novel method to assess antiretroviral target trough concentrations

1

Novel Method to Assess Antiretroviral Target Trough Concentrations Using In 1

Vitro Susceptibility Data 2

3

Edward P. Acosta1*, Kay L. Limoli2, Lan Trinh2, Neil T. Parkin3, Jennifer R. King1, 4

Jodi M. Weidler2, Ighovwerha Ofotokun4, and Christos J. Petropoulos2 5

6

1 Division of Clinical Pharmacology, University of Alabama at Birmingham School of 7

Medicine, Birmingham, AL; 8

2 Monogram Biosciences, Inc., South San Francisco, CA; 9

3 Data First Consulting Inc., Menlo Park, CA; 10

4 Emory University, Atlanta, GA. 11

12

*Corresponding author: 13

Edward P. Acosta, Pharm.D. 14

University of Alabama at Birmingham, School of Medicine 15

Division of Clinical Pharmacology, 1670 University Blvd., VH 258 16

Birmingham, AL 35294-0019 17

Phone: 205-934-2655 18

Fax: 205-934-6201 19

E-mail: [email protected] 20

Running title: Antiretroviral trough concentrations 21

22

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00691-12 AAC Accepts, published online ahead of print on 10 September 2012

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ABSTRACT 23

Durable suppression of HIV-1 replication requires the establishment of antiretroviral 24

drug concentrations that exceed the susceptibility of the virus strain(s) infecting the 25

patient. Minimum plasma drug concentrations (Ctrough) are correlated with response, but 26

determination of target Ctrough values are hindered by a paucity of in vivo concentration-27

response data. In the absence of these data, in vitro susceptibility measurements, 28

adjusted for serum protein binding, can provide estimations of suppressive in vivo drug 29

concentrations. We derived serum protein binding correction factors (PBCF) for 30

protease inhibitors, non-nucleoside reverse transcriptase inhibitors, and an integrase 31

inhibitor by measuring the effect of a range of human serum concentrations on in vitro 32

drug susceptibility measured with the PhenoSense® HIV assay. PBCFs corresponding 33

to 100% HS were extrapolated using linear regression, and ranged from 1.4 for 34

nevirapine to 77 for nelfinavir. Using the mean 95% inhibitory concentration (IC95) of 35

≥1200 drug-susceptible viruses we calculated protein-bound IC95 (PBIC95) values. 36

PBIC95 values were concordant with the minimum effective Ctrough concentrations that 37

were established in well-designed pharmacodynamic studies (e.g. indinavir, saquinavir, 38

and amprenavir). In other cases, the PBIC95 values were notably lower (e.g. darunavir, 39

efavirenz and nevirapine) or higher (nelfinavir, etravirine) than existing target 40

recommendations. The establishment of PBIC95 values as described here provides a 41

convenient and standardized approach for estimating the minimum drug exposure that 42

is required to maintain viral suppression and prevent the emergence of drug resistant 43

variants, particularly when in vivo concentration-response relationships are lacking. 44

Keywords: HIV-1, susceptibility, protein binding, trough concentration.45


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INTRODUCTION 46

Fully suppressive antiretroviral therapy (ART) for human immunodeficiency virus type 1 47

(HIV-1) infection requires the administration of drug combinations that target multiple 48

sites on one or more proteins required for viral replication. Approved antiretrovirals 49

(ARVs) include nucleoside/nucleotide and non-nucleoside reverse transcriptase 50

inhibitors (NRTIs and NNRTIs), protease inhibitors (PIs), entry inhibitors, and integrase 51

strand-transfer inhibitors (INSTIs). With the exception of the NRTIs, which require 52

intracellular phosphorylation, plasma drug concentrations are correlated with drug 53

efficacy. At the same time, high drug concentrations are associated with excess toxicity. 54

55

To durably suppress HIV replication in infected patients, ARV concentrations must 56

reach and be maintained at levels that exceed the susceptibility of the virus to that drug. 57

Treatment response is often hampered by the failure to achieve sufficient drug exposure 58

(i.e., poor adherence, drug interactions), reduced drug susceptibility (i.e. viral drug 59

resistance), or both. Drug concentrations within patients vary over time and, due to ease 60

of sampling, are generally characterized by minimum (trough) concentrations (Ctrough) 61

immediately prior to administration of the next scheduled dose. Drug concentrations 62

also vary considerably between individual patients as a result of differences in 63

absorption, distribution, metabolism, and excretion. In addition, each drug 64

characteristically binds to human plasma proteins to different extents. Furthermore, the 65

susceptibility of HIV-1 variants, even in patients not previously exposed to drug therapy, 66

varies over a range that is unique to each drug (23, 24, 47). 67

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In vivo clinical pharmacodynamic data are available for some, but not all, ARVs. 69

