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Augmented inhibition of CYP3A4 in human primary hepatocytes by ritonavir solid drug nanoparticles Philip John Martin, Marco Giardiello, Tom O McDonald, Darren L Smith, Marco Siccardi, Steven P Rannard, and Andrew Owen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00204 • Publication Date (Web): 08 Sep 2015 Downloaded from http://pubs.acs.org on September 14, 2015

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Molecular Pharmaceutics

Augmented inhibition of CYP3A4 in human primary hepatocytes by ritonavir solid drug nanoparticles Philip Martin,1 Marco Giardiello,2 Tom O McDonald,2 Darren Smith,3 Marco Siccardi,1 Steven P Rannard2,* and Andrew Owen1,* 1

Department of Molecular and Clinical Pharmacology, University of Liverpool, Block H, 70 Pembroke Place, Liverpool, L69 3GF, UK 2

3

Department of Chemistry, University of Liverpool, Crown Street, L69 3BX, UK

Department of Applied Sciences, University of Northumbria at Newcastle, Ellison Building, NE1 8ST.

KEYWORDS: Ritonavir, CYP3A4, Pharmaco-enhancer, Nanoparticles, Hepatocytes

ABSTRACT: Ritonavir is a protease inhibitor utilized primarily as a pharmaco-enhancer with concomitantly administered antiviral drugs including other protease inhibitors. However, poor tolerance, serious side effects and toxicities associated with drug-drug interactions are common during exposure to ritonavir. The aim of this work was to investigate the impact of nanoformulation on ritonavir pharmacological properties. Emulsion-templated freeze drying techniques were used to generate ritonavir (10 wt%) solid drug nanoparticle formulations. A total of 68 ritonavir formulations containing various mixtures of excipients were assessed for

ACS Paragon Plus Environment

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inhibition of CYP3A4 in baculosomes and primary human hepatocytes. Accumulation and cytotoxicity was assessed in HepG2 (hepatocytes), Caco-2 (intestinal), THP-1 (monocytes), ATHP-1 (macrophage) and CEM (lymphocytes). Transcellular permeation across Caco-2 cells was also assessed. From 68 solid drug nanoparticle formulations tested, 50 (73.5%) for baculosome and 44 (64.7%) for human primary hepatocytes exhibited enhanced CYP3A4 inhibition relative to an aqueous ritonavir solution. 61 (89.7%) and 49 (72%) solid drug nanoformulations had higher apical to basal permeation across Caco-2 cells than aqueous solution of ritonavir after 60 minutes and 120 minutes, respectively. No significant difference in cellular accumulation was observed for any solid drug nanoparticle for any cell type compared to aqueous ritonavir. However, incubation with the vast majority of solid drug nanoparticle formulations resulted in lower cytotoxicity of ritonavir than detected with an aqueous solution. This data provides in vitro proof of concept for improved inhibition of CYP3A4 by ritonavir through formation of solid drug nanoparticles. Nanodispersions also showed enhanced permeability across Caco-2 cells lower cytotoxicity across hepatic, intestinal and immune cell types compared to an aqueous solution of ritonavir.

INTRODUCTION Ritonavir (RTV) is a human immunodeficiency virus (HIV) protease inhibitor (PI) and is used routinely in combination with other PIs in the treatment of HIV infection.1, 2 However, RTV is not used for its PI activity, but is almost exclusively used as a “booster” to increase pharmacokinetic exposure of co-administered PIs such as lopinavir (LPV) and darunavir (DRV).3,

4

Although the precise mechanisms are unclear, there remains discordance between

acute and chronic effects arising from ritonavir (RTV) exposure.5, 6 After acute exposure, RTV is known to inhibit the activity of proteins involved in drug disposition such as Cytochrome P450


2

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3A4 (CYP3A4) and P-glycoprotein (P-gp).7, 8 CYP3A4 and P-gp act synergistically with phase II conjugating enzymes (e.g. glutathione S-transferases) in the detoxification and clearance of xenobiotics.9-12 After chronic exposure, RTV is also capable of inducing various clearance mechanisms, collectively making drug-drug interactions with RTV common.13,14 In humans, CYP3A4 plays a crucial role in the detoxification and elimination of approximately 50% of all clinically used drugs.15 In addition, CYP3A4 is the most abundant enzyme both in the intestine and liver16 with the small intestine containing the highest expression of CYP3A4 followed by the liver16,

