Preclinical Pharmacokinetics and Tissue Distribution of Long-Acting ...

1

Preclinical Pharmacokinetics and Tissue Distribution of Long-Acting 1

Nanoformulated Antiretroviral Therapy 2

Nagsen Gautam1, Upal Roy2, Shantanu Balkundi2, Pavan Puligujja2, Dongwei Guo2, 3

Nathan Smith2, Xin-Ming Liu2, Benjamin Lamberty2, Brenda Morsey2, Howard S. Fox2, 4

JoEllyn McMillan2, Howard E. Gendelman2, and Yazen Alnouti1 # 5

6

1 Department of Pharmaceutical Sciences, College of Pharmacy, University of 7

Nebraska Medical Center, Omaha, NE 68198-6025, USA 8

2 Departments of Pharmacology and Experimental Neuroscience and, University of 9

Nebraska Medical Center, Omaha, NE 68198-5215, USA 10

Running title: Pharmacokinetics of nanoformulated antiretrovirals 11

12

# Corresponding author: 13

Yazen Alnout i 14

Department of Pharmaceutical Sciences, College of Pharmacy 15

University of Nebraska Medical Center 16

986025 Nebraska Medical Center, Omaha, NE 68198-6025 17

Phone: 402-559-4631 18

Fax: 402-559-9543 19

E mail: [email protected] 20

21

22

Copyright © 2013, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00267-13 AAC Accepts, published online ahead of print on 22 April 2013

on January 31, 2018 by guesthttp://aac.asm

.org/D

ownloaded from

http://aac.asm.org/

2

Abstract 23

Long-acting injectable nanoformulated antiretroviral therapy (nanoART) was 24

developed with the explicit goal of improving medicine compliance and for drug 25

targeting of viral tissue reservoirs. Prior nanoART studies completed in humanized 26

virus-infected mice demonstrated sustained antiretroviral responses. However, the 27

pharmacokinetic (PK) and tissue distribution of nanoART were not characterized. To 28

this end, PK and tissue distribution of nanformulated atazanavir (ATV) and ritonavir 29

(RTV) injected subcutaneously or intramuscularly in mice and monkeys were 30

evaluated. Fourteen days after injection, ATV and RTV levels were up to 13-, 41- and 31

4500- fold higher than those resulting from native drug administration in plasma, 32

tissues, and at the site of injection, respectively. At 10, 50, 100, and 250 mg/kg 33

nanoART doses, more and less than proportional increases in plasma and tissue levels 34

with dose relationships were demonstrated with ATV and RTV. Multiple-dose 35

regimens showed serum and tissue concentrations up to 270-fold higher than native 36

drug throughout eight-weeks of study. Importantly, nanoART was localized in non-37

lysosomal compartments in tissue macrophages creating intracellular depot sites. 38

Reflective data were obtained in representative rhesus macaque studies. We conclude 39

that nanoART demonstrates enhanced blood and tissue antiretroviral drug levels over 40

native drugs. The sustained and enhanced PK profile of nanoART, at least in part, is 41

the result of the sustained release of ATV and RTV from tissue macrophases and at 42

the site of injection. 43

44

45


.org/D

ownloaded from

http://aac.asm.org/

3

Introduction 46

The development of effective antiretroviral therapy (ART) has transformed 47

human immunodeficiency virus (HIV) disease into a long-term and manageable 48

disorder (1). Infected patients can live well past their fifth, sixth and seventh decades 49

of life (2). The shortcomings for ART are, notably, associated with viral resistance and 50

rebounds that may occur despite long periods of undetectable virus in blood (3). This 51

can occur despite the presence of therapeutic plasma drug levels, which is attributed to 52

activation of latent virus hidden in anatomical and intracellular reservoirs (4). As ART 53

regimens are commonly ineffective in reaching viral sanctuaries, viral replication occurs 54

continuously at low levels as a result of ineffective antiretroviral penetrance into viral 55

reservoirs including the lymphoid and central nervous system reservoirs (5), which 56

allows HIV to circumvent eradication (6, 7). Thus, life-long treatment is needed in 57

order to suppress the virus and to enable the patient to remain clinically asymptomatic 58

(8). 59

Patients’ compliance with medications intake plays yet another crucial role for 60

disease management (9), which becomes even more challenging with complex ART 61

regimens that require long-term adherence for often substantive pill burdens (10). 62

Drug-regimen compliance has also been identified as a critical risk factor for viral 63

resistance (11, 12). In attempts to overcome such limitations, long-acting 64

nanoformulated ART (nanoART) was developed to achieve steady state drug levels 65

with infrequent dosing (13, 14). Moreover, as HIV-infected individuals serve as 66

vehicles for viral transmission, long acting formulations could also serve to decrease 67

viral spread (15, 16). As ART leads to spectrum of toxicities and drug-drug interactions 68


.org/D

ownloaded from

http://aac.asm.org/

4

causing added disease morbidities, these may be overcome by improved viral 69

suppression and reduced toxicities through nanoART (17). 70

While previous reports demonstrated that mononuclear phagocytes (MP; 71

monocytes and macrophages) can act as reservoirs and transporters of HIV-1, these 72

cells could also potentially serve to facilitate drug uptake, transport, and release of 73

nanoART (18-21). Using MP cell culture systems, it was shown that uptake and 74

release of nanoART into and from monocyte-derived macrophages (MDM) are 75

sustained at levels equal to or beyond the effective concentrations (EC50) with limited 76

cytotoxicity (22). This was achieved after optimizing the shape, size, and charge of the 77

nanoparticles for cell entry and release of two commonly administered protease 78

inhibitors, atazanavir (ATV) and ritonavir (RTV) (23, 24). After passing in vitro 79

screening, ATV and RTV nanoformulations were selected for in vivo pharmacokinetic 80

(PK) studies (13). While the pilot efficacy studies in humanized virus-infected mice 81

demonstrated effective and sustained antiretroviral responses, dosing regimens and 82

tissue and cell biodistributions remained incomplete (13, 14, 25). To these ends, we 83

characterized the pharmacokinetics and biodistribution of nanoART in mice and in 84

monkeys after subcutaneous (SC) administration. Results showed clear improvements 85

in the PK profile over native (unformulated) drug at various dosing regimens. 86

Intracellular nanoART reservoirs associated with endosomal MP compartments (23) in 87

tissues paralleled what had previously been demonstrated in in vitro studies (18) and 88

resulted in sustained and enhanced systemic drug levels in vivo. These preclinical 89

studies may further enable the development of nanoART for clinical intervention (26, 90


.org/D

ownloaded from

http://aac.asm.org/

5

27). Such formulations would provide advantages in PK properties and patient 91

compliance over what is now established by conventional native drug regimens (9). 92

Materials and methods 93 94 Chemicals 95

Free base RTV was obtained from Shengda Pharmaceutical Co. (Zhejiang, 96

China). ATV-sulfate was purchased from Gyma Laboratories of America Inc. 97

(Westbury, NY, USA). Lopinavir (LPV) was purchased from Toronto Research 98

Chemicals Inc. (North York, Ontario, Canada). HPLC-grade methanol, acetonitrile, 99

ammonium acetate, acetic acid, propylene glycol and phosphate buffered saline- 1X 100

were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Isoflurane was obtained 101

from Halocarbon Product Corporation (River Edge, NJ, USA). BD- 28G-1/2 insulin 102

syringes were obtained from Becton Dickinson and Company (Franklin Lakes, NJ, 103