Efficient collection of these data is difficult and ideally performed early in the drug 70

development process. Alternative methods of incorporating ARV pharmacokinetics into 71

therapeutic decision-making are being explored. In vitro phenotypic drug susceptibility 72

testing of individual patient viruses is now widely available, and generates information 73

that can be used to calculate an inhibitory quotient (IQ), defined as the ratio between 74

the Ctrough and the drug concentration that inhibits in vitro replication by a defined 75

percentage (e.g. IC50 or IC95) (27, 35, 44, 57). Derivatives of the IQ, including the 76

genotypic IQ (GIQ, or Ctrough divided by the number of resistance-associated mutations 77

for a given drug) have also been evaluated (36). Several studies have attempted to 78

define the optimal IQ required to produce long-term viral suppression; in some cases IQ 79

has been retrospectively linked to clinical outcome (15, 34, 42, 43, 56), while in others, 80

direct relationships between IQ and viral load response were not observed (5, 13). 81

82

For most ARV drugs, little to no in vivo concentration-response data have been 83

generated, or these data are inconsistent with clinical observations. Collectively, there is 84

insufficient agreement in the field regarding the determination of the optimal ARV target 85

trough concentrations in the absence of concentration-response data. We conducted 86

the current study to address this deficiency by: 1) assessing the activity of PIs, NNRTIs, 87

and an INSTI in a standardized in vitro phenotypic drug susceptibility assay 88

(PhenoSense® HIV, Monogram Biosciences) in the presence of human serum (HS), 2) 89

establishing drug specific serum protein binding correction factors (PBCFs), and 3) 90


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approximating the optimum target trough concentrations for currently available PIs, 91

NNRTIs, and INSTIs. 92

93

METHODS 94

Determination of drug activity in the presence of human serum 95

The PhenoSense® HIV assay (Monogram Biosciences, South San Francisco, CA) was 96

performed as described (48) with the following modifications. For all PIs except 97

darunavir and atazanavir, each drug was prepared at 10 times the final concentration 98

using complete media containing 10% fetal bovine serum (FBS) without HS. Fifteen 99

microliters (µL) of the 10X drug stocks were mixed with 85 µL of complete media 100

containing 10% FBS and 0%, 25%, 50%, or 75% pooled HIV-negative HS, or 90% HS 101

plus 10% FBS, in 96-well plates. Fifty µL of trypsinized, transfected (virus-producing) 102

cells were added to the plates containing 100 µL drug and media, which had been re-103

suspended in the corresponding media (i.e. with or without HS at 25-90%). Thus the 104

concentration of HS present during virus particle formation was 22.5%, 45.0%, 67.5%, 105

or 81%; FBS was present at 10% for all conditions. Viral stocks were harvested 106

approximately 48 hours after transfection, and 100 µL was used to infect fresh 293 cell 107

cultures (target cells) that had been plated in a volume of 50 µL in media containing 108

10% FBS. During the period of time when darunavir and atazanavir were evaluated, 109

several modifications to the PhenoSense assay were implemented, resulting in subtle 110

differences in final HS concentrations compared to the procedure described above. 111

These changes resulted in final HS concentrations during virus production being 21.7%, 112

43.5%, 65.2%, or 76.5% during virus production. For the purposes of this report these 113


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minor differences (3-6%) in final HS concentration was ignored during analysis. For 114

NNRTIs and raltegravir, virus produced in the presence of the same range in HS 115

concentrations as used for most of the PIs, but in the absence of drugs, was used, and 116

the drugs were added at the time of infection. Three lots of HS (Intergen, Purchase, NY) 117

were used during the course of these experiments. The HS contained an average of 118

0.77 mg/mL alpha-1 acid glycoprotein, and 44.6 mg/mL albumin. This level of alpha-1 119

acid glycoprotein is consistent with values previously reported in HIV-negative subjects 120

(29). 121

122

Approximately 72 hours after infection, target cells were lysed and luciferase activity 123

was measured to assess virus replication in the presence or absence of drugs at each 124

concentration of HS. IC50 and IC95 values were determined as described (48). Between 125

2 and 32 (mean = 8.5) replicates of susceptibility assays were performed for each virus, 126

using either a drug sensitive reference virus (NL4-3) or a well-characterized multi-drug 127

resistant virus (MDRC-4) that exhibits reduced susceptibility to all licensed reverse 128

transcriptase and protease inhibitors. 129

130

Calculation of Protein Binding Correction Factors and Target Trough Concentrations 131

