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In vitro characterization of ritonavir drug products and correlation to human in vivo performance Hao Xu, Socrates Vela, Yi Shi, Patrick Morroum, and Ping Gao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00552 • Publication Date (Web): 12 Sep 2017 Downloaded from http://pubs.acs.org on September 24, 2017

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Molecular Pharmaceutics

In vitro characterization of ritonavir drug products and correlation to human in vivo performance

Hao Xu1, Socrates Vela1, Yi Shi1, Patrick Marroum2, Ping Gao1* 1 NCE-Formulation Sciences, Drug Product Development, 2 Clinical Pharmacology and Pharmacometrics Abbvie Inc. 1 North Waukegan Road, North Chicago, IL 60064

Manuscript is intended for submission to Mol. Pharm. *Corresponding author: E-mail: [email protected] Phone: 847-938-4532

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ABSTRACT Ritonavir (RTV) is a weakly basic drug with a pH-dependent solubility. In vitro dissolution and supersatuation behaviors of three Norvir® oral products including the Tablet, Powder and Solution were investigated by two biorelevant dissolution methods with pH alteration: a twostage dissolution test and a biphasic dissolution-partition test. The two-stage dissolution test revealed a high degree of supersaturation of RTV from these products accompanied by the occurrence of liquid-liquid phase separation (LLPS) in biorelevant dissolution media. Higher, stable apparent RTV concentrations were observed in the FeSSIF-V2 as compared to those in the FaSSIF-V2, suggesting a food effect with higher exposure in the fed state. This is inconsistent with the evaluation in vivo. The biphasic test revealed significantly lower degrees of supersaturation of RTV in the aqueous media from these dosage forms as compared to results of the two-stage dissolution test. RTV concentrations in octanol at 6 hours obtained from the tablet and powder with the use of the biorelevant media are consistent with corresponding in vivo AUC and Cmax under the fasting and moderate fat fed (MFF) states, predicting the food effect. The underlying mechanisms responsible for the food effect are also proposed. Fractional partition profiles of RTV obtained in octanol from these three Norvir® oral products are in agreement with the corresponding fractional absorption profiles in vivo under both the fasting and MFF states. This study revealed a complex interplay among the dissolution, precipitation, and partition processes from these formulations that dictate the oral exposure of RTV. KEYWORDS: ritonavir, absorption, dissolution, in vitro models, IVIVR, Wagner-Nelson method, partition, biphasic, BCS, food effect, relative bioavailability,


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INTRODUCTION

Many active pharmaceutical ingredients are classified as weak bases. Regardless of the salt form, special attention should be paid to oral administration of weakly basic drugs with low pKa value(s) and a high dose number. A weak base drug with low pKa values (≤ 7) may possess sufficient dissolution in the acidic environment of the stomach. However, this could yield a supersaturated state as the drug transits into the small intestine due to the low solubility of the un-ionized form with a tendency to precipitate. Investigations of precipitation events in the GI tract in vivo have been explored for several weakly basic drugs including ketoconazole, itraconazole, posaconzole, and dipyridamole [1-7]. All of these drugs possess a pH-dependent solubility and undergo a supersaturated state accompanied by significant precipitation in the small intestine due to the elevated pH. These studies demonstrated significant variation of the oral bioavailability due to pH change as a result of co-administration of other drugs (e.g., proton pump inhibitors), the presence of food (e.g., fasting or fed state), and pH alteration beverages. The concept of sustaining the supersaturation as an effective formulation approach for improving bioavailability of poorly water soluble drugs has been broadly explored in the last decade [8-13]. In particular, enabling technologies such as the amorphous solid dispersion (ASD) have been widely applied in the pharmaceutical industry [9-15]. The scheme shown in Figure 1 proposed by Taylor et al. illustrates possible pathways of poorly water soluble drugs when the ASD is introduced into the aqueous media [15]. As a supersaturated solution of the drug is achieved and unstable with respect to its amorphous and crystalline forms, then a liquid-liquid-phase separation (LLPS) could occur [15-17]. The LLPS may serve as a reservoir to sustain the “amorphous solubility” in the supersaturated state, replenishing the depleted drug concentration due to permeation. A major challenge for developing supersaturatable formulations is to control the rate and degree of supersaturation with the use of polymeric precipitation inhibitor (PPI) [817]. While studying precipitation or crystallization in vivo is not practical, the possibility of using biorelevant in vitro tests in combination with the PK modeling and simulation has been explored. Characterization of in vitro dissolution, and, in particular, characterization of the



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supersaturated state with emphasis on polymer-modulated interactions, are highly desirable in order to optimize such supersaturation based formulations [8-17]. Diverse two-compartment dissolution methods with pH alteration have been utilized to simulate the dissolution and transit from the gastric compartment to the intestinal compartment in vivo [14, 18-35]. Representative two-compartment dissolution methods include a transfer model with both donor and acceptor compartments reported by Kostewicz et al [24], an artificial stomachduodenum model developed by Hawley et al [25-29] , and a gastrointestinal simulator (GIS) incorporates a jejunum compartment as the third chamber reported by Amidon et al. [33-35]. In contrast, a two phase dissolution-partition test method with pH alteration, referred to as the biphasic test, has been developed in order to simulate a dynamic environment in which three processes of drug dissolution, precipitation and partition occur simultaneously. A practical biphasic system combining an USP IV apparatus (with a flow cell) and an USP II dissolution apparatus and its application for the evaluation of several poorly soluble drugs were first reported by Gao and his co-workers in 2009 [36]. The biphasic test method permits dissolution in the aqueous media (with pH alteration) under the non-sink condition and simultaneous partition of the dissolved drug into an organic phase that acts as an “absorption compartment”. The partition of the drug is driven by the free drug concentration in the aqueous phase and this is to mimic the absorption in vivo. Therefore, the concentration-time profile of the drug in the organic phase may serves as an output for establishing in vitro and in vivo relationships (IVIVR). Applications of the biphasic method to BCS II drugs including AMG517 [36], celecoxib [37], ibuprofen [38], ABT-072 [39], fenofibrate [40] and related formulations have been reported with IVIVR. Multiple investigations of biphasic tests for several poorly water soluble drugs demonstrated its broad utility for drug product development [41-45]. RTV (molecular structure shown in Figure 2) is a HIV protease inhibitor. It is a very weak base with pKa values of 1.8 and 2.6 [46-47]. It has a high aqueous solubility at pH < 1 and extremely low solubility at pH 4-7. RTV is an antiretroviral medication and is often used along with other medications to treat HIV/AIDS [48-51]. Commercial RTV products under the brand name of Norvir® include solution (80 mg/mL), tablet (100 mg strength), and powder (100 mg/unit). The solution contains 43.2% (v/v) ethanol and 26.0% (v/v) propylene glycol with the presence of 4 ACS Paragon Plus Environment

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surfactant polyoxyl 35 castor oil [48]. Both the tablet and powder formulations are based on an ASD containing PVP-VA copolymer. RTV and related prototype ASD formulations were well studied [16-17, 52-56]. These results revealed that the relationship between supersaturation and solubilization of RTV from related ASDs is complex and highly media dependent. The purpose of this work was to evaluate two biorelevant in vitro tests including the two-stage dissolution test and the biphasic test using RTV commercial products (e.g., Norvir® tablet, powder, and solution) as a model drug product. This was to characterize dissolution and precipitation behaviors of RTV in the aqueous media from the ASD (e.g., tablet and powder) and the solution formulations. The in vitro profiles of three RTV formulations obtained from both dissolution methods were compared to pharmacokinetic data in human subjects. Based on the in vitro characterization, we propose the underlying mechanisms that account for a food effect of the tablet and powder formulations observed in vivo.