USA). Cremophore EL and poloxamer 188 (P188) were obtained from Sigma-Aldrich 104

(St. Louis, MO, USA) and ethyl alcohol from Acros Organics (NJ, USA) 105

Preparation and characterization of nanoART 106

NanoART RTV and ATV were prepared with polymer excipients by high-107

pressure homogenization as described previously (28). These formulations consisted 108

of crystalline drug surrounded by a thin layer of a P188 surfactant (29). Drug loading 109

was analyzed by high performance liquid chromatography (HPLC-UV) [11] and by 110

ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) 111

(30). These formulations were screened for cell uptake, retention, release and 112


.org/D

ownloaded from

http://aac.asm.org/

6

antiretroviral activity using human monocyte-derived macrophages as described 113

previously (18, 22, 28). 114

Mouse studies 115

Eight-week-old, healthy male Balb/C mice were purchased from Charles River 116

Laboratories (Wilmington, MA). Sterilized 7012 Teklad diets (Harlan, Madison, WI) 117

were used for mice, and water was provided ad libitum. Mice were housed in the 118

University of Nebraska Medical Center (UNMC) laboratory animal facility according to 119

the American Animal Association and Laboratory Animal Care guidance. All 120

procedures were approved by the Institutional Animal Care and Use Committee at the 121

University of Nebraska Medical Center (UNMC) at set forth by the National Institutes of 122

Health (NIH). 123

(A) Acute single- and multiple- dose administration 124

Acute single- and multiple-dose PK studies were performed at 10 mg/kg of 125

either nanoART or native (unformulated) ATV and RTV. ATV and RTV nanoART were 126

manufactured separately and the two formulations were mixed together before 127

administration to animals. The acute dose study encompassed a single dose (day 0), 128

and three-dose (days 0, 3, and 7) administration. Mice receiving the single dose were 129

sacrificed on day 1 and day 14, whereas mice receiving the three doses were 130

sacrificed on day 7 (before the administration of the 3rd dose on day 7) and on day 14. 131

Each group consisted of five mice. NanoART doses were suspended in phosphate 132

buffered saline (PBS) the native drug doses were suspended in a mixture of ethanol-133

Cremophor EL- propylene glycol-water (43-5-20-32 v/v). The injection volume was a 134

125 μl for both nanoART and native drug. Blood samples were collected at 0.5, 1, 2, 4, 135


.org/D

ownloaded from

http://aac.asm.org/

7

8 hrs and days 1, 2, 3, 7, 10, and 14. Tissue samples of liver, kidneys, spleen, lungs, 136

brain, and skin from the site of injection (~100 mg) were collected on days 1, 7, and 14. 137

In addition, dose-escalation studies were performed at 10, 50, 100, and 250 138

mg/kg nanoART doses with equivalent designs of dose administration and sample 139

collection as described above for the acute multiple-dose study. 140

(B) Chronic-Dose Administration 141

The-chronic pharmacokinetic studies were performed at 50 mg/kg nanoART or 142

with native (unformulated) drugs. Eight groups of mice (N=6) were dosed with either 143

nanoART or native drug, eight doses each, on days 0, 3, 7, 14, 21, 28, 35, 42 (3 doses 144

in the 1st week, followed by weekly dosing for another 5 weeks), over a period of six 145

weeks. Blood samples were collected on days 1, 3, 7, 8, 10, 14, 15, 17, 21, 22, 24, 28, 146

29, 31, 35, 36, 38, 42, 43, 45, 49, 50, 52, and 56. Every week, one group of mice from 147

each arm of the study was sacrificed and tissues including liver, kidneys, spleen, lung, 148

brain, and skin from the site of injection were collected. After dosing, mice were 149

returned to their home cage, and cage-side observation was performed on the day of 150

dosing and at least daily for the remainder of the study. 151

Monkey Studies 152

Rhesus macaques were purchased from PrimGen (Hines, IL), and tested 153

negative for SIV, SRV-type D and Cercopithecine herpesvirus 1 virus. All protocols 154

and procedures were performed under approval of the Institutional Animal Care and 155

Use Committee of UNMC following NIH guidelines. 156

Rhesus macaques were used as a non-human primate (NPH) model. Animals 157

were anesthetized with 10 mg/kg of ketamine, administered intramuscularly, prior to 158


.org/D

ownloaded from

http://aac.asm.org/

8

experimental procedures and bleeding. Blood was drawn from the femoral vein, and 159

plasma was obtained by centrifugation of EDTA-treated blood. Pharmacokinetic 160

studies were performed at 50 mg/kg dose of nanoART in animals weighing 3.5 kg 161

(N=2). Prior to injection, 10mg/ml nanoART was suspended in sterile PBS. NanoART 162

was injected as a subcutaneous bolus into the nape of the neck. One monkey was 163

given a single dose of 50 mg/kg dose of nanoART (day 0), while a second monkey was 164

given three-doses (days 0, 3, and 7) of 50 mg/kg dose of nanoART. 165

Blood and tissue collection 166

For mice, blood samples (100 µl) were collected from the facial vein using a 167

sterile 0.5 mm goldenrod animal lancet (MEDIpoint, Inc., Mineola, NY). Blood drops 168

were collected into serum separator tubes (BD Microtainer Tubes). Serum was 169

separated by centrifugation of blood samples at 1500 × g for 10 min at 4°C within one 170

hr of sample collection and stored at -80°C until analysis. Tissue samples were stored 171

at -80 ºC until analyzed by LC-MS/MS. For monkeys, blood samples were collected 172

from the femoral vein using a 21G syringe, into an EDTA-treated tube on days 3, 7, 10, 173

14 and 38 after dose administration. Plasma was separated by centrifugation at room 174

temperature for 20 minutes at 900 × g and stored at -80°C until analysis by LC-MS/MS. 175

Sample preparation and analysis 176

Serum, plasma and tissue sample preparation and analysis were performed as 177

previously described (30). Briefly, about a 100 mg of tissues of interest were 178

homogenized in deionized H2O (1:4 (w/v)). One mL of ice-cold acetonitrile was added 179

to 100 µL serum or tissue homogenate samples pre-spiked with 10 µL internal 180


.org/D

ownloaded from

http://aac.asm.org/

9

standard (IS: 2.0 µg/mL lopinavir, 200 ng/ml final concentration). Samples were 181

vortexed for 3 min, shaken continuously for 15 min, and centrifuged at 16,000 × g for 182

10 min. The supernatant was aspirated, evaporated under vacuum at room 183

temperature, reconstituted in 100 µL of 50% methanol in H2O, and sonicated for 5 min. 184

After centrifugation at 16,000 × g for 10 min, 10 µL of each sample was used for LC–185

MS/MS analysis using a Waters ACQUITY UPLC (Waters, Milford, MA) coupled to an 186

Applied Biosystems 4000 QTRAP quadrupole ion trap hybrid mass spectrometer 187

(Applied Biosystems, Foster City, CA). For drug analysis in liver cells, each cell 188

fraction was suspended in H2O to a final cell concentration of 10 million/ml. A 100 µl 189

aliquot of each cell suspension pre-spiked with 10 µl internal standard was extracted as 190

described for serum. 191

Pharmacokinetic analysis 192

Mean serum drug concentrations were calculated per treatment group for 193

different doses. The pharmacokinetic parameters were derived using 194

noncompartmental analysis of averaged serum concentration vs. time profiles, using 195