A PBCF corresponding to 45% HS was defined as the fold-increase in experimentally 132

determined IC50 in the presence of 45% HS compared to normal complete medium 133

(10% FBS). The PBCF was calculated within each batch, then averaged across 134

replicates from multiple batches. The PBCF for 100% HS was derived by linear 135

regression analysis (Prism 5.0, GraphPad, San Diego, CA) based on fold-changes in 136


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IC50 in the presence of 22.5%, 45.0%, 67.5%, or 81% HS vs. 0% HS; the PBCF was 137

extrapolated from the Y-intercept when X = 100. R-squared values for the linear 138

regression (log10[IC50] vs. % HS) were between 0.82 and 0.99, except for SQV (0.59) 139

and TPV (0.61). Second and third-order polynomial fitting was inferior for all drugs (data 140

not shown). 141

142

Mean IC50 and IC95 values of wild-type viruses for each drug were derived from the 143

susceptibility measurements of clinical isolates tested between 2009-2010, as compiled 144

in the Monogram Biosciences database. An isolate was defined as wild type in protease 145

(PR) and reverse transcriptase (RT) if drug-selected mutations in PR and RT as 146

previously described (47) were not detected (N=6,537). For raltegravir, drug selected 147

mutations were defined as any change from the wild-type subtype B amino acid 148

sequence at the following positions: H51, T66, E92, F121, G140, Y143, Q146, S147, 149

Q148, S153, N155, and R263 (N=1200). The PR and RT genotypes of these viruses 150

were not available. The protein-bound IC50 or IC95 values (PBIC50 or PBIC95, 151

respectively) were computed by multiplying the mean IC50 or IC95 values by the 152

corresponding PBCF for each drug. All Ctrough values except that for raltegravir are 153

derived from the individual drug manufacturers’ prescribing information (1-3, 6-9, 17, 19, 154

38, 54, 55). The Ctrough for raltegravir was taken from the intensive PK profiles of 155

patients treated with raltegravir twice daily in the QDMRK study (51). 156

157

RESULTS 158


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Drug susceptibility was determined using the PhenoSense assay (48) under standard 159

conditions (10% FBS) and in the presence of 23 to 81% HS. In some cases, because of 160

the cytostatic effects of HS on cultured cells, infectivity (i.e. luciferase signal) was too 161

low to accurately determine the IC50 at 81% HS. Assays were performed using a drug 162

susceptible reference virus (NL4-3) and a well-characterized multi-drug resistant 163

reference virus (MDRC-4). Representative inhibition curves generated using NL4-3 are 164

shown in Figure 1 for a PI (darunavir, Fig. 1A), NNRTI (efavirenz, Fig. 1B), and INSTI 165

(raltegravir, Fig. 1C). IC50 values of all drugs for both the drug-susceptible and resistant 166

viruses at increasing HS concentrations are plotted in Figure 2. As expected, the 167

inhibition curves (Figure 1) shifted toward higher drug concentrations (i.e. to the right) 168

as the percentage of HS increased, although the magnitude of this shift was specific for 169

each drug. For example, the IC50 for saquinavir and tipranavir between 23 and 81% HS 170

remained relatively constant, while the IC50 for most other drugs increased 171

incrementally. Since the relationship between IC50 and HS concentration for most drugs 172

was fairly linear between 23 and 81% HS, the IC50 at 100% HS was estimated using 173

linear regression. 174

175

The ratio between the estimated IC50 at 100% HS (mimicking the in vivo situation) and 176

the IC50 under standard in vitro conditions is referred to as the PBCF. A PBCF for 45% 177

HS can also be calculated using the experimental data. Table 1 lists the PBCF values 178

for 100% HS and 45% HS derived here, along with the 50% HS correction factors 179

derived in a previous study (40), as well as estimations of protein binding derived from 180

in vitro biochemistry studies as reported by the various drug manufacturers. Correction 181


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factors for 45% HS (this work) and 50% HS (40) were highly correlated for the six drugs 182

where both values were available. The PBCF values that were independently 183

determined in both studies also correlated with the magnitude of protein binding for 184

each drug (Table 1). Previously reported PBCFs for atazanavir and amprenavir (13.4 185

and 7.4, respectively) are similar to the values reported here, in spite of the fact that 186

these older studies used only purified AAG and HSA and not complete serum (14, 49). 187