17

. In vitro screens are used as standard across the pharmaceutical

industry to determine the degree of CYP3A4 inhibition. Typically, these assays use liver microsomes or human primary hepatocytes.18 Human primary hepatocytes have been suggested to be optimal for the time-dependent study of CYP3A4 inhibition19, 20 being recognized as the gold standard for prediction in both preclinical species and humans.21, 22 Formation of solid drug nanoparticles (SDNs) has attracted recent attention as an approach to overcome issues with water solubility and/or bioavailability, either for licensed drugs or drugs in development. Current commercially available SDN products have almost exclusively been developed by the physical fracturing of large diameter solids into SDNs and have been successful for oral administration, providing clinically-relevant increases in bioavailability and efficacy.23, 24 SDNs have recently attracted attention in HIV therapy for provision of long-acting depot formulations,25,

26

tissue/cellular targeting27,

28

and increasing bioavailability after oral

dosing.29 Although RTV is widely used in second line combination antiretroviral therapies it is associated with numerous toxicities, particularly following chronic exposure.30, 31 Therefore, any strategy that reduces RTV dose while maintaining “boosting” efficacy is of clear importance.


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Therefore, the aim of this study was to assess the potential benefits of SDN formulation for improving CYP3A4 inhibition by RTV. A total of 68 RTV SDN formulations where synthesized from binary combinations of polymers with surfactants using emulsion-templated freeze drying.32 All were characterized for surface charge (zeta potential; mV), size (Z-average diameter; nm) and polydispersity (PDI). All dispersions were characterized for their cellular accumulation in HepG2 (modelling hepatocytes), Caco-2 (modelling intestinal epithelium), THP-1 (modelling monocytes), A-THP-1 (modelling phagocytic cells) and CEM (modelling lymphocytes). Transcellular permeation across Caco-2 cells was also assessed along with cytotoxicity in all cell lines. Finally, relationships between particle properties or excipients used with inhibition of CYP3A4 activity in cell-free (baculosome) and cell-based (cryopreserved human primary hepatocytes) were assessed.

MATERIALS AND METHODS Materials. All chemicals were purchased from Sigma-Aldrich, UK, unless otherwise stated. Polymers and surfactants used include (molecular weights of small molecule surfactants shown to nearest 1 g/mol). Polymers: poly(ethylene glycol) (PEG, 1000 g/mol), poly(ethylene oxide)80block-poly(propylene oxide)27-block-poly(ethylene oxide)80 (Pluronic® F68, MW = 8400 g/mol), poly(ethylene oxide)101-block-poly(propylene oxide)56-block-poly(ethylene oxide)101 (Pluronic® F127, MW = 12600 g/mol), poly(vinyl alcohol)–graft-poly(ethylene glycol) copolymer (KollicoatTM, MW = 45000 g/mol), poly(vinyl alcohol) (80% hydrolyzed PVA, MW = 9500 g/mol), poly(vinyl pyrolidone) (PVP K30, MW = 40000 g/mol), hydroxypropyl cellulose (HPC, MW = 80000 g/mol), hydroxypropylmethyl cellulose (HPMC, MW = 10000 g/mol), hydrolyzed gelatin (HG, MW = 1980 g/mol), sodium carboxymethyl cellulose (NaCMC, MW = 90000 g/mol). Surfactants: sodium deoxycholate (NaDC, MW = 414 g/mol), sodium caprylate


4

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(NaCap, MW = 166 g/mol), α-tocopherol poly(ethylene glycol) succinate (Vit-E-PEG, MW = 1000 g/mol), Sisterna 11 (sucrose stearate, MW = 608 g/mol), Sisterna 16 (sucrose palmitate, MW = 580 g/mol), sodium 1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate (AOT, MW = 444 g/mol)), poly(ethyleneoxide)35 modified castor oil (Cremophor EL, MW = 2500 g/mol), polyethylene