WinNonlin Professional software (version 5.1). Peak serum concentration (Cmax), time 196

to reach Cmax (Tmax), and area under the serum concentration versus time curve (AUC) 197

were calculated. Mean tissue concentrations were calculated and expressed as ng/g 198

tissues. 199

NanoART cell localization studies 200

(I) Labeling nanoformulations 201


.org/D

ownloaded from

http://aac.asm.org/

10

CF633-labeled P188 was synthesized for the preparation of fluorescence-202

labeled nanoART. Briefly, P188 was activated with an eight-fold molar ratio of p-203

toluenesulfonyl chloride in dichloromethane (DCM; Acros Organics, Thermo Fisher 204

Scientific, Waltham, MA) at room temperature, and purified by ether precipitation. The 205

tosylated product was converted to azido-P188 by incubation with a six-fold molar ratio 206

of sodium azide in N,N-dimethylformamide (Sigma-Aldrich) at 100°C for 6 h, and 207

purification with DCM/saline extraction. Azido-P188 was then reduced to amine-P188 208

using a four-fold molar ratio of triphenylphosphine in tetrahydrofuran at room 209

temperature, and purified by ether precipitation. Polymers from all steps were further 210

purified using a Sephadex LH-20 column (GE Healthcare, Waukesha, WI) before the 211

next reaction step. Finally, amine-P188 was reacted with the succinimidyl ester of 212

CF633 (Sigma-Aldrich) in dimethyl sulfoxide at room temperature. The crude product 213

was purified with a Sephadex LH-20 column to remove free CF633. A mixture 214

consisting of 30% CF633-labeled P188 and 70% P188 was used to manufacture 215

fluorescence-labeled nanoART using high-pressure homogenization as described (28). 216

(II) Animals, liver cell isolations and flow cytometric tests 217

Eight-week old male Balb/cJ mice (Jackson Labs, Bar Harbor, ME) were 218

administered 250 mg/kg each ATV/RTV as CF633-labeled nanoART by SC injection. 219

Twenty-four hours later liver cells were isolated by in situ collagenase perfusion of 220

mouse liver using a modification of previously published methods (31-33). Isolated 221

cells were dispersed and washed in Krebs-Ringer bicarbonate buffer (Sigma-Aldrich) 222

containing 100 mM HEPES (KRH), 2 mM Ca++ and 2% bovine serum albumin (BSA). 223

Hepatocytes were separated from non-parenchymal cells by centrifugation at 50 x g for 224


.org/D

ownloaded from

http://aac.asm.org/

11

5 min. The resulting cell pellet was resuspended in KRH/2 mM Ca++/2% BSA. The cells 225

were counted and viability determined by trypan blue exclusion. Drug content in the 226

cells was determined using LC-MS/MS. The 50 x g supernatant was centrifuged at 227

600 x g for 5 min and the resulting cell pellet was resuspended in 1 ml KRH/ 2 mM 228

Ca++ /2% BSA. The cells were counted and viability determined by trypan blue 229

exclusion. The cell suspension was centrifuged at 400 x g for 5 min and the pellet 230

resuspended in 100 µl MACS buffer (0.5% BSA, 2 mM EDTA in 1x phosphate buffered 231

saline). Cells were incubated with 10 µl mouse CD11b MicroBeads per 106 cells 232

(Miltenyl Biotec Inc, Auburn, CA) for 15 min at 4 ˚C, followed by 10 µl of CD11b-PE 233

(Miltenyl Biotec Inc) for 15 min at 4 ˚C. Cells were washed with buffer by centrifugation 234

at 300 x g for 10 min and resuspended in 1 ml MACS buffer. CD11b positive cells 235

were selected using a MACS LS column and autoMACS Separator (Miltenyl Biotec 236

Inc). Drug content in CD11b-positive (CD11b+) and CD11b-negative cells was 237

determined using LC-MS/MS. Co-localization of CD11b-PE antibody and CF633-238

labeled nanoART was determined following MACS column CD11b+ selection using a 239

Fluorescence Activated Cell Sorting (FACS) Diva system (BD Immunocytometry 240

Systems, Mountain View, CA). The percentages of CD11b+ and CF633 positive cells 241

were determined from the gate set on viable cells. 242

(III) Confocal microscopy 243

CD11b+ selected cells were imaged using confocal microscopy to visualize co-244

localization of CF633-nanoART and CD11b+ staining. Subcellular localization of the 245

CF633-nanoART was determined by incubating CD11b+ selected cells with 75 nM 246

Lysotracker Green DND-26 (Molecular Probes/Life Technologies, Grand Island, NY) 247


.org/D

ownloaded from

http://aac.asm.org/

12

for 60 min at 37˚C. The cells were pelleted and resuspended in dye-free RPMI 1640 248

medium. CF633-nanoART/CD11b+ cells and CF633-nanoART/Lysotracker Green 249

cells were visualized using a 63x oil immersion lens on a LSM710 confocal microscope 250

(Carl Zeiss Microimaging Inc, Thornwood, NY) with 3x zoom and analyzed with Zeiss 251

AIM software. 252

Results 253

Acute Dose Administration in Mice 254

ATV and RTV serum concentrations vs. time profiles following subcutaneous 255

administration of nanoART and native drug from the acute single- and multiple-dose 256

PK studies are shown in Figure 1. After administering the first dose of native ATV and 257

RTV at 10 mg/kg, serum concentrations increased gradually up to 4 hr (Cmax ATV 2222 258

ng/ml, and RTV 790 ng/ml) then declined within 48 hr to 4.3 and 4.7 ng/ml for ATV and 259

RTV, respectively. One day after the administration of the 2nd dose on day 3 (i.e. day 260

4), ATV and RTV concentrations were 2.5-fold higher than those at day 2, and on day 261

7, before the 3rd dose administration, serum levels were 3-fold lower than those on day 262

4. Three days after the administration of the 3rd dose on day 7 (i.e. day 10), drug levels 263

were 2-fold and 3-fold higher compared to those on day 7 for ATV and RTV, 264

respectively. By day 14, serum levels were 6- and 13-fold lower than those after the 3rd 265

dose on day 7 for ATV and RTV, respectively (Figure 1. A, C). In contrast, after the 266

first dose administration of nanoART (ATV and RTV) at 10 mg/kg, the serum 267

concentrations increased gradually up to 1 hr (Cmax ATV 561 ng/ml, and RTV 1103 268

ng/ml) and then declined to about 15 ng/ml within 24 hr for both ATV and RTV. After 269

that, nanoART drug levels remained nearly constant up to 14 days. ATV and RTV 270


.org/D

ownloaded from

http://aac.asm.org/

13

concentrations in tissues after nanoART and native drug administration are shown in 271

Table 1. ATV and RTV tissue levels after nanoART treatment were always higher than 272

those after native-drug administration. One day after the 1st dose administration, ATV 273

and RTV levels were up to 506-fold higher in tissues obtained from mice treated with 274

nanoART compared to those treated with equimolar doses of the native drug. ATV and 275

RTV were undetectable in most tissues by the 1st week of native-drug treatment, 276

whereas they remained detectable for at least two weeks after nanoART treatment. 277

After nanoART multiple dose administration (days 0, 3, and 7), ATV and RTV levels on 278

days 7 and 14 were 9- 41 fold higher in the liver, and were 4-26 fold higher in the 279

kidney, compared to the native drug. Drug concentrations at the site of injection were 280