188

To derive in vivo target trough concentrations for patients infected with drug susceptible 189

virus, PBCFs were applied to mean IC50 and IC95 values from clinically-derived HIV-1 190

strains. Over 6500 results from viruses lacking detectable amino acid substitutions that 191

are non-polymorphic and selected under ARV drug pressure (47) were identified in a 192

database of linked phenotypic and genotypic assay results. Fewer results were 193

available for raltegravir (n=1200). The total plasma drug concentration expected to 194

inhibit wild-type virus in vivo by 50% (PBIC50) or 95% (PBIC95) was calculated from 195

these data (Table 2). Although it is not known whether the PBIC50 or PBIC95 most 196

accurately reflects the optimal target trough concentration for these agents, given the 197

considerable inter- and intra-patient variability in Ctrough, a conservative approach is to 198

use the highest of these (i.e. the PBIC95). The corresponding optimal IQ95 (Ctrough 199

divided by PBIC95) for an effective ARV drug should thus be 1 or greater. In fact, this is 200

the case for all of the drugs that we examined except nelfinavir (IQ95=0.26). For 201

comparison, the currently proposed target drug concentrations from the Department of 202

Health and Human Services (DHHS) guidelines (46) are also listed in Table 2. 203

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DISCUSSION 205

The results generated in this study add to the current body of knowledge relating ARV 206

drug target trough concentrations to drug efficacy and more specifically demonstrate the 207

importance of serum protein binding when relating in vitro drug susceptibility 208

measurements to plasma drug concentrations. As expected, the ARV drugs that exhibit 209

the lowest levels of protein binding (indinavir, amprenavir, atazanavir, nevirapine and 210

raltegravir) have the smallest PBCFs (Table 1). Notably, lopinavir also has a relatively 211

low PBCF, despite reports of high (98-99%) serum protein binding. This apparent 212

discrepancy has been reported by others and may be attributed to differential binding 213

properties of bovine serum proteins in the tissue culture medium (26, 28). 214

215

The PBCFs reported here agree well with values previously published by Molla et al. for 216

the subset of PIs that were tested in both studies (40). This earlier study employed a 217

multiple replication cycle inhibition assay using MT4 cells, HIV-1 IIIB, and a colorimetric 218

readout for cytopathic effects. In contrast, our study was conducted using a single cycle 219

inhibition assay, replication defective recombinant viruses, and a luciferase reporter 220

readout (48). Our methodology permits assessments of protein binding effects on drug 221

susceptibility measurements at higher HS concentrations than replication-competent 222

systems because of the shorter amount of time that cells are in contact with high HS 223

concentrations (2-3 days). In most cases, we were able to assess drug susceptibility in 224

the presence of 81% HS, although the luciferase signal was considerably reduced at 225

this high concentration. 226

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Our results for several PIs are also consistent with previously derived in vivo 228

pharmacokinetic/pharmacodynamic relationships, particularly amprenavir, indinavir, and 229

saquinavir. In a dose-finding study of amprenavir, a fitted sigmoid maximum effect 230

(Emax) curve between amprenavir Ctrough and HIV-1 RNA response demonstrated a 231

significant relationship (52). The plasma concentration required to produce 50% of the 232

maximum response (EC50) was 87 ng/mL. By using the reported Hill coefficient, we 233

were able to estimate the EC95 (320 ng/mL) value as well. Current DHHS guidelines 234

recommend an amprenavir target Ctrough of 400 ng/mL (46). Using the PBCF determined 235

in this study, our estimate of amprenavir PBIC95 is 358 ng/mL (Table 2), which is 236

strikingly similar to the recommended Ctrough value. 237

238

In a small subset of pediatric patients receiving indinavir, stavudine, and didanosine, a 239

significant inhibitory Emax relationship between the indinavir Ctrough and change in HIV-1 240

RNA from baseline to week 24 was described (16). The maximum reduction in HIV-1 241

RNA (2.0 log10 copies/mL) corresponded to an EC50 of 80 ng/mL. In a separate study in 242

adult patients treated with indinavir in combination with zidovudine and lamivudine, the 243

indinavir Ctrough levels at week 4 were significantly associated with changes in HIV-1 244

RNA from baseline to week 4 (43). Based on an Emax model, an EC90 was estimated at 245

110 ng/mL. Current DHHS guidelines recommend a target Ctrough of 100 ng/mL (46). We 246

estimated the indinavir PBIC95 to be 73 ng/mL (Table 2), which again is similar to in vivo 247

estimations. 248

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A significant correlation between saquinavir Ctrough and sustained viral load suppression 250

has been observed in children (21). The minimum mean saquinavir Ctrough associated 251

with durable viral load suppression was 200 ng/mL. Additional support for this target 252

comes from the modeling of saquinavir pharmacokinetics/pharmacodynamics following 253

saquinavir monotherapy in HIV-positive patients (18). Following administration of 1200 254

mg saquinavir three times daily (soft gel formulation), both area-under-the-curve (AUC) 255

and Ctrough measures were related to peak reduction in plasma HIV-1 RNA. The median 256

