Martinson et al, CROI 2004). Objective: Overall, to evaluate the selection of resistant variants of HIV-1 following the use of intrapartum dose of NVP therapy in infected mothers and their infants. Specifically, to determine the level of NVP resistance in mothers and infants 6 months postpartum, and examine the patterns of mutations persisting in mothers and infants 6 months postpartum. Methods: Prospective cohort of pregnant women enrolled at 32–38 weeks gestation in S Africa. Single-dose NVP given to both mother and infant. Visit 1–6 weeks postpartum; Visit 2–6 months postpartum. Results: 623 women enrolled; median time from NVP to delivery 6 hours; 1st follow-up visit—462 mother and 482 infants; with median time of 7 weeks (IQR 6–10); 2nd follow-up visit—90% with a median time of 6 months (IQR: 5–7). 98% of patients had subtype C virus. Resistance assay performed at baseline in 232 of 623; in 458 at Week 6 and in 157 at Month 6. 38% of mothers had persistent resistance at 6 weeks vs 42% in infants, and resistance persisted at 6 months in 14% of mothers vs 26% of infants. Resistance persisted despite maternal CD4 at 6 months (> or <200). Number of mutations detected declined from genotype on Week 6 vs Month 6 follow-ups (f/u). K103N persisted in mothers and Y181C persisted in infants between 6 weeks and 6 months f/u. VL was significantly higher in mothers with persistent resistance (81,300c/mL) and lower CD4 count (386 c/mL) than those w/o resistance at Week 6 (VL: 19,850 c/mL and CD4 of 538/mm3). Cressey TR, et al: Perinatal HIV Prevention Trial (PHPT-2) (Thailand) was conducted prospectively in 1844 women and their infants (non-breast feeding). Mothers received ZDV 300 mg BID starting at gestation Week 28; NVP 200 mg QD or placebo was given to the mother during labor, while ZDV 300 mg Q3h was continued; NVP 6 mg x1 at 48–72 h post delivery or placebo given to the infant in addition to ZDV 2 mg/kg QID. Addition of single intrapartum dose of NVP to ZDV prophylaxis was extremely efficacious in reducing risk of HIV perinatal transmission. Maternal and infant NVP+ZDV prophylaxis resulted in transmission rates comparable to using ARV during pregnancy. Based on PHPT-2 results, the ministry of Public Health of Thailand modified their prevention of MTCT guidelines to include ZDV at gestation Week 28 + NVP at labor to mother and child. Objective: Assess NVP drug levels at onset of labor, delivery, 10 days and 42 days postpartum. Methods: n=110; NVP plasma levels measured using HPLC (detection limit 50 ng/mL); 2 samples/patient collected; NVP IC50 = 3–30 ng/mL. Results: Median age 27; weight 63 kg, CD4: 409/mm3; VL 3.89 c/mL. NVP concentration remained detectable in 56% of patients through Day 15–21. In some women, this means that NVP levels will remain above IC50 for up to 4 weeks. 1. Morris L, et al. XV IAC, Bangkok 2004, #ThOrB1353; 2. Cressey TR, et al. ibid, #ThOrB1352

