Hydroxypropyl Methylcellulose Mediated Precipitation Inhibition of

Article pubs.acs.org/molecularpharmaceutics

Hydroxypropyl Methylcellulose Mediated Precipitation Inhibition of Sirolimus: From a Screening Campaign to a Proof-of-Concept Human Study Marija Petruševska,† Miha Homar,† Boštjan Petek,† Aleksander Resman,† Darko Kocjan,‡ Uroš Urleb,§ and Luka Peternel*,† †

Sandoz Development Center Slovenia, Pharmaceutical and Biological Profiling, Lek Pharmaceuticals d.d., 1526 Ljubljana, Slovenia EN-FIST Centre of Excellence, 1000 Ljubljana, Slovenia § Sandoz International, Global Product Development, 83607 Holzkirchen, Germany ‡

ABSTRACT: The aim of this study was to develop a sirolimus (BCS class II drug substance) solid oral dosage form containing a precipitation inhibitor, which would result in an improved sirolimus absorption in humans compared to the formulation containing nanosized sirolimus without a precipitation inhibitor, i.e., Rapamune. The selection of the precipitation inhibitor was based on the results of a screening campaign that identified two “hit” excipients: HPMC 603 (i.e., Pharmacoat 603) and Poloxamer 407. However, in a confirmatory precipitation inhibitor study using biorelevant media (Fa/FeSSIF) HPMC 603 more effectively inhibited sirolimus precipitation than Poloxamer 407. In the PAMPA assay, HPMC 603, but not Poloxamer 407, significantly increased the flux of the sirolimus across the membrane lipid layer. Additionally, a differential scanning calorimetry (DSC) and an infrared (IR) spectroscopy study revealed that interactions between the sirolimus and HPMC 603 were developed that could lead to the observed precipitation inhibition effect. Based on the above data, two formulations with HPMC 603-coated sirolimus particles were developed, namely, formulation A (d (0.5) = 0.21 μm) and formulation B (d (0.5) = 1.7 μm). A human pharmacokinetic study outlined that significantly higher AUC and Cmax were obtained for formulations A and B in comparison to Rapamune. This result could be attributed to the HPMC 603 (Pharmacoat 603) mediated sirolimus precipitation inhibition resulting in improved sirolimus absorption from the gastrointestinal tract in humans. KEYWORDS: absorption, pharmacokinetic, precipitation inhibition, screening, supersaturation

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INTRODUCTION Drug substances belonging to the Biopharmaceutical Classification System (BCS) class II represent a significant challenge in the development of a formulation with the desired absorption properties.1−3 The rate limiting step of BCS class II drug absorption is either the drug solubility or the dissolution rate in the lumen of the gastrointestinal tract (GIT). A variety of formulation approaches have been developed to tackle this challenge, including lipid-based formulations, nano/microsizebased delivery systems, self-emulsifying delivery systems, cosolvents, and amorphous solid dispersions.1,2,4−6 The goal of these approaches is to ensure an adequate fraction of a dissolved drug in the GIT lumen and consequently a satisfactory flux across the intestinal epithelia.1,7 Such a formulation would result in an increased extent of absorption as long as the transport mechanisms over the epithelial membrane do not represent the rate limiting step for drug absorption. However, when the drug is released in the GIT from these formulations,1,2,4−6 drug precipitation can occur, subsequently leading to a decreased fraction of the dissolved drug and a decreased flux across the intestinal epithelia. © XXXX American Chemical Society

Difficult-to-make formulations are typically represented by BCS class II drug substances, which represent the lowest bioequivalence success rate among the BCS drug classes.8,9 The BCS class II drug sirolimus is a macrolide with an immunosuppressive activity showing a low aqueous solubility of 2.6 μg/mL and log P > 5, which makes sirolimus a challenging drug substance for the development of an oral formulation.7,10 According to the BCS, the rate limiting step of drug absorption is drug permeability, solubility, or dissolution rate.3 Regarding sirolimus, it cannot be straightforwardly predicted whether solubility or dissolution rate is the rate limiting step of its oral absorption. The formulation approaches comprising sirolimus have focused on combination of improved sirolimus solubility and dissolution rate (e.g., cyclodextrins, solubilizing agents, micro/nanosizing, solid dispersions) to improve sirolimus oral absorption. On the other hand, in Received: November 9, 2012 Revised: April 8, 2013 Accepted: April 15, 2013