46- fold 2500-fold higher at day 14 after 3 doses of nanoART compared to those 281

resulting from the native drug administration, for ATV and RTV, respectively. After 282

single-dose administration, on day 14, nanoART ATV and RTV concentrations in the 283

site of injection were 2900-4454 fold higher compared to the native drug (Table 1). 284

In the dose-escalation study, ATV and RTV serum concentration vs. time 285

profiles of nanoART are shown in Figure 2. At all four dose levels of nanoART, serum 286

concentrations increased gradually up to 8 hr and then remained relatively constant 287

from day 1 to day 14. The AUC values of serum ATV were 2.9-, 12.4-, and 62.3- fold 288

higher compared to the 10 mg/kg dose, after 50, 100, and 250 mg/kg doses, 289

respectively. Similarly, serum RTV AUC values were 3-, 5.3-, and 12.8- fold higher 290

compared to the 10 mg/kg dose, after 50, 100, and 250 mg/kg doses, respectively 291

(Table 2). Tissue concentrations of ATV and RTV after 14 days of nanoART 292

administration at 10, 50, 100, and 250 mg/kg doses in mice are shown in Table 3. A 293


.org/D

ownloaded from

http://aac.asm.org/

14

trend similar to that in serum was also seen in tissues. ATV tissue concentrations on 294

day 14 were 4-8, 24-616, and 41-933 folds higher compared to the 10 mg/kg dose, 295

after 50, 100, and 250 mg/kg doses, respectively. Similarly, RTV concentrations were 296

4-5, 9-14, and 17-44 folds higher compared to the 10 mg/kg dose, after 50, 100, and 297

250 mg/kg doses, respectively (Table 3). 298

Chronic Dose Administration in Mice 299

Serum concentration vs. time profiles of ATV and RTV after chronic dose 300

administrations are shown in Figure 3. This multiple-dose chronic PK study was 301

performed to compare the PK and toxicity profiles of nanoART with those of the native 302

drug at steady state conditions. Three doses were administered the 1st week (days 0, 303

3, and 7) followed by weekly injections for an additional 5 weeks, i.e. total 6 weeks. 304

Serum and tissue samples were collected for an additional 2 weeks after the last dose 305

in week 6, i.e. total 8 weeks. The serum concentrations of ATV and RTV declined 3-14 306

fold by the end of each dosing interval of the native drugs. In addition, serum levels fell 307

more than 6-fold for ATV and 71-fold for RTV within two weeks after the last dose 308

administration in week 6. In contrast, nanoART ATV and RTV serum levels were 309

nearly constant starting at week 2 and throughout the experiment (Figure 3 A and B). 310

Pharmacokinetic parameters of nanoART and native drug after the last dose 311

administration on day 42 are shown in Table 4 (A), and the ratio of nanoART/native 312

drug levels in serum and tissues by the end of study on day 56 are shown in Table 4 313

(B). The AUC, mean residence time (MRT), and t0.5 were 2-, 1.4, and 4.6- fold higher 314

for nanoART ATV compared to native ATV. Similarly, the AUC, MRT, and t0.5 were 315

1.7-, 2.2, and 2.8- fold higher for nanoART RTV compared to native RTV. In contrast, 316


.org/D

ownloaded from

http://aac.asm.org/

15

Clearance (Cl) was 7.9-, and 2.6- times faster with the native ATV and RTV compared 317

to nanoART. The concentrations of ATV and RTV in liver, kidney, spleen, lung, brain, 318

and site of injection, obtained from the same study are shown in Figures 4 and 5. By 319

the end of the experiment, ATV concentrations in the various tissues were 2-13 folds 320

higher compared to the native drug (Figure 4 and Table 4 B). Similarly, RTV 321

concentrations in the various tissues were 5-270 folds higher compared to the native 322

drug (Figure 5 and Table 4 B). NanoART ATV and RTV concentrations in the site of 323

injection were also 8-27 fold higher compared to the native drug (Figure 4 and 5). 324

PK of nanoART in Monkeys 325

ATV and RTV plasma concentration vs. time profiles of nanoART in monkeys 326

are shown in Figure 6. One monkey was injected with a single dose on day 0 and 327

another monkey was injected with 3 doses at days 0, 3, and 7. Plasma concentrations 328

of nanoART ATV and RTV were detected up to 38 days. In addition, after multiple 329

dosing (3 doses) in monkeys, plasma levels of both ATV and RTV were sustained at 330

levels higher than 100 ng/ml for at least 14 days. 331

nanoART in Intracellular Reservoirs 332

FACS sorting of liver non-parenchymal cells from CF633-P188-ATV/RTV treated 333

mice incubated with CD11b antibody and collected using MACS® cell separation 334

columns is shown in Figure 7A. Co-localization of nanoART (red) inside CD11b 335

positive cells (green) in the liver (i.e. Kupffer cells), rather than CD11b negative cells 336

(i.e. hepatocytes, endothelial cells) is shown in Figure 7 B. Figure 7 C demonstrates 337

that intracellular concentrations of ATV and RTV after nanoART administration to mice 338


.org/D

ownloaded from

http://aac.asm.org/

16

were more than 6-fold higher in CD11b positive cells compared to other types of liver 339

cells including hepatocytes. Intracellular nanoART (red) were localized outside the 340

lysosomal compartment (green) (Figure 7 D). 341

342

Discussion 343

Serum concentrations of ATV and RTV after three doses of nanoART at days 0, 344

3, and 7 were in average 1.1-13 fold higher than those resulting from equi-molar doses 345

of native drug throughout the 14-days experiment. By the end of the experiment on 346

day 14, nanoART ATV and RTV levels were 7 -13 fold higher than those resulting from 347

native drug administration (P value < 0.05) (Figure 1. A, C). The enhanced and 348

sustained serum levels of ATV and RTV after nanoART administration were more 349

prominent after multiple dose administration at days 0, 3, and 7 (Figure 1. A, C) and 350

that phenomenon did not exist after single dose administration (Figure 1. B, D). 351

Similar to the multiple-dose effect on serum levels, the enhanced and sustained tissue 352

accumulation of ATV and RTV associated with nanoART was more prominent after 353

multiple (3 doses) rather than single-dose administration. For example, concentrations 354

in the liver were 9-fold higher for ATV and 41-fold higher for RTV at day 14 after 3 355

doses of nanoART compared to those after 3 doses of native drugs (Table 1). In 356

contrast, 14 days after a single dose administration of nanoART, both RTV and ATV 357

concentrations in the liver were only about 7-fold higher compared to a single dose of 358

native drugs (Table 1). These data clearly demonstrate that higher serum and tissue 359

levels could be attained and sustained for a longer period of time after multiple 360

administrations of nanoART. The requirement of multiple-dosing for nanoART to 361


.org/D

ownloaded from

http://aac.asm.org/

17

demonstrate full effect in enhancing the PK profile of anti-HIV drugs may be related to 362

the activation of monocytes/macrophages in tissues, which contribute to the uptake 363

and slow release of nanoART as discussed later. 364

NanoART ATV and RTV concentrations at the site of injection were up to 4454 365

fold higher compared to native drug (Table 1). Therefore, depots of nanoART are 366

formed at the site of SC injection, which enables sustained drug release over a long 367

period of time. This provides one mechanism to explain the sustained and enhanced 368

blood and tissue PK of nanoART. 369

The dose escalation study showed that serum levels of both ATV and RTV 370

increased in a non-linear pattern with the dose. In this experiment, near-steady state 371

serum concentrations were achieved by day 14 (Figure 2). The nonlinear PK behavior 372

of ATV and RTV were clearly demonstrated at higher doses in the dose escalation 373

studies. ATV AUC levels increased in a more than proportional pattern with increasing 374

the dose, whereas RTV AUC levels increased in a less than proportional pattern with 375

increasing the dose (Table 2). Similarly, ATV serum concentrations on day 14 376

increased in a more than proportional pattern with increasing the dose, whereas RTV 377

serum levels increased in a less than proportional pattern with increasing the dose 378