(Solutol®

glycol15-hydroxystearate

poly(ethyleneoxide)20

sorbitan

monolaurate

HS

15,

MW

=

345

g/mol),

(Tween®

20,

MW

=

1230

g/mol),

poly(ethyleneoxide)20 sorbitan monooleate (Tween® 80, MW = 1300 g/mol), poly(ethylene glycol) hexadecyl ether (Brij 58, MW = 1124 g/mol), alkyl(C12-16) dimethylbenzylammonium chloride (Hyamine®, MW = 448 g/mol) and cetyl trimethylammonium bromide (CTAB, MW = 364 g/mol). Acetonitrile (ACN, MW = 41.05 g/mol), RTV (MW = 720.9 g/mol), Hepatocyte Complex Media (HCM) ascorbic acid, transferrin, human recombinant growth factor (rhEGF), insulin, gentamycin sulfate amphotericin, hydrocortisone 21 hemisuccinate, bovine serum albumin-fatty acid free (BSA- FAF) were all obtained from Lonza, UK, foetal bovine serum (FBS: Bio-Whittaker, Berkshire, UK),

3

H-labelled RTV (Moravek Biochemicals, USA),

Dulbecco’s modified eagle’s medium (DMEM), RPMI-1640, Phorbol 12-myristate 13 acetate (PMA), ATP cell viability assay; CellTiter-Glo® (Promega, UK), Hank’s Balanced Salt Solution (HBSS) Ultima Gold liquid scintillation cocktail fluid (Meridian, UK), Vivid® CYP450 Screening Kit (Invitrogen, UK), dimethyl sulfoxide (DMSO). Ritonavir solid drug nanoparticle manufacture. RTV (10 wt%) loaded SDNs were formulated using a previously described emulsion-templated freeze-drying approach.

29, 32-34

Briefly, SDNs were prepared using a 10 mg/mL stock solution of RTV in chloroform, a 22.5 mg/mL of polymer stock solution and a 22.5 mg/mL stock solution of surfactant. Stock solutions were added in the following proportion; 100µl RTV: 267µl polymer and 133µl surfactant.


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Therefore, the solid mass ratio was; 10% RTV, 60% polymer and 30% surfactant in a 1:4 oil to water mixture. The mixtures were emulsified using a probe sonicator (UP400S, Hielscher, Germany) fitted with an H3 titanium probe and operated at 20% amplitude for 7 seconds followed by immediate cryogenic freezing in liquid nitrogen. The polymer and surfactant combinations used to generate the 68 different SDNs are detailed in Supplemental Table 1. Samples were then lyophilized (Virtis benchtop K series freeze-dryer) for 42 hours resulting in a dry porous product. Lyophilized samples were sealed in individual glass vials and stored at ambient temperature until required. Where required, 3H-labelled RTV (Moravek Biochemicals, USA) was added to the chloroform stock solution to generate radioactive RTV SDNs to assist in pharmacological assays such as accumulation and permeation. Characterization of ritonavir solid drug nanoparticle dispersions. Immediately prior to analysis, each of the 68 SDN samples were separately diluted in water. 1mL of the resultant dispersion was separately dispensed into plastic disposable cuvettes. SDN size (Z-average diameter (Dz; nm)), charge (zeta potential (ζ; mV)) and polydispersity index (PDI) were determined by dynamic light scattering (DLS) at 25°C using a Malvern Zetasizer Nano ZS equipped with a 4 mW He–Ne, 633 nm laser. Malvern Zetasizer software version 6.20 was used for data analysis. Zeta potential measurements were also carried out at 1 mg/mL, 25˚C, and an initial pH of 6.5, using disposable capillary zeta cells. Dz, ζ and PDI measurements were obtained as an average of 3 individual measurements and were obtained using the instrument’s automatic optimization settings. Scanning electron microscopy images were recorded using a Hitachi S-4800 FE-SEM at 3 kV. Glass coverslips were stuck onto the aluminum stubs with carbon tabs. The sample (dispersed to 0.5 mg/mL with regards to RTV) was added to the surface