A

dx.doi.org/10.1021/mp300641h | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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bariatric population (a condition that is characterized with reduced gastric volume in the range of 100 to 175 mL) sirolimus exhibited a reduction in drug absorption after the surgery.11 This latter observation could emphasize that sirolimus absorption is solubility limited. In 1999, the first commercially available sirolimus product was an oral solution, Rapamune (Wyeth, USA) with a sirolimus concentration of 1 mg/mL in Phosal 50 PG (a dispersion of 50% phosphatidylcholine in a propylene glycol/ethanol carrier) and Tween 80.12 The reported oral bioavailability of this formulation was 14%.12 In 2002, a sirolimus tablet was launched on the market under the same product name Rapamune. This formulation was developed by using NanoCrystal technology resulting in a satisfactory dissolution rate, improved product stability, and an oral bioavailability of 17%.13−16 As this technology is based on crystal nanoparticles (top-down approach) it can be considered as a high energy system.17 In addition to the reduction of the particle size (i.e., NanoCrystal technology) sirolimus was also the subject of several other formulation approaches, including solid dispersions,14 liposomal formulations,18 inclusion complexes with cyclodextrins,19 and nanoparticle albumin-bound technology.20 All these approaches represent valuable technological sirolimus reformulation strategies with the aim of improving its absorption and consequently its efficacy profile in humans.16,21−23 The alternative formulation approach to crystal sirolimus nanoparticles in Rapamune24 is the incorporation of the sirolimus precipitation inhibitor in the final solid dosage form. This approach could also be used for some of the already mentioned approaches (e.g., solid dispersions). The rationale for such an approach is that the expected maximum concentration of sirolimus in the GIT lumen is 3 times higher than its thermodynamic solubility (i.e., 2 mg/250 mL), which could lead to a sirolimus precipitation in the GIT lumen.1 We hypothesized that certain excipients incorporated in the test formulation could inhibit the sirolimus precipitation in the GIT lumen, which could consequently result in an increased flux across the intestinal epithelia. Since effective precipitation inhibitors cannot be straightforwardly predicted, sirolimus “hit” precipitation inhibitors were identified by the screening campaign, followed by the confirmation study using biorelevant incubation media. In order to confirm that effectiveness of sirolimus precipitation inhibitors is translated into increased flux across intestinal epithelia, the parallel artificial membrane permeability assay was approached. At the molecular level interactions between sirolimus and “hit” precipitations inhibitors were characterized by DSC and IR, which altogether convinced us to proceed to develop a solid dosage form containing sirolimus and HPMC 603 (Pharmacoat 603). Following dissolution studies, the proof-of-concept human pharmacokinetic (PK) study confirmed the superiority of the developed formulation containing HPMC 603 (Pharmacoat 603), which showed a significantly higher AUC and Cmax in comparison to the reference product Rapamune.

Figure 1. Structure of sirolimus.

Table 1. List of Excipients (That Is, Precipitation Inhibitors (PIs)) Tested in the High-Throughput (HT) Precipitation Inhibitor Screening Assay classification group

nonproprietary name

brand name (manufacturer) Tween 80 (BASF, Germany) Tween 20 (BASF, Germany) Cremophor RH40 (BASF, Germany)

nonionic surfactant

polysorbate 80 polysorbate 20 polyoxyl 40 hydrogenated castor oil polyoxyl 35 castor oil

nonionic surfactant nonionic surfactant

poloxamer 188 poloxamer 407

nonionic surfactant

PEG 1500 lauric glycerides PEG 400 caprylic/ capric glycerides sodium lauryl sulfate

nonionic surfactant nonionic surfactant nonionic surfactant

nonionic surfactant ionic surfactant cellulosic polymer cellulosic polymer cellulosic polymer polyvinyl pyrrolidone polymer polyvinyl pyrrolidone polymer copolymer

polyvinyl pyrrolidone polymer polymer Mowiol 4-88 (Sigma-Aldrich, Belgium) sugar alcohol

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MATERIALS AND METHODS Materials and Reagents. The sirolimus (Figure 1) was obtained from Biocon (India). The tested excipients (i.e., precipitation inhibitors) and their suppliers are given in Table 1. The excipients used for the preparation of the test formulations A and B are shown in Table 2. The commercial sirolimus tablet Rapamune was supplied by Wyeth Medica

hydroxypropyl methylcellulose hydroxypropyl methylcellulose hydroxypropyl cellulose polyvinyl pyrrolidone K30 polyvinyl pyrrolidone K25 vinylpyrrolidonevinyl acetate copolymer polyvinyl pyrrolidone K17 polyvinyl alcohol

