Efficacy, safety and pharmacokinetics of atazanavir (200mg twice daily) plus raltegravir (400mg twice daily) dual regimen in the clinical setting

This is a pre-copyedited, author-produced PDF of an article

accepted for publication in Journal of Clinical Virology

following peer review. The version of record J Clin Virol.

2016 Dec 1;87:30-36. doi: 10.1016/j.jcv.2016.11.015.

[Epub ahead of print] is available online at:

http://www.journalofclinicalvirology.com/article/S1386-6532(16)30613-8/abstract

Efficacy, safety and pharmacokinetics of atazanavir (200 mg twice daily) plus raltegravir (400

mg twice daily) dual regimen in the clinical setting

Letizia Marinaroa_{,∗, Andrea Calcagno}a_{, Diego Ripamonti}b_{, Giovanni Cenderello}c_{, Veronica} Pirriatorea_{, Laura Trentini}a_{, Bernardino Salassa}a_{, Caterina Bramato}a_{, Giancarlo Orofino}d_{, Antonio}

D’Avolioa_{, Marco Rizzi}b_{, Giovanni Di Perri}a_{, Stefano Rusconi}e_{, Stefano Bonora}a

a Unit of Infectious Diseases, Department of Medical Sciences, University of Torino, Ospedale Amedeo di Savoia, Torino, Italy b Infectious Diseases Unit, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy c Department of Infectious Diseases, Ente Ospedaliero Ospedali Galliera, Genova, Italy d Department of Infectious Diseases, “Divisione A”, Ospedale Amedeo di Savoia, Torino, Italy e Unit of Infectious Diseases, University of Milano, Ospedale Luigi Sacco, Milano, Italy

ABSTRACT

Background: Unboosted atazanavir with raltegravir has been investigated at 300 mg twice daily

showing frequent hyperbilirubinemia and selection of resistance-associated mutations.

Objectives: Atazanavir 200 mg twice daily could increase tolerability and plasma exposure.

Study design: Patients on atazanavir/raltegravir (200/400 twice daily), with self-reported adherence

> 95% and no concomitant interacting drugs were retrospectively evaluated.

Results: 102 patients [72.5% male, age 46.4 years (42-54), BMI 24 Kg/m2 _{(22-26)] were included.} CD4+ T lymphocytes were 417 cell/µL (302-704) and 76 patients (74.5%) had HIV-RNA <50 copies/ml. After 123 weeks 18.6% patients showed virological failure and 3.9% discontinued for intolerance. Available genotypes showed selection of major integrase (7/10 patients) and protease resistance-associated mutations (5/13 patients). In patients switching with dyslipidemia (n=67) total, LDL cholesterol and triglycerides significantly decreased. Patients switching with eCRCL<60 ml/min (n=27) had no significant changes while patients with eCRCL> 60 ml/min showed significant decrease (-9.8 ml/min, p=0.003) at 96-weeks. Atazanavir and raltegravir trough concentrations were 321 ng/ml (147-720) and 412 ng/ml (225-695). Self-reported non-adherence 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55

(n=4) was significantly associated with virological failure (p=0.02); patients with virological success had borderline longer previous virological control (33 vs. 18 months, p=0.07).

Discussion: Switch to atazanavir/raltegravir was safe and well tolerated allowing optimal drugs’

plasma exposure. However, a concerning rate (18.6%) failed with newly selected mutations and stopped ATV/RAL because of DDI and intolerance issues or were lost to follow-up. This regimen might be considered in selected patients, without history of protease inhibitors failure or HBV infection, showing optimal adherence and prolonged suppression.