MATERIALS AND METHODS Materials Norvir® commercial drug products evaluated in this study including the oral solution (80 mg/mL), 100 mg tablet, powder (100 mg/unit), as well as crystalline powders of RTV substance were obtained in house. Ethanol, octanol, acetonitrile, maleic acid, sodium hydroxide, acetic acid, sodium acetate and sodium chloride were purchased from Sigma (St. Louis, MO). The FaSSGF, FaSSIF-V2 and FeSSIF-V2 powders were purchased from Biorelevant (UK). The FeSSGF (pH 5.0) medium was prepared following a published recipe with whole milk [57]. The v/v ratio of the buffer to milk was 1:1 and the final pH of the mixture was adjusted to 5.0 with 1 N HCl after mixing.

Solubility and liquid-liquid phase separation (LLPS) The solubilities of RTV crystal Form I and II were determined in FaSSGF (pH 1.6), FeSSGF (pH 5.0), FaSSIF-V2 (pH 6.5) and FeSSIF-V2 (pH 5.8). Approximately 5 mg of RTV was loaded to each clear glass vials containing 3 mL of the biorelevant medium and shaken for 24 hours (in 5 ACS Paragon Plus Environment


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FeSSGF for 6 hours) at 37 °C. After the samples from the FaSSGF, FaSSIF-V2 and FeSSIF-V2 were centrifuged for 5 min at 21,000 x g force, supernatant (300 µl) was pipetted out and mixed with an equal amount of acetonitrile (300 µl). Samples were then analyzed by an Agilent 1100 HPLC instrument. Because the FeSSGF contained whole milk, samples from this medium (1 mL) were first centrifuged for 5 min at 21,000 x g force. The supernatant (350 µL) was pipetted out and mixed with an equal amount of acetonitrile (350 µL). The mixture was centrifuged again for 5 min at 21,000 x g force to spin down the precipitated protein. Samples of 600 µL were pipetted and analyzed by HPLC. The LLPS onset concentration was detected by monitoring the light scattering effect at high wavelength using a UV probe (Rainbow®, Pion Inc.). An ethanol solution containing 10 mg/mL RTV was gradually introduced to each biorelevant medium at 37 °C. The RTV concentration was considered as the LLPS onset concentration when a sharp increase of the UV absorption at 700 nm was observed [15]. The total amount of ethanol introduced into the aqueous media was less than 1% (v/v) for all experiments.

HPLC Assay Determination of RTV aqueous concentration was performed on an Agilent 1100 HPLC system with a 4.6 x 50 mm 2.5 micron XBridgeTM C18 column (Waters) and a gradient mobile phase of two buffers (A: water with 0.1% formic acid; B: acetonitrile with 0.1% formic acid; 2% – 40% B at 0 – 1 min; 40% - 90% B at 1 – 7 min). The flow rate of the mobile phase was 1 mL/min. RTV concentrations were determined by the peak areas of the HPLC peak at 240 nm. All solubility results were carried out in triplicate.

Two-stage dissolution test The two-stage dissolution test is schematically shown in Figure 3A. The drug product was loaded to the USP I apparatus equipped with a mesh #10 rotating basket. Initially, the vessel contained 30 mL FaSSGF at 37 ºC and the basket was rotated at 200 rpm for 30 min. Samples were collected from this medium at the end of this stage as the time point of 0 min, followed by an addition of 120 mL FaSSIF-V2 (×1.25). Samples (500 µL) were taken at 10, 40, 70, 100, 130, 210 and 360 min. and centrifuged for 5 min at 24,000×g force. The supernatant (110 µL) was pipetted out and mixed with a diluent (acidified 50%/v acetonitrile, 220 µL) and then analyzed 6 ACS Paragon Plus Environment

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for RTV concentration. In addition, solution samples were taken from the aqueous media during the dissolution test and examined for suspended particles by a polarized light microscopy equipped with a 1st order red filter.

Biphasic dissolution test The biphasic dissolution-partition test is schematically shown in Figure 3B. This test included an USP II dissolution apparatus in combination with an USP IV apparatus (Sotax) [36-37]. A standard CE 7 flow cell (USP IV apparatus, Sotax) with a diameter of 2.2 cm was used in this study. The USP II apparatus has six vessels and each contained 200 mL aqueous dissolution media (Table 2) and 200 mL octanol above the aqueous phase. The two phases were allowed to saturate with each other by mixing for 30 min prior to use. Initially, individual RTV products were loaded into the flow cell filled with the FaSSGF (or FeSSGF). The dissolution medium (a total volume 41 mL in the flow cell and tubing) was circulated in this closed-loop for 30 min. at a fixed flow rate. This was considered as “the gastric stage”. Then, the medium in the flow cell was connected to the aqueous medium of 200 mL FaSSIF-V2 (x1.2) (or FeSSIF-V2 x1.2) in the USP II vessel through a loop of Teflon tubing (Figure 3B). A dual paddle mounted on a regular USP II apparatus was rotated at 60 revolutions-per-minute (RPM) in order to provide sufficient mixing in both aqueous and organic phases. Each biphasic test was run for 6 hours. The RTV concentration-time profile in octanol was obtained using a UV probe (Rainbow®, Pion Inc.) at 240 nm. Partitioning of species from the biorelevant media (i.e., lecithin, bile salt, fat from milk) into octanol may occur due to their hydyophobicity (and solubility). Therefore, the presence of these species in octanol is expected to contribute to the UV absorption spectra in octanol. Determination of RTV by direct UV absorbance was considered not a reliable approach due to interference of these species partitioning from the biorelevant media (even though their concentrations were very low). Instead, the 2nd derivative of UV absorbance was employed for quantitation of RTV concentration. The UV absorption profiles of both the blank (as Control) and the RTV formulations (as Test) were always obtained in parallel. The 2nd derivatives of UV absorbance from the Control and the Test were carefully compared. The wavelength of 268 nm was selected for quantitation of RTV under the 2nd 7 ACS Paragon Plus Environment


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derivative mode based on (1) no signals from the Control, (2) RTV exhibiting strong absorption, and (3) the calibration curve covering a wide range of concentrations with linearity. The drug concentration in octanol was regularly calibrated by HPLC as a routine practice. Aqueous samples were collected from the dissolution medium at predetermined time points and centrifuged for 5 min at 21,000 x g force. Supernatants were diluted with an equal amount of ethanol, and analyzed for RTV content by an Agilent 1100 HPLC system. A water bath, which jacketed the dissolution vessel and the flow cells, was maintained at 37 ± 0.2 ºC. The flux of RTV into octanol, J(t), the change in mass per unit time and area across the wateroctanol interface was calculated using the equation: J(t) = dM/(dt.A) = (V/A) x dC/dt, where dM/dt is the change in mass (mg) per unit time (min), A (cm2) is the interfacial area between water and octanol, V (mL) the volume of octanol, and dC/dt (µg/mL.min) the slope of the drug concentration-time profile . The flux (µg/min.cm2 ) within a time window selected was calculated by the slope of the corresponding concentration-time profile in octanol multiplying the volume of octanol (200 mL) and divided by the interfacial area of 84.9 cm2.