Kollidon K17 (BASF, Germany) Elvanol (DuPont)

mannitol

D-mannitol

polyethylene glycol

polyethylene glycol Mw = 6000

alcohol

propylene glycol

polyvinyl alcohol

polyvinyl alcohol graft copolymer polyethylene glycol graft copolymer α-cyclodextrin

polyethylene glycol graft copolymer cyclodextrin polyethylene glycol

Cremophor EL (SigmaAldrich, Belgium) Lutrol F68 (BASF, Germany) Lutrol F127 (BASF, Germany) Gellucire 44/14 (Gattefosse, Germany) Labrasol (Gattefosse, Germany) Texapon K15 (Cognis, The Netherlands) Pharmacoat 606, (Harke, Germany) Pharmacoat 603, (Harke, Germany) Klucel EF (Hercules Inc., Germany) Kollidon K30 (BASF, Germany) Kollidon K25 (BASF, Germany) Kollidon VA 64, (BASF, Germany)

polyethylene glycol Mw = 400

(Sigma-Aldrich, Belgium) poly(ethylene glycol) Mn 6000 (Sigma-Aldrich, Belgium) propylene glycol (SigmaAldrich, Belgium) Kollicoat IR (BASF, Germany)

α-cyclodextrin (Merck, Germany) poly(ethylene glycol) Mn 400 (Sigma-Aldrich, Belgium)

(Ireland). SIF Powder (Phares AG, Switzerland) was used for the preparation of fasted state simulated intestinal fluid (FaSSIF) and a fed state simulated intestinal fluid (FeSSIF) B



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Table 2. Qualitative Composition of Rapamune, Formulation A and Formulation B Rapamune24 excipient

formulation A and B function

lactose sucrose

filler in inert core filler

talc

glidant, lubricant in inert core

magnesium stearate shellac glyceryl monooleate calcium sulfate

lubricant in inert core water barrier coating of the inert core component of shellac coat component of shellac coat

poloxamer 188

stabilizer for nanoparticles and potential precipitation inhibitor filler, binder

polyethylene glycol 8000 polyethylene glycol 20 000 PVP K25 L-alpha tocopherol carnauba wax microcrystalline cellulose titanium and iron oxides

excipient (manufacturer) ̌ sucrose (Zito Šumi d.o.o., Slovenia) low substituted hydroxypropyl cellulose (JRS Pharma, Germany) sodium stearyl fumarate (Moehs Cantab, Spain) titanium dioxide (Europe SRL, Italy) PVP K25 (Kollidon K25, BASF, Germany) colloidal silicon dioxide (Degussa, Germany) polyethylene glycol 400 (Clariant GmbH, Germany)

function filler binder and potential precipitation inhibitor lubricant pigment binder and potential precipitation inhibitor glidant component of color coat

hydroxypropyl cellulose (Klucel EF, Hercules, USA)

coating agent and potential precipitation inhibitor

brown iron oxide (Italy SPA, Italy) yellow iron oxide (Italy SPA, Italy)

pigment pigment

hydroxypropyl methylcellulose, (Pharmacoat 603, Harke, Germany) hydroxypropyl methylcellulose (Pharmacoat 606, Harke, Germany)

stabilizer for nanoparticles and potential precipitation inhibitor coating agent and potential precipitation inhibitor

coating agent binder and potential precipitation inhibitor antioxidant polishing agent filler pigments

buffer according to the manufacturer’s procedure. In blank Fa/ FeSSIF, lecithin and sodium taurocholate are not present. Dimethyl sulfoxide (DMSO), methanol (CH3OH), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), sodium chloride (NaCl), sodium hydroxide (NaOH), citric acid (C6H8O7), and acetic acid (CH3COOH) were purchased from Merck (Germany). Ultrapure ion exchanged water was used for the chromatographic analysis. All the chemicals were of an analytical grade except the methanol, which was of a chromatographic reagent grade. In Vitro High-Throughput (HT) Precipitation Inhibitor Screening Method. The in vitro HT precipitation inhibitor method was performed as described by Petrusevska et al.25−27 Twenty-three excipients (Table 1) at three different concentrations (0.1% (w/v), 0.01% (w/v), and 0.001% (w/v)) were evaluated as potential inhibitors of the sirolimus precipitation out of a supersaturated solution. The excipients were chosen in a way that covered a wide range of physicochemical and structural properties. Briefly, sirolimus was prepared as a stock solution (4.54 × 104 μg/mL) in DMSO, and the potential precipitation inhibitors were dissolved at 0.1% (w/v), 0.01% (w/v), and 0.001% (w/v) in a McIlvaine buffer pH 6.8. The sirolimus and the precipitation inhibitor’s solutions were dispensed into the wells of 96-well microtiter plates using a Freedom EVO 150 pipettor (Tecan, Switzerland). The solution resulted in nonsink conditions, which are suggested to be used in the evaluation of drug precipitation inhibition.28 The Freedom EVO 150 was equipped with three temperature controlled incubators, each accommodating four microtiter plates. The sealed microtiter plates were incubated for 6 h at 37 °C, 7 Hz. Samples were withdrawn at 30, 90, 180, and 360 min and filtered through a hydrophilic PVDF 0.22 μm pore size microtiter plate (Millipore AG, Switzerland) using a multiscreen vacuum manifold (Millipore AG, Switzerland). Prior quantification samples were diluted 20 times with a mobile phase in order to stabilize the samples.