(Figure 2). In addition, a similar pattern of nonlinearity was also demonstrated in 379

tissue concentrations (Table 3). 380

This non-linear PK behavior of RTV and ATV has been reported in several 381

occasions and is attributed to inhibition/induction of the enzymes and transporters 382

involved in the elimination of these drugs (34, 35). nanoART ATV and RTV showed a 383


.org/D

ownloaded from

http://aac.asm.org/

18

similar nonlinear PK trend as the native drugs, which suggests that nanoART did not 384

interfere with the metabolism/distribution of these drugs. In addition, all four doses 385

resulted in the highest ATV and RTV concentrations in liver and spleen, whereas they 386

were barely detectable in brain (Table 3). This indicates that even though nanoART 387

did improve the PK profile of anti-HIV drugs, it did not improve their limited permeability 388

across the blood brain barrier. Protease inhibitors are known substrates for P-389

glycoprotein (PGP), which limit their oral absorption and CNS penetration. The use of 390

formulation/additives that can inhibit PgP is established known approach to enhancing 391

the CNS penetration of PgP substrates (36, 37). The current study does not 392

demonstrate any nanoART advantage in enhancing the CNS penetration of ATV and 393

RTV. However, other modified nanoART formulations were shown to have a marked 394

effect on improving the CNS penetration of ART (25). 395

In the 8-weeks chronic-dose administration study, concentrations of ATV and 396

RTV declined after each dose administration of the native drugs. In contrast, nanoART 397

ATV and RTV serum levels were nearly constant starting at week 2 and throughout the 398

experiment. Therefore, ATV and RTV serum concentrations at steady state were 399

maintained constant and at higher levels at weekly dosing compared to native 400

formulations. Moreover, nanoART ATV and RTV serum levels remained constant for 401

at least two weeks after the last dose administration in week 6 (Figure 3 A, B). The 402

pharmacokinetic parameters clearly demonstrate that nanoART resulted into longer t0.5 403

and MRT of both ATV and RTV due to 3-7-fold decrease in Cl (Table 3A). With the 404

exception of brain, ATV and RTV levels in tissues were up to 270-fold higher after 405

nanoART treatment compared to native drug throughout the 8-week period of the study 406


.org/D

ownloaded from

http://aac.asm.org/

19

(Figure 4 and 5, Table 3B). In addition, similar to the trend in serum, ATV and RTV 407

concentrations in tissues declined exponentially after the last dose administration of 408

native drug in week 6, whereas tissue levels remained constant for at least 2 weeks 409

after the last dose administration of nanoART. 410

In general, tissue binding, especially for lipophilic drugs, is either equal to or 411

higher than plasma protein binding. This is evident for protease inhibitors such as ATV 412

due to its high volume of distribution (~80 L) compared to body water (38, 39). In 413

addition, our data shows that tissue: serum concentration ratio is as high as 18.1 414

(Figures 3, 4, and 5), also indicating high affinity to tissues. These tissue 415

concentrations represent total drug concentration (bound and unbound). Assuming 416

tissue binding is at least as strong as plasma protein binding, then these tissue 417

concentrations are expected to be mostly in the bound form. However, we have 418

previously shown that total tissue (spleen) concentrations of ATV and RTV are 419

inversely proportional to viral load in humanized mice (13, 14). In addition, it has been 420

shown that total drug concentration in tissues (ileum and rectum) are inversely 421

proportional to viral load in humans (40). The half maximum effective concentration for 422

ATV and RTV was reported in the range of 2.6-5.3 nM and 22-130 nM, respectively 423

(41, 42) and the tissue concentration resulting from nanoART exceeded these 424

thresholds in mouse liver, kidney, and spleen. 425

After multiple dosing (3 doses) in monkeys, plasma levels of both ATV and RTV 426

were sustained at levels higher than 100 ng/ml for at least 14 days. At day 38, plasma 427

levels of ATV and RTV resulting from multiple dosing were up to 22- fold higher than 428

those after a single dose administration (Figure 6. A, B, respectively). Similar to the 429


.org/D

ownloaded from

http://aac.asm.org/

20

results in mice, the enhanced and sustained plasma accumulation of ATV and RTV 430

associated with nanoART was more prominent after multiple (3 doses) rather than 431

single-dose administration. After SC administration, slight inflammation at the injection 432

site was observed on day 2, followed by swelling, and development of hard nodules at 433

the injection sites on day 14. Histopathological analysis of biopsies from the injection 434

site showed granuloma formation and crystallization of the nanoART formulation. To 435

overcome these problems, the intramuscular (IM) route was used for nanoART 436

administration. After IM single dose administration at 50 mg/kg, ATV and RTV plasma 437

concentrations up to day 14 were 2.5-5 fold higher than those resulting from SC 438

administration on day 14 (data not shown). In contrast to the SC route, there were no 439

local reactions observed at the site of IM injection. In addition, nanoART administration 440

resulted in normal blood cell counts and serum chemistry profiles. 441

NanoART clearly resulted in enhanced blood and tissue levels of ATV and 442

RTV in vivo. One obvious mechanism for the sustained PK of nanoART is the 443

sustained release of ATV and RTV from the site of injection. Table 1, Figures 4 and 5 444

demonstrate that ATV and RTV levels at the site of injection were up to 4454- fold, and 445

2883-fold higher, respectively, after acute and chronic-dose administration of nanoART 446

compared to native drug treatment. Similarly, other tissues/organs clearly contribute to 447

the sustained PK profile associated with nanoART, where tissue levels of ATV and 448

RTV were up to 13-fold and 270-fold higher, respectively, after nanoART treatment 449

compared to native drug treatment. 450

Cell-mediated drug delivery is a novel concept that employs intracellular 451

recycling and late endosomaes as reservoirs for drugs (22, 26, 27). This was 452


.org/D

ownloaded from

http://aac.asm.org/

21

demonstrated in vitro through the uptake via clathrin-coated pits and the sustained 453

release of both ATV and RTV from endosomal-encased nanoART in macrophages 454

(18). It was also shown that nanoART is efficiently internalized by macrophages, 455

where it is stored in endosomal reservoirs that protect the drug from degradation (23). 456

The internalized particles released the drug slowly and ART activity against HIV-1ADA 457

were detected for 2 weeks (22). Evidence is now provided that monocytes and 458

macrophages serve as a major cellular reservoir for nanoART in vivo as well as “Trojan 459

Horses” for drug delivery (Figure 7). In mice, the CD11b is expressed in macrophages 460

and granulocytes (43). Localization of nanoART in these cells suggest that, nanoART 461

accumulates selectively inside Kupffer cells of liver, which may also be the case with 462

macrophages in other tissues. Prolonged release from deep cellular reservoirs inside 463

tissues may be responsible for the sustained and enhanced pharmacokinetic profile 464

produced by nanoART. 465

In summary, we have shown that weekly dosing of nanoART maintains 466

therapeutic plasma levels of ATV and RTV at steady state. The effect of nanoART in 467

improving the PK profile of anti-HIV drugs is due to the sustained release of these drug 468

from intracellular depots in tissues and in the site of injection. 469

470

Acknowledgements 471

This work was supported by the National Institutes of Health [Grant DA028555-01]. 472