Wagner-Nelson (W-N) method The W-N method was used to calculate the fraction absorption-time profile of RTV from pharmacokinetic data in human subjects [58]: t

F

W-N (t)

=

⌠ C(t) dt + C(t) ke ⌡ 0

∞

⌠ ke ⌡ C(t) dt 0

•

C(t):

drug concentration in human plasma

•

ke :

elimination rate constant

•

FW-N(t): absorbed drug fraction

Where the elimination rate constant (ke) was estimated from the mean value of the pharmacokinetic profile and used in the calculation of the fraction of RTV absorbed-time profiles. As either individual or mean pharmacokinetic profiles can be deconvoluted by the W-N method, we chose to use mean plasma drug concentration-time profiles based on scientific 8 ACS Paragon Plus Environment

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literature on this subject [58-60]. The calculation of the fraction of drug absorbed - time profiles was performed with the use of the W-N equation using Excel (Microsoft).

RESULTS AND DISCUSSION The pH-solubility profile of RTV is shown in Figure 2B. RTV crystalline Form I and II possess ~ 400 µg/mL at pH 1.0 and show a significant reduction of solubility at pH 6.8 [46-47]. Form II has an equilibrium solubility of ~2 µg/mL at pH 6.8. The equilibrium solubility of crystalline RTV and LLPS concentrations in biorelevant media (e.g., FaSSGF, FeSSGF, FaSSIF-V2, FeSSIF-V2) were determined and are reported in Table 2. Assessment of supersaturation, LLPS and precipitation in the two-stage dissolution test In the simulated fasting media The two-stage dissolution test was conducted with a sequential use of the FaSSGF and FaSSIFV2 media (Figure 3A). The solution formulation was not tested under this condition due to a lack of the PK data. RTV concentration-time profiles observed in the FaSSIF-V2 are shown in Figure 4A. The very first data point reported at t = 0 in Figure 4A was obtained in the FaSSGF (pH 1.6) at the “gastric dissolution stage” immediately prior to adding the FaSSIF-V2. Due to a low pH value of the FaSSGF, apparent RTV concentrations of ~ 230 µg/mL and 420 µg/mL were observed from the Tablet and Powder, respectively. It is worth noting that the drug content in these samples after centrifugation may inevitably include the free drug, solubilization by bile salt micelles, LLPS and solid particles in submicron size. Therefore, the RTV concentration determined in this study is referred to as the apparent concentration. After the FaSSIF-V2 was added at the second stage, the pH value of the dissolution media was elevated from 1.6 to 6.5. As expected, a very rapid decrease of RTV concentration was observed within 10 min. The first sample obtained after addition of FaSSIF-V2 yielded the RTV concentration of ~ 60 µg/mL from both the tablet and powder formulations and these concentrations remained unchanged during the course of 6 hours. Samples were taken from the FaSSIF-V2 medium at 6 hours and examined under a polarized light microscope (PLM) with a magnification of 500X. PLM photos of samples from the tablet and powder formulations are shown in Figures 4B and 4C. Suspended particles in the range of 9 ACS Paragon Plus Environment


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a few microns are observed in both samples without birefringence, suggesting an absence of crystalline material. Compared to the tablet formulation, the sample from the powder contained more condensed particles.

In the simulated fed media The dissolution study of the solution, tablet and powder was performed by the two-stage dissolution test in a similar manner with the use of the FeSSGF and FeSSIF-V2. RTV concentration-time profiles are shown in Figure 5A. The solution exhibited a high apparent RTV concentration of ~ 520 µg/mL in the FeSSGF immediately prior to adding the FeSSIF-V2 and a sharp decrease to ~190 µg/mL after 10 min. This concentration was remained unchanged during the rest of the 6 hours. In contrast, the tablet and powder exhibited a lower apparent RTV concentration of ~50 µg/mL in the FeSSGF that increased to ~160 µg/mL after adding the FeSSIF-V2 around 10 min. These apparent RTV concentrations remained unchanged for the duration of the experiment. Samples were also taken from the FeSSIF-V2 at 40 min. and 6 hours, respectively. PLM photos of these samples from the tablet, powder and solution formulations at 40 min are shown in Figures 5B-C-D and PLM photos of samples at 6 hours are shown in Figures 5E-F-G. Consistent with the samples from the FaSSIF-V2 (Figures 4B-C), suspended particles with the size of few microns were observed in Figures 5B-C-D without exhibiting birefringence. However, a few needle shaped crystals, consistent with RTV Form II, were present in all three samples (Figures 5E-F-G). These observations differ from observations of samples in the FaSSIF-V2 (Figures 4B-C). Compared to the tablet and powder formulations, the solution showed more particles of smaller size in the dissolution medium (not visible in the PLM). As evidenced by these PLM photos, RTV-LLPS occurred in biorelevant media. This apparent RTV concentration of 60 µg/mL in the FaSSIF-V2 from the tablet and powder (Figure 4A) is consistent with its LLPS concentration of ~40 µg/mL determined separately (Table 2). Similarly, apparent RTV concentrations of the tablet, powder and solution in the FeSSIF-V2 are approximately 180-200 µg/mL (Figure 5A), also in agreement with the LLPS in the FeSSIF-V2 (~ 180 µg/mL , Table 2). These results suggest that RTV LLPS concentration, or the amorphous solubility in the medium, dictates the maximum concentration of RTV. 10 ACS Paragon Plus Environment