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of the coverslip and blotted dry. The samples were gold coated for 1 minute at 15 µA using a sputter-coater (EMITECH K550X) prior to imaging. Culture of cryopreserved human primary hepatocytes and cell lines. Cryopreserved human primary hepatocytes (Lonza, UK) were removed from liquid N2 and rapidly warmed to 37°C before transfer into a conical centrifuge tube containing Hepatocyte Complex Media (HCM: containing; ascorbic acid (0.1% v/v), transferrin (0.1% v/v), human recombinant growth factor (rhEGF) (0.1% v/v), insulin (0.1% v/v), gentamycin sulfate amphotericin (0.1% v/v), hydrocortisone 21 hemisuccinate (0.1% v/v) and bovine serum albumin (BSA-FAF (fatty acid free: FAF) (2% v/v)) supplemented with 2% sterile filtered foetal bovine serum (FBS: BioWhittaker, Berkshire, UK). The resulting cell suspensions were then centrifuged at 50 x g and 4°C for 3 minutes (Heraeus Multifuge 3SR+; Thermo Scientific, UK). The cell pellet was then suspended in 10 mL pre-warmed (37°C) HCM and cell viability was determined by trypan blue exclusion and showed these cells to be typically > 95% viable for all subsequent experiments. HepG2 (hepatic) and Caco-2 (intestinal) cells were purchased from American Type Culture Collection (ATCC; USA) and maintained in Dulbecco’s modified eagle’s medium (DMEM; Sigma; Dorest, UK). Media was supplemented with 10% sterile filtered FBS for HepG2 cells and 15% FBS for Caco-2 cells. THP-1 (monocyte) and CEM (lymphocyte) cells were purchased from European Collection of Cell Culture (ECACC; Porton Down, UK) and grown in RPMI1640 (Sigma; Dorest, UK) supplemented with 10% sterile filtered FBS. THP-1 and CEM cells were routinely sub-cultured when a density of 1 x 106 cells/mL was achieved. THP-1 cells were activated to macrophage like cells (A-THP-1) by the addition of Phorbol 12-myristate 13 acetate (PMA: Sigma; Dorest, UK) to final concentration of 10nM in THP-1 culture medium (RPMI1640 supplemented with 10% sterile filtered FBS). Cells were then incubated at 37°C and 5%


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CO2 for 7 days prior to use to allow differentiation from monocytes to moncyte derived macrophages (MDM). Cell count and viability was determined by Trypan Blue exclusion assay. Cytotoxicity of ritonavir in HepG2, Caco-2, THP-1, A-THP-1 and CEM cells. HepG2, Caco-2, THP-1, CEM, A-THP-1 cells were separately seeded at a density of 2.5 x 104 cells / 100µl in their cell type specific media into each well of a 96 well plate (NunclonTM, Denmark) and incubated for 24 hours at 37oC and 5% CO2. Media was then aspirated and replaced with media containing either 0.1, 1, 10, 100, 500 or 1000µM final concentration of RTV as a nanodispersion or as an aqueous solution, then incubated for a further 24 hours at 37°C and 5% CO2. All cell cytotoxicity analyses were determined using the ATP cell viability assay (CellTiter-Glo® Reagent (Promega, UK)) as follows. All reagents were made fresh and in accordance with the manufacturer’s instructions and allowed to equilibrate to room temperature before use. Post 24 hours incubation the 96 well plates were removed from the incubator and the plate and its contents allowed too equilibrate to room temperature for approximately 30 minutes. After 30 minutes, 100µl CellTiter-Glo® Reagent was added to the media and cells. The contents were then mixed for 2 minutes on an orbital shaker to induce cell lysis. The plate was then incubated at room temperature for 10 minutes to stabilize the luminescent signal. Luminescence was subsequently measured using a Tecan GENios plate reader (Tecan; Austria). Cellular accumulation of ritonavir in HepG2, Caco-2, THP-1, A-THP-1 and CEM cells. HepG2, Caco-2, THP-1, CEM or A-THP-1 cells were separately seeded into each well of a 6 well plate at a density of 5 x 106 cells per well in cell type specific routine cell culture media and incubated at 37°C and 5% CO2 for 24 hours. Plates were then removed from the incubator and the media aspirated. Cells were washed twice with pre-warmed HBSS (37°C) then replaced with pre-warmed (37°C) HBSS containing either 10µM (final concentration) an aqueous solution


