European Journal of Pharmaceutical Sciences 74 (2015) 77–85

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier .com/ locate/e jps

Novel oral formulation approach for poorly water-soluble drug usinglipocalin-type prostaglandin D synthase

http://dx.doi.org/10.1016/j.ejps.2015.04.0120928-0987/� 2015 Elsevier B.V. All rights reserved.

Abbreviations: L-PGDS, lipocalin-type prostaglandin D synthase; API, activepharmaceutical ingredient; BCS, biopharmaceutical classification system; SEM,scanning electron microscope; SE-HPLC, size exclusion high-performance liquidchromatography; CD, circular dichroism; SGF, simulated gastric fluid; FaSSGF,fasted state-simulated gastric fluid; FaSSIF, fasted state-simulated intestinal fluid;SIF, simulated intestinal fluid; SHR, spontaneously hypertensive rat; SBP, systolicblood pressure.⇑ Corresponding author at: Laboratory of Biological Macromolecules, Graduate

School of Life and Environmental Sciences, Osaka Prefecture University, 1-1Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan. Tel.: +81 72 254 9473; fax:+81 72 254 9921.

E-mail address: inuit@bioinfo.osakafu-u.ac.jp (T. Inui).

Masashi Mizoguchi a,b, Masatoshi Nakatsuji a, Haruka Inoue a, Keisuke Yamaguchi a, Atsushi Sakamoto c,Koichi Wada b, Takashi Inui a,⇑a Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japanb Department of Chemistry, Manufacturing and Control, Kobe Pharma Research Institute, Nippon Boehringer Ingelheim Co., Ltd., 6-7-5, Minatojima-minamimachi, Chuo-ku,Kobe, Hyogo 650-0047, Japanc Department of Pharmacokinetics and Nonclinical Safety, Kobe Pharma Research Institute, Nippon Boehringer Ingelheim Co., Ltd., 6-7-5, Minatojima-minamimachi, Chuo-ku,Kobe, Hyogo 650-0047, Japan

a r t i c l e i n f o

Article history:Received 21 October 2014Received in revised form 10 April 2015Accepted 14 April 2015Available online 20 April 2015

Chemical compounds studied in this article:Telmisartan (PubChem CID: 65999)

Keywords:Drug-delivery carrierL-PGDSPoorly water-soluble drugSolubilityOral absorptionSpray drying

a b s t r a c t

Lipocalin-type prostaglandin D synthase (L-PGDS), a member of the lipocalin superfamily, possesses thefunction of forming complexes together with various small lipophilic molecules. In this study, we chosetelmisartan as a model drug due to its pH dependent poor water solubility, and developed and character-ized a novel solubilized formulation of telmisartan using a complex formulation with L-PGDS. The solidstate of the complex formulation was prepared using a spray-drying process. The spray-dried formulationof telmisartan/L-PGDS powder showed a typical spray-dried particle without any change in the sec-ondary and tertiary structures of L-PGDS. Furthermore, the complex formulation showed a high rateand level of drug release in pH 1.2, 5.0, and 6.8 solutions in comparison with the active pharmaceuticalingredient (API) and commercial product. To validate the potential for oral administration of the telmis-artan/L-PGDS complex in vivo, the pharmacokinetic and pharmacodynamic profiles were assessed inspontaneous hypertensive rats. An animal study revealed that the complex formulation led to a signifi-cant improvement of AUC and Cmax as compared with API, and the prolonged pharmacologic effect onblood pressure reduction was comparable with the commercial product. These results, taken together,demonstrate that this novel approach is feasible for the solubilized solid oral formulation and it can beapplied to poorly water-soluble drugs to enhance oral bioavailability.

� 2015 Elsevier B.V. All rights reserved.

1. Introduction differences in oral bioavailability due to formulation factors can

Insolubility or poor solubility in water of drug candidate is one ofthe most serious problems in pharmaceutical development. Whenthe absorption of a drug is limited by its solubility, marked

be observed. However, new drug entities tend to have a lower sol-ubility using the current drug discovery approach (i.e., high-throughput screening, combinatorial chemistry) (Lipinski, 2000;Lipinski et al., 2001). These active pharmaceutical ingredients(APIs) belong to class II and IV drugs of the biopharmaceutical clas-sification system (BCS) that are insoluble in water (Amidon et al.,1995), and about 65% of new drug candidates are classified in thisclass (Ku and Dulin, 2012). The development of drugs which includepoorly soluble compounds is a serious bottleneck for the pharma-ceutical industry. Telmisartan (Fig. 1A) is an antagonist of theangiotensin II type-1 receptor that is indicated for the treatmentof hypertension (Wienen et al., 2000). Telmisartan is categorizedas a BCS class II compound, and its solubility is quite low withinthe physiological gastrointestinal pH range (Tran et al., 2008).

As solutions to improve the low solubility of compounds, vari-ous technologies have been investigated from several aspects

Fig. 1. Structures of telmisartan and L-PGDS [molecular mass: 18777.7]. (A)Chemical structure of telmisartan [relative molecular mass: 514.6]. (B) Three-dimensional structure of human L-PGDS (PDB code: 3O2Y).

78 M. Mizoguchi et al. / European Journal of Pharmaceutical Sciences 74 (2015) 77–85

(Kawabata et al., 2011; Fahr and Liu, 2007). The addition of solubi-lizers such as organic solvents, surfactants, lipids, cyclodextrin, andpH modifiers is considered as a beneficial approach to solubilityimprovement (Gao and Shi, 2012; Loftsson and Brewster, 2012).However, formulations including solubilizers need the quantitativeand qualitative optimization of compounds to overcome the stabil-ity problem (Strickley, 2004).