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Investigation by Taylor’s group indicated that RTV is a slow crystallizer and has a long induction time for crystal nucleation (>2 h) [15-17]. Thus, LLPS is kinetically favored at high supersaturations relative to crystallization. The LLPS concentration of RTV was reported to be 37.2 - 39.8 µg/mL in water (pH 7.4) and comparable to the predicted amorphous solubility [15]. In addition, Taylor et al. reported that RTV LLPS concentration is not affected by the presence of polymers including PVP, PAA, HPMC, HPMCAS, and CAAdP. These facts indicate that the polymer is not substantially incorporated into the RTV-rich colloidal phase [17]. Brandl et al. reported that the dissolution rate of RTV from ASD extrudates was dramatically increased with the presence of 1 mM Na-taurocholate in the phosphate buffer (pH 7.3) and small particles in the size range of 40–50 nm were generated from PVP-VA based extrudates and stable for several hours [52-53]. Another study by the same group revealed three types of nano-sized colloid particles from RTV (and LPV) PVP-VA based ASDs by the asymmetrical field-flow fractionation analysis, including drug-rich particles, PVP-VA particles and surfactant/silica particles [61]. Recently Harmon et al reported the formation of nano-sized drug/surfactant particles from PVP-VA based ASDs in the aqueous media and revealed the underlying mechanism [62]. Although different terms including the nano/micro-dispersion, nanodroplet, LLPS, and glass-liquid phase separation (GLPS) were used in these manuscripts, these descriptions may be more or less similar in nature as it is difficult to differentiate them unless detailed comparison is provided). Our study results of the RTV tablet and powder based on PVP-VA ASD described above are in agreement with these observations. Rodriguez et al. reported that RTV self-organized into various phases as a result of the supersaturation created in aqueous solutions [54]. The lyotropic liquid crystal (LLC) vesicles of RTV slowly transformed to the polymorphs of RTV, Form I and finally Form II. Amorphous RTV in aqueous suspension also underwent a transformation to a mesophase of similar morphology. PLM photos of samples collected in the FeSSIF-V2 from all three RTV products by the two-stage dissolution test exhibited crystalline RTV needles at 6 hours, in agreement with this previous investigation. Assessment of supersaturation, LLPS and precipitation in the biphasic test



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In the simulated fasting media The hydrodynamic conditions (e.g., the flow rate and dual paddle speed) of the biphasic test play a significant role in the dissolution and partition kinetics, and, therefore, affect the drug concentration profiles observed in both aqueous and octanol phases [38, 40]. In this work, profiles of all the three RTV formulations were obtained with a fixed dual paddle speed of 60 RPM and the flow rate varying at 5, 10 and 15 mL/min (data at 10 and 15 mL/min not reported). The RTV amount-time profiles in octanol at different flow rates were compared to the corresponding fraction absorption-time profiles (obtained by the W-N method). In vitro profiles in octanol obtained at the flow rate of 5 mL/min were identified as the best match to those absorption profiles in vivo and are selected for discussion. RTV apparent concentration-time profiles of the tablet and powder obtained in the FaSSIF-V2 are reported in Figures 6A. As RTV from each formulation was initially dissolved in the FaSSGF (pH 1.6) that was then mixed with the FaSSIF-V2 (pH 6.5), this resulted in a high degree of supersaturation in the FaSSIF-V2. The tablet exhibited an apparent peak concentrations of ~ 50 µg/mL around 20 min, then rapidly declined to ~20 µg/mL within 40 min and gradually decreased to ~10 µg/mL during the course of the study. In contrast, the powder exhibited a much higher apparent peak concentration of 200 µg/mL around 20 min, rapidly declined to ~40 µg/mL around 40 min and gradually decreased to ~10 µg/mL. Note that the RTV LLPS is ~ 40 µg/mL in the FeSSIF-V2 (Table 2). The apparent RTV concentrations at > 40 µg/mL of the tablet and powder in this medium (Figure 6A) were observed only within a time window of less than the initial 60 min, indicative of a rapid removal of RTV by partition and precipitation. The RTV concentration-time profiles in octanol for the tablet and powder are plotted in Figure 6B. These profiles exhibited a low flux between 0 to ~ 40 min from the tablet and 0 to ~100 min. for the powder, respectively (Figures 6B), indicating a low partition flux associated with a low free drug concentration. A rapid decrease of the partition flux of RTV and reaching a concentration plateau in octanol were observed for the tablet around 180 min. and for the powder around 270 min. (Figure 6B). Reaching a concentration plateau in octanol indicates that the free RTV concentration in the aqueous medium is diminishing. At 6 hours, the RTV concentration in octanol from the tablet and powder reached ~310 µg/mL and 330 µg/mL, respectively. These 12 ACS Paragon Plus Environment

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concentrations result in a total amount of 62 mg and 65 mg of RTV partitioned into octanol (also reported in Table 3), representing about 65% of the entire dose (100 mg). RTV concentration-time profiles of the tablet and powder observed in octanol overlapped during the initial stage (time 0 to 100 min.), but exhibited significantly different flux afterwards (Figure 6B). The apparent flux of RTV into octanol of the tablet and powder between the time region of 100 to 200 min. is estimated. The flux of RTV of the powder ( ~ 3.3 µg/min.cm2) is significantly lower than that (~4.9 µg/min.cm2) of the tablet. Both the tablet and powder are made from the same ASD material. The powder contains granules with d50 of ~350 um that possess a large surface area. Thus, the powder exhibited a more rapid dissolution and yielded a high degree of supersaturation in the FaSSGF-V2 (pH 1.6). Sequentially, an extensive amount of RTV precipitated upon adding the FaSSIF-V2 (pH 6.4), as compared to that for the tablet (Figure 6A). This led to a lower apparent flux of the powder due to sluggish dissolution of RTV precipitates as compared to those of the tablet (Figure 6B).

In the simulated fed media Apparent RTV concentration–time profiles for all three products obtained in the FeSSIF-V2 (pH 5.8) after exposure to the FeSSGF (pH 5.0) are shown in Figure 7A. A rapid decrease of apparent concentrations from ~50 µg/mL to ~5 µg/mL in about 100 min. was observed with the solution formulation. As the solution contains 43% ethanol and 26% propylene glycol, a loss of RTV solubilization capacity upon mixing with the aqueous medium is expected, resulting in a significant amount of precipitation. Initially, the powder exhibited a low apparent concentration of 20 µg/mL that increased to its peak concentration of ~ 30 µg/mL around 50 min., and then gradually decreased to ~10 µg/mL at 180 min. Similarly, an apparent RTV concentration of ~10 µg/mL was observed for the tablet and increased to ~ 20 µg/mL between 50 and 200 min. and gradually decreased to < 10 µg/mL. Overall, the much lower peak concentrations of RTV for the tablet and powder compared to the LLPS concentration of 180 µg/mL in the FeSSIF-V2 (Table 2) clearly indicate a low degree of supersaturation. Noticed that apparent RTV concentrations from both the tablet and powder were significantly lower than the LLPS concentration of ~ 180 µg/mL because of insufficient dissolution in the medium.



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The RTV concentration-time profiles in octanol for the solution, tablet and powder are shown in Figure 7B. The solution shows the highest flux (~ 5.4 µg/min.cm2) of RTV into octanol among these three products and is attributed to a high degree of supersaturation. The tablet exhibited a slightly lower flux (~ 4.9 µg/min.cm2). The flux of the powder (~1.4 µg/min.cm2) was consistently low during the whole course. As the powder possesses significantly larger surface area than the tablet, it is expected to show comparable or even higher partition flux than that of the Tablet. The significantly lower flux for the powder than those of the tablet and solution is reflective of its lower RTV free drug concentration in the FeSSIF-V2 and this is most likely due to a rapid precipitation of RTV and conversion from the amorphous state to the crystalline Form II (as revealed in the two-stage dissolution test, Figure 5). At 6 hours, RTV concentrations in octanol from the tablet, powder and solution formulations reached ~260, 205 and 260 µg/mL , corresponding to a total amount of 52, 41 and 52 mg RTV partitioned into octanol (also reported in Table 3), respectively.