Key words: atazanavir, raltegravir, HIV, NRTI-sparing antiretroviral therapy,

treatment-experienced patients. 56 57 58 59 60 61 62 63 64

BACKGROUND

The astonishing success of antiretroviral therapy (ART) is limited by long-term toxicity, suboptimal adherence, drug-drug interactions (DDI) and multidrug resistant HIV strands. Recommended regimens include three drugs with nucleoside reverse transcriptase inhibitors (NRTIs) as backbone [1]. NRTIs- and ritonavir (RTV)- sparing regimens provide an interesting choice to reduce drugs’ toxicity and improve tolerability in ART-experienced patients. There is growing interest in NRTIs-sparing dual regimens containing HIV integrase inhibitors (INI) and protease inhibitors (PIs) [2,3] as well as for lamivudine containing dual regimens [4,5]. Raltegravir (RAL) associated to unboosted atazanavir (ATV) represents an option for NRTI- and RTV-sparing maintenance therapy. Raltegravir is mainly metabolized through glucuronidation by uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), secondarily by UGT1A3 and UGT1A9 and is not a cytochrome P450 (CYP) substrate [6]. Atazanavir 300 mg boosted by RTV 100 mg showed efficacy in treatment-naïve [7-9] and -experienced subjects [10-13]; ATV is mainly metabolized by CYP3A, inhibits CYP3A and UGT1A1. Unboosted ATV dosed 400 mg once daily (qd) showed drug exposure below recommended concentration at the end of dosing interval (Ctrough, 150 ng/mL) [1,14] in up to 50% subjects [15-18]. Splitting 400 mg daily dosing of ATV in 200 mg twice daily (bid) may favour adequate plasma exposures [19]. Studies in healthy volunteers and HIV-infected subjects [20-24] showed RAL AUC increased by 40-55% through ATV-mediated UGT1A1 inhibition; given its safe toxicity profile, RAL dosage adjustment is not suggested. ATV Cmin was reduced by 29% with concentrations remaining however above EC90 for wild-type HIV-1 (14 ng/mL). The efficacy of this combination has been studied as initial and maintenance strategy with different dosing. A comparative study in treatment-naïve patients with RAL 400 mg and ATV 300 mg bid showed efficacy comparable to standard therapy [25]. Nevertheless the study was stopped at 24 weeks due to high rates of RAL resistances (in those who failed) and hyperbilirubinemia. Small-sized studies in experienced subjects showed viral efficacy and improved tolerability with ATV 200 mg and RAL 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

400 mg both dosed bid [26,27]. Persistent viral suppression was reported in 90% of patients treated with ATV 400 mg qd plus standard dose of RAL at 48 weeks [28]. A recent study showed efficacy (100% at 48 weeks) and good safety profile in twenty-five experienced patients randomized to RAL 400 mg plus ATV 300 mg bid for four weeks followed by RAL 800 mg plus ATV/ritonavir 300/100 mg qd for four weeks or vice versa [29]. The lowering effect of RAL co-administration could be counterbalanced by increased plasma exposure of ATV given 200 mg bid (higher Cmin, lower Cmax), avoiding hyperbilirubinemia observed with higher doses [25].

OBJECTIVE

Aim of our study was to further investigate efficacy, safety and pharmacokinetics of the dual NRTI-and RTV-sparing combination of ATV 200 mg NRTI-and RAL 400 mg, both given bid in clinical setting.

STUDY DESIGN

HIV-1 infected patients receiving ATV 200 mg and RAL 400 mg, both bid, were retrospectively evaluated. Inclusion criteria were: treatment with ATV+RAL from at least three months, self-reported adherence of 95% or more, no concomitant interacting drugs. Subjects were outpatients of Infectious Diseases Department of Torino University, Genova University, Ospedale Galliera, Ospedale Papa Giovanni XXIII of Bergamo and Milano University, Ospedale Luigi Sacco. We could identify approximately 95% (200) of total patients starting ATV+RAL in study period from drugs registry of each Hospital Pharmacy involved in the study. Patients receiving ATV+RAL and responding to inclusion criteria were identified and data were collected from clinical records and referent physicians between December 2012 and February 2013. Informed consent request was waived by Hospitals due to the retrospective study design. A subgroup of patients underwent an intensive pharmacokinetic (PK) study. Study regimen was introduced because of toxicity, tolerability, or resistance issues. Virological response was considered as last HIV viral load <50 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112

copies/mL without previous confirmed viral rebound above 50 copies/mL. ATV and RAL resistance associated mutations (RAMs) were calculated according to Stanford University database.