Lipocalin-type prostaglandin D synthase (L-PGDS, Fig. 1B) is amulti-functional protein which plays the role of a catalyzer forthe isomerization of prostaglandin H2, a scavenger for reactive oxy-gen species, and an extracellular transporter for small lipophilicmolecules as a member of the lipocalin superfamily (Nagataet al., 1991; Toh et al., 1996; Fukuhara et al., 2012). Recently, weshowed that L-PGDS acted as a scavenger of biliverdin, a metabo-lite of hemoglobin that accumulates in the cerebrospinal fluid ofaneurysmal subarachnoid hemorrhage patients (Inui et al., 2014).The structure of L-PGDS exhibited the typical lipocalin fold, con-sisting of an eight-stranded, antiparallel b-barrel and a long a-helixassociated with the outer surface of the barrel. The interior of thebarrel formed a hydrophobic cavity opening into the upper endof the barrel, the size of which was larger than those of other lipo-calins (Shimamoto et al., 2007; Miyamoto et al., 2010; Zhou et al.,2010). We demonstrated that L-PGDS could bind to a large varietyof lipophilic molecules such as heme metabolites, retinoids, thy-roids, steroids, flavonoids, and saturated fatty acids in thehydrophobic cavity (Kume et al., 2012; Inui et al., 2003). Usingthe function of L-PGDS, we have already evaluated the feasibilityof L-PGDS as a novel drug-delivery carrier to improve the solubilityof the poorly water-soluble molecules mentioned in the previousarticle (Fukuhara et al., 2012).

Proteins in liquid formulations are generally at a greater risk ofchemical and physical instability (Wang, 1999; Mahler et al.,2005). Therefore, L-PGDS needs to be prepared in dry form in orderto increase the stability and apply it to multiple dosage forms. Thecommon process to produce dried protein is lyophilization andspray drying. Lyophilization has less of a thermal influence on pro-tein than other drying processes. However, the lyophilization pro-cess is energy-intensive. Furthermore, the process involves time-consuming steps such as freezing followed by drying under lowpressure, and there are high production costs involved (Wang,2000). On the other hand, the spray-drying process utilizes heatto evaporate micro-dispersed droplets created by atomization ofa continuous liquid feed. Therefore, the one-step drying processleads to a significantly shorter operation time and cost-effectivedehydration (Cal and Sollohub, 2010).

In the present study, we show the application of L-PGDS as anovel drug-solubilizing carrier for a solid oral formulation usingtelmisartan as a model compound. The solid state of the telmisar-tan and L-PGDS complex formulation was produced using thespray-drying technique, and the physicochemical properties of

the produced particle were characterized. Finally, the in vitro andin vivo performance of the developed formulation was assessed.

2. Materials and methods

2.1. Materials

Telmisartan and the commercial product (Micardis�) were sup-plied by Boehringer Ingelheim GmbH & Co. KG (Ingelheim,Germany). Jet-milling of telmisartan was carried out by A–O jetmill (Seishin Enterprise Co., Ltd, Osaka, Japan). The injector airpressure and the grinding air pressure were 7.5 bar, and powderfeed rate was set to 3 g min�1 at room temperature. All otherchemicals were of analytical grade.

2.2. Purification of recombinant human L-PGDS

The recombinant human C65A/C167A (e280 = 25,900 M�1 cm�1)-substituted L-PGDS mutant, in which a catalytic residue of cysteinewas substituted to alanine to get rid of an enzymatic activity of L-PGDS was used in this study. And the 22 N-terminal amino acidresidues corresponding to the putative secretion signal peptide ofL-PGDS were truncated. C65A/C167A-substituted L-PGDS wasexpressed in Escherichia coli BL21 (DE3) (TOYOBO, Osaka, Japan)(Kume et al., 2012). Site-directed mutagenesis was performed usingthe QuikChange™ site-directed mutagenesis kit (StratageneCalifornia, La Jolla, California, USA). The mutated L-PGDS wasexpressed as a glutathione S-transferase fusion protein. The fusionprotein was bound to a glutathione–Sepharose 4B column (GEHealthcare Bio-Sciences, Little Chalfont, UK) and incubated over-night with thrombin (16.5 units mL�1 column bed volume) torelease L-PGDS at room temperature. The protein was further puri-fied by gel filtration chromatography with HiLoad 26/600 Superdex75 (GE Healthcare Bio-Sciences) in 5 mM Tris–HCl (pH 8.0). Thepurified L-PGDS of 100–200 mg was routinely obtained from 1 Lof culture.

2.3. Solubility study

An excess amount of telmisartan was weighed in a 2-mL micro-tube with 1 mL of aqueous solution containing several kinds of buf-fer medium in the presence of L-PGDS or without L-PGDS. Sealedmicrotubes were shaken with a Rotator RT-50 (TAITEC, Saitama,Japan) for 2 h at 37 �C, followed by filtration through a 0.45-lm fil-ter (EMD Millipore, Billerica, Massachusetts, USA). After incuba-tion, the sample solution was diluted with methanol andcentrifuged at 3000 rpm for 10 min. Then, 20 lL of filtered super-natant (0.45-lm filter) was injected into a high-performance liquidchromatography (HPLC, Waters Corporation, Milford,Massachusetts, USA) equipped a YMC Pack ODS-AM column(150 � 4.6 mm I.D., 5 lm, YMC Co., Ltd., Kyoto, Japan) to analyzethe amount of telmisartan. The mobile phase, a mixture of metha-nol and 2% (w/v) ammonium phosphate (pH 3.0) (70:30, v/v), waseluted at a flow rate of 1 mL min�1. The chromatogram was mon-itored at 297 nm.