Comparison of the two biorelevant dissolution tests In the FaSSIF-V2, the apparent RTV concentrations for both the Tablet and Powder were maintained at ~60 µg/mL without noticeable variation within 6 hours by the two-stage dissolution test (Figure 4A). This is significantly higher than those (10 to 25 µg/mL) obtained in the same medium by the biphasic test (Figure 6A). In the FeSSIF-V2, the constant apparent RTV concentrations for all three formulations were observed around ~180 to 200 µg/mL within 6 hours by the two-stage dissolution test (Figure 5A). Much lower apparent RTV concentrations of less than 50 µg/mL for the three formulations are obtained by the biphasic test in the same medium with a gradual decrease to 5 µg/mL at 6 hours (Figure 7A). The significant difference of the apparent RTV concentrations in the same medium for these products between the two biorelevant dissolution tests is of interest and worth commenting on. Overall, an overestimation of the degree and duration of supersaturation of RTV in the same aqueous media from the two-stage dissolution test is apparent. The apparent RTV concentration is dictated by the aqueous media used in the two-stage dissolution test and this is limited to its amorphous solubility (or the LLPS concentration) by removal of the portion of dissolved RTV exceeding the LLPS through precipitation. Thus, the same apparent RTV concentration was 14 ACS Paragon Plus Environment

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observed for the three products and maintained without variation. In contrast, due to the presence of the octanol phase acting as an “absorption compartment”, a constant desupersaturation by removal of dissolved RTV through partition was occurring simultaneously in addition to the precipitation. As a result, complex kinetic equilibria among dissolution, precipitation and partition were established in the aqueous media during the entire course of the test. Thus, the lower degree of apparent supersaturation of RTV in the aqueous media evidenced by their low apparent concentrations is a realistic reflection of such dynamics. Amidon et al. have repeatedly pointed out that the single aqueous based dissolution tests with pH alteration under non-sink condition, in general, may lead to an overestimation of the degree and duration of supersaturation of poorly water soluble drugs in vitro [33-35]. In particular, for BCS class II drugs in the human GI tract, dissolved drug will be quickly absorbed and the drug concentration in the human intestinal lumen will be lower than one observed in the in vitro dissolution chamber. Therefore, the introduction of the absorption compartment such as the biphasic system would be a superior approach for characterization of the supersaturation based formulations. Comparison between absorption and partition profiles An absolute bioavailability of 60-80% was estimated for RTV in humans and the tablet and powder formulations are bioequivalent under the fasting state. The pharmacokinetic performance of these RTV tablet, powder and solution formulations at a single dose of 100 mg was evaluated in healthy human subjects under the fasting and MFF states [63-64]. The mean plasma concentration-time profiles of RTV after oral administration of the Tablet under the fasting and MFF states are plotted in Figure 8A [63]. Mean plasma concentration-time profiles of RTV after oral administration of the powder and solution under the fasting and MFF states are plotted in Figure 8B [64]. Relative bioavailabilities of these Norvir® products are reported in Table 1. The pharmacokinetic performance of the solution under the fasting condition was not available for comparison. A food effect was observed from both the tablet and powder with ~ 20% reduction of AUC and 23-40% reduction of Cmax in human subjects under the MFF state as compared to those under the fasting state (Figure 8 and Table 3).



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The % fraction of drug absorbed- time profiles of these three RTV products were obtained through deconvolution corresponding pharmacokinetic profiles by the W-N method and are shown in Figure 9. Approximately 90% of the absorbed RTV for the tablet and powder formulations was obtained in 3 hours under the fasting state. A much slower absorption of RTV occurred under the MFF state with approximately 65% absorption in 5 hours. The fractional partition profiles of RTV in octanol were obtained by normalizing the measured concentrations against 350 µg/mL, which is the maximum concentration for these dosage forms at 6 hours. The fractional partition profile of each RTV formulation with the FaSSIF-V2 (or the FeSSIF-V2) is plotted with the corresponding fractional absorption profile under the fasting (or MFF) state in vivo as shown in Figure 10. Under the fasting state, the in vitro fractional partition profile and in vivo fractional absorption profile of the tablet and powder show a similar shape and extent, but are separated by a time shift of ~ 50 - 100 min. (Figures 10A and 10B). The fractional partition-time profiles of RTV show a lag time of ~40 min. for the tablet and a lag time of ~100 min. for the powder. In contrast, under the MFF state, the in vitro and in vivo profiles of the tablet and powder overlap reasonably well as shown in Figures 10C and 10D. The fractional partition profile for the solution are separated from the fractional absorption profile with a time shift varying between 20 and 120 min. (Figure 10E). Overall, the shape of the fractional partition profiles of RTV products in biorelevant media closely matched the fractional absorption profiles under the fasting and MFF states. Assessment of food effect Under the fasting condition, the fractional absorption profiles of RTV for the tablet and powder reach about 90% absorption around 3 hours (Figure 9). Under the MFF state, the tablet, powder and solution are bioequivalent and reach a plateau of approximately 65 -70% (relative to the fasted state) around 5 hours (Figure 10). The longer time for reaching the absorption plateau under the MFF state is attributed to a slower gastric emptying time. These studies showed a food effect for the tablet and powder formulations due to a significant reduction of AUC and Cmax under the MFF state versus the fasting state (Table 3)[63-64].


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It is a common practice to assess the food effect of BCS II drugs by comparing their concentration profiles in the biorelevant media to assess the effect [18-23]. This is based on an implicit assumption that the solubilization of the drug would increase the bioavailability. The two-stage dissolution test revealed significantly higher apparent RTV concentrations of ~ 180200 µg/mL in the FeSSIF-V2 (Figure 5A) as compared to those (~50 µg/mL ) in the FaSSIF-V2 (Figure 4A), suggesting a food effect with a higher exposure in the fed state. However, this is inconsistent with in vivo results. The failure of the two-stage dissolution test with respect to the prediction of a food effect is most likely related (1) a lack of reliable free drug concentration determination (a non-trivial task) in such media, and (2) more importantly, a lack of the “absorption compartment” of this test, leading to overestimation of supersaturation as discussed above. The RTV concentration profiles of the tablet in octanol associated with either the FaSSIF-V2 or the FeSSIF-V2 are shown in Figure 11A. They are superimposable within the time window from 0 to 150 min. and exhibit a similar decrease of the partition flux around 180 min. and beyond. The lower final RTV concentration with the FeSSIF-V2 than the final RTV concentration in the FaSSIF-V2 (260 µg/mL) reveals a lower extent of partition in the presence of higher concentrations of bile salt. Similarly, the RTV concentration-time profiles of the powder formulation in octanol between the FaSSIF-V2 and FeSSIF-V2 are shown in Figure 11B for comparison. Significantly different partitioning of RTV was observed from these two aqueous media, leading to highly different RTV concentrations in octanol at 6 hours. The amounts of RTV partitioned into octanol at 6 hours from these products are reported in Table 3 along with the relative AUC and Cmax obtained under the fasting and MFF states. For the tablet, the ratio of RTV partitioned into octanol from the FaSSIF-V2 and FeSSIF-V2 is 1 : 0.80 and this agrees with the ratio of AUC (1 : 0.80) and the ratio of Cmax (1: 0.77) between the fasting and MFF states (Table 3). Similarly, for the powder, the ratio of partitioned RTV into octanol from the FaSSIF-V2 and the FeSSIF-V2 is 1 : 0.62 and this also agrees with the ratio of AUC (1 : 0.78) and ratio of Cmax (1: 0.61) between the fasting and MFF states (Table 3). Overall,



