1. ART drug resistance mutations Implications for TDF use Phyllis Kanki and Beth Chaplin Harvard PEPFAR Harvard School of Public Health

2. Drug resistance to current d4t or ZDV based regimens? –Implications for TDF in alternative or second line regimens What do we know about drug resistance to TDF as first line regimens? –HIV subtype issues Preliminary data on TDF resistance (K65R) transmission potential Baseline and toxicity issues with TDF

3. K65R Mutation of Lysine to Arginine in Codon 65 of the reverse transcriptase. It was first identified in response to TDF; only drug resistance mutation for TDF It confers resistance to TDF and also causes cross resistance to other NRTIs (ABC, ddI, FTC, 3TC) except zidovudine. In subtype B virus it is uncommon in patients who are not on a regimen containing TDF

4. Drug resistance in d4t or AZT based first line regimens “Options for 2nd line ART regimens whose initial regimen of d4T+3TC+NVP fails” 92-95% of mulit-drug resistance to NRTIs and NNRTIs (89% M184V) TAMs (37%), K65R (6%), Q151M (8%) (Sungkanuparph et al, CID 2007:44-447)

5. 194 Nigerian patients on a regimen of d4T-3TC- NVP/EFV in virologic failure Virologic failure defined as VL >2000 copies/ml with history of 6 month regular drug pickup. 10/194 (5.2%) had the K65R mutation Preliminary data on drug resistance mutations in Nigerian patients on non-TDF regimens

6. Possible Implications: Drug resistance to TDF (K65R) observed in non- TDF based regimens was not predicted. Coupled with M184V will effectively limit options for alternative or second line regimens.

7. What do we know about drug resistance to TDF as first line regimens? No significant difference in efficacy measured by viral suppression

8. Preliminary data from Nigeria: -Evaluated drug resistance in patients initiating TDF first line regimens in virologic failure -4 of 10 TDF virologic failures had K65R -K65R was accompanied by multi-drug resistance mutations including M184V and TAMS What do we know about drug resistance to TDF as first line regimens?

10. Which mutations are the first to appear? # of patients with this mutation Patients with only one mutation NNRTIs: NVP EFV NRTIs: 3TC d4T ZDV

11. “Early” and “Late” NNRTI Mutations early mutation mutations that seem to appear later in the course of resistance

12. Implications: Use of TDF in first line may lead to high rates of resistance (K65R) TDF resistance mutations would occur late Therefore, AZT could still be employed in alternative and second line regimens

13. Botswana : Increased In vitro resistance to TDF in subtype C virus (Wainburg et al) Nigeria: Genotype data -Subtype CRF 02 increased development of resistance to TDF - Subtype 06 decreased development of resistance to TDF What do we know about drug resistance to TDF as first line regimens? HIV subtype issues

14. Emergence of Resistance to Tenofovir (TDF) in Subtype C Compared to Other Subtypes in Vitro SubtypeWeeks of selection 71220–2535–40 C (BG-05)wtK65R C (Mole 18)wtK65R C (BG-15)wtK65R, A62V C (4742)wtK65RK65R, M41L B (n=4)wt AE (n=2)wt A (n=2)wt G (n=1)wt HIV-2wt 12-23 AIDS 20:F9-F13,2006

15. LUTH Lagos State N = 47 NIMR Lagos State N = 58 68Military Lagos State N = 43 JUTH Plateau State N = 123 UMTH Borno State N = 31 UCH Oyo State N = 39 G CRF06 A CRF13 F2 RECD CRF02 G CRF06 CRF11A REC CRF02 G A CRF06 REC CRF02 G CRF06 A F2REC CRF02 G CRF06 REC CRF02 G CRF06 REC

16. K65R by subtype SubtypeK65R% Subtype G3/873.4% CRF_02 A/G7/769.2% CRF_060/90%

17. Frequency of Mutations Associated with NRTI Resistance Grouped by Subtype M41LE44DD67NK70RV118IL210WT215YFK219QE Multi-nRTI Resistance Didanosine L74V K65R TenofovirK65R LamivudineK65RM184VI EmtricitabineK65RM184VI ZidovudineM41LE44DD67NK70RV118IL210WT215YFK219QE K65RStavudineM41LE44DD67NK70RV118IL210WT215YFK219QE AbacavirY115FM184VK65R L74V Multi-NRTI Resistance 151 Complex A62VV75IF77LF116YQ151M Multi-NRTI Resistance Ins. Complex M41LA62V69insK70RL210WT215YFK219QE A62V 2/87 5/76 0/9 V75I 9/87 5/76 0/9 F77L 3/87 5/76 0/9 F116Y 2/87 3/76 0/9 Q151M 3/87 5/76 0/9 Subtype: G CRF02 CRF06 M41L 14/87 6/76 4/9 E44D 3/87 1/76 0/9 K65R 3/87 7/76 0/9 D67N 18/87 10/76 5/9 K70R 25/87 11/76 4/9 L74V 2/87 0/76 0/9 Y115F 2/87 2/76 0/9 V118I 6/87 2/76 1/9 M184V 72/87 65/76 7/9 L210W 4/87 3/76 1/9 T215F 11/87 13/76 3/9 K219Q 12/87 6/76 3/9 Subtype: G CRF02 CRF06 T215Y 13/87 14/76 0/9 M184I 2/87 3/76 1/9 K219E 6/87 4/76 0/9