2.4. Spray drying of telmisartan/L-PGDS complex solution

Based on the solubility study, the complex of L-PGDS andtelmisartan was prepared as a 1:1 M ratio. The complex solutionwas filtered with a 0.45-lm filter (EMD Millipore) before thespray-drying process. Dried L-PGDS or the telmisartan/L-PGDScomplex was prepared with Mini Spray Dryer B-290 (BÜCHILabortechnik AG, Flawil, Switzerland). Solution was delivered tothe water-cooled nozzle (0.7-mm liquid orifice internal diameter)

M. Mizoguchi et al. / European Journal of Pharmaceutical Sciences 74 (2015) 77–85 79

at a flow rate of 3 mL min�1, sprayed at an inlet air temperature(Tinlet) of 90 or 120 �C and an outlet air temperature of 40–43 or60–62 �C. The drying air volumetric flow rate was set at35 m3 h�1, and the atomizing air volumetric flow rate at600 L h�1. The produced powders were collected through a high-efficiency cyclone in a glass container, and stored at �20 �C.

2.5. Morphological analysis

Powder samples were mounted on the brass stub using double-sided adhesive tape and were made electrically conductive bycoating with gold (18 nm min�1) in a vacuum (4 kPa) using theMSP-mini Magnetron Sputter (Vacuum Device Co., Ltd., Ibaraki,Japan) for 45 s at 26 mA. The scanning electron microscope (SEM)images were analyzed at 15 kV accelerating voltage with an imageanalysis system (Miniscope� TM3000, Hitachi High-TechnologiesCorporation, Tokyo, Japan). Micrographs at 6000� magnificationwere presented.

2.6. Particle size measurement

Particle size measurements were examined with a SympatecHELOS/RODOS (Sympatec GmbH, Clausthal-Zellerfeld, Germany)equipped with a vibratory feeder and R1 lens (0.1–35 lm). The pri-mary pressure was manually set using the adjustment valve in therange of 0.2–4.5 bar, and three measurements were taken usingfreshly loaded powder. The particle size distribution (d10, d50 andd90) was calculated using the Fraunhofer theory.

2.7. Size exclusion high-performance liquid chromatography (SE-HPLC)

Size exclusion chromatography was used to evaluate theamount of soluble protein aggregates in the spray-dried powdersafter dissolution. Analysis was performed on a Waters HPLC sys-tem with a TSK3000SWXL column (300 � 7.8 mm, Tosoh, Tokyo,Japan) and UV detection at 280 nm. The mobile phase consistedof 0.1 M disodium hydrogen phosphate dehydrate and 0.1 Msodium sulfate, and was adjusted with phosphoric acid to pH 6.8.The flow rate was 1 mL min�1 and the injection volume was50 lL with a protein concentration of approximately 5 mg mL�1.Samples were analyzed in duplicate.

2.8. Circular dichroism (CD) measurement

CD measurements were performed with a J-820 spectropo-larimeter (Jasco, Tokyo, Japan). The temperature of the samplesolution in the cuvette was controlled at 25.0 �C by a Peltier PTC-423 L thermo-unit (Jasco). The path length of the optical quartzcuvette was 1.0 mm for far-UV range CD measurements at 200–260 nm, and 10 mm for near-UV range CD measurements at250–350 nm. The sample concentration for the far-UV range andnear-UV range was 5 and 40 lM in 5 mM Tris–HCl (pH 8.0), respec-tively. The data are expressed as the molar residue ellipticity (h).

2.9. Dissolution testing

Dissolution testing of the spray-dried telmisartan/L-PGDS com-plex in several media [simulated gastric fluid (SGF) including pep-sin, McIlvaine buffer pH 5.0, and 0.05 M phosphate buffer pH 6.8]was implemented for 60 min at 37 �C with a miniaturized dissolu-tion apparatus (lDiss Profiler™, pION, Billerica, Massachusetts,USA) due to the lack of sample availability. The applied amountof telmisartan was 0.67 mg in 15 mL of test medium, which isequivalent in concentration to 40 mg of telmisartan in 900 mL oftest medium. The commercial product was milled by mortar andpestle before the testing. An appropriate amount of the produced

powder was added to the test medium directly on stirring with amagnetic stirrer at 300 rpm. Sample solution was withdrawn atsampling points, and the amount of telmisartan assayed with theHPLC method is described in Section 2.3.

2.10. In vitro digestion study

2.10.1. Preparation of biorelevant mediaFasted state-simulated gastric fluid (FaSSGF) and fasted state-

simulated intestinal fluid (FaSSIF) were prepared from simulatedintestinal fluid (SIF) powder (Biorelevant.com, Croydon, Surrey,UK) (Vertzoni et al., 2005; Galia et al., 1998).

2.10.2. Digestion study in FaSSGFAliquots (180 lL) of FaSSGF including 3.2 mg mL�1 pepsin were

placed in 1.5-mL microcentrifuge tubes and incubated in a waterbath at 37 �C for 180 min. L-PGDS of 20 lL (3.8 mg mL�1) wasadded to each of the FaSSGF vials to start the digestion reaction.NaOH (3.75 N) of 20 lL was added to each vial to stop the reaction.The samples were then mixed with loading dye with b-mercap-toethanol and resolved on polyacrylamide gels with sodium dode-cyl sulfate (SDS). Proteins were visualized by Coomassie BrilliantBlue staining.