Data obtained from the precipitation inhibition screening campaign were analyzed as follows. The precipitation inhibitor (PI) index was calculated at each sampling time point as the ratio of sirolimus concentration in the presence of the potential precipitation inhibitor with respect to the sirolimus concentration in the absence of the precipitation inhibitor. The stability of the supersaturated concentrations (ΔDS) was calculated as the ratio of the sirolimus concentration in the presence of the potential precipitation inhibitor at 30, 90, and 180 min with respect to the sirolimus concentration in the presence of the precipitation inhibitor at 360 min. In the interpretation of the results obtained from the precipitation inhibitor screening assay, the ΔDS and PI index should be considered. For example, in samples with the ΔDS value close to 1, the concentration of dissolved sirolimus is stable within the measured time period. In this very same sample the PI index value would then provide information about the effectiveness of the excipient-mediated precipitation inhibition. Effect of Selected Excipients on the Thermodynamic Solubility of Sirolimus. An excess amount of crystalline sirolimus was added to the McIlvaine buffer pH 6.8 (20 mL) with or without the dissolved excipients (i.e., precipitation inhibitor), followed by incubation at 550 rpm, 37 °C on a Thermomixer (Eppendorf, Germany). After 24 h, samples were withdrawn and filtered through a hydrophilic PVDF 0.22 μm pore size microtiter plate (Millipore AG, Switzerland) using a multiscreen vacuum manifold (Millipore AG, Switzerland). Prior quantification samples were diluted 20 times with a mobile phase in order to stabilize the samples. “Hit” Confirmation: Precipitation Inhibition Effect of Excipients on the Sirolimus Concentration in Fa/FeSSIF. In the screening campaign the identified precipitation inhibitors of the sirolimus were further evaluated in the biorelevant dissolution media.29 FaSSIF and FeSSIF were prepared by utilizing SIF powder (Phares AG, Switzerland) resulting in the dissolution media described by Galia et al.30 The identified “hit” precipitation inhibitors, HPMC 603 and Poloxamer 407, were C



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were either a pure sirolimus (or excipient) or a physical mixture of the sirolimus and the excipient in the mass ratios as described above. The enthalpy of the sirolimus peak was measured and renormalized to the amount of the present sirolimus (i.e., the enthalpy divided by the relative sirolimus content in the mixture). Infrared Spectroscopy (IR). The infrared spectra were measured using a Thermo Nicolet Nexus FT-IR spectrometer (USA) driven by the Nicolet Omnic software. The samples were mixed thoroughly with potassium bromide (1/100 = sample/KBr). A pressure of 5 tons was applied for 5 min in a hydraulic press to compress the powder in order to prepare potassium bromide disks. The spectra were recorded at a resolution of 2 cm−1, from 4000 to 600 cm−1, at a scan repetition of 32. Preparation of Sirolimus Test Formulation for Pharmacokinetic Studies. The sirolimus was suspended in a solution of HPMC 603 dissolved in water. The resulting suspension was milled in a ball mill (Coball mill MS-18, FrymaKoruma, Switzerland) with zirconium beads (0.6−0.8 mm diameter) to obtain sirolimus particles with d (0.5) = 0.21 μm (formulation A) and d (0.5) = 1.7 μm (formulation B) (the particle size measured using a Mastersizer 2000, Malvern, U.K.). The milled suspension was further mixed with water, a part of sucrose, and PVP K25. The resulting mixture was sprayed onto the remaining sucrose in a fluid bed apparatus (Glatt GPCG 3, Glatt, Germany) to form granules. The apparatus was assembled in the top spray setup with the 1.2 mm nozzle (Düsen-Schlick, Germany) in the upper position. The dried granules were mixed with sodium stearyl fumarate, colloidal silicon dioxide, and low substituted hydroxypropyl cellulose and compressed into tablet cores on a rotary tablet press (Killian LX 10, Killian, Germany). Pharmacoat 606, hydroxypropyl cellulose (Klucel EF), and polyethylene glycol 400 were dissolved in water, followed by the addition of titanium dioxide and iron oxides to form a homogeneous suspension. The tablet cores were coated with the resulting suspension in a perforated drum (O’Hara Labcoat I, O’Hara Technologies, Canada). For the preparation of the sirolimus test formulations (A and B) prevention of nanoparticle agglomeration was achieved by the incorporation of HPMC 603 (Pharmacoat 603), which in addition to its potential precipitation inhibitor effect acts also as a nanoparticle stabilizer. This resulted in stable nanomilled dispersion for a period of 1 month. In order to reduce the potential agglomeration, nanomilled dispersions were converted to dry form (granulate) one day after milling, thus further reducing the likelihood of agglomeration and crystal growth. The composition of the test formulations A and B is shown in Table 2. Dissolution Studies. The dissolution of formulation A, formulation B, and Rapamune tablets was conducted in a USP dissolution apparatus Erweka DT 4000 (Erweka Instruments, Germany) under nonsink conditions using 500 mL of FaSSIF, FaSSIF v2,32 or water with 0.15% SLS. The paddle speed was set to 100 rpm with stationary baskets 10 mesh, at 37 °C. At selected time intervals (15 min, 30 min, and 60 min) aliquots of 5 mL were withdrawn from each of the dissolution vessels and filtered through a 0.2 μm hydrophilic PVDF membrane filter. The concentration of the dissolved sirolimus was determined by the UPLC−UV method. Pharmacokinetic Studies. The study was performed by a contract research organization PharmaNet/i3 (Princeton,