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of RTV radiolabeled with 0.1µCi 3H or 10µM (final concentration of RTV) SDN radiolabeled with 0.1µCi 3H RTV. After 60 minutes incubation at 37°C and 5% CO2, 100µl extracellular sample was removed and placed into an empty 5mL scintillation vial. The remaining media was aspirated from the well and cells washed twice in ice cold HBSS. The ice cold HBSS was aspirated and replaced with 500µl of tap water and the plates incubated for 24 hours at -20°C. 4 mL of Ultima Gold liquid scintillation cocktail fluid was added to all intracellular and extracellular samples and radioactivity detected as DPM using a Perkin Elmer 3100TS scintillation counter. Average cell volumes for each cell type were determined using the Scepter 2.0 Handheld Automated Cell Counter (Millipore, USA) and volumes used to calculate intracellular concentrations. A cellular accumulation ratio was then calculated as the ratio of intracellular/extracellular concentrations. Transcellular permeation of ritonavir across Caco-2 cells. 3.5 x 104 Caco-2 cells were seeded on to 6.5mm 0.4µm polycarbonate membrane transwell inserts (Sigma-Aldrich, UK) and propagated to a monolayer over a 21 day period, yielding transepithelial electrical resistance (TEER) values of >1300 Ω. 10µM of RTV SDN or 10µM aqueous solution of RTV (including 0.1µCi radiolabeled drug) was added to the apical chamber of 4 wells and the basolateral chamber of 4 wells to quantify transport in both apical to basolateral (A > B) and basolateral to apical (B > A) direction. Samples from apical and basolateral chambers were taken at 60 minutes intervals to a maximum of 240 minutes and transferred to scintillation tubes. 4 mL of Ultima Gold liquid scintillation cocktail fluid (Meridian, UK) was added to all samples and radioactivity detected as DPM using a 3100TS scintillation counter (Perkin Elmer, USA). Apparent permeability coefficient (Papp) was determined by the amount of compound transported over time using equation below.