Pharmacokinetic evaluation

ATV and RAL plasma concentrations were measured by HPLC-UV/PDA-validated method using a solid phase extraction procedure with lower quantification limit 46.8 ng/mL and of 23.4 ng/mL respectively, lower detection limit of 11.7 ng/mL for both drugs [30]. Samples collected 10 to 14 hours after drug intake were considered trough concentrations (Ctrough). Patients undergoing 12-hour intensive ATV and RAL PK evaluation were instructed to take tablets at 8 a.m. and 8 p.m. Blood samples were obtained at the end of dosing interval (8 a.m.), 0.5, 1, 2, 3, 4, 8, 12 hours after morning dose given with standard light meal. Plasma was separated within 1 hour from drawing and samples stored at -70°C until analyzed. PK parameters were calculated with a non-compartmental model using the software Kinetica (vers. 5.0, Thermo Scientific, MA, USA). Area under the concentration-time curve (AUC) over 12 hours (AUC12) was calculated by the linear-log trapezoidal rule. Maximum concentration (Cmax), Tmax, C12h (concentration at 12h post-dose) and C0 (concentration pre-dose) were obtained.

Statistical analysis

Quantitative variables were described as median (interquartile range, IQR), categorical ones by number (percentage). Non-parametric tests were used for all analysis and tests are specified in the text. Factors with p<0.2 at univariate analysis were compared through binomial logistic regression for multivariate analysis. Data analysis was performed using SPSS software for Mac (version 20.0, IBM Corp. Released 2011. Armonk, NY: IBM Corp).

113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135

RESULTS

Of 200 patients in ATV/RAL in the study period in different centers, 102 patients responding to inclusion criteria were enrolled. Subjects excluded were taking ATV/RAL for less than three months, or were assuming potential interacting drugs (mainly proton pump inhibitors or antacids), showed incomplete compliance to study visits or timing required for blood samples. Subjects were 72.5% male, aged 46,4 years (42-53.7) (Table 1). CD4+ cell count was 417 cell/µL (302-704) and 76 patients (74.5%) had HIV-RNA below 50 copies/ml. In patients with measurable viral replication (n=26), HIV-RNA was 525 copies/mL (82-10.462). Thirty-four patients (33.3%) were HCV-coinfected (7 with liver cirrhosis). Patients had been previously treated for 102 months (37-138). Last regimen was boosted PI-based in 81 patients (79.4%), mainly boosted ATV (60, 58.8%), NRTIs-based (3, 2.9%), NNRTIs-based (11, 10.7%), INI-based (7, 6.8%). Eight (7.8%) and 12 patients (11.7%) had previous virological failure to NNRTIs and PIs, respectively. Switch to ATV/RAL was due to toxicity in 61 subjects (59.8%) 35 of which (57.3%) showed renal abnormalities, 17 (27.8%) dyslipidemia and or lipodystrophy, 6 (9.8%) gastro-intestinal intolerance. In ten patients (9.8%) change of therapy was motivated by failure to previous treatment and in 8 (7.8%) by regimen simplification.

Ninety-nine patients (97%) completed 12 weeks of study treatment. At last observation (123 weeks, 70-170), 59 subjects (57.8%) were on treatment with self-reported adherence of 95% or more in 55 (93,2%). Seventy-nine patients (77.5%) showed viral load <50 copies/mL. Seventeen patients discontinued due to DDI (4, 3.9%), toxicity or intolerance (4, 3.9%); 9 patients (8%) changed therapy while suppressed (7 for unspecified reason, 1 for anti-HCV treatment, 1 for antineoplastic treatment (Figure 1).

Nineteen patients (18.6%) failed. Available genotype showed selection of major INI-RAMs in 7/10 patients and PI-RAMs in 5/13 patients (Table 2). Selection of major INI-RAMs was reported in 7/10 patients). Three patients with INI-RAMs had accompanied newly selected PI-RAMs (1 with 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160

previous PI failure, 1 was RAL-experienced). Three patients with PI-RAMS did not experience previous PI failure and pre-switch genotype was unavailable. After changing therapy all these subjects showed complete suppression at follow up.

Viral load was <50 copies/mL in 81/90 subjects (90%) that completed 48 weeks of follow-up and 51/62 subjects (82.3%) that reached 96 weeks follow-up; CD4 cell increase from baseline was 39 cell/μL (16-152) and 62 cell/μL (32-163). At last observation CD4 cell count was 519 /μL (378,7-740) and CD4 gain from baseline was 65 cell/μL (39-181).