We would like to acknowledge the assistance of Dr. Carol Casey, Department of 473

Internal Medicine, University of Nebraska Medical Center, in developing the methods 474


.org/D

ownloaded from

http://aac.asm.org/

22

for isolation of parenchymal and non-parenchymal liver cells. We would also like to 475

acknowledge the University of Nebraska Medical Center Cell Analysis Facility for their 476

assistance in developing the methods for isolation of liver CD11b positive cells and 477

confocal imaging of dye-labeled cells. We would also like to acknowledge David 478

Muirhead, for histopathological analysis. 479

480

References 481

1. Ellis R, Langford D, Masliah E. 2007. HIV and antiretroviral therapy in the 482

brain: neuronal injury and repair. Nat Rev Neurosci 8:33-44. 483

2. 2012. Summary report from the Human Immunodeficiency Virus and Aging 484

Consensus Project: treatment strategies for clinicians managing older 485

individuals with the human immunodeficiency virus. J Am Geriatr Soc 60:974-486

979. 487

3. Pasternak AO, de Bruin M, Jurriaans S, Bakker M, Berkhout B, Prins JM, 488

Lukashov VV. 2012. Modest Nonadherence to Antiretroviral Therapy Promotes 489

Residual HIV-1 Replication in the Absence of Virological Rebound in Plasma. J 490

Infect Dis 206:1443-1452. 491

4. Coiras M, Lopez-Huertas MR, Alcami J. 2010. HIV-1 latency and eradication 492

of long-term viral reservoirs. Discov Med 9:185-191. 493

5. Rao KS, Ghorpade A, Labhasetwar V. 2009. Targeting anti-HIV drugs to the 494

CNS. Expert Opin Drug Deliv 6:771-784. 495

6. Anabwani G, Navario P. 2005. Nutrition and HIV/AIDS in sub-Saharan Africa: 496

an overview. Nutrition 21:96-99. 497


.org/D

ownloaded from

http://aac.asm.org/

23

7. Piot P, Bartos M, Ghys PD, Walker N, Schwartlander B. 2001. The global 498

impact of HIV/AIDS. Nature 410:968-973. 499

8. Thaker HK, Snow MH. 2003. HIV viral suppression in the era of antiretroviral 500

therapy. Postgrad Med J 79:36-42. 501

9. Rathbun RC, Lockhart SM, Stephens JR. 2006. Current HIV treatment 502

guidelines--an overview. Curr Pharm Des 12:1045-1063. 503

10. Crowe S, Zhu T, Muller WA. 2003. The contribution of monocyte infection and 504

trafficking to viral persistence, and maintenance of the viral reservoir in HIV 505

infection. J Leukoc Biol 74:635-641. 506

11. Fogarty L, Roter D, Larson S, Burke J, Gillespie J, Levy R. 2002. Patient 507

adherence to HIV medication regimens: a review of published and abstract 508

reports. Patient Educ Couns 46:93-108. 509

12. Rathbun RC, Farmer KC, Stephens JR, Lockhart SM. 2005. Impact of an 510

adherence clinic on behavioral outcomes and virologic response in treatment of 511

HIV infection: a prospective, randomized, controlled pilot study. Clin Ther 512

27:199-209. 513

13. Roy U, McMillan J, Alnouti Y, Gautum N, Smith N, Balkundi S, Dash P, 514

Gorantla S, Martinez-Skinner A, Meza J, Kanmogne G, Swindells S, Cohen 515

SM, Mosley RL, Poluektova L, Gendelman HE. 2012. Pharmacodynamic and 516

antiretroviral activities of combination nanoformulated antiretrovirals in HIV-1-517

infected human peripheral blood lymphocyte-reconstituted mice. J Infect Dis 518

206:1577-1588. 519


.org/D

ownloaded from

http://aac.asm.org/

24

14. Dash PK, Gendelman HE, Roy U, Balkundi S, Alnouti Y, Mosley RL, 520

Gelbard HA, McMillan J, Gorantla S, Poluektova LY. 2012. Long-acting 521

nanoformulated antiretroviral therapy elicits potent antiretroviral and 522

neuroprotective responses in HIV-1-infected humanized mice. AIDS 26:2135-523

2144. 524

15. Swindells S, Flexner C, Fletcher CV, Jacobson JM. 2011. The critical need 525

for alternative antiretroviral formulations, and obstacles to their development. J 526

Infect Dis 204:669-674. 527

16. Novitsky V, Essex M. 2012. Using HIV viral load to guide treatment-for-528

prevention interventions. Curr Opin HIV AIDS 7:117-124. 529

17. Sterling TR, Pham PA, Chaisson RE. 2010. HIV infection-related tuberculosis: 530

clinical manifestations and treatment. Clin Infect Dis 50 Suppl 3:S223-230. 531

18. Nowacek AS, Miller RL, McMillan J, Kanmogne G, Kanmogne M, Mosley 532

RL, Ma Z, Graham S, Chaubal M, Werling J, Rabinow B, Dou H, Gendelman 533

HE. 2009. NanoART synthesis, characterization, uptake, release and toxicology 534

for human monocyte-macrophage drug delivery. Nanomedicine (Lond) 4:903-535

917. 536

19. Nowacek A, Gendelman HE. 2009. NanoART, neuroAIDS and CNS drug 537

delivery. Nanomedicine (Lond) 4:557-574. 538

20. Dou H, Grotepas CB, McMillan JM, Destache CJ, Chaubal M, Werling J, 539

Kipp J, Rabinow B, Gendelman HE. 2009. Macrophage delivery of 540

nanoformulated antiretroviral drug to the brain in a murine model of neuroAIDS. 541

J Immunol 183:661-669. 542


.org/D

ownloaded from

http://aac.asm.org/

25

21. Dou H, Destache CJ, Morehead JR, Mosley RL, Boska MD, Kingsley J, 543

Gorantla S, Poluektova L, Nelson JA, Chaubal M, Werling J, Kipp J, 544

Rabinow BE, Gendelman HE. 2006. Development of a macrophage-based 545

nanoparticle platform for antiretroviral drug delivery. Blood 108:2827-2835. 546

22. Nowacek AS, Balkundi S, McMillan J, Roy U, Martinez-Skinner A, Mosley 547

RL, Kanmogne G, Kabanov AV, Bronich T, Gendelman HE. 2011. Analyses 548

of nanoformulated antiretroviral drug charge, size, shape and content for uptake, 549

drug release and antiviral activities in human monocyte-derived macrophages. J 550

Control Release 150:204-211. 551

23. Kadiu I, Nowacek A, McMillan J, Gendelman HE. 2011. Macrophage 552

endocytic trafficking of antiretroviral nanoparticles. Nanomedicine (Lond) 6:975-553