dissolved at 0.1% (w/v) in Fa/FeSSIF. The sirolimus was prepared as a DMSO stock solution (4.54 × 104 μg/mL). 2.5 μL of the stock solution was added to 247.5 μL of Fa/FeSSIF containing 0.1% (w/v) “hit” precipitation inhibitors. The experiment was performed in 96-well plates, which after the addition of the sirolimus stock solution were sealed with an adhesive foil and incubated at 37 °C, 7 Hz for 24 h. Samples were withdrawn at 0.5, 1, 1.5, 2, and 24 h, filtered through a hydrophilic PVDF 0.22 μm pore size microtiter plate (Millipore AG, Switzerland) using a multiscreen vacuum manifold (Millipore AG, Switzerland). The prior quantification samples were diluted 20 times with mobile phase in order to stabilize the samples. Parallel Artificial Membrane Permeability Assay (PAMPA). The impact of the “hit” precipitation inhibitors on the sirolimus flux across the artificial lipid membrane was studied in a Gentest precoated PAMPA plate system which was used according to the manufacturer’s procedure (Becton Dickinson, USA).31 The sirolimus was prepared as a stock solution in DMSO (4.54 × 104 μg/mL), followed by a 100times dilution with the “hit” precipitation inhibitors’ solution (i.e., HPMC 603 and Poloxamer 407) (0.1% (w/v)) in a McIlvaine buffer pH 6.8. The donor wells were then loaded with 300 μL of the supersaturated sirolimus solutions with or without the “hit” precipitation inhibitors. Each acceptor well was loaded with 200 μL of the blank McIlvaine buffer pH 7.4. The acceptor plate was then placed on the 96-well receiver plate, and the resulting PAMPA sandwich was incubated at 37 °C without shaking. The sirolimus content was determined in the acceptor well at 20 min, 40 min, 60 min, 90 min, and 120 min. One well was used for one time point only. The potential impact of the tested excipient on the PAMPA membrane integrity was checked by the impact of the excipients on the permeability of the low permeability marker FITC-dextran. The amount of FITC-dextran was measured by the plate reader Infinite M1000 (Tecan, Switzerland) at λexc/em = 495/515 nm. The determined FITC-dextran permeability values were 0.05), while Poloxamer 407 (0.1% (w/v)) increased the sirolimus solubility 3.3 times (ANOVA, p < 0.01). This result could outline the different mechanism mode of the sirolimus precipitation inhibition. Since the tested concentration of Poloxamer 407 was above the critical micelle concentration,46,47 the increased fraction of the dissolved sirolimus in the precipitation inhibition screening campaign and in the thermodynamic solubility assay was most probably the result of a sirolimus entrapment within the Poloxamer 407formed micelles. On the other hand, the HPMC 603 mediated F



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Figure 4. (A) The impact of “hit” excipients HPMC 603 (0.1%) and Poloxamer 407 (0.1%) on the flux of sirolimus in the parallel artificial membrane permeability assay (PAMPA). The results are shown as linear regression lines (R2 > 0.8243, p < 0.05) obtained from the concentration− time profiles (n = 4). (B) The slopes from the linear regression lines are shown as a mean ± SEM (n = 4), *p < 0.05 one-way ANOVA in comparison to the control group (McIlvaine buffer pH 6.8).