2.10.3. Sequential digestion study (FaSSGF followed by FaSSIF)L-PGDS of 20 lL (3.8 mg mL�1) was added to each of the 180 lL

of FaSSGF including 3.2 mg mL�1 pepsin vials, and the digestionreaction was performed at 37 �C for 60 min. The digested solutionof 100 lL was added to each of the 900 lL of FaSSIF with10 mg mL�1 pancreatin. Each sample was incubated at 37 �C for240 min, and the digestion reaction was stopped by placing thetube in a boiling water bath for 5 min. The samples were thenmixed with loading dye with b-mercaptoethanol. Proteins wereseparated by sodium dodecyl sulfate polyacrylamide gel elec-trophoresis (SDS–PAGE) and visualized by Coomassie BrilliantBlue staining.

2.11. Pharmacokinetic study

Male spontaneously hypertensive rats (SHR, 9 weeks of age,Japan SLC Inc., Shizuoka, Japan) were housed under a 12-h light–dark schedule with free access to food and water for 1 week torecover from the stress of transportation. All procedures used inthis study complied with policies of the Osaka PrefectureUniversity Animal Care and Use Committee (Approval No. 25-80).

2.11.1. Sample administration and blood samplingEighteen rats were randomly divided into 3 groups (n = 6, per

group). Jet-milled telmisartan was administered orally as a PBS(pH 7.4) suspension at a dose of 4 mg kg�1 telmisartan(5 mL kg�1). And, the commercial product and telmisartan/L-PGDS complex were administered orally as a PBS (pH 7.4) solutionjust after the preparation at a dose of 4 mg kg�1 telmisartan(5 mL kg�1). The dose of telmisartan used in this study was a 40-mg dose for a 60-kg human based on calculation with body surfacearea as a factor to convert a dose for translation from rats tohumans (Reagan-Shaw et al., 2008). Blood samples (300 lL) werecollected from the tail vein at pre-dose (0 h), 0.25, 0.5, 1, 2, 4, 8,12, 24, and 48 h after oral administration. Collected samples werecentrifuged at 10,000 rpm for 5 min to harvest serum, and stored at�20 �C until analysis.

2.11.2. LC–MS/MS analysis of serum concentration of telmisartanThawed 50 lL of serum and 50 lL of Probenecid solution as

internal standards were mixed, and 200 lL of acetonitrile/metha-nol/water (50/45/5, v/v) was added for protein precipitation.

80 M. Mizoguchi et al. / European Journal of Pharmaceutical Sciences 74 (2015) 77–85

After centrifugation at 4000 rpm for 5 min, 200 lL of supernatantwas mixed with 200 lL of 10 mM ammonium acetate/acetonitrile(50/50, v/v) and analyzed with LC–MS/MS. In the HPLC part, usingan Agilent 1100 series HPLC (Agilent, Santa Clara, California, USA)equipped Xbridge BEH 300 C18 (2.1 mm I.D. � 50 mm length,3.5 lm, Waters Corp.), the sample was separated under a gradientcondition which consists of 10 mM ammonium acetate (Solvent A)and acetonitrile (Solvent B) at a flow rate of 0.4 mL min�1. Thegradient condition was configured as 0–1.0 min; 50% Solvent A,1.0–2.0 min; 50–10% Solvent A, 2.0–3.0 min; 10–50% Solvent Aand 3.0–5.0 min; 50% Solvent A. The separated sample was placedinto the MS/MS part API4000™ (AB SCIEX, Framingham,Massachusetts, USA) equipped with TurboIonSpray as an ionsource. Telmisartan and Probenecid were monitored by multiplereaction monitoring of the transitions of m/z 516.1 ? 276.3 andm/z 286.2 ? 243.8, respectively.

2.12. Pharmacodynamic study

Male SHR (9 weeks of age, Japan SLC Inc.) were housed under a12-h light–dark schedule with free access to food and water for1 week to recover from the stress of transportation. All proceduresused in this study complied with the institutional policies for thecare and use of laboratory animals (Approval No. 140328A).

Twenty-four rats were randomly assigned to 4 groups (n = 6,per group) based on the systolic blood pressure (SBP). The samesamples as in the pharmacokinetic study were orally administeredand PBS (pH 7.4) was administered as a control. The SBP and heartrate were measured by the noninvasive tail-cuff method using MK-2000 (Muromachi Kikai Co., Ltd., Tokyo, Japan) at pre-dose (0 h),0.5, 1, 2, 4, 8, 12, 24, and 48 h after administration.

2.13. Statistical analysis

Data were statistically evaluated using one-way ANOVA fol-lowed by Dunnett’s test. The results were considered significantat the 5% significance level (p < 0.05).

3. Results

3.1. Solubility study

3.1.1. Evaluation of solubility enhancement by L-PGDSFirstly, we evaluated the solubility enhancement of telmisartan

by the addition of L-PGDS in the several kinds of buffer medium(Table 1). The solubility of telmisartan (0.26–3.12 lg mL�1) was sig-nificantly enhanced approximately 260-fold (816–834 lg mL�1)with 25 mg of L-PGDS in the all evaluated mediums, and therewas no effect of buffer species and ion strength on the solubilityenhancement.

Table 1Solubility of telmisartan at 37 �C in different pH buffers, and in the presence ofL-PGDS.

Medium 25 mg L-PGDS lg mL�1

SGF (pH 1.2) � 257McIlvaine buffer (pH 5.0) � 0.26

+ 82650 mM phosphate buffer (pH 6.8) � 0.22

+ 8345 mM Tris–HCl buffer (pH 8.0) � 3.12

+ 816Sorenson’s buffer (pH 9.0) � 44.1

3.1.2. Determination of complexation rateWe checked the solubility profiles of telmisartan in 5 mM Tris–

HCl buffer solution (pH 8.0) with the various amounts of L-PGDS at37 �C (Fig. 2) and Table 2. The solubility of telmisartan rose withthe higher levels of L-PGDS, and a linear increase was observedafter incubation for 120 min (r2 = 0.987). From the solubility pro-file, the apparent stability constant was calculated as1.73 � 105 M�1, and the complexation rate was approximately1:1 M ratio.