994. 554

24. Nowacek AS, McMillan J, Miller R, Anderson A, Rabinow B, Gendelman 555

HE. 2010. Nanoformulated antiretroviral drug combinations extend drug release 556

and antiretroviral responses in HIV-1-infected macrophages: implications for 557

neuroAIDS therapeutics. J Neuroimmune Pharmacol 5:592-601. 558

25. Kanmogne GD, Singh S, Roy U, Liu X, McMillan J, Gorantla S, Balkundi S, 559

Smith N, Alnouti Y, Gautam N, Zhou Y, Poluektova L, Kabanov A, Bronich 560

T, Gendelman HE. 2012. Mononuclear phagocyte intercellular crosstalk 561

facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to 562

human brain endothelial cells. Int J Nanomedicine 7:2373-2388. 563

26. Kabanov AV, Gendelman HE. 2007. Nanomedicine in the diagnosis and 564

therapy of neurodegenerative disorders. Prog Polym Sci 32:1054-1082. 565


.org/D

ownloaded from

http://aac.asm.org/

26

27. Gendelman HE, Kabanov A, Linder J. 2008. The promise and perils of CNS 566

drug delivery: a video debate. J Neuroimmune Pharmacol 3:58. 567

28. Balkundi S, Nowacek AS, Veerubhotla RS, Chen H, Martinez-Skinner A, 568

Roy U, Mosley RL, Kanmogne G, Liu X, Kabanov AV, Bronich T, McMillan 569

J, Gendelman HE. 2011. Comparative manufacture and cell-based delivery of 570

antiretroviral nanoformulations. Int J Nanomedicine 6:3393-3404. 571

29. Balkundi S, Nowacek AS, Roy U, Martinez-Skinner A, McMillan J, 572

Gendelman HE. 2010. Methods development for blood borne macrophage 573

carriage of nanoformulated antiretroviral drugs. J Vis Exp. 574

30. Huang J, Gautam N, Bathena SP, Roy U, McMillan J, Gendelman HE, 575

Alnouti Y. 2011. UPLC-MS/MS quantification of nanoformulated ritonavir, 576

indinavir, atazanavir, and efavirenz in mouse serum and tissues. J Chromatogr 577

B Analyt Technol Biomed Life Sci 879:2332-2338. 578

31. Casey CA, Kragskow SL, Sorrell MF, Tuma DJ. 1987. Chronic ethanol 579

administration impairs the binding and endocytosis of asialo-orosomucoid in 580

isolated hepatocytes. J Biol Chem 262:2704-2710. 581

32. Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, 582

Schwabe RF. 2007. TLR4 enhances TGF-beta signaling and hepatic fibrosis. 583

Nat Med 13:1324-1332. 584

33. Liu W, Hou Y, Chen H, Wei H, Lin W, Li J, Zhang M, He F, Jiang Y. 2011. 585

Sample preparation method for isolation of single-cell types from mouse liver for 586

proteomic studies. Proteomics 11:3556-3564. 587


.org/D

ownloaded from

http://aac.asm.org/

27

34. Foisy MM, Yakiwchuk EM, Hughes CA. 2008. Induction effects of ritonavir: 588

implications for drug interactions. Ann Pharmacother 42:1048-1059. 589

35. Havlir DV, O'Marro SD. 2004. Atazanavir: new option for treatment of HIV 590

infection. Clin Infect Dis 38:1599-1604. 591

36. Schroeder U, Sommerfeld P, Ulrich S, Sabel BA. 1998. Nanoparticle 592

technology for delivery of drugs across the blood-brain barrier. J Pharm Sci 593

87:1305-1307. 594

37. Bansal T, Akhtar N, Jaggi M, Khar RK, Talegaonkar S. 2009. Novel 595

formulation approaches for optimising delivery of anticancer drugs based on P-596

glycoprotein modulation. Drug Discov Today 14:1067-1074. 597

38. Schmidt S, Gonzalez D, Derendorf H. 2010. Significance of protein binding in 598

pharmacokinetics and pharmacodynamics. J Pharm Sci 99:1107-1122. 599

39. Barrail-Tran A, Mentre F, Cosson C, Piketty C, Chazallon C, Gerard L, 600

Girard PM, Taburet AM. 2010. Influence of alpha-1 glycoprotein acid 601

concentrations and variants on atazanavir pharmacokinetics in HIV-infected 602

patients included in the ANRS 107 trial. Antimicrob Agents Chemother 54:614-603

619. 604

40. Cohen J. 2011. HIV/AIDS research. Tissue says blood is misleading, confusing 605

HIV cure efforts. Science 334:1614. 606

41. Robinson BS, Riccardi KA, Gong YF, Guo Q, Stock DA, Blair WS, Terry BJ, 607

Deminie CA, Djang F, Colonno RJ, Lin PF. 2000. BMS-232632, a highly 608

potent human immunodeficiency virus protease inhibitor that can be used in 609


.org/D

ownloaded from

http://aac.asm.org/

28

combination with other available antiretroviral agents. Antimicrob Agents 610

Chemother 44:2093-2099. 611

42. Kempf DJ, Marsh KC, Denissen JF, McDonald E, Vasavanonda S, Flentge 612

CA, Green BE, Fino L, Park CH, Kong XP, et al. 1995. ABT-538 is a potent 613

inhibitor of human immunodeficiency virus protease and has high oral 614

bioavailability in humans. Proc Natl Acad Sci U S A 92:2484-2488. 615

43. Arnaout MA. 1990. Structure and function of the leukocyte adhesion molecules 616

CD11/CD18. Blood 75:1037-1050. 617

618

619


.org/D

ownloaded from

http://aac.asm.org/

29

Table 1. Tissue concentrations of ATV and RTV, after acute single- and multiple- dose 620

nanoART and native-drug administration at 10mg/kg in mice (N=5, Mean ± SEM) 621

Tissue

Day of tissue collection

ATV

RTV

Native drug NanoART Native drug NanoART

Conc. (ng/g) Conc. (ng/g) Conc. (ng/g) Conc. (ng/g)

Liver Day1 after single dose 13.2 ± 4.2 1623.5 ± 1377 83.4 ± 15.2 115.8 ± 19.2

Day7 after two doses 1.4 ± 0.7 21.7 ± 2.6 1.3 ±0.1 40.2 ± 3.9

Day14 after three doses 4.4 ± 1.6 39.7 ± 11.2 1.6 ± 0.4 65.0 ± 8.5

Day14 after single dose 1.3 ± 0.6 7.4 ± 0.7 1.6 ± 0.5 14.2 ± 1.6

Spleen Day1 after single dose 4.3 ± 2.1 2177.4 ± 1482 61.4 ± 19.2 109.2 ± 30.5

Day7 after two doses * 2.7 ± 0.7 * 35.2 ± 6.9

Day14 after three doses * 6.7 ± 3.7 * 39.1 ± 4.4

Day14 after single dose * 1.3 ± 1.1 * 11.7 ± 1.6

Lung Day1 after single dose 2.2 ± 0.6 345.7 ± 140.4 23.9 ± 4.0 20.2 ± 2.1

Day7 after two doses * 3.2 ± 0.4 * 8.4 ± 1.3

Day14 after three doses * 5.2 ± 1.3 * 13.6 ± 2.5


Kidney Day1 after single dose 4.5 ± 1.0 101.1 ± 54.6 54.6 ± 9.6 75.6 ± 18.8

Day7 after two doses 1.5 ± 0.2 7.6 ± 2.0 1.4 ± 0.2 37.7 ± 2.9

Day14 after three doses 1.3 ± 0.7 5.8 ± 0.8 2.0 ± 1.2 52.1 ± 5.5


Brain Day1 after single dose * 2.3 ± 0.5 1.3 ± 0.3 1.4 ± 0.2

Day7 after two doses * * * *

Day14 after three doses * * * *

Day14 after single dose * * * *

Conc. (µg/g) Conc. (µg/g) Conc. (µg/g) Conc. (µg/g)