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the relative amount of RTV partitioned into octanol at 6 hours appears to correlate with the relative in vivo AUC and Cmax under the fasting and MFF states. In addition, the profiles obtained from the biphasic test provide an insight of the underlying mechanism of the food effect. Two primary factors may be accountable for the food effect with a higher exposure in the fasting state vs. the MFF state. First, the dissolution of RTV in the “stomach compartment”, that is either in the FaSSGF (pH 1.6) or FeSSGF (pH 5.0) media, plays a dominate role. As RTV solubility is pH-dependent, it rapidly dissolves in the FaSSGF (pH 1.6) to a high concentration. When the FaSSGF transitions to the FaSSIF-V2 (pH 6.5), a high degree of supersaturation of RTV is achieved, enhancing its partition flux (e.g., absorption in vivo). In comparison, less dissolution of RTV in the FeSSGF (pH 5.0) results in a low degree of supersaturation in the FeSSIF-V2. Secondly, solubilization of RTV in GI fluids under the MFF state also plays a key role. The solubility of crystalline RTV in water is 1 µg/mL, 7.4 µg/mL in the FaSSIF-V2 and 18.5 µg/mL in the FeSSIF-V2 (Table 2). An increase of RTV solubility in the FaSSIF-V2 and the FeSSIFV2 are directly associated with solubilization by bile salts in the media. Solubilization of RTV by micelles reduces the free drug fraction and results in lower driving force for absorption in vivo. Although the tablet possesses a similar flux from the FeSSIF-V2 and FaSSIF-V2, the extent of RTV partitioning into octanol is noticeably reduced as reflected by the final RTV concentration in octanol (Figure 11A, Table 3). For the powder formulation, a more severe reduction of the flux (from 5.2 to 1.5 µg/min.cm2) is indicative of a low supersaturation and thus, a reduction of the final RTV concentration in octanol at 6 hours (from 330 to 205 µg/mL ) when the dissolution medium changes from the FaSSIF-V2 to the FeSSIF-V2 (Figure 11B, Table 3). As discussed above, the significantly lower supersaturation of RTV for the powder is most likely due to transformation of the precipitate to the crystalline Form II. Several publications revealed that the in vitro flux across artificial membranes or Caco-2 cell lines is solely driven by the free drug fraction, while solubilization of the poorly soluble drug by surfactants and/or bile salts would reduce the free drug fraction and, therefore, the flux [15-17, 65-66]. Difference of the flux of BCS II drugs determined by these in vitro dissolutionpermeation test is considered to have direct relevance to the exposure in vivo [15-17, 65-66]. 18 ACS Paragon Plus Environment

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Our results of the tablet and powder using the biphasic test support the mechanistic hypothesis from the in vitro dissolution-partition tests.

Examining the in vitro-in vivo relationship A Levy plot is a common way to estimate the time shift and scaling factor. The Levy plots of the in vitro and in vivo results from the RTV formulations under the fasting and MFF states are shown in Figures 12A and 12B. Time scaling factors of 0.76 and 0.93 and a time shift of -37 min and +36 min were applied for fasting and MFF states, respectively. With the correction of both the time scaling factor and the time shift, the fraction of RTV absorbed is plotted against the partitioned RTV and shown in Figure 13, yielding a correlation coefficient (R2) of 0.93. All data points are within the lines representing ± 20% error of the fraction absorption. The mechanistic model to describe the drug transport phenomenon associated with the two-phase dissolution system indicates that the physiological relevance of this test method is based on the in vitro partitioning rate coefficient, kp (equal to (AI/Va)*PI) approximates the in vivo absorption rate coefficient, ka (equal to (A/V)*Peff) [38]. It is recognized that the biphasic test may possess an AI/Va in the range of 0.1 to 0.5 depending on the experimental apparatus and set up; this is approximately an order of magnitude smaller than the corresponding physiological A/V in vivo [38]. However, the value of PI of drug substances from water to octanol is reported to be in a wide range of 2 - 30 x 10-4 cm/s (pending on the hydrodynamic condition) with several BCS II drugs including piroxicam, nimesulide, valproic acid, ibuprofen and felodipine (see Tables 3 and 7 in ref 38). These PI values are approximately an order of magnitude larger than the typical high permeability Peff in vivo. Provided that a significantly higher PI relative to the Peff in vivo is achievable, the biphasic test may possess an in vitro kp of a given drug that would be similar (or equal) to its in vivo ka. The current experimental set up of the biphasic test appears reasonably simulating the three parallel kinetic processes (e.g., dissolution, precipitation, and partition) that may occur in vivo. In particular, approx. 50-70% of RTV dose was obtained in octanol at 6 h and this is comparable to the absolute bioavailability of 60-80% of RTV in human. Consistently, a “Level A” type of IVIVC is obtained with these diversified formulations (Figure 11). Without a comprehensive mathematical model to account for the complex, dynamic processes encountered in the biphasic test, these preliminary results may suggest that the in vitro partition rate, kp, is comparable to the in vivo absorption rate, ka (if not exactly) in the case of RTV. 19 ACS Paragon Plus Environment


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CONCLUSION In this study, three commercial RTV products of RTV (the tablet, powder and solution) were evaluated using two biorelevant in vitro dissolution tests. As RTV is a weak base, the pH alteration involved in both dissolution tests is considered to be of critical importance. When RTV was dissolved in the FaSSGF (pH 1.6) and transitioned to the FaSSIF-V2 (pH 6.4), supersaturation of RTV was observed from these products accompanied by the appearance of LLPS. However, the degree of supersaturation of RTV is significantly overestimated by the twostage dissolution test. Higher exposure for the tablet and powder formulations is predicted based on higher apparent RTV concentrations in the FeSSIF-V2 as compared to those in the FaSSIFV2. This is inconsistent with the in vivo results obtained in human. In contrast, a significantly lower degree of supersaturation of RTV in the aqueous media was observed for the tablet, and powder by the biphasic test as compared to their counterparts by the two-stage dissolution test. This is primarily attributed to a constant removal of dissolved RTV in the aqueous media by partitioning to octanol. The partitioned amounts of RTV into octanol at 6 hours for the tablet and powder agree with the relative AUC and Cmax in human subjects under the fasting and MFF states. Further, the fractional partition-time profiles of RTV obtained by the biphasic tests are in agreement with the corresponding fractional absorption-time profiles. The tablet formulation of RTV, among the three dosage forms tested, seems to possess an optimal dissolution rate in the aqueous phase coupled with an adequate partitioning rate into the organic phase. This yields a relatively low degree of supersaturation and de-supersaturation of RTV (with minimal precipitation) in the aqueous media as compared to those from the powder and solution formulations. The flux of the tablet revealed by the biphasic test appears insensitive to the pH alteration between the FaSSIF-V2 and FeSSIF-V2 media. This study reveals that the interplay among the dissolution, precipitation and partition processes of the three RTV formulations ultimately dictates the oral exposure.