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ௗொ

ܲܽ‫ = ݌݌‬ቀ ቁ ቀ ௗ௧

ଵ

஺஼బ

Page 10 of 45

ቁ

Where (dQ/dt) is the amount per time (nmol/sec), A is the surface area of the filter and C0 is the starting concentration of the donor chamber (10 µM). Cell-free CYP3A4 inhibition using baculosomes. The effect of SDNs on CYP3A4 activity was determined using the Vivid® CYP450 Screening Kit (Invitrogen, UK) as per manufacturer’s instructions. Briefly, 50µl of Master Pre-Mix (4850µl of Vivid® CYP450 reaction buffer (2x) and 100µl of Regeneration System) plus 50µl of CYP3A4 was added to each well of a 96 well plate (Nunclon, Denmark). Immediately prior to the experiment, each of the 68 SDNs were separately dispersed in sterile distilled H2O at a concentration of 20000nM RTV to form the SDN stock. A RTV aqueous stock solution was also prepared at the same concentration using DMSO as a vehicle. 50µl of SDN or aqueous RTV stock were then separately added to each individual well of the 96 well plate to give final concentrations of 10000, 1000, 500, 100, 10, 1, 0.1 or 0.01nM. Finally, 10µl of fluorescent substrate consisting of 885µl of Vivid® CYP450 reaction buffer, 15µl of reconstituted substrate and 100µl of NADP+, was added to each well prior to incubation for 2 minutes. Fluorescent readings were then taken every 5 minutes to endpoint (30 minutes) at ambient temperature using a Fluoro4 Tecan GENios Genosis plate reader (Magellan, Austria) with excitation wavelength of 530 nm and emission wavelength of 585 nm. The rate (relative fluorescent units per minute; RFU/min) of substrate turn-over was then calculated in the presence and absence of RTV SDNs or aqueous solution (B) direction at 1 and 2 hour time points. At 1 hour, the mean ± standard deviation of Papp A>B was 7.1 ± 4.3 cm / sec compared to 2.7 cm / sec for the RTV aqueous solution. Of the 68 SDNs tested, 61 exhibited a higher RTV Papp A > B than when incubated with an aqueous solution at 1 hour. At 2 hours, the mean ± standard deviation of Papp A>B was 13.3 ± 9.2 cm / sec compared to 8.2 cm / sec for the RTV aqueous solution. Of the 68 SDNs tested, 49 exhibited a higher RTV Papp A > B than when incubated with an aqueous solution at 2 hours. Summary of CYP3A4 inhibition in baculosomes and primary human hepatocytes. A summary of the inhibitory effects (IC50 nM) for the 68 different SDN formulations on baculosome derived CYP3A4 or human primary hepatocyte CYP3A4 activity are presented in Supplemental Table 1. For inhibition of baculosome CYP3A4, 50 (73.5%) of the 68 SDN formulations exhibited enhanced CYP3A4 inhibition relative to a RTV aqueous solution ( -15mV (suboptimal charge). (C) Difference in ritonavir-mediated inhibition (IC50; nM) of baculosome CYP3A4 between nanoparticles with Z-average diameter between 300 and 900nm (optimal size) versus 900nm (suboptimal size). (D) Difference in ritonavir-mediated inhibition (IC50; nM) of CYP3A4 in primary human hepatocytes between nanoparticles with Z-average diameter between 300 and 900nm (optimal size) versus 900nm (suboptimal size). (E) Combined association of optimal charge and size on ritonavir-mediated inhibition (IC50; nM) of baculosome CYP3A4. (F) Combined association of optimal charge and size on ritonavir-mediated inhibition (IC50; nM) of CYP3A4 in primary human hepatocytes. (G) Correlation between Z-average diameter (nm) and selectivity index (Cytotoxicity IC50 / Inhibition of CYP3A4 in primary human hepatocytes IC50). (H) Correlation between zeta potential (mV) and selectivity index (Cytotoxicity IC50 / Inhibition of CYP3A4 in primary human hepatocytes IC50). Data are given as mean ± SEM for different rSND dispersions. Influence of excipients on CYP3A4 inhibition. We have previously published data illustrating the effects of the excipient used in the present study on the activity of CYP3A4 from cell-free baculosome incubations36. Therefore, we postulated that excipients known to interfere with CYP3A4 activity might influence the resulting RTV-mediated inhibition of CYP3A4. A single component analysis of the inhibition of CYP3A4 in primary human hepatocytes was first


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conducted, not taking into consideration that excipients were present in binary combinations (Figure 5A). In this analysis, SDNs containing excipients known to activate CYP3A4 generally exhibited higher RTV-mediated CYP3A4 inhibition in primary human hepatocytes (Figure 5A; red data). Similarly, SDNs containing excipients shown to inhibit CYP3A4 generally exhibited lower RTV-mediated CYP3A4 inhibition in primary human hepatocytes (Figure 5A; green data). Data were next analyzed to assess the combination of excipients. Data showing the IC50 for RTV-mediated inhibition of CYP3A4 in primary hepatocytes for SDNs with each successful excipient binary combination (Figure 5B). Previous knowledge of the impact of excipients on CYP3A4 activity were then used to color code the individual binary combinations according to whether they contain two excipients known to exert activation of CYP3A4 (Figure 5B; dark red data), one excipient known to exert activation and one inert excipient (Figure 5B; light red data), two inert excipients (Figure 5B; white data), one excipient known to activate and one known to inhibit CYP3A4 (Figure 5B; yellow data), one excipient known to exert inhibitory effects and one inert excipient (Figure 5B; light green data), or two excipients known to inhibit CYP3A4 (Figure 5B; dark green data). Good segregation of the data according to used excipients was evident (Figure 5B) and SDN dispersions with at least one CYP3A4 activator were less potent than SDN dispersions with either an activator and an inhibitor excipient (P = 0.01), at least one inhibitor excipient with an inert excipient (P = 0.0004) or two inert excipients (P = 0.06; Figure 5C).