Site of

Injection

Day1 after single dose 87.9 ± 16.6 1121.6 ± 401 48.0 ± 14.4 984.4 ± 436

Day7 after two doses 427.9 ± 135 2771.9 ± 429 62.7 ± 26.8 2654.0 ± 417

Day14 after three doses 42.3 ± 17.4 1968.0 ± 149 0.6 ± 0.2 1514.3 ± 207

Day14 after single dose 0.2 ± 0.2 1064.8 ± 134 0.2 ± 0.1 581.3 ± 94.6

* values are < LLOQ


.org/D

ownloaded from

http://aac.asm.org/

30

Table 2. AUC(0-last) values for ATV and RTV after three doses of nanoART at 10, 50, 622

100, and 250 mg/kg in mice (N=5) 623

624 625

Doses (mg/kg) ATV RTV

10 4908 8024

50 14146 23784

100 61032 42666

250 306029 102617


.org/D

ownloaded from

http://aac.asm.org/

31

Table 3. Tissue concentrations of ATV and RTV on day 14 after nanoART multiple-626

dose (days 0, 3, and 7) administration at 10, 50, 100 and 250 mg/kg in mice 627

(N=5, Mean ± SEM) 628

629

630

Tissues 10 mg/kg 50 mg/kg 100 mg/kg 250 mg/kg

Conc. (ng/g) Conc. (ng/g) Conc. (ng/g) Conc. (ng/g)

nanoART-ATV

Liver 39.7 ± 11.2 181.6 ± 19.3 1166.7 ± 355 6834.5 ± 2164

Spleen 6.7 ± 3.7 54.0 ± 24.0 4144.8 ± 672 6414.9 ± 3631

Lung 5.2 ± 1.3 18.1 ± 4.5 900.9 ± 787 215.5 ± 43

Kidney 5.8 ±0.8 23.6 ± 4.1 138.7 ± 26.2 2867.0 ± 1575

Brain * 2.2 ± 1.4 4.2 ± 0.9 17.2 ± 6.2

nanoART-RTV

Liver 65.0 ± 8.5 331.9 ± 51.3 595.0 ± 106 2855.0 ± 754

Spleen 39.1 ± 4.4 169.0 ± 24.2 331.6 ± 32.1 651.3 ± 27.4

Lung 13.6 ± 2.5 59.6 ± 10.3 189.2 ± 54.6 243.2 ± 27.6

Kidney 52.1 ± 5.5 281.1 ± 39.3 487.9 ± 35.4 1366.0 ± 258.7

Brain * 2.9 ± 0.3 6.9 ± 1.2 12.3 ± 1.3


.org/D

ownloaded from

http://aac.asm.org/

32

Table 4. (A) Pharmacokinetic parameters of ATV and RTV after the last dose 631

administration (day 42) of the chronic-dose study, (B) ratio of nanoART/native 632

ATV and RTV by end of the study on day 56 (N=6). 633

634

A: Pharmacokinetic parameters

Parameters nanoART-ATV Native-ATV nanoART-RTV Native-RTV

AUClast (ng.h/ml) 12592.8 6164.4 13586.4 8028.0

t0.5 (h) 1152.5 253.3 230.1 83.6

Vβ (l/kg) 1106.8 1933.0 771.4 731.4

CL (l/h/kg) 0.67 5.29 2.32 6.06

MRT0-∞ (h) 171.6 120.9 142.5 63.5

B: Ratio of nanoART/native drug

ATV RTV

Serum 4.2 13.5

Liver 12.9 270.8

Kidney 4.1 34.3

Lung 3.2 13.7

Spleen 5.7 38.4

Brain 2.1 5.1

Site of injection 7.5 27.2


.org/D

ownloaded from

http://aac.asm.org/

33

Figure Legends 635

Figure 1: Serum concentration vs. time profiles from the acute dose studies after (A) 636

three ATV doses at days 0, 3, and 7, (B) a single ATV dose at day 0, (C) 637

three RTV doses at days 0, 3, and 7, (D) a single RTV dose. Data shows 638

Mean ± SEM (N= 5) and dose = 10 mg/kg. 639

Figure 2: (A) ATV and (B) RTV serum concentration vs. time profiles after 10, 50, 100 640

and 250 mg/kg nanoART administration on days 0, 3, and 7 in mice (N=5, 641

Mean ± SEM). 642

Figure 3: Serum concentration vs. time profiles of (A) ATV and (B) RTV after multiple 643

dose administration of nanoART and native drugs, at 50mg/kg in mice (N=6, 644

Mean ± SEM). 645

Figure 4: Concentration vs. time profiles of ATV in (A) liver, (B) kidney, (C) spleen, (D) 646

lung, (E) brain, and (F) site of injection, after multiple dose administration of 647

nanoART and native drug for 6 weeks. Doses were administered on days 0, 648

3, 7, 14, 21, 28, 35 and 42 at 50mg/kg. Samples were collected right before 649

dose administration on days 7, 14, 21, 28, 35 and 42 in mice (N=6, Mean ± 650

SEM). 651

Figure 5: Concentration vs. time profiles of RTV in (A) liver, (B) kidney, (C) spleen, (D) 652

lung, (E) brain, and (F) site of injection, after multiple dose administration of 653

nanoART and native drug for 6 weeks. Doses were administered on days 0, 654

3, 7, 14, 21, 28, 35 and 42 at 50mg/kg. Samples were collected right before 655


.org/D

ownloaded from

http://aac.asm.org/

34

dose administration on days 7, 14, 21, 28, 35 and 42 in mice (N=6, Mean ± 656

SEM). 657

Figure 6: (A) ATV and (B) RTV plasma concentration vs. time profile after 50 mg/kg 658

nanoART administration on days 0, 3 and 7 in Rhesus macaques (N=2). 659

Figure 7: In vivo co-localization of nanoART in CD11b positive cells of the liver and 660

storage in non-lysosomal compartments. Male Balb/cJ mice were treated SC 661

with 250 mg/kg ATV/RTV (1:1 drug ratio) coated with CF633-modified P188. 662

Liver cells were isolated 24 hours later by in situ collagenase digestion. (A) 663

FACS sorting of liver non-parenchymal cells from CF633-P188-ATV/RTV 664

treated mice incubated with CD11b antibody and collected using MACS® cell 665

separation columns. (B) Confocal microscopy of nanoART-loaded non-666

parenchymal cells following CD11b positive cell purification showing 667

localization of nanoART (red) in CD11b positive cells (green) (bar = 20 µm; 668

inset bar = 5 µm). (C) ATV and RTV levels in various liver cell types following 669

cell separation using differential centrifugation and CD11b positive MACS® 670

cell separation (data from a representative experiment are shown). Drug 671

levels were quantitated by LC-MS/MS (bld = below detection limit). (D) 672

Confocal microscopy of nanoART-loaded (red) non-parenchymal cells 673

incubated with Lysotracker Green showing localization of nanoART outside of 674

lysosomal (green) compartments (bar = 10 µm). 675

676


.org/D

ownloaded from

http://aac.asm.org/


.org/D

ownloaded from

http://aac.asm.org/

Preclinical Pharmacokinetics and Tissue Distribution of Long-Acting ...

Documents

Transcript of Preclinical Pharmacokinetics and Tissue Distribution of Long-Acting ...