group (ANOVA, p > 0.05). However, HPMC 603 significantly increased the sirolimus flux across the lipid bilayer (ANOVA, p < 0.01), which is most probably related to the HPMC 603 mediated increase of sirolimus concentrations in the donor compartment.42,48 In the PAMPA assay the same incubation buffer was used as in the screening campaign, where at 0.1% (w/v) HPMC 603 and Poloxamer 407 showed a comparable precipitation inhibition effect. However, only HPMC 603 showed a significant increase of sirolimus flux across the lipid bilayer (Figure 4). The observed higher flux across the lipid bilayer is most probably the outcome of the different mechanism mode responsible for providing free sirolimus molecules in the solution. The presence of Poloxamer 407 above its critical micelle concentration46,47 resulted in a decreased fraction of free sirolimus, which is available for permeation across the PAMPA membrane. On the other hand, the ability of HPMC 603 to reduce the crystallization rate43−45 could lead to a higher fraction of free sirolimus available for permeation across the PAMPA membrane. A similar conclusion has been recently described by Miller et al.,49−51 where the permeation rate of progesterone across the PAMPA and the rat jejunum perfusion model was not increased in the presence of the surfactants sodium lauryl sulfate and sodium taurocholate, even though a significant increase in the solubility was observed. On the other hand, the presence of HPMC−acetyl succinate significantly increased the permeation rate of progesterone across the PAMPA and the rat jejunum perfusion model.49 Differential Scanning Calorimetry (DSC) and an Infrared (IR) Spectroscopy Study. The thermogram of pure sirolimus exhibited an endothermic peak at 191 °C without the presence of any endothermic or exothermic events prior to reaching the melting point temperature. When the sirolimus was mixed with different amounts of HPMC 603 and Poloxamer 407, the enthalpy of the normalized melting peak was reduced, depending on the relative amount of the added precipitation inhibitor. The relationship between the normalized enthalpy and the fraction of sirolimus in the excipient followed sigmoidal shape, which was described using a nonlinear regression model (Table 3). At the low fraction of the sirolimus in HPMC 603 and Poloxamer 407 the normalized enthalpy decreased close to 0 J/g. Moreover, even at equal mass ratios of sirolimus in the excipient, normalized enthalpy is in comparison to pure sirolimus significantly reduced. These

precipitation inhibition alters the crystallization rate by adsorbing to the crystal’s surface providing temporal diffusion resistance.42,44,45 This is reflected in the inability of HPMC 603 to increase the thermodynamic solubility of the sirolimus. The “hit” sirolimus precipitation inhibitors identified in the screening campaign were further evaluated in a physiologically more relevant dissolution medium, namely, Fa/FeSSIF (Figure 3). The concentration of sirolimus in the Fa/FeSSIF group at the 24 h time point was still 1.2 times (FaSSIF) and 2.1 times (FeSSIF) higher in comparison to the blank Fa/FeSSIF group (i.e., without the presence of sodium taurocholate and lecithin). These results demonstrated that sirolimus supersaturation can be created and maintained to a certain degree in the absence of precipitation inhibitors and that the solubilizing components in Fa/FeSSIF also increase the sirolimus thermodynamic solubility. The solubilizing components of Fa/FeSSIF inhibited the sirolimus precipitation to the extent that the addition of Poloxamer 407 did not cause any additional effects of sirolimus precipitation inhibition. However, in contrast to Poloxamer 407 a significant decrease of sirolimus precipitation in comparison to the Fa/FeSSIF media was caused by HPMC 603 (ANOVA, p < 0.01) (Figure 3). This observation could be explained by the different mechanism mode of the HPMC 60342,44,45 and Poloxamer 40746,47 mediated precipitation inhibition. In addition, a recently published study proposed that a thin HPMC (Methocel E15LV) polymer film can indeed inhibit drug surface crystallization.39 Interestingly, the HMPC 603-mediated precipitation inhibition of sirolimus was more pronounced in FeSSIF than FaSSIF, which could indicate the synergistic precipitation inhibition effect of HPMC-603 and sodium taurocholate and/ or lecithin.40 Parallel Artificial Membrane Permeability Assay (PAMPA). The need to assess the precipitation inhibitor effect in an absorptive environment was recently reported by Bevernage et al.48 where less loviride precipitate was observed when the possibility for compound permeation across the membrane is included in the in vitro assay. In order to investigate the impact of the supersaturated sirolimus solution on the flux across the lipid bilayer, a PAMPA permeability assay was performed. Figure 4 shows the sirolimus flux across the lipid bilayer in the presence of 0.1% (w/v) HPMC 603 and 0.1% (w/v) Poloxamer 407. Even though the increase of the flux was observed in the presence of Poloxamer 407, it was not considered significantly higher in comparison to the control G