18. Implications: HIV subtype may influence (increase) development of K65R More studies need to evaluate potential impact of geographic (subtype) implications for TDF use However, even with K65R subtype differences - AZT could still be employed in alternative and second line regimens

19. Preliminary data from Nigeria: - Clustering of TDF resistance (K65R) -3 possible cases of TDF resistance(K65R) - where resistance seen prior to ART Preliminary data on TDF resistance (K65R) transmission potential

20. K65R T69del V75I F77L Q151M K219R -- NNRTIs K65R T69del V75I F77L Q151M K219R -- NNRTIs d4T-3TC-NVPAZT-TDF-LPV/r Responsive to a regimen of Kaletra (LPV/r), AZT and TDF G

21. K65R Q151M -- NNRTIs K65R V75I F77L Y115F F116Y Q151M -- NNRTIs K65R V75I F77L F116Y Q151M -- NNRTIs d4T-3TC-NVPAZT-TDF-LPV/r This patient may represent a case of transmitted resistance. CRF02

22. Another case of transmitted resistance? K65R F116Y Q151M M184V -- NNRTIs K65R T69I F77L F116Y Q151M M184V -- NNRTIs AZT-3TC- NVP AZT-3TC- TDF-LPV/r CRF02 Surveying for possible cases of transmitted resistance...

23. Surveillance is needed to evaluate possibility of transmission of drug resistant viruses TDF-first line regimens might have reduced efficacy in the face of transmitted resistant virus (K65R) but we need much more data In our small numbers patients still responded to TDF-second line even with the drugs resistance mutations (K65R) Implications:

24. Baseline and toxicity issues with TDF Reports of renal dysfunction in TDF-treated patients raise concerns about the potential for nephrotoxicity despite its excellent safety profile in clinical trials; particularly in patients with other risk factors for renal dysfunction, or deranged baseline renal function.

25. Baseline and toxicity issues with TDF Preliminary data from Nigeria: Baseline chemistries suggest that abnormal renal function will be low -- TDF could be used in the majority of patients. Serum CR and creatinine clearance compromised in patients on TDF- regimens

26. Laboratory values at baseline (n= 25,747)

27. TEMPORAL CHANGES IN RENAL FUNCTION ASSOCIATED WITH THE USE OF TENOFOVIR DISOPROXIL FUMARATE (TDF) IN HIV- INFECTED NIGERIAN ADULTS Agbaji O 1, Agaba P 1, Sule H 1, Ojoh R 1, Audu E 1, Sani M 1, Akintunde L 1, Taiwo B 2, Idoko J 1, Murphy R² Kanki P 3 1 AIDS Prevention Initiative in Nigeria Plus, Jos University Teaching Hospital, Jos, Nigeria; 2 Division of Infectious Diseases, Northwestern School of Medicine, Chicago, IL, USA; 3 Harvard School of Public Health Boston, MA, USA. Clinical/laboratory data for 84 on TDF-regimen and 102 on other NRTI regimens evaluated at 12 months Creatinine clearance (CLcr) estimated using the Cockcroft-Gault equation. Changes in serum creatinine and CLcr from baseline for each patient were compared between the TDF-treated patients and those in the non-TDF NRTI group. Multivariate analysis to control for other factors.

28. Results Serum creatinine increased by 23% (97.8 ± 25.9) and 3% (89.0 ± 26.2) in the TDF and non- TDF NRTI arms, at 48 weeks (p=0.02). Greater mean decrease in CLcr reduction from baseline in TDF (14.7 ± 44.4 ml/L) versus the non-TDF NRTI (10.4 ± 37.7 ml/L) arm at 48 weeks (p=0.001). In multivariate analyses, variables predictive of reduced CLcr were TDF use (p=0.005), age (p=0.002) and male gender (p=0.004).

29. TDF-regimens associated with a small, but statistically significant renal compromise compared with non-TDF- regimens. CLcr however remained within normal range. Assessment of renal function prior to initiation of tenofovir therapy is recommended for all patients. Therefore, frequent monitoring of renal function may not be necessary in patients with baseline normal renal function. Implications:

30. Future Directions Are second line therapies containing tenofovir effective over the long-term for resistant (K65R) patients? - How do failure rates compare to patients with other mutation patterns? - Does TDF in a 2nd line regimen impact efficacy? Testing partners where available and examining cases of transmitted resistance.

31. P. Kanki B. Chaplin R. Murphy J-L Sankalé S. Meloni A-Dieng Sarr S. Calves J. Hosseini W. Odutolu P. Okonkwo E. Ekong T. Jolayemi J. Samuels S. Ochigbo P. Akande B. Aluko B. Taiwo K. Scarsi K. Hurt J. Idoko O. Idigbe I. Adewole D. OIaleye C. Okany S. Akanmu S. Ogunsola W. Gashau M. Garbati R. Nkado D. Owujekwe H. Muktar S. Garko J. Abah Harvard PEPFAR R. Marlink T. Gaolathe J. Mukhema N. Ndwapi P.J. Burns P. Mwala K. Mukendi J. Puvimanasinghe M. Mine C. Bussmann M. Essex V. Novitsky M. Wainburg