On the other hand, in the case of the preparation of the telmis-artan and L-PGDS complex carried out at room temperature, thesame enhanced concentration of telmisartan was observed, but24 h were required to achieve the concentration. When the volumeof L-PGDS solution which includes the same amount of L-PGDS waschanged from 1 to 100 mL, there was no significant difference inthe solubility enhancement of telmisartan as compared with thecase at 37 �C. Based on this result, we decided to prepare thetelmisartan/L-PGDS at 37 �C for further studies, and 10–25 mg mL�1 of L-PGDS was employed for the spray-drying process.

3.2. Physical characterization of spray-dried telmisartan/L-PGDScomplex

3.2.1. Spray-drying process of L-PGDS solutionAll spray-drying runs showed a high yield (50–90%). Only a

small amount of powdery deposit was observed, and the insidewall of the drying chamber was clean. Therefore, the marked par-ticle loss was mainly caused by fine particles passing through thecyclone into the exhausted air. The dried particles were obtainedfrom the cyclone and collecting vessel.

3.2.2. Morphological and particle size analysesThe surface morphology of the spray-dried L-PGDS powder and

spray-dried telmisartan/L-PGDS complex powder was observedwith SEM (Fig. 3). Jet-milled telmisartan was the agglutinate mix-ture of flattened fine particles with a wide size distribution(d10 = 0.18 lm, d50 = 1.30 lm, d90 = 5.05 lm, Fig. 3A). The spray-dried L-PGDS and telmisartan/L-PGDS complex at 90 �C of Tinlet

had a smooth surface and typical spherical shape, with a narrowsize distribution (d10 = 0.26 lm, d50 = 2.60 lm, d90 = 6.16 lm,Fig. 3B and C). In the case of the powder spray-dried L-PGDS at

Fig. 2. Relationship between solubility enhancement and complexation in 5 mMTris–HCl buffer (pH 8.0). The complex was prepared at 37 �C for 120 min.

Table 2Solubility profile of telmisartan with several concentrations of L-PGDS in 5 mM Tris–HCl buffer (pH 8.0).

Solubility (lM)

L-PGDS 0 270 800 1330Telmisartan 45 474 1120 1590

Fig. 3. SEM images of (A) jet-milled telmisartan, (B) spray-dried L-PGDS powder (Tinlet = 90 �C), (C) spray-dried telmisartan/L-PGDS complex powder (Tinlet = 90 �C), and (D)spray-dried L-PGDS powder (Tinlet = 120 �C). Each bar represents 10 lm.

M. Mizoguchi et al. / European Journal of Pharmaceutical Sciences 74 (2015) 77–85 81

120 �C of Tinlet, however, dimple-type particles were observed(Fig. 3D).

3.2.3. Reconstitution of spray-dried L-PGDSThe spray-dried L-PGDS powder was reconstituted with 5 mM

Tris–HCl (pH 8.0), and the reconstituted solution was applied toSE-HPLC to verify the soluble aggregate of L-PGDS. The chro-matogram indicated only a single peak of L-PGDS, and the solubleaggregation was not observed in the reconstituted L-PGDS solution(data not shown). The structure of reconstituted L-PGDS was mon-itored with CD measurement. The reconstituted solutions of spray-dried L-PGDS produced at 90 and 120 �C of Tinlet had the same spec-trum as the control in the far- and near-UV regions, respectively(Fig. 4A and B). These results showed that the secondary and ter-tiary structures of L-PGDS were not altered by the spray drying.

3.2.4. In vitro dissolution behaviorSmall-scale dissolution testing was performed to evaluate the

in vitro dissolution behavior of the spray-dried telmisartan/L-PGDS complex (Fig. 5). Jet-milled telmisartan and the

Fig. 4. CD spectra in (A): far-UV and (B): near-UV region of intact L-PGDS solution (black)Tinlet = 120 �C (red). (For interpretation of the references to colour in this figure legend,

commercial product were measured as a control of the telmisar-tan/L-PGDS complex. The dissolution level of jet-milled telmisar-tan in SGF with pepsin (pH 1.2) was 90% of the applied totalamount of the drug (Fig. 5A), while the solubility at pH 5.0 and6.8 was 4–5% at 60 min, respectively (Fig. 5B and C). The commer-cial product improved the solubility of telmisartan, and a 100% dis-solution level was achieved at both pH 1.2 and 6.8 (Fig. 5A and C).However, the maximum drug release from the commercial productwas only 16% at pH 5.0 at 60 min (Fig. 5B). In contrast, the complexformulation of telmisartan/L-PGDS significantly enhanced the sol-ubility of telmisartan, and achieved 100% dissolution within10 min in all evaluated test media. Furthermore, the L-PGDS for-mulation maintained the enhanced solubility of telmisartan inthe presence of pepsin for up to 2 h (data not shown).

3.3. In vitro digestibility of L-PGDS under simulated gastrointestinalconditions

An in vitro digestion study was implemented with the simu-lated gastrointestinal medium to verify the stability of L-PGDS in

, reconstituted L-PDGS dried at Tinlet = 90 �C (blue) and reconstituted L-PGDS dried atthe reader is referred to the web version of this article.)

Fig. 5. Dissolution profiles of telmisartan formulations in (A) SGF with pepsin (pH1.2), (B) McIlvaine buffer (pH 5.0), and (C) 0.05 M phosphate buffer (pH 6.8). s:telmisartan, h: commercial product and �: spray-dried telmisartan/L-PGDS com-plex. Final concentration of telmisartan: 0.044 mg�1 mL. Each bar represents themean ± SD of 3 independent experiments.