8 Response to therapy in women receiving single-dose NVP for MTCT
269 women started d4T, 3TC, NVP postpartum 221 NVP-exposed, 48 not NVP-exposed intrapartum Genotype postpartum (median 12 days [range 10−14]) K103N (21%) G190A (10%) Y181C (4%) Subsequent response to triple therapy (<50 c/mL) 33 68 36 52 25 38 10 20 30 40 50 60 70 80 Baseline 3 months 6 months % with VL <50 copies/mL Mother-to-child transmission (MTCT) and the resistance associated with single-dose NVP strategies used to interrupt MTCT, are important issues. In resource-constrained parts of the world, where treatment is available, perhaps the most popular treatment regimen is a combination pill that contains d4T, 3TC and NVP. Protecting this regimen is important as subsequent regimens are more costly and much more difficult for patients to access. Jourdain et al examined whether women given NVP to interrupt MTCT experienced problems with later treatment with this triple drug combination. 221 women who received NVP as part of MTCT interruption and 48 who did not receive NVP were followed. Significant rates of resistance were seen in the women who received NVP during delivery. Other studies that have looked at earlier administration of NVP have shown resistance rates increased by about 75%; however, this tends to disappear quickly. 61 patients receiving NVP had detectable plasma concentrations more than 3 weeks postpartum. This may be related to CYP 2B6 enzymes, especially in persons of African descent who carry a particular enzyme genotype that seems to lead to very delayed clearance of NNRTIs. What does that translate into in terms of treatment efficacy? Response to 3-drug therapy using a <50 copies/mL assay is shown in the bar graph. Women who have never received NVP had good virologic responses at the end of 3 and 6 months. In contrast, women who had previously received NVP and had no detectable mutations prior to start of therapy, and the women receiving NVP and had one of these mutations, had dramatically lower response rates. This has engendered a lot of work to try to identify ways to protect NVP and develop better strategies for interruption of MTCT. No NVP NVP no mut NVP + mut Nonexposed Exposed; no NNRTI mutations Exposed; NNRTI mutations Jourdain G, et al. N Engl J Med 2004;351:229–40

9 Testes de Resistência para o HIV-1
Genotipagem Detecta mutações no gene pol gene (RT/prot) Produz lista de mutações Interpretação (falta de conhecimento) Fenotipagem Cultiva o vírus na presença de cada ARV Quantifica perda de susceptibilidade comparados ao WT Interpretação (“cut-off”) Fenotipagem virtual

10 Nomenculatura das Mutações
M 184 V gtg atg aminoácido normalmente encontrado nesta posição posição na molécula da TR aminoácido encontrado neste vírus mutante


12 Performance da Fenotipagem virtual
Perez-Elias et al. Antivir Ther Dec;8(6): Mazzotta et al J Acquir Immune Defic Syndr Mar 1;32(3):

13 susceptibilidade à ARV
Pressão seletiva dos ARV Mutação Diminuição de susceptibilidade ao ARV Aumento de susceptibilidade à ARV Resistência cruzada

14 Pressão seletiva do Saquinavir
N88S Diminuição de susceptibilidade ao Saquinavir Aumento de susceptibilidade ao Amprenavir Resistência cruzada ao Nelfinavir

15 Pressão seletiva do Efavirenz
G190A Diminuição de susceptibilidade ao Efavirenz Aumento de susceptibilidade à Delavirdina Resistência cruzada ao Nevirapina

16 susceptibilidade ao AZT,
Pressão seletiva do 3TC M184V Diminuição de susceptibilidade ao 3TC Aumento de susceptibilidade ao AZT, Tenofovir Resistência cruzada ao DDC

17 Fold Change in NRTI Susceptibility as a Function of Number of TAMs with or without M184V
10,000 AZT 100 TDF 100 d4T 1000 10 10 Fold Change in NRTI 100 10 1 1 1 -1 -1 -1 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 100 3TC 10 ddI 100 ABC Fold Change in NRTI 10 10 1 1 1 -1 -1 -1 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 Number of TAMs M184wt Whitcomb JM. 9th CRO; 2002; Seattle, Washington. Abstract 569. M184V

18 Perda da atividade dos ITRN raramente é total com a emergência de mutantes de resistência

19 Reverse Transcriptase Inhibitor Interruption
(continued Protease Inhibitor Therapy) 5 of 5 patients had an immediate and durable increase in viremia (> 0.5 log) Deeks et al

20 Deeks et al Interruption of RTI
No change in phenotype during first 16 weeks Reversion of M184V occurred late in 3 patients and was associated with increased viremia in 2 patients Deeks et al

21 Complete Treatment Interruption vs. 3TC Monotherapy (n = 50)
Interruption of all drugs in patients with drug-resistant HIV results in rapid increase in viremia Detectable viremia on 3TC-based regimen, CD4 > 500 Castagna et al. International AIDS Conference, Bangkok, 2004 (Abstract WeOrB1286.)