Treatment was generally well tolerated, 4 patients (3,9%) however switched for toxicity or intolerance (1 CK increase, 1 liver function test increase, 1 gastro-intestinal symptoms, 1 asthenia). Biochemical analysis at baseline, W48 and W96 are shown in Table 3. Significant reduction in triglycerides at week 48 (-27 mg/dL) and week 96 (-28.5 mg/dL) was observed while HDL cholesterol significantly increased at week 48 (+3 mg/dL). AST and ALT decreased (-3 and -4 U/L, respectively) while fasting glucose levels increased (+5 mg/dL) as well as plasma creatinine (+0.03 mg/dL) at week 96. In patients switching with eCRCL<60 ml/min (n=27) no significant changes were noted while patients with baseline eCRCL> 60 ml/min showed a significant decrease (-9.8 ml/min, p=0.003) at 96 weeks. In patients switching with dyslipidemia (n=67) total, LDL cholesterol and triglycerides (-30 mg/dL, p=0.001, -19.5 mg/dL, p=0.02, -51 mg/dL, p=0.002 respectively, Wilcoxon test) significantly decreased at week 96.

Pharmacokinetics

108 samples were available in 61 patients [2 (1-2) samples per patient]. ATV and RAL Geometric means (GM) trough concentration were 321 ng/mL (147-720, IQR) and 412 ng/ml (225-695, IQR) respectively, with 48 subjects (78.6%) showing ATV values above suggested minimum effective concentration (MEC) of 150 ng/mL. One patient had ATV plasma concentrations below detection limit (11.7 ng/mL). 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184

Twenty-one patients completed the PK study. All were Caucasians, 80% males, mean age (±SD) was 46 (±8.7) years. Median weight and Body Mass Index were 69 kg (62-76) and 23.8 (20.6-26) respectively. Five patients were HCV co-infected with compensated liver disease.

Pharmacokinetic parameters are described in Table 4. Five patients (24%) had ATV Ctrough below 150 ng/ml; no subject had RAL Ctrough below IC95% (15 ng/mL).

No significant difference was noted in patients with or without virological success as for ATV Ctrough or for prevalence of exposure above MEC, presence of more than one ATV RAMS, viral load below 50 copies/mL at baseline, previous virological failure to NNRTIs and PIs. In patients with ATV self-reported non-adherence (n=4) this was significantly associated with virological failure (p=0.02) while longer duration of previous suppression showed a trend towards virological response [33 months (12-68) vs. 18 months (2-35), p=0.07].

DISCUSSION

We report data from the largest cohort of experienced patients (102 patients) treated with ATV 200 mg plus RAL 400 mg, both given bid, with a prolonged time of observation (123 weeks). A consistent number of subjects stopped ATV/RAL (24, 24%) for DDI, toxicity or intolerance issues. Nineteen patients experienced a virological failure. Available genotype showed selection of major INI- and PI-RAMs with newly selected PI-RAMs. In our study, 6/8 failing patients had viral isolates bearing mutations at positions N155 (table 2) which retain “near wild-type” susceptibility to dolutegravir31_.

The rate of selection of RAL-associated RAMs reported in our study is similar to those observed in SPARTAN study [25] highlighting the low virological barrier of this regimen. These findings suggest selecting patients to this unconventional therapy carefully: highly adherent subjects, with history of prolonged viral suppression and not taking concomitant interacting drugs are best candidates. 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208

Favorable safety and tolerability profile of ATV plus RAL was confirmed: only four patients switched for toxicity/intolerance, no serious adverse event was reported. Good lipid profile associated to RAL and ATV was confirmed: after two years of treatment 67 dyslipidemic patients showed significant decrease of triglycerides, total cholesterol and LDL cholesterol. Those were mainly receiving boosted PIs and tenofovir (TDF) was frequently discontinued without noticing lipid abnormalities [32]: raltegravir favorable lipid profile was here confirmed [33].

As for renal function, patients switching with eGFR<60 ml/min (n=27) did not show progression in kidney impairment while a significant reduction in eGFR was observed in patients with baseline levels > 60 ml/min. This worrisome observation may be explained by increasing patients’ age and continuous ATV use, associated with altered renal function and interstitial nephritis [34,35].