Fig. 6. SDS–PAGE analysis of the (A) in vitro digestion study in FaSSGF and (B)sequential digestion study in FaSSGF followed by FaSSIF. M: molecular weightmarker. Digestion reaction was performed at 37 �C.

Fig. 7. The time-profile of the serum concentration of telmisartan in SHR after oraladministration (4 mg kg�1 telmisartan). s: jet-milled telmisartan, h: commercialproduct and �: telmisartan/L-PGDS complex. Each bar represents the mean ± SD of6 independent experiments.

82 M. Mizoguchi et al. / European Journal of Pharmaceutical Sciences 74 (2015) 77–85

the gastrointestinal environment (Fig. 6). Although the minor andtime-dependent digestion of L-PGDS (Mr = 19,000) under thesimulated gastric condition was observed, the major structure ofL-PGDS was stable in the environment for 180 min (Fig. 6A). Thestructure of L-PGDS was completely changed to the digested format 30 min after exposure under the gastric conditions.

On the other hand, the digested L-PGDS in FaSSGF was rapidlydegraded in the simulated intestinal environment, and there wasno corresponding band for L-PGDS at 1 min after digestion inFaSSIF (Fig. 6B).

Table 3Pharmacokinetic parameters for telmisartan formulations after oral administration.

AUC0–48h

(ng h ml�1)Cmax

(ng ml�1)Tmax

(h)T1/2

(h)

Jet-milled telmisartan 947 ± 435 66.0 ± 27.4 4.0 9.3Commercial product 4820 ± 836* 296 ± 70.5* 4.0 12.1Telmisartan/L-PGDS

complex5610 ± 548* 348 ± 100* 1.3 9.8

Values (AUC0–48h, Cmax) are expressed as the mean ± SD of 6 experiments.* Significant difference from jet-milled telmisartan (P < 0.05).

3.4. In vivo evaluation of solubilized formulation of telmisartan withL-PGDS complex

3.4.1. Pharmacokinetic studyThe concentration of telmisartan in serum was quantified to

evaluate the in vivo behavior of the orally administered telmisar-tan/L-PGDS complex with SHR (Fig. 7). The calculated pharmacoki-netic parameters are listed in Table 3. Jet-milled telmisartanshowed the lowest values for all parameters, and telmisartanwas not detected at 48 h after oral administration. The telmisar-tan/L-PDGS complex significantly improved the in vivo behaviorof telmisartan in the area under the serum concentration–time

curve from 0 to 48 h (AUC0–48h), maximum concentration (Cmax),and time to maximum concentration (Tmax). Especially, AUC0–48h

of the complex formulation was 5.9-fold that of the jet-milled

Fig. 8. Change in systolic blood pressure (DSBP). (A): time-profile of DSBP after oral administration (4 mg kg�1 telmisartan). d: control, s: jet-milled telmisartan, h:commercial product and �: telmisartan/L-PGDS complex. The bar of the control is expressed as mean ± SE of 6 independent experiments, and bars of the others represent themean ± SE of 5 independent experiments. (B) Comparison of the area under the curve of DSBP (mmHg h).

M. Mizoguchi et al. / European Journal of Pharmaceutical Sciences 74 (2015) 77–85 83

telmisartan, and Tmax of the complex formulation was significantlyshorter than in jet-milled telmisartan and the commercial product.And, the significant difference for elimination half-life (T1/2) inthese three formulations was not observed.

3.4.2. Pharmacodynamic studyNext, we investigated the antihypertensive effect of the complex

formulation of telmisartan/L-PGDS, and the effect was comparedwith jet-milled telmisartan and the commercial product bymonitoring the change in the systolic blood pressure (DSBP) aftera single oral administration (Fig. 8). An increase in SBP at 0.5 h afteradministration was observed in each sample group, and the samplegroup data were analyzed in 5 subjects. All 3 formulations showed acomparable reduction of SBP (Fig. 8A). However, based on thecalculated area under the curve of DSBP, the commercial productand telmisartan/L-PGDS complex formulation had a tendency toreduce SBP more than jet-milled telmisartan in comparisonwith the control (Fig. 8B). In addition, the commercial productand telmisartan/L-PGDS complex decreased SBP immediately(jet-milled telmisartan: �11.4 ± 5.5 mmHg, commercial product:�17.4 ± 4.7 mmHg and telmisartan/L-PGDS complex: �18.2 ± 6.7mmHg at 0.5 h after administration). Although the effect ofjet-milled telmisartan returned to the basal level at 48 h afteradministration, the commercial product and complex formulationretained the effect (jet-milled telmisartan: 5.6 ± 6.3 mmHg, com-mercial product: �4.6 ± 7.9 mmHg and telmisartan/L-PGDS com-plex: �8.4 ± 6.3 mmHg at 48 h after administration). Furthermore,there was no significant change in the heart rate compared withthe control with all formulations (data not shown). These resultsdemonstrated that the telmisartan/L-PGDS complex possessed adrug potency almost equal to the commercial product.