22 Complete Treatment Interruption vs. 3TC Monotherapy (n = 50)
3TC monotherapy exerts a persistent anti-HIV effect (even in presence of M184V) This may be due to fitness or residual drug effect

23 Barreira genética para seleção de mutações
“Proximidade” genética para aquisição de resistência completa aos anti-retrovirais Número de mutações necessárias para diminuição do efeito ARV (IP) Facilidade na seleção de determinadas mutações de resistência (ddI, d4T, TDF) Perfil (tipo) de mutações (associações de ARV)

24 Barreira genética e número de mutações
Efeito quantitativo Efeito qualitativo Fenotipagem ITR IP Associação de IP Genotipagem

25 Pacientes Virgens de tratamento
Pelo menos 5 mutações para aumento de IC50 superior a 3,0 na fenotipagem. 1 10 100 1000 10,000 Cmax BMS-008—ATV 400 mg PK margem de segurança Concentração plasmática média (SD) no Steady State (ng/mL) Percentil 75% Mediana  14 ng/mL Percentil 25% 2 4 6 8 10 12 14 16 18 20 22 24 Tempo (h)

26 Barreira genética e facilidade com que a mutação é selecionada
Alta ddI TDF Intermediária AZT d4T ABV Baixa 3TC Nevirapina

27 Barreira genética e perfil (tipo) de mutações (associações de ARV) 1
ABC/3TC/TDF em 36 pacientes; 12 meses 12 falhas virológicas 11/12 com ambas K65R e M184V 1 com M184V Nível sérico drogas normal Low Genetic Barrier to Resistance Is a Possible Cause of Early Virologic Failures in Once-Daily Regimen of Abacavir, Lamivudine, and Tenofovir: R Landman et al. 11th CROI

28 ARV + replicação viral Piora da Resistência Melhora do
Susceptibilidade Resistência Cruzada Melhora do “fitness”

29 Probabilidade de aquisição de mutações em pacientes com falha estável (n = 98)
Any (n=98) NRTI (n=96) NNRTI (n=28) PI (n=68) BL and new mutations in patients receiving ARV class % patients on each ARV class with mutations BL mutations New mutations Edwards D, et al. 44th ICAAC, Washington 2004, #H-176

30 New mutations by log10 HIV RNA slope
Probabilidade de aquisição de mutações em pacientes com falha estável (n = 98) New mutations by log10 HIV RNA slope 0.00 0.05 0.10 0.15 0.20 0.25 0.30 -0.80 -0.60 -0.40 -0.20 0.40 0.60 0.80 HIV RNA (log10 c/mL) slope per month P-value = 0.01 Acquired mutations per person-month

31 ARV + replicação viral Piora da Resistência Melhora do
Susceptibilidade Resistência Cruzada Melhora do “fitness”

32 Tendências na resistência do HIV-1
1 classe Proporção de resist. 2 classes 3 classes n = Dados da BC, Canada, J. Montaner, 2004

33 Freqüência de resistência em pacientes com múltiplas falhas
94.7% 43.2% 58,2% 21.5% 42.2% 34.4% Sucupira, Antiviral Therapy 6:

34 Falha decorrente do número insuficiente de drogas
Proporção com sucesso virológico Consistent with the previous slide showing greater virologic response at week 24 in patients with greater baseline PSS scores, this slide shows that background regimens with additional number of active drugs delay time to virologic failure. PSS:” Phenotypic Susceptibility Score”, uma estimativa do número de drogas ativas

35 Tipranavir IP não peptídico – ação contra cepas resistentes a outros IPs 500 mg 2 vezes ao dia com 200 mg RTV 2 vezes ao dia Estimulante do p450 Queda de CV = 1,5 log Diarréia, náuseas, vômitos. 10V, 13V, 20M/R/V, 33F, 35G, 36I, 43T, 46L, 47V, 54A/M/V, 58E, 69K, 74P, 82L/T, 83D, e 84V (RESIST) Até 4 mutações – sensível 5-9 mutações – 2 “fold resistance” >= 10 mutações – 4 “fold resistance”