24 hour AUC (AUC24) was 20 µgxh/mL, which corresponded to 85% of the maximum 257

effect (EC85); the Ctrough at this dose was 216 ng/mL (18). Current DHHS guidelines 258

recommend a target Ctrough of 100 to 250 ng/mL (46). We calculated the saquinavir 259

PBIC95 to be 234 ng/mL (Table 2), which is highly concordant with clinically established 260

values. 261

262

There is controversy regarding the optimum Ctrough for efavirenz. The reported 263

therapeutic range (lower to upper exposure targets) for efavirenz is 1,000 to 4,000 264

ng/mL (37). These data come from a study that explored the relationships of EFV 265

exposure with efficacy and development of central nervous system (CNS) side effects. 266

Since the discordance between this pharmacodynamic study and our in vitro results is 267

considerable, it is worth noting that this was an exploratory field trial where all patients 268

received the same EFV dose and not a well-controlled, broad dose-ranging study. In 269

addition, EFV concentrations were determined using blood samples that were collected 270

on average 14 hours post-dosing (range of 8-20 hours). Although EFV has a relatively 271

long half-life, actual Ctrough values were not determined or estimated. Furthermore, 272


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treatment adherence was not considered, which may account for the broad range of 273

EFV concentrations (125-15,230 ng/mL) that were observed. Mutations in cytochrome 274

P450 (CYP) 2B6 are also associated with EFV exposure (22), but in the absence of 275

race/ethnicity data, it is not possible to infer the contribution of genetics to the inter-276

patient variability in EFV concentrations in the study. Also, as discussed by the authors, 277

there was considerable overlap in EFV concentrations among patients that experienced 278

treatment failure vs. those that did not. Lastly, drug concentrations were determined 3 to 279

18 months after initiating EFV therapy - well past the time at which EFV-associated 280

neurologic symptoms have been correlated to plasma concentrations (10). Our 281

estimation of the PBIC95 for efavirenz is 126 ng/mL, which is well below the average 282

Ctrough of 1800 ng/mL in patients receiving 600 mg once daily and below the proposed 283

EFV lower concentration threshold (1000 ng/mL). The decay rate of EFV relative to its 284

PBIC95 in one patient who discontinued treatment with EFV, stavudine, and lamivudine 285

has been described (50). In this patient, plasma HIV-1 RNA was <50 copies/mL at the 286

time of treatment discontinuation and viral rebound was detected 21 days later; 287

coincident with the time that the EFV plasma concentration fell below 50-100 ng/mL. 288

Although these data represent a single patient, they suggest that the target EFV Ctrough 289

may be well below 1000 ng/mL and are consistent with our estimation of EFV PBIC95 290

described here. In light of these findings, re-examination of the generally accepted 291

therapeutic concentration range of 1000-4000 ng/mL for EFV seems warranted. 292

293

In vivo exposure-response relationships for a number of the ARVs are not well defined. 294

Suggested Ctrough targets for some of these agents are based either on in vitro 295


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susceptibility data alone or studies that compare plasma drug concentrations to virologic 296

outcomes (30, 31). For example, in HIV-NAT 017, virologic outcome was assessed in 297

PI-naïve children treated with lopinavir/ritonavir (230/57.5 mg/m2) plus saquinavir (50 298

mg/kg) twice daily (4). The 2 children with lopinavir Ctrough < 1000 ng/mL both 299

experienced virologic failure (HIV RNA >400 copies/mL) at week 24, compared to a 300

single virologic failure among the remaining 17 children with lopinavir Ctrough >1000 301

ng/mL. This finding suggests that the target Ctrough of lopinavir is approximately 1000 302

ng/mL in PI-naïve patients. Similarly, plasma drug concentrations and the risk of 303

virologic failure was evaluated in a cohort of 189 HIV-infected, NNRTI-naïve patients 304

treated with standard dose nevirapine in combination with other agents (11). In 305

multivariate analysis, a nevirapine plasma concentration less than 3000 ng/mL was 306

associated with a 5-fold increased risk of virologic failure, suggesting that a nevirapine 307

Ctrough of 3000 ng/mL or greater should be maintained to obtain optimum virologic 308

outcomes. Using similar approaches, target Ctrough values of 150 ng/mL and 20,500 309

ng/mL, respectively, have been proposed for ritonavir-boosted atazanavir (atazanavir/r) 310

and tipranavir (tipranavir/r) (20, 45); however neither study was sufficiently powered to 311

define threshold therapeutic concentrations. These values are higher than the PBIC95 312

measures that we determined in this study (60 ng/mL and 3902 ng/mL, respectively; 313