Liver test, in particular bilirubinemia, showed no significant change. This may result in better compliance compared to that observed with ATV 300 mg bid in SPARTAN where hyperbilirubinemia and related lower tolerability could have determined high rate of virological failure [25]. Importance of patients’ adherence doesn’t require to be emphasized but, in case of lower drug exposure or drugs’ number reduction an increased compliance may be even more desirable [36]: in this cohort, self-reported non-adherence, although infrequent, was associated with virological failure. More refined instruments for adherence assessing in order to understand the link between such behavior and selection of resistance-associated mutations are warranted.

Concern in using unboosted ATV has been reported due to potential insufficient ATV plasma exposure (below target concentration of 150 ng/mL) when administered with raltegravir. However the entity of DDI on ATV concentrations is lower than the effect reported for TDF [37]. The influence of TDF on ATV concentrations was questioned in clinical setting [38,39] and didn’t lead to failure in observational studies using unboosted ATV [11]. Basing on current knowledge, genetic background may explain a higher part of ATV exposure variability rather than interaction with TDF or raltegravir [40,41]. To ensure adequate ATV exposure we splitted the dose giving ATV 200 mg 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233

bid rather than increasing the dose. This approach optimized drug exposure in a PK study [19] involving 10 HIV infected subjects in unboosted ATV and TDF containing regimen: 60% in ATV 400 mg qd showed C12h lower than MEC, compared to 20% in ATV 200 mg bid. Our results are consistent as 13/61 patients (21.3%) had ATV GM Ctrough lower than MEC. No difference was noted in virological efficacy with ATV Ctrough above or below MEC or 100 ng/mL.

Target RAL concentration predicting virological failure has not been identified [42-44]. This suggests that plasma levels with prescribing dose are higher than needed. Being RAL well tolerated, dose adjustment in case of positive DDI (such as for RAL and ATV) is unrequired. However data in naïve patients advise reduced efficacy with lowest RAL Cmin in patients receiving RAL 800 mg qd but not with RAL 400 mg bid[45]. All our patients had RAL Ctrough above wild type IC95% (RAL GM Ctrough 412 ng/ml, 225-695), with high inter- and intra-patient variability of RAL pharmacokinetic [40] with exposure above target concentrations both for ATV and RAL; RAL AUC GM 0-12 (9085 ng.h/ml) was at least 1,3 folds higher than reported [43,45].

Several limitations must be highlighted: the retrospective study design entailed collecting data from clinical records in selected population after therapy was already started and a control group was not identified. Data recall was limited for some important elements; genotype test was available for certain patients only, follow up was uneven. Costs should be considered: ATV and RAL are among most expensive antiretrovirals in Italy [46].

In conclusion, experienced patients could benefit from change to ATV (200 mg bid) plus RAL as a well-tolerated maintenance option when drugs-related toxicities have to be managed with a NRTI-and RTV-sparing regimen. Nevertheless, not only a concerning rate of patients failed with newly selected RAL and ATV but also a high percentage of subjects stopped study regimen for DDI, intolerance/toxicity issues or were just lost to follow-up. This observation represents an important limitation potentially introducing a major bias.

234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257

Finally, our findings suggest considering ATV/RAL in strictly selected patients showing optimal adherence, long history of virological suppression and without previous PI failure.

ACKNOWLEDGEMENTS

Competing Interests:

L. Marinaro has received travel grants or speaker’s honoraria from Abbvie, Bristol-Myers Squibb (BMS), Merck Sharp &Dohme (MSD) and Janssen-Cilag.

A. Calcagno has received travel grants or speaker’s honoraria from Abbott, Bristol-Myers Squibb (BMS), Boehringer-Inghelheim , Merck Sharp &Dohme (MSD), Janssen-Cilag, Viiv.

M. Rizzi S. has received travel grants or speaker’s honoraria Boehringer-Inghelheim, Bristol-Myers Squibb, Gilead-Sciences, Merck Sharp & Dome, Viiv.

G. Di Perri has received grants, travel grants and consultancy fees from Abbott, Boehringer-Inghelheim, BMS, Gilead-Sciences, GSK, MSD, Pfizer, Roche and Tibotec (Johnson & Johnson). S. Rusconi has received grants, travel grants and consultancy fees from BMS, MSD, GSK, ViiV, Gilead, Janssen, Abbvie, Pfizer.