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The experimental set up and the hydrodynamic condition of the biphasic test have a significant effect upon drug concentration profiles in both aqueous and octanol phases. Identification of the optimal hydrodynamic condition is a prerequisite to achieve a meaningful IVIVC. The biphasic test employed in this study appears reasonably simulating the dynamic processes (e.g., dissolution, precipitation, and partition) that may occur in vivo. Preliminary analysis may suggest that the in vitro partition rate, kp, is comparable to the in vivo absorption rate, ka (if not exactly) in the case of RTV.

ACKNOWLEDGEMENTS AND DISCLOSURE We thank Drs. Eric Schmitt, John Morris and Devalina Law for their thorough review and comments on this manuscript and Dr. Joerg Rosenberg for his input and scientific insight on this project. H.X. thanks for the financial support from the AbbVie Postdoctoral Research Program. This study was funded by AbbVie Inc. AbbVie participated in the study design, research, data collection, analysis and interpretation of data, as well as writing, reviewing, and approving the publication. Hao Xu, Yi Shi, Socrates Vela, Patrick Marroum and Ping Gao are AbbVie employees and may own AbbVie stock.



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46. Law, D.; Krill, S.L.; Schmitt, E.A.; Fort, J.J.; Qiu, Y.; Wang, W.; Porter, W.R. Physicochemical considerations in the preparation of amorphous ritonavir-poly(ethylene glycol) 8000 solid dispersions. J. Pharm. Sci. 90(8):1015-1025 (2001). 47. Law, D.; Schmitt, E.A.; Marsh, K.C.; Everitt, E.A.; Wang, W.; Fort, J.J.; Krill, S.L. Qiu, Y. Ritonavir–PEG 8000 amorphous solid dispersions: In vitro and in vivo evaluations. J. Pharm. Sci. 93(3): 563-570 (2004). 48. Physician insert Norvir® drug product. 49. Kempf, D.J.; Marsh, K.C.; Denissen, J.F.; McDonald, E.; Vasavanonda S, Flentge CA, Green BE, Fino L, Park CH, Kong XP, et al. ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans. Proc. Natl. Acad. Sci. USA. 92(7): 2484-2488 (1995). 50. Hsu, A.; Granneman, G.R.; Bertz, R.J. Ritonavir. Clinical pharmacokinetics and interactions with other anti-HIV agents. Clin. Pharmacokinet. 35(4): 275-291(1998). 51. VIEKIRA XR™ Physician insert. 52. Tho, I.; Liepold, B.; Rosenberg, J.; Maegerlein, M.; Brandl, M.; Fricker, G. Formation of nano/micro-dispersions with improved dissolution properties upon dispersion of ritonavir melt extrudate in aqueous media. Eur. J. Pharm. Sci. 40(1): 25-32 (2010). 53. Kanzer, J.; Hupfeld, S.; Vasskog, T.; Tho, I.; Holig, P.; Magerlein, M.; Fricker, G.; Brandl, M. In situ formation of nanoparticles upon dispersion of melt extrudate formulations in aqueous medium assessed by asymmetrical flow field-flow fractionation. J. Pharm. Biomed. Anal. 53(3): 359-365 (2010). 54. Rodriguez-Spong, B.; Acciacca, A.; Fleisher, D.; Rodriguez-Hornedo, N. pH-induced nanosegregation of ritonavir to lyotropic liquid crystal of higher solubility than crystalline polymorphs. Mol. Pharm. 5(6): 956-967 (2008). 55. Indulkar, A.S.; Mo, H.; Gao, Y.; Raina, S.A.; Zhang, G. Z.Z.; Taylor, L.S. Impact of micellar surfactant on supersaturation and insight into solubilization mechanisms in supersaturated solutions of atazanavir. Pharm. Res. 34(6): 1276-1295 (2017). 56. Lu, J.; Ormes, J.D., Lowinger, M.A.; Mann, K.P.; Xu, W.; Litster, J.D.; Taylor, L.S. Maintaining supersaturation of active pharmaceutical ingredient solutions with biologically relevant bile salts. Cryst. Growth. Des. 17: 2782-2791 (2017). 57. Jantratid, E.; Janssen, N.; Reppas, C.; Dressman, JB. Dissolution media simulating conditions in the proximal human gastrointestinal tract: an update. Pharm. Res. 25(7):16631676 (2008). 58. Wagner, J.G. Estimation of theophylline absorption rate by means of the Wagner-Nelson equation. J. Allergy. Clin. Immunol. 78(4 Pt 2): 681-688 (1986). 26 ACS Paragon Plus Environment

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59. Suarez-Sharp, S.; Li, M.; Duan, J.; Shah, H.; Seo, P. Regulatory experience with in vivo in vitro correlations (IVIVC) in new drug applications. AAPS J. 18(6): 1379-1390 (2016). 60. Ostrowski, M.; Wilkowska, E.; Baczek, T. The influence of averaging procedure on the accuracy of IVIVC predictions: immediate release dosage form case study. J Pharm. Sci. 99(12): 5040-5045 (2010). 61. Kanzer, J.; Hupfeld, S.; Vasskog T.; Tho, I.; Peter Hölig, P.; Mägerlein, M.; Fricker, G.; Brandl, M. In situ formation of nanoparticles upon dispersion of melt extrudate formulations in aqueous medium assessed by asymmetrical flow field-flow fractionation. J. Pharm. Bio. Aanly. 53: 359-365 (2010). 62. Harmon, P.; Galipeau,K.; Xu, W.; Brown, C.; Wuelfing, WP. Mechanism of DissolutionInduced Nanoparticle Formation from a Copovidone-Based Amorphous Solid Dispersion Mol. Pharm. 13: 1467-1481 (2016). 63. Ng, J.; Klein, C.E.; Chui, Y.L.; Awni, W.M.; Morris, J.B.; Podsadecki, T.J.; Cui, Y.; Bernstein, B.; Kim, D. The effect of food on ritonavir bioavailability following administration of ritonavir 100 mg film-coated tablet in healthy adult subjects. J. Inter. AIDS Soc.11(Suppl 1): 247 (2008). 64. Salem, A.H.; Chiu, Y.L.; Valdes, J.M.; Nilius, A.M.; Klein, C.E. A novel ritonavir paediatric powder formulation is bioequivalent to ritonavir oral solution with a similar food effect. Antivir. Ther. 20(4): 425-432 (2015). 65. Buckley, ST.; Frank, KJ.; Fricker, G.; Brandl, M. Biopharmaceutical classification of poorly soluble drugs with respect to ‘‘enabling formulations”. Eur. J. Pharm. Sci. 50: 8-16 (2013). 66. Stewart, AM.; Grass, ME.; Mudie, DM.; Morgen, MM.; Friesen, DT.; Vodak, DT. Development of a Biorelevant, Material-Sparing Membrane Flux Test for Rapid Screening of Bioavailability-Enhancing Drug Product Formulations. Mol. Pharm. 14: 2032−2046 (2017)



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Table 1. Norvir® formulations tested in this work.