Table 2). One possible explanation is related to the use of cohort studies, which are 314

likely to include intermittently adherent patients, to define minimum effective Ctrough 315

values for antiretroviral drugs. Unlike carefully conducted, wide dose ranging 316

pharmacokinetic/pharmacodynamic studies, target drug concentrations estimated from 317

cohort studies are based on variations in inter-patient concentrations for a given dose 318


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administered to many individuals. Consequently, this approach generates insufficient 319

data to accurately populate the low end of a true concentration-response curve, and will 320

tend to over-estimate the threshold drug concentrations. 321

322

The PBIC95 calculated for nelfinavir (3865 ng/mL) in this study is considerably higher 323

than the measured (1000 ng/mL) or recommended target (800 ng/mL) Ctrough (46). This 324

discrepancy may be at least partly explained by the observation that nelfinavir is 325

metabolized in the liver, producing metabolites including M8 (32), which itself exhibits 326

antiviral activity comparable to the parent drug (58). M8 concentrations approach 30% 327

of the parent drug concentration, and this metabolite exhibits slightly less serum protein 328

binding than nelfinavir (58). Since nelfinavir is not metabolized to M8 in vitro, our 329

estimate of nelfinavir PBIC95 in this study is likely higher than the true in vivo value, and 330

thus the effective IQ95 is likely to be underestimated. 331

332

The IQ95 for raltegravir (1.2) is lower than expected for a highly efficacious drug. A 333

recent presentation reported a median plasma Ctrough in healthy volunteers of 82 ng/mL 334

(n=45), which agrees well with our estimates of PBIC95 but is 30% higher than the value 335

reported in the drug label (33). In vitro studies have shown that dissociation of 336

raltegravir from the pre-integration complex occurs more slowly than dissociation of the 337

pre-integration complex itself, meaning that raltegravir binding is essentially irreversible 338

(25). Results from a 48-week, once daily (800 mg) vs. twice daily (400 mg) raltegravir 339

study (51) showed that the once daily arm was virologically inferior to the twice daily 340

arm. The average C12h, derived from intensive pharmacokinetic assessments, was 257 341


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nM (114 ng/mL) in the 400 mg twice daily group vs. 40 nM (17.8 ng/mL) in the 800 mg 342

once daily group. Our results indicate that the raltegravir PBIC95 is 94 ng/mL (211 nM), 343

which may in part explain why the once daily group was inferior. Relationships between 344

Ctrough and HIV-1 RNA response for two additional INSTI candidates have been 345

demonstrated in wide dose-ranging early phase monotherapy trials (12, 53). 346

347

The application of in vitro susceptibility data to in vivo PBIC95 predictions has several 348

limitations. The HS conditions used in the infectivity assays described here may not 349

accurately mimic the situation in vivo, i.e. drug binding in human blood. Inter- and intra-350

individual variability in binding protein concentration encountered in vivo are not taken 351

into account using this approach. However, our study was performed using two lots of 352

HS, each pooled from multiple donors, to minimize the possibility of lot-specific artifacts. 353

In addition, in vitro determinations of IC50 or IC95 values for individual drugs do not 354

capture synergistic, antagonistic or additive effects among different drugs that may 355

occur in vivo. Furthermore, the use of a simple linear regression model to extrapolate 356

PBCFs for 100% HS from the experimentally-determined IC50 values in the presence of 357

23 - 81% HS may not be equally applicable to all drugs. Most importantly, there is a 358

paucity of in vivo pharmacodynamic data with which our results can be validated. 359

360

Collectively, the available in vivo concentration-response data, which are considered the 361

gold standard from the pharmacodynamic perspective, correlate strikingly well with the 362

PBIC95 values we have derived from in vitro susceptibility and PBCF measurements, 363

with the exceptions of the cohort studies described above. Consequently, we propose 364


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that this parameter is an appropriate surrogate measure of target Ctrough for antiretroviral 365

drugs (excluding those that require intracellular activity, i.e. NRTI) in lieu of formal early-366

phase pharmacodynamic assessments. Ideally, concentration-response relationships 367

for ARV drugs should be defined early in the development process. Phase III trials (or 368

Phase IV post-marketing studies) are not the appropriate time to define drug target 369

concentrations because their design requires that all participants either receive one 370

given dosage or one of several dosages within a narrow range. Late Phase I or early 371

Phase II trials should incorporate wide dose-finding designs encompassing dosages 372

that exceed 10-fold differences (preferably larger) in order to adequately populate 373

concentration-response curves. In the absence of clearly defined concentration-374

response relationships, identifying precise target drug exposure is not possible. 375