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1670 cm−1) stretch peaks (Figure 5B). In the physical mixture of sirolimus and HPMC 603 the sirolimus characteristic stretch peaks were also identified (Figure 5C). In the solid dispersion, sirolimus characteristic peaks were also recorded, but they were, in comparison to crystalline sirolimus, significantly widened (Figure 5D). This latter result outlined that in the solid dispersion specific interactions between the sirolimus and HPMC 603 are developed and it cannot be ruled out that these interactions are responsible for the observed HPMC 603 mediated precipitation inhibition of the sirolimus.13 Dissolution and Pharmacokinetic Study. The results obtained from the precipitation inhibition studies and the PAMPA assay suggest that HPMC 603 effectively inhibits the sirolimus precipitation and increases the flux of the sirolimus across the lipid bilayer. A DSC and IR spectroscopy study gave additional evidence that interactions between the sirolimus and HPMC 603 are developed. Therefore, two formulations containing HPMC 603 were prepared (Table 2). Formulation A (sirolimus particles with d (0.5) = 0.21 μm) most probably differed from Rapamune (sirolimus particles below 0.4 μm according to ref 24) due to the presence of HPMC 603, while formulation B (sirolimus particle size d (0.5) = 1.7 μm) differed from Rapamune also due to the larger sirolimus particle size. Besides these differences the technological procedure for the

Table 3. DSC Results from the Nonlinear Fit Model of the Physical Mixtures between the Sirolimus and HPMC 603 (Pharmacoat 603) at Different Weight Ratios of 1/1, 1/2, 1/ 5, 1/10, 2/1, 5/1, and 10/1a

sirolimus/Poloxamer 407 sirolimus/HPMC 603 (Pharmacoat 603)

top

bottom

Log dH 50

R2

72.89 78.27

−0.000409 1.577

58.90 44.20

0.9487 0.9165

a

Top and bottom are the plateau values of the normalized sirolimus enthalpy (J/g); Log dH50 is the normalized sirolimus enthalpy between the top and the bottom of the sigmoidal curve; R2, goodness of fit.

changes are most probably a result of sirolimus interaction with melted precipitation inhibitor (HPMC 603 or Poloxamer 407). In order to provide additional evidence of interactions between the sirolimus and HPMC 603, solid dispersions containing sirolimus and HPMC 603 were prepared followed by infrared (IR) spectra recording. The FT-IR spectra of a pure drug, polymer alone, physical mixtures, and vacuum-dried mixture were recorded (Figure 5). The sirolimus (Figure 1) contains six CO and four CC double bonds, resulting in the characteristic CC (1680−1640 cm−1) and CO (1760−

Figure 5. (A) IR spectra of HPMC 603, (B) IR spectra of pure sirolimus, (C) IR spectra of the physical mixture of the sirolimus and HPMC 603 (1/ 10), (D) IR spectra of the sirolimus−HPMC 603 (1/10) solid dispersion (vacuum-dried). H



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Figure 6. In vitro dissolution profiles of the sirolimus released from Rapamune, formulation A, and formulation B. The results are shown as mean ± SEM (n = 2).

Table 4. Sirolimus Pharmacokinetic Parameters Obtained after Oral Administration of Rapamune and Formulations A/B to Fasted Human Volunteersa

a

formulation

Cmax (pg/mL)

Tmax (h)

AUC72 (pg h/mL)

AUC12 (pg h/mL)

Rapamune formulation B formulation A

7655 ± 1896 *12616 ± 2867 *19401 ± 3781

2.02 ± 0.54 1.39 ± 0.91 1.42 ± 0.91

132957 ± 34628 140056 ± 27265 151243 ± 38907

51089 ± 15754 *64793 ± 12878 *78405 ± 17889

Results are shown as mean (n = 11) ± SD. ANOVA with Dunnett’s post-test, *p < 0.05 vs Rapamune.