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Figure 5. Effect of excipient choice on inhibition of CYP3A4 in primary human hepatocytes. (A) Single component analysis showing inhibition of CYP3A4 in primary hepatocytes (IC50;


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nM) according to different excipients used in formation of SDNs. Data colored red illustrate excipients that have previously been shown to augment CYP3A4 activity whereas data colored green illustrate excipients previously shown to inhibit CYP3A4 activity.36. (B) Dual component analysis showing inhibition of CYP3A4 in primary hepatocytes (IC50; nM) according to different excipient combinations used in formation of SDNs. Data colored dark red illustrate SDN dispersions with both excipients previously shown to augment CYP3A4 activity. Data colored light red illustrate SDN dispersions with one excipient previously shown to augment CYP3A4 activity. Data colored dark green illustrate SDN dispersions with both excipients previously shown to inhibit CYP3A4 activity. Data colored light green illustrate SDN dispersions with one excipient previously shown to inhibit CYP3A4 activity. Data colored yellow illustrate SDN dispersions with one excipients previously shown to inhibit CYP3A4 and one excipient previously shown to augment CYP3A4 activity. (C) Mean ± SEM inhibition of CYP3A4 in primary hepatocytes (IC50; nM) according to whether excipients used have previously been shown to influence CYP3A4 activity. To further probe the association with excipients previously shown to influence CYP3A4 activity, SDNs were segregated based on which polymer or surfactant they contained. For polymer excipients, an independent variable was created (polymer effect) whereby a value of -1 was given for use of a polymer previously shown to activate CYP3A4, a value of 1 was given for inclusion of a polymer previously shown to inhibit CYP3A4, and a value of 0 was assigned to those SDNs containing excipients known not to influence CYP3A4. Another independent variable (surfactant effect) was created in the same way with the same value assignments based on known CYP3A4 interactions of the surfactant. Subsequently, multiple linear regression was


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conducted using the new polymer and surfactant independent variables and RTV-mediated inhibition (IC50) in either baculosomes or primary human hepatocytes as the dependent variable. For RTV-mediated inhibition in baculosomes, the final model (R2 = 0.11; PANOVA = 0.03) included the constant (Beta = 4.0; P < 0.0001), polymer effect (Beta = -3.2; P 0.03) and surfactant effect (Beta = -1.4; P = 0.09). Similarly for primary human hepatocytes, the final model (R2 = 0.14; PANOVA = 0.03) included the constant (Beta = 7.7; P < 0.0001), polymer effect (Beta = -5.6; P 0.02) and surfactant effect (Beta = -3.0; P = 0.03). Taken collectively, these data indicate that only 11% and 14% of the variability in CYP3A4 inhibition could be explained by prior knowledge of the excipients used for baculosomes and primary human hepatocytes, respectively.

Discussion RTV has gained wide utility as a pharmaco-enhancer, effectively boosting efficacy of coadministered antiretrovirals through inhibition of drug efflux transporters and CYP3A4 via competitive37 or mixed competitive-/non-competitive mechanisms.38 Although the use of RTV has a beneficial effect on bioavailability and clearance of co-administered drugs, it is also associated

with

numerous

drug–drug interactions

with

concomitant

medications.39-41

Additionally, toxicities and poor tolerance associated with chronic exposure to RTV have justified investigation into strategies to avoid RTV and the development of alternative pharmacoenhancers. For example, atazanavir is frequently administered off-label without boosting and cobicistat has been developed as a mechanism-based pharmaco-enhancer for PIs. Interestingly, both cobicistat and RTV interact directly at the heme iron of CYP3A442 and deactivate CYP3A4 in a time- and concentration-dependent manner.43 Cobicistat has also been shown to inhibit certain drug transporters leading to reduced creatinine renal tubular secretion.44 However, despite


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associated toxicity and drug-drug interaction problems, RTV is also being investigated as a “boosting” agent for hepatitis C virus PIs.45 Therefore, any strategy for reducing or eliminating RTV dose is of clear benefit. In recent years, nanomedicine approaches have provided several technological platforms including polymeric micelles,46 solid lipid nanoparticles,47 liposomes48 and solid dispersions49 aimed at increasing dissolution and oral bioavailability of poorly water-soluble drugs,50,