Drug Product

Strength (mg/unit)

Tablet

100 mg

Powder

100 mg/pkg

Oral Solution

80 mg/ml

Relative F% (P.E.) Description

Ref. Fasting

MFF

ASD in PVP-VA copolymer matrix

100 (REF)

80

60

Same composition of ASD of the tablet, milled into powder of ~350 µm

100 (REF)

78

61

N/A

74

Solution contains alcohol, propylene glycol and polyoxyl 35 castor oil.


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Table 2. Biopharmaceutical properties of RTV

Description

Value

M.W.

720.9 g/mol

Log D (pH 6.8)

4.3 [47]

Papp (Caco-2)

~17 x 10 cm/s [47]

pKa (weak bases)

1.8, 2.6 [47]

Aq. Solubility (pH 1)

400 µg/mL [46]

Aq. Solubility (pH 6.8)

1 µg/mL [46]

Solubility in FaSSGF

~500 µg/mL

Solubility in FeSSGF

6.58 ± 0.89 µg/mL

Solubility in FaSSIF-V2

7.4 ± 1.1 µg/mL

LLPS in FaSSIF-V2

~40 µg/mL

Solubility in FeSSIF-V2

18.5 ± 1.9 µg/mL

LLPS in FeSSIF-V2

~180 µg/mL

Solubility in Octanol

19 ± 2 mg/mL

-6



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Table 3. The amount of RTV partitioned into octanol from the FaSSIF-V2 or FeSSIF-V2. The ratio of the RTV partitioned amounts are compared with the ratio of AUC and Cmax under the fasting and MFF states [60-61].

RTV Product

Amount of RTV (mg) partitioned into octanol at 6 hours

In Vitro In Vivo Ratio of RTV Ratio of AUC Amount Fasting : Fed

Fasting : MFF

Ratio of Cmax Fasting : MFF

Fasting Fed (FaSSGF/FaSSIF-V2)(FeSSGF/FeSSIF-V2) Tablet 62 52

1 : 0.84

1 : 0.80

1 : 0.77 [60]

Powder

66

41

1 : 0.62

1 : 0.78

1 : 0.61 [61]

Solution

N/A

52

N/A

N/A

N/A


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Figure 1. Schematic relationships of crystalline, amorphous and highly supersaturated solutions generated by dissolution of an ASD (obtained from ref. 15 with permission).



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Figure 2. (A) Molecular structure of RTV and (B) the solubility of crystalline Forms I and II versus solution pH.


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Figure 3. Schematic depiction of the biorelevant dissolution tests used in this work A) The two-stage dissolution test, and B) the biphasic dissolution-partition test.



Figure 4. (A) Apparent RTV concentration-time profiles obtained in the FaSSIF-V2 from the tablet and powder formulations obtained by the two-stage dissolution test. Error bars represent one std. dev. of n = 3; (B) PLM photo of the sample taken from the FaSSIF-V2 with (B) the tablet and (C) the powder at 6 hours.

600

A

(FaSSIF-V2)

500 Concentration (µg/mL)

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400

Tablet Powder

300 200 100 0 0

100

200

300

400

Time (min)


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Figure 5. (A) Apparent RTV concentration-time profiles obtained in the FeSSIF-V2 from the tablet, powder and solution formulations obtained by the two-stage dissolution test. Error bars represents one std. dev. of n = 3. PLM photo of the sample taken from the FeSSIF-V2 of (B) the tablet at 40 min; (C) the powder at 40 min.; (D) the solution at 40 min.; (E) the tablet at 6 hours; (F) the powder at 6 hours; and (G) the solution at 6 hours. Replaced with a new Figure A (remove the title in the figure and change the Y-scale to 600 ug/mL)

A

600

FeSSIF-V2

500

Concentration (µg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


400

Tablet Powder

300

Solution

200 100 0 0

100

200

300

400

Time (min)



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Figure 6. (A) Apparent RTV concentration-time profiles in the FaSSIF-V2 from the tablet; and powder and (B) cumulative RTV concentration-time profiles in octanol from the tablet and the powder obtained from the biphasic test. Error bars represented one std. dev. of n = 3.

B

Octanol phase

400 350

Concentration (ug / ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


300 250 200 150

Tablet

100

Powder

50 0 0

100

200

300

400

Time (min)



Figure 7. (A) Apparent RTV concentration-time profiles in the FeSSIF-V2 from the tablet, powder, and solution formulations and (B) cumulative RTV concentration-time profiles in octanol from the tablet, powder and solution obtained from the biphasic test. Error bars represented one std. dev. of n = 3.

A

B

400

Octanol Tablet

350


600


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Powder

300

Aqueous phase Tablet Powder

500

Solution 400 300 200 100 0

Solution

0

100

250

200

300

400

Time (min)

200 150 100 50 0 0

100

200

300

400

Time (min)


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Figure 8. Mean RTV plasma concentration-time profiles obtained in humans from (A) the tablet under fasting and MFF states from ref [63], and (B) the powder and solution formulations under fasting and MFF states from ref [64].



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Figure 9. Fractional absorption–time profiles from the tablet, powder and solution formulations in healthy human subjects under the fasting and MFF states. Solid lines are the best fit to data points and do not have scientific meaning.


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Figure 10. Comparison of the fractional absorption-time profiles of RTV from (A) the tablet, and (B) the powder under the fasting condition.; (C) the tablet, (D), the powder and (E) the solution under the MFF state with corresponding fractional partition-time profiles in octanol with either the FaSSIF-V2 or the FeSSIF-V2.



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Figure 11. Cumulative RTV concentration-time profiles in octanol from (A) the tablet and (B) the powder associated with the simulated fasting and fed media. Error bars represented one std. dev. of n = 3.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 12. Levy plots of three RTV formulations under (A) the fasting and (B) the MFF states.



Figure 13. The fractional absorption-time profile of RTV from three RTV formulations are plotted against the corresponding fractional partition-time profile under either the fasting or MFF states.

IVIVC

1.2

Fraction absorbed in vivo

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1

y = 0.9614x + 0.0041 R² = 0.9327

0.8 0.6 Tablet-fasting 0.4

Powder-fasting Tablet-MF

0.2

Powder-MF Solution-MF

0 0

0.2

0.4

0.6

0.8

1

1.2

Fraction partitioned in vitro


Page 45 of 45

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279x215mm (200 x 200 DPI)

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