S. Bonora has received grants, travel grants and consultancy fees from Abbott, Boehringer-Inghelheim, BMS, Gilead-Sciences, GSK, MSD, Pfizer and Janssen-Cilag.

Other authors have no potential conflict of interest to declare.

Funding:

No other sources of funding to declare Ethical approval: Not required 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281

REFERENCES

Markowitz M, Morales-Ramirez JO, Nguyen B-Y, et al. Antiretroviral activity, pharmacokinetics, and tolerability of MK-0518, a novel inhibitor of HIV-1 integrase, dosed as monotherapy for 10 days in treatment-naive HIV-1-infected individuals. J Acquir Immune Defic Syndr. 2006;43:509–515.

44. Grinsztejn B, Nguyen B-Y, Katlama C, et al. Safety and efficacy of the HIV-1 integrase inhibitor raltegravir (MK-0518) in treatment-experienced patients with multidrug-resistant virus: a phase II randomised controlled trial. Lancet. 2007;369:1261–1269.

45. Rizk ML, Hang Y, Luo WL,et al. Pharmacokinetics and pharmacodynamics of once-daily versus twice-daily raltegravir in treatment-naive HIV-infected patients. Antimicrob Agents Chemother. 2012 Jun;56(6):3101-6.

46. Rizzardini G, Restelli U, Bonfanti P, et al. Cost of human immunodeficiency virus infection in Italy, 2007-2009: effective and expensive, are the new drugs worthwhile? Clinicoecon Outcomes Res. 2012;4:245-52.

Reprint requests should be directed to Letizia Marinaro, Department of Infectious Diseases, University of Torino c/o Ospedale Amedeo di Savoia,

C.so Svizzera 164 10159, Torino, Italy Tel +390114393980, fax +39011439388 letizia.marinaro@gmail.com

Table 1. Baseline characteristics of study participants. 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306

Note. Variables are described as median (interquartile range) or number (percentage).

ARV: antiretroviral; PI: Protease Inhibitors; NNRTI: Non Nucleoside Reverse Transcriptase Inhibitors; NRTI: Nucleo-side/-tide Reverse Transcriptase Inhibitors; INI: Integrase Inhibitors

Patients

(n=102)

Gender, male, n (%) 74 (72.5)

Age, years,(IQR) 46.4 (42-53.7)

BMI, Kg/m2_{, (IQR) 24 (22-26)}

Duration of HIV infection, months, (IQR) 160.2 (218.7-86.2)

Chronic HCV hepatitis, n (%) 34 (33.3)

Chronic HBV hepatitis,n (%) 8 (7.8)

Cirrhosis, n (%) 7 (6.9)

Nadir CD4, n/μL (IQR) 186 (84.5-250)

Baseline CD4, n/μL (IQR) 417 (302.5-704)

HIV RNA <50 copies/mL, n (%) 76 (74.5)

HIV RNA (in pts.withHIV RNA>50

copies/mL), copies/mL 525.5 (82.5-10.462)

Previous ARV duration (months) 102 (37-138)

Last ARV: - PI 81 (79.4%) - NNRTI 11 (10.7%) - NRTI 3 (2.9%) - INI 7 (6.8%) 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333

Table 2. Mutations at baseline and at failure.

Pt

BASELINE FAILURE

PI RAMs RAL experienced PI prev failure

Weeks on treatment

PI RAMs RAL RAMs

1 na Yes No 127 na na 2 na No No 117 na na 3 77I No No 22 na na 4 13V, 60E, 62V, 63P, 77I No No 24 13V na 5 na No No 150 36I, 62V, 63P, 93L N155H 6 na No No 74 36I, 88NS, 13V, 60E, 64V No 7 35G, 36I, 62V, 63P No Yes 47 36I, 50L, 60E, 62V, 63P G140GS, N155H 8 16E, 36I, 62V, 63P, 93L Yes Yes 105 na na 9 10V Yes No 59 10V, 13V, 33I, 63P N155H 10 na No No 67 54L, 71I, 62V, 77I, 90M na 11 13V, 62V, 63P, 77I No Yes 133 na na 12 na Yes No 70 na na 13 10I, 13V, 20R, 36I, 53L, 62V, 63P, 71V, 73S, 90M No Yes 110 10I, 13V, 20R, 36I, 53L, 62V, 63P, 71V, 73S, 90M na 14 na No No 36 No 97A 15 na No No 70 No 155H 16 na No No 95 No 148R 17 na No No 140 No 151I, 155H, 63R 18 na No No 176 No No 19 na No No 21 No 155H

PI: protease inhibitors, RAL: raltegravir, RAMs: resistance-associated mutations, “na”: not available. 334 335 336 337 338 339

Table 3. Biochemical analysis at baseline, week 48 and week 96.