Figure 7. The correlation of the sirolimus in vitro results with in vivo human pharmacokinetic data (AUC12, black symbols, and Cmax, white symbols). The following parameters were correlated against AUC12 and Cmax: (A) flux determined in the PAMPA assay; (B) precipitation inhibition determined in the high-throughput (HT) screening campaign; (C) precipitation inhibition in FaSSIF expressed as AUC24; (D) percentage dissolved at 60 min in FaSSIF. The results are shown as mean + SEM (n = 4).

preparation of the final dosage form was also different. Formulations A and B were prepared by granulating the sirolimus suspension followed by compression into tablets, whereas Rapamune is prepared by directly coating sirolimus nanosuspension onto inert cores.24 Dissolution experiments were conducted under nonsink conditions as they better predict the in vivo performance of the precipitation inhibitors than sink conditions.28,41 Dissolution method represents a standard methodology for characterization

of solid dosage form. The most appropriate dissolution media for the classical dissolution testing were previously developed, and the presented dissolution experimental conditions were considered to demonstrate the highest degree of discrimination under nonsink conditions. To ensure proper hydrodynamic conditions (i.e., proper mixing), a combination of USP1 and USP2 dissolution apparatus (i.e., paddle method with stationary basket) was used with the aim to use mild hydrodynamic conditions and to avoid cone effect.52 Since sirolimus has a low I



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solubility in physiological pH range,7,10 surfactants should be added to the dissolution medium to ensure adequate fraction of dissolved compound. In addition to the SLS containing media, dissolution experiments were also performed in FaSSIF and FaSSIF v2, since the human pharmacokinetic study was performed with fasted subjects. In the FaSSIF media, the percentage of dissolved sirolimus was higher than in FaSSIF v2 and the SLS containing dissolution media (Figure 6). The difference between the FaSSIF and FaSSIF v2 media could be attributed to the higher amounts of lecithin in FaSSIF than in FaSSIF v2.32 The general conclusion is that, under all the experimental conditions, formulation A showed a faster dissolution rate than formulation B and Rapamune. Most probably the combination of small sirolimus particles and the presence of HPMC 603 in formulation A resulted in a faster sirolimus dissolution rate in comparison to the formulation B and Rapamune. Formulation B resulted in slightly lower but comparable dissolution profile to Rapamune despite the presence of a significantly larger sirolimus particle size in formulation B (Figure 6). The results of the human fasted PK study outlined a significantly higher Cmax and AUC of formulations A and B in comparison to Rapamune (ANOVA, p < 0.01) (Table 4). Since the formulations A and B contained the same amount of HPMC 603, the higher Cmax and AUC in the case of formulation A could be attributed to the smaller sirolimus particles size. This conclusion is also in line with the dissolution data, where a higher sirolimus dissolution rate was observed for formulation A than for B (Figure 5). On the other hand, the dissolution experiments failed to predict a significantly higher Cmax and AUC of formulation B versus Rapamune (Table 4). Several other studies describing the performance of the precipitation inhibitors also outlined the rather poor correlation between the results obtained in the dissolution experiments and in vivo pharmacokinetic data.28,40,53−55 For example, in vitro dissolution profiles of itraconazole solid dispersion containing HPMC and Eudragit E100 poorly correlated with the observed in vivo pharmacokinetic profiles in healthy human volunteers.55 In contrast to the poor correlation of the dissolution data with the human PK study outcome, the correlation of the precipitation inhibition data and the flux across the PAMPA membrane with the human Cmax and AUC data was very good (Figure 7). This observation emphasized that, for the characterization of the precipitation inhibitors, alternative methodologies with predictive potential should be applied in combination with the conventional dissolution methodologies in order to correctly predict the in vivo effect of the precipitation inhibitor. However, this conclusion does not imply that the dissolution studies do not represent the key experimental step in the biopharmaceutical characterization of the precipitation inhibitor containing the drug formulation, but rather proposes that the further optimization of the dissolution methodologies beyond the nonsink conditions is highly desired.

formulations A and B is very low, the sirolimus particles are in close contact with HPMC 603, thus creating a desired microenvironment for maintaining the supersaturated solution and preventing sirolimus precipitation. However, the benefit of the improved sirolimus absorption on patient therapy would need to be confirmed in a clinical trial.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +38615803315. Fax: +386 1 5683517. E-mail: luka. [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AUC, area under the curve; BCS, Biopharmaceutical Classification System; DMSO, dimethyl sulfoxide; DSC, differential scanning calorimetry; FaSSIF, fasted state simulated intestinal fluid; FeSSIF, fed state simulated intestinal fluid; FT, Fourier transform; GIT, gastrointestinal tract; HPMC 603, hydroxypropyl methyl cellulose (i.e., Pharmacoat 603); IR, infrared; PI, precipitation inhibitor; PVDF, polymer of vinylidene fluoride; PVP, polyvinylpyrrolidone; SIF, simulated intestinal fluid; SLS, sodium lauryl sulfate

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