376

The derivation of PBCFs that can be applied to IC50 data generated by routine 377

phenotypic assays such as PhenoSense HIV also provides a useful way to relate these 378

in vitro concentrations to total in vivo drug exposure for applications including 379

therapeutic drug monitoring and calculation of IQ values for viruses with reduced drug 380

susceptibility. 381

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ACKNOWLEDGMENTS 383

We gratefully acknowledge the contributions of the Monogram Clinical Reference 384

Laboratory for performing PhenoSense and GeneSeq assays, and Mojgan Haddad 385

(Monogram Biosciences) for assistance with retrieval of IC50 and IC95 data from the 386

Monogram database and regression analysis. Development of the Monogram 387

PhenoSense and GeneSeq Integrase assays was supported by a grant from the 388

NIH/NIAID, SBIR-AT 5 R44 AI057074. EA is the recipient of NIH grant R01 AI05869. 389

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combination with ritonavir administered at 600 and 100 milligrams, respectively, every 559

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Raritan, NJ. 578

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57. Winston, A., and S. Khoo. 2008. Clinical application of the inhibitory quotient: is there a 584

role in HIV protease inhibitor therapy? Curr Opin HIV AIDS 3:608-11. 585

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immunodeficiency virus protease inhibitor nelfinavir in humans: structural 588

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45:1086-93. 590

591

592 593


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FIGURE LEGENDS 594

595

Figure 1. Effect of human serum on in vitro drug susceptibility. Representative 596

PhenoSense® inhibition curves showing the effect of adding human serum at 22.5% 597

(red diamonds, solid line), 45.0% (green diamonds, solid line), or 67.5% (red squares, 598

dotted line), compared to standard (10% FBS) conditions (blue squares, dotted line). 599

The drug susceptible (NL4-3) reference virus was used. A: darunavir (DRV); B: 600

efavirenz (EFV); C: raltegravir (RAL). Vertical dotted lines denote the IC50. 601

602

Figure 2. Relationship between ARV drug IC50 and human serum concentration. 603

Mean IC50 values from replicate susceptibility assays +/- SD are shown. A: susceptibility 604

of the drug susceptible (NL4-3) reference virus to protease inhibitors (PIs); B: 605

susceptibility of the multi-drug resistant (MDRC-4) reference virus to PIs. Results for 606

nelfinavir with MDRC-4 are not shown since human serum concentrations shifted the 607

IC50 beyond the dynamic range of the assay. APV: amprenavir (closed circles, solid 608

line); ATV: atazanavir (open squares, solid line); DRV: darunavir (closed triangle, dotted 609

line); IDV: indinavir (closed diamonds, solid line); LPV: lopinavir (open circles, dotted 610

line); NFV: nelfinavir (filled squares, dotted line); SQV: saquinavir (open triangles, 611

dotted line); TPV: tipranavir (open diamonds, solid line). C: susceptibility of the drug 612

susceptible (NL4-3) reference virus to NNRTIs and raltegravir; D: susceptibility of the 613

multi-drug resistant (MDRC-4) reference virus to NNRTIs. Since the multi-drug resistant 614

virus contains the wild-type integrase region, the results for this virus with raltegravir are 615


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included in panel C. EFV: efavirenz (filled squares); ETR: etravirine (filled triangles); 616

NVP: nevirapine (filled circles); RAL: raltegravir (open circles). 617


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Table 1. Protein Binding Correction Factors. 618

Protein Binding Correction Factors

Druga

45% HS, this

workb

100% HS, this

workc 50% HS, Molla et al.(41)d % Bounde

APV 4.8 ± 2.1 11 ± 0.6 8.5 ± 3.3 90%

ATV 5.0 ± 1.1 9.3 ± 0.2 nd 86%

DRV 6.5 ± 1.3 14 ± 0.2 nd 95%

IDV 2.0 ± 0.7 3.5 ± 0.07 2.2 ± 1.1 60%

LPV 5.5 ± 1.8 9.9 ± 1.0 8.8 ± 4.3 98 - 99%

NFV 27 ± 14 77 ± 8.5 39 ± 11 >98%

RTV 12 ± 5.9 20 ± 3.9 25 ± 10 98 - 99%

SQV 14 ± 10 17 ± 6.8 34 ± 14 98%

TPV 11 ± 4.2 15 ± 2.7 nd >99.9%

EFV 11 ± 3.6 27 ± 1.2 nd 99.5 - 99.75%

ETR 13 ± 4.2 33 ± 2.7 nd 99.9%

NVP 1.3 ± 0.3 1.4 ± 0.1 nd 60%

RAL 1.5 ± 0.4 2.1 ± 0.03 nd 83% a APV: amprenavir; ATV: atazanavir; DRV: darunavir; IDV: indinavir; LPV: lopinavir; 619