4. Discussion

According to the chemical structure of telmisartan, the com-pound is readily ionizable, and the solubility is pH-dependent(Fig. 1A and Table 1). The inherent solubility profile applies tothe solubilization, and microenvironmental pH modification hasbeen investigated as the solubilization approach (Nakatani et al.,2004). Although the solubilization approach with pH modificationis an effective method, this method can apply to ionizable API as aprerequisite. In addition, the formulation including pH modifierhas a high risk of chemical instability and poor manufacturabilitycaused by the acidifier/alkalizer (Taniguchi et al., 2014; Badawyand Hussain, 2007). Therefore, careful qualitative and quantitativeselections of pH modifiers are required. Furthermore, the solubility

improvement by formation of a solid dispersion using a pH modi-fier incorporating a common pharmaceutical solubilizer such as anorganic solvent, cyclodextrin, and surfactant has also been demon-strated. However, a series of solubilizing approaches requires thecombination of several solubilizers to achieve adequate solubilityof telmisartan, and the all marketed formulations of telmisartanare mainly formulated with solid dispersion technique using pro-portionally high concentration of strong alkalizer such as sodiumhydroxide and meglumine or combinations thereof (Sangwai andVavia, 2013; Marasini et al., 2013; Zhong et al., 2013).

In the present study, we showed that the solubility of telmisar-tan is enhanced in the presence of L-PGDS, and that the complexformulation is the first order increase with respect to the amountof L-PGDS with the simple preparation method (Fig. 2). Recently,we reported that the driving force responsible for complex formu-lation between L-PGDS and the guest compound is considered tobe hydrophilic and hydrophobic interaction adjusted byenthalpy–entropy compensation (Kume et al., 2014). Althoughthe solubilizing capacity of L-PGDS may be limited by the size ofguest compounds due to the size of the L-PGDS cavity, the capabil-ity for the current development of low-molecular-weight drug can-didates could be sufficient. Indeed, L-PGDS can bind and solubilizethe hydrophobic guest compounds up to approximately Mr = 850(Inui, T., unpublished observation). On the other hand, thein vitro dissolution study revealed that the telmisartan/L-PGDScomplex showed a significantly faster dissolution rate than jet-milled telmisartan, and reached 100% in the physiological pHrange. Interestingly, the complex formulation improved the extentof dissolution for telmisartan, and the improved solubility wasmaintained in SGF (Fig. 5A). Moreover, the main structure of L-PGDS should be stable under the simulated gastric conditions,but low-level digestion of L-PGDS with pepsin was observed(Fig. 6A). The digested fragment might be the flexible fragmentin both the N- and C-terminal regions (Miyamoto et al., 2010),and the central antiparallel b-barrel and long a-helix region thatform the hydrophobic cavity would be maintained. These resultssuggest that the complex formulation is maintained in a low pHenvironment in the presence of pepsin. The pH environment inthe gastrointestinal tract varies due to several factors such asaging, food, and the influence of administered drugs (Moriharaet al., 2001; Charman et al., 1997; Hunt et al., 2005), and variablebioavailability and unexpected drug absorption are observed dueto pH variation. Hence, this consistent dissolution behavior underphysiological pH conditions should be beneficial.

The spray-drying process robustly produced the stable driedprotein powder that maintained the secondary and tertiary struc-tures with the standard process parameters such as Tinlet, the

84 M. Mizoguchi et al. / European Journal of Pharmaceutical Sciences 74 (2015) 77–85

atomizing air volumetric flow rate, and liquid concentration(Fig. 4). In addition, the narrow particle size distribution ofproduced powder demonstrated that the spray-drying process ofL-PGDS formulation achieved the atomizing of well-disperseddroplet without the additives such as surfactants to prevent thegeneration of aggregates. There was a morphologic difference inthe dried particles due to the increase of Tinlet (Fig. 3D). The rateof droplet evaporation may affect the morphological properties ofthe particles (Vehring, 2008). A fast drying rate will promote theformation of a more viscous film in the initial phase of drying.Because the water pressure increases rapidly inside the droplet,the film tends to burst. Therefore, the fast drying conditions resultin a large fraction of the particles containing dimples or holes. Thespray-drying process in general may alter the secondary structureof protein because of the removal of water molecules that arerequired to form hydrogen bonds to stabilize the structure. Thiscan be prevented by adding excipients, e.g., carbohydrates, whichare capable of forming hydrogen bonds with the protein (Shoyeleet al., 2011; Hulse et al., 2009; Schule et al., 2008). In this study,the produced powder was stored at �20 �C until each analysis toevaluate the characteristics precisely without the potential degra-dation derived from temperature and humidity. However, a stabil-ity study of the complex should be conducted to evaluate the effectof process parameters and the addition of water-replacing agents,such as sucrose and trehalose, on the quality of spray-dried L-PGDSfor bulk storage.

In the pharmacokinetic study, the telmisartan/L-PDGS complexsignificantly enhanced the in vivo behavior of jet-milled telmisar-tan, and, in particular, AUC0–48h and Cmax were enhanced 5.9- and5.3-fold, respectively (Table 3). Tmax of the complex formulationwas significantly shorter than jet-milled telmisartan and the com-mercial product. The dissolution profile of the commercial productin McIlvaine buffer pH 5.0 showed the incomplete dissolution andthe moderate precipitation behavior after supersaturation (Fig. 5B).On the other hand, the in vitro sequential digestion experimentsrevealed the rapid release of telmisartan from the telmisartan/L-PGDS complex by the digestion of L-PGDS in the simulatedintestinal environment (Fig. 6B). Therefore, the significant shorterTmax of complex formulation was considered that the release oftelmisartan from the complex formulation was faster than that ofthe commercial product. These results suggest that the spray-driedcomplex formulation enhanced oral exposure and the absorptionrate.