37 83% of patients presenting at least 1 log10 drop in VL

38 ARV + replicação viral Piora da Resistência Melhora do
Susceptibilidade Resistência Cruzada Melhora do “fitness”

39 Fitness viral Capacidade replicativa (em determinado meio ambiente)
Mutações de resistência produzem deficiência replicativa (redução do fitness viral) Mutações adicionais produzem uma recuperação do fitness viral Entretanto, o vírus com melhor fitness na presença de ARVs é o vírus mutante

40 Capacidade Replicativa Relativa do HIV-1 do tipo selvagem e mutante.
Wild-type K65R K65R+M184V 100% 100 Replication Capacity (% of Wild-Type) 80 60 53% 40 24% 20 White et al., AAC, 2002 1 ViroLogic single-cycle replication capacity assay

41 RT mutations before and after 12w ARV interruption (54)

42 Protease mutations before and after 12w ARV interruption (54)

43 Single genotypic tests underestimate resistance
Results: Historical genotype Most recent genotype Difference Lamivudine (M184 V/I) 58.8% 25.5% 33.3% Other NRTIs 46.0% 27.7% 18.3% NNRTIs 38.5% 24.5% 14.0% PIs 27.9% 15.6% 12.3% Harrigan PR, Wynhoven B, Brumme ZL, et al., Infect Dis ;191(8):1325–30.

44 Qual a persistência de mutações de resistência em pacientes com resistência primária?

45 Primary resistance mutation persists!
Delaugerre C et al. Antivir Ther Jun;9(3):415-21 Brenner B et al. AIDS Aug 20;18(12): Barbour JD et al. AIDS Aug 20;18(12): Wainberg MA, et al. Paper presented at: XIV International AIDS Conference, 2002; Barcelona - Spain. Little SJ et al. Paper presented at: 11th Conference on Retroviruses and Opportunistic Infections, 2004; San Francisco, CA.

46 Efeito na resposta ART em paciente com infecção com resistência primária
JAMA 1999;282:1142

47 Primary Resistance did not affect short term virologic efficacy among pregnant women
Retrospective analyses 20% of primary ARV resistance (8 women) M184V (4), K103N (2), and L90M (2) Viral load bellow detection at labor! (importance of HAART for preventing MTC transmission) Gasparotto et all 2005

48 Prevalência de Resistência Primária na UK (n=2410)
NRTI NNRTI PI Any class 5 10 15 20 25 1996-7 1998 1999 2000 2001 2002-3 Year of sample Prevalence (%) 316 312 360 453 502 467 Prevalence (%) Pillay D, et al. HIV 7, Glasgow 2004, #PL5.3

49 Primary Resistance: San Francisco Clinics
TrueGene HIV-1 Genotyping Kit (Visible Genetics, Inc) NRTI NNRTI Genotypic Resistance (% patients) Slide #7: Primary Resistance: San Francisco Clinics Grant and colleagues monitored recently-infected and drug-naïve patients for trends in primary genotypic resistance.1 San Francisco from June 1996 through June 2001. Primary genotypic resistance.1 Overall: increased from 16.7% to 27.6% (P=0.003). NNRTI class: 0% to 17% (P=0.003). PI-resistance prevalence remained relatively stable compared with NNRTIs and NRTIs. Likelihood of transmissibility of HIV-1 resistant to different classes of drugs was: NNRTI>NRTI>PI>3 drugs. 1996 to 1998: prevalence of genotypic resistance with NRTI decreased from 25% to 6%, before significantly increasing to 20% in 2001 (P=0.03). These data illustrate that primary resistance is highly prevalent and increasing significantly, especially for NNRTIs, in San Francisco.1 Reference Grant RM, Hecht FM, Warmerdam M, et al. Time trends in primary HIV-1 drug resistance among recently infected persons. JAMA. 2002;288; >2 drugs PI >3 drugs (n=12) (n=28) (n=53) (n=41) (n=96) (n=29) Grant RM, et al. JAMA. 2002;288:

50 Drug-Resistant HIV in Treatment-Naïve Patients
IC50 >10-fold increase via PhenoSense HIV (ViroLogic) NNRTI PI Phenotypic Resistance (% patients) Slide #6: Drug-Resistant HIV in Treatment-Naïve Patients Little and colleagues retrospectively assessed the prevalence of transmitted drug-resistant virus in treatment-naïve patients with primary HIV infection.1 High-level resistance (measured by the PhenoSense assay [Virologic]) was defined as a >10-fold increase in IC50 of the patient’s virus as compared with the control virus. Samples were taken between May 1995 and June 2000 in 10 North American cities. The proportion of new HIV infections that involve drug-resistant virus is increasing.1 Significant increases were also seen for NRTI, NNRTI, and PI drugs as well as for combination of 2 or more drugs. These data demonstrate that transmission of resistant virus to all classes of ART is on the increase in recently HIV-infected patients.1 Reference Little SJ, Holte S, Routy J-P, et al. Antiretroviral-drug resistance among patients recently infected with HIV. N Engl J Med. 2002;347: NRTI (n=11) (n=56) (n=101) (n=97) (n=90) (n=23) Little SJ, et al. N Engl J Med. 2002;347:

51 % with reduced susceptibility % Subjects with reduced susceptibility
Prevalence of resistance from 40 US cities in 2003 in ART-naïve patients 317 HIV+ ART-naïve subjects PT/GT determined GT: 14% with mutation Reduced susceptibility is most prevalent with NNRTI class % with reduced susceptibility to any specific drug 20 16 12 % Subjects with reduced susceptibility 8 Demographics Mean age years % male 92% Mean CD4+ (copies/mL) 277 Race White 55% Black 33% Hispanic/Asian/other 12% HIV-1 clade B 97% Transmission route Homosexual 74% Heterosexual 28% Iv use/transfusion/other 6% PUT SPECIFIC MUTATIONS IN NOTES 4 d4T ZDV DLV EFV NVP APV IDV NFV RTV SQV Ross L, et al. 44th ICAAC, Washington 2004, #H-173

52 Primary resistance in Santos, Brazil
RECENT INFECTION GROUP Resistant Wild Type Positive PCR Negative PCR TOTAL OF RESISTANT PI 3 19 22 3 3/22 (13.6%) NRTI 5 17 22 3 5/22 (22.7%) NNRTI 22 22 3 PI & RTI 19 6 0/19 (0%) PI or RTI 7 12 19 6 7/19 (36,8%) ESTABLISHED INFECTION GROUP Resistant Wild Type Positive PCR Negative PCR TOTAL OF RESISTANT PI 4 58 62 3 4/62 (6.4%) NRTI 12 45 57 8 12/57 (21.0%) NNRTI 2 55 57 8 2/57 (3.5%) PI & RTI 2 56 8 2/56 (3.6%) PI or RTI 14 42 56 8 14/56 (25.0%)

53 Efeitos das Mutações de Resistência na capacidade replicativa
11 9 5 17 capacidade replicativa * *mutções Wrin T, 40th ICAAC 2000

54 Viral fitness Paradigm 1: resistance mutations produce a replicative impairment (reduction in viral fitness) Paradigm 2: Increased number of mutations restores the viral fitness

55 Tratamento ARV CV Limite de detecção tempo

56 Conclusões Resistência aos ITRN podem estar relacionados a diminuição de incorporação ou excisão. Mutações de resistência selecionadas por um anti-retroviral podem levar a diminuição de susceptibilidade a este anti-retroviral, resistência cruzada ou aumento de susceptibilidade a anti-retrovirais ainda não utilizados. Barreira genética para resistência do HIV aos ARV refere-se à facilidade com que ocorre a perda de susceptibilidade aos ARV. Tem relação com número e tipo mutações e com tipo de associação ARV

57 Conclusões (cont.) Mutações de resistência selecionadas por um anti-retroviral podem seguir “caminhos mutacionais” diferentes. A alteração de susceptibilidade natural (resistência primária) ou induzida (resistência secundária) aos diferentes tipos e subtipos do HIV ainda não está totalmente definida. Persistência na supressão viral incompleta leva risco de resistência ampla, resistência cruzada e recuperação do “fitness” viral.

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