NFV: nelfinavir; SQV: saquinavir; TPV: tipranavir; EFV: efavirenz; ETR: etravirine; 620

NVP: nevirapine; RAL: raltegravir. 621

b 45% HS values (actual) represents averages ± SD derived from 3 to 6 assays 622


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c 100% HS values (extrapolated) represents averages ± SE derived from linear 623

regression 624

d mean ± SD of ratios between 50% HS and control for all viruses listed in Table 1 from 625

ref (41). nd, not done. 626

e All percent bound values are from product prescribing information for each drug (1-3, 627

6-9, 17, 19, 38, 39, 54, 55). 628

629


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Table 2. Protein-bound IC95 and IQ95 Values 630

Druga Dose, mgb

MW

(g/mol)

Ctrough

(ng/mL)c PBCFd

WT IC50

(nM)e

WT IC95

(nM)e

WT IC50

(ng/mL)

WT IC95

(ng/mL)

PBIC50

(ng/mL)

PBIC95

(ng/mL)

DHHS

target46

(ng/mL)

IQ95

(Ctrough/PBIC95)

APV 700/100 bid 505 2120 11 10 62 5.3 31 60 358 400 5.9

ATV 300/100 qd 705 800 9.3 2.4 9.2 1.7 6.5 15.9 60 150 13

DRV 600/100 bid 548 3500 14 0.71 3.4 0.4 1.9 5.3 25 naf 138

IDV 800/100 bid 614 1300 3.5 7.0 34 4.3 21 15.2 73 100 18

LPV 400/100 bid 629 5500 10 5.0 27 3.1 17 31 168 1000 33

NFV 1250 bid 664 1000 77 16 75 11 50 819 3865 800 0.26

SQV 1000/100 bid 767 400 17 4.7 18 3.6 14 60 234 100-250 1.7

TPV 500/200 bid 603 15,670 15 88 433 53 261 796 3902 naf 4.0

EFV 600 qd 316 1800 27 4.2 15 1.3 4.7 36 126 1000 14

ETR 200 bid 435 297 33 2.2 8.2 0.9 3.5 31 116 naf 2.6

NVP 400 qd 266 4500 1.4 120 952 32 253 46 366 3000 12

RAL 400 bid 444 114 2.1 8.0 100 3.6 44 7.5 94 na 1.2

a APV: amprenavir; ATV: atazanavir; DRV: darunavir; IDV: indinavir; LPV: lopinavir; NFV: nelfinavir; SQV: saquinavir; 631

TPV: tipranavir; EFV: efavirenz; ETR: etravirine; NVP: nevirapine; RAL: raltegravir. 632


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b prescribed dose and frequency (bid: twice daily; qd: once daily); for protease inhibitors other than NFV the amount of co-633

dosed with ritonavir is indicated after the slash. 634

c all Ctrough values are taken from the individual drug manufacturer’s prescribing information (1-3, 6-9, 17, 19, 38, 54, 55) 635

except raltegravir which was from reference (51). 636

d protein binding correction factor at 100% HS, extrapolated from experimental data described above 637

e wild-type (WT) virus IC50 and IC95 data are the means of a large collection of independent determinations from clinical 638

samples lacking drug-selected mutations in PR or RT (for PIs and NNRTIs, n=6537) or IN (for RAL, n=1200). 639

f not applicable: no target drug concentration has been determined for PI naïve patients; for PI-experienced patients, the 640

recommended Ctrough values for darunavir, tipranavir, and etravirine are 550, 20, 500, and 52 ng/mL, respectively (46) 641


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drug concentration (nM)

% in

hib

itio

n

40

60

80

100

0

20

1.00.10.01 10 100

B. EFV


% in

hib

itio

n40

60

80

100

-40

-20

0

20

1.0

0.1

0.01 10 100 1000

A. DRV

40

60

80

100

0

20

1.00.1 10 100 1000


% in

hib

itio

n

C. RAL

0.001

0.1


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Drug Sensitive Virus

0 20 40 60 80 1000.1

1

10

100

1,000

10,000APV

ATV

DRV

LPV

NFV

IDV

TPV

SQV

Percent Human Serum

IC50 (nM)

0 20 40 60 80 1001

10

100

1,000

10,000EFV

NVP

ETR

RAL

Percent Human Serum

IC50 (nM)

Drug Resistant Virus

0 20 40 60 80 1000.1

1

10

100

1,000

10,000

Percent Human Serum

IC50 (nM)

0 20 40 60 80 1001

10

100

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