Baseline Week 48 Week 96

Median (IQR) Median (IQR) Change from BL p Median (IQR) Change from BL p

Total Cholesterol 193 (153-234) 184 (163.5-219.5) 0 (-23 - +23) 0.8 200 (163.5-223.7) 0.5 (-32.7 - +16.5) 0.3 HDL Cholesterol 41 (33-50) 44.5 (38.7-58) +3 (-1 - +10) 0.01 47 (38-58) +2 (-4 - +7.7) 0.08 LDL Cholesterol 113 (86-140) 110 (95-138.5) 0 (-17 - +15) 0.8 114.5 (97-144) 3 (-29 - +22) 0.7 Triglycerides _(115-221)158 _(83-169.5)123 _{(-89 -+17)}-27 0.01 _(88.2-198)129.5 _{(-83.5- +21.5)}-28.5 0.02 Total Bilirubin _(1-3)1 _(2-3)2 _{(-1 - +1)}0 0.3 _(1-2.9)2 _{(-0.9 - +1)}0 0.5 Indirect Bilirubin 1 (1-2.5) 1 (1-2) 0 (-1 - +1) 0.8 1 (0.7-2) 0 (-1 - +1) 0.9 AST _(22-46)30 _(21-41.5)27 _{(-9.5 - +8)}-1 0.8 _(20.7-44.2)26 _{(-12 - +4)}-3 0.06 ALT 36 (22-59) 33 (18.7-56.2) +1 (-9.2 - +12) 0.6 26 (19-46.2) -4 (-17 - +7.2) 0.04 Fasting Glucose 91 (82-100.5) 89.5 (82-102.7) +0.5 (-0.5 - +9.2) 0.16 90 (84.2-101.7) +5 (-3.7- +12.7) 0.03 Creatinine 0.9 (0.7-1.3) 1.1 (0.8-1.3) 0 (-0.1 - +0.1) 0.9 0.9 (0.7-1.3) +0.03 (-0.07 - +0.17) 0.06

Laboratory tests normality ranges were: LDH (<248 U/L), Total Cholesterol (<190 mg/dL), HDL Cholesterol (>43 mg/dL), LDL Cholesterol (<115 mg/dL), Triglycerides (<180 mg/dL), Total Bilirubin (0.30-1 mg/dL), Indirect Bilirubin (0.1-1 mg/dL), AST (10-31 U/L), ALT (10-31 U/L), GGT (9-40 U/L), Fasting Glucose (75-100 mg/dL), Creatininemia (0.5-0.95 mg/dL). Estimate Creatinine Clearance (eCRCL) was calculated with the Modification of Diet in Renal Disease (MDRD) formula. 340 341 342 343 344 345 346 347 348 349

Table 4. Pharmacokinetic parameters expressed as median (coefficient of variation, in percentage and interquartile range).

C0

(ng/mL) (ng/mL)Cmax (ng/mL)C12 (ng h/mL)AUC0-12 (hours)Tmax half-life(hours)

Atazanavir 305 (60) (55-772) 1062 (65) (202-3944) 227 (76) (31-815) 6257 (56) (894-18605) 2 (2-3) 3.5 (3-5) Raltegravir 426 (99) (56-2223) 2403 (66) (116-8361) 132 (131) (21-1166) 9085 (57) (728-25674) 2 (2-3) 3.5 (3-5)

“C0” and “C12” are plasma concentration before and 12 hours after drug intake; “Cmax” is the

maximal concentration; “AUC0-12” is the area under the curve between 0-12 hours; “Tmax” is the

time to maximal concentration.

Figure 1. Study population, reasons for starting ATV 200 mg bid + RAL 400 mg bid (ATV+RAL) and

treatment outcomes. Data are expressed in numbers (percentage).

351 352 353 354 355 356 357 358 359 360 361 362 363 364 365

366 367