51

avoiding first-pass metabolism, pre-systemic metabolism and efflux mechanisms.52, 53 Currently, best practice for HIV interventions requires oral administration as the chronic nature of this blood-borne disease effectively rules out daily parenteral dosing. The development of SDNbased intramuscular and subcutaneous depot injections for long-acting delivery of antiretrovirals has shown recent promise; SDNs are also being considered for intravenous injection and, therefore, studies that indicate enhanced or detrimental behavior derived from the particulate nature of the SDNs are important in assessing both the long-term opportunities and potential safety concerns from such strategies.54 With a large of range of chemical and physical outcomes available from different SDN manufacturing techniques, it is clear that a wide diversity of SDN sizes, zeta potentials and surface chemistries must be studied against a broad spectrum of relevant pharmacological outcomes. In this study, we have utilized emulsion-templated freeze-drying technology29 to generate 68 different SDNs (RTV 10%w/w) from a wide diversity of formulations containing binary combinations of commonly used pharmaceutical polymers and surfactants. These pharmaceutical excipients were selected to enable control of particle characteristics i.e. Z-average diameter (nm), zeta potential (mV) and polydispersity and represent clinically relevant materials that would be expected to be used in therapeutic product development. The adsorption of the excipients to the


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surface of the hydrophobic SDNs is hypothesized to impact the interaction with cells, tissues, proteins and enzymes. The library of SDNs were subsequently assessed for their transcellular permeation across Caco-2 cells to establish opportunities for nanoformulation under oral dosing conditions, cellular accumulation and potential to augment inhibition of CYP3A4. The majority of SDNs were found to enhance CYP3A4 inhibition in baculosomes (i.e. cellfree) and human primary hepatocytes. More specifically, from the 68 different formulations generated, 50 (73.5%) significantly enhanced baculosome derived CYP3A4 (cell free) inhibition and 44 (64.7%) significantly enhanced CYP3A4 inhibition in primary human hepatocytes relative to the effects of an aqueous solution of RTV. Furthermore, the vast majority of these SDNs had comparable cellular accumulation compared to an aqueous solution of RTV but with significantly less cytotoxicity for HepG2 (hepatic), Caco-2 (intestinal), THP-1 (human acute monocytic), ATHP-1 (activated monocyte derived macrophages) or CEM (human T lymphocytes). The increase in activity of the SDNs over free drug in experiments under cell-free conditions suggests a general benefit from the particulate nature of the SDNs rather than an effect mediated directly by the excipients or any modified surface chemistry. The exact nature of this enhancement is not clear; however, it is possible that the SDNs are able to access the binding site of CYP3A4, or efficiently direct larger concentrations of free molecule to the binding sites. The enhancement of inhibition in primary human hepatocytes suggests that the critical penetration into cells is also occurring for the SDNs, but the decrease in the number of SDNs able to demonstrate an enhanced activity under these conditions would imply mediation of cell accumulation by SDN surface chemistry, resulting from the different excipients used. Actual drug accumulation was highly comparable between SDNs and free drug molecules but it is important to note that the assays utilized to quantify drug concentrations do not discriminate


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between the physical nature of the drug compound within cells. Our previous studies using hydrophobic FRET dye pairs34 to form combination SDNs, clearly showed the potential for cellular uptake of intact SDNs and we postulate that this is the reason for the observation of comparable drug accumulation but reduced toxicity from SDNs when compared to free drug; the SDNs enter the cells predominantly as nanoparticles, presenting a low molecular concentration of RTV, which then display an altered cellular distribution and enhanced CYP3A4 inhibition, despite the apparent comparable molar concentration of drug being present. It is important to ensure that the effects and hypotheses around nanoparticle-derived benefits are not purely derived from excipient effects alone as the nanoparticulate nature of the SDNs may not be key to the observed behavior. Statistical analysis was conducted to determine relationships between specific SDN characteristics i.e. Z-average diameter, polydispersity or zeta potential and pharmacological endpoints. Consistent with previous studies,55, 56 we determined negatively charged nanoparticles (

Augmented Inhibition of CYP3A4 in Human ... - ACS Publications

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