The pharmacodynamic study showed no significant differencein the reduction of SBP between all formulations (Fig. 8A). Theadministered dose may achieve the dose range for a saturatedreduction effect. Therefore, a comparison study with a lower dosestrength could reveal the difference between formulations forthe reduction of SBP. However, the complex formulation led tothe improvement of the antihypertensive effect on comparing thetotal DSBP area with jet-milled telmisartan (Fig. 8B). One of theclinical advantages of telmisartan is its long-acting effect due tothe delayed dissociation from angiotensin II type-1 receptor(Wienen et al., 2000). The complex formulation prolonged thereduction effect of API as well the commercial product, and theeffect was observed at 48 h after oral administration. Thisprolonged effect corresponded to the results obtained from thepharmacokinetic study that detected telmisartan in the serum at48 h after the administration of telmisartan/L-PGDS formulation.In addition, the prolonged effect of telmisartan can achieve asignificant antihypertensive effect without rebound on repeateddosing (Wienen and Schierok, 2001).

The results of animal studies suggest that L-PGDS deliverstelmisartan to the absorption site while maintaining the complexformed in an adverse environment for protein (i.e., low pH,presence of protease). The details of the delivery mechanism of

L-PGDS are still unclear, and elucidation of the mechanism is a sub-ject for a future study. This study demonstrated that L-PGDS is apotent drug-delivery carrier for the solid oral formulation, and thisdrug-delivery system using biodegradable material can be an alter-native approach for the current investigation of solubilizing tech-niques, such as with supersaturable formulations (Xu and Dai,2013). Moreover, this fine dry powder produced by spray dryingcould be inhaled into the lung as an aerosol.

5. Conclusions

The solubilization of telmisartan was achieved by a simple com-plex formulation method with L-PGDS. The dried powder of thesolubilizing formulation was successfully produced by spray-dry-ing. The solubilizing effect of L-PGDS demonstrated a consistentin vitro dissolution profile in the physiological pH range andin vivo behavior comparable with the commercial product. Thisstudy demonstrated that L-PGDS is a potent drug-delivery carrierfor the solid oral formulation.

Acknowledgments

The authors thank Drs. A. Liesener, T. Matsumaru, O. Ishibashi,and S. Kume for their helpful discussions, and Mrs. M. Kohno andS. Tsuge, and Ms. M. Saito for their technical assistance. Theauthors are also grateful to the New Drug Research Center, Inc.for their contribution to the experiments. This work was supportedby Grants 25242046 (to T.I.) from Grants-in-Aid for ScientificResearches (A) and 02120076 (to T.I.) as Grants-in-Aid forScientific Research on Innovative Areas.

References

Amidon, G.L., Lennernas, H., Shah, V.P., Crison, J.R., 1995. A theoretical basis for abiopharmaceutic drug classification: the correlation of in vitro drug productdissolution and in vivo bioavailability. Pharm. Res. 12, 413–420.

Badawy, S.I., Hussain, M.A., 2007. Microenvironmental pH modulation in soliddosage forms. J. Pharm. Sci. 96, 948–959.

Cal, K., Sollohub, K., 2010. Spray drying technique. I: hardware and processparameters. J. Pharm. Sci. 99, 575–586.

Charman, W.N., Porter, C.J., Mithani, S., Dressman, J.B., 1997. Physiochemical andphysiological mechanisms for the effects of food on drug absorption: the role oflipids and pH. J. Pharm. Sci. 86, 269–282.

Fahr, A., Liu, X., 2007. Drug delivery strategies for poorly water-soluble drugs.Expert Opin. Drug Deliv. 4, 403–416.

Fukuhara, A., Yamada, M., Fujimori, K., Miyamoto, Y., Kusumoto, T., Nakajima, H.,Inui, T., 2012. Lipocalin-type prostaglandin D synthase protects againstoxidative stress-induced neuronal cell death. Biochem. J. 443, 75–84.

Fukuhara, A., Nakajima, H., Miyamoto, Y., Inoue, K., Kume, S., Lee, Y.H., Noda, M.,Uchiyama, S., Shimamoto, S., Nishimura, S., Ohkubo, T., Goto, Y., Takeuchi, T.,Inui, T., 2012. Drug delivery system for poorly water-soluble compounds usinglipocalin-type prostaglandin D synthase. J. Control. Release 159, 143–150.

Galia, E., Nicolaides, E., Horter, D., Lobenberg, R., Reppas, C., Dressman, J.B., 1998.Evaluation of various dissolution media for predicting in vivo performance ofclass I and II drugs. Pharm. Res. 15, 698–705.

Gao, P., Shi, Y., 2012. Characterization of supersaturatable formulations forimproved absorption of poorly soluble drugs. AAPS J. 14, 703–713.

Hulse, W.L., Forbes, R.T., Bonner, M.C., Getrost, M., 2009. Influence of protein onmannitol polymorphic form produced during co-spray drying. Int. J. Pharm. 382,67–72.

Hunt, R.H., Armstrong, D., James, C., Chowdhury, S.K., Yuan, Y., Fiorentini, P.,Taccoen, A., Cohen, P., 2005. Effect on intragastric pH of a PPI with a prolongedplasma half-life: comparison between tenatoprazole and esomeprazole on theduration of acid suppression in healthy male volunteers. Am. J. Gastroenterol.100, 1949–1956.

Inui, T., Ohkubo, T., Emi, M., Irikura, D., Hayaishi, O., Urade, Y., 2003.Characterization of the unfolding process of lipocalin-type prostaglandin Dsynthase. J. Biol. Chem. 278, 2845–2852.

Inui, T., Mase, M., Shirota, R., Nagashima, M., Okada, T., Urade, Y., 2014. Lipocalin-type prostaglandin D synthase scavenges biliverdin in the cerebrospinal fluid ofpatients with aneurysmal subarachnoid hemorrhage. J. Cereb. Blood FlowMetab. 34, 1558–1567.

Kawabata, Y., Wada, K., Nakatani, M., Yamada, S., Onoue, S., 2011. Formulationdesign for poorly water-soluble drugs based on biopharmaceutics classificationsystem: basic approaches and practical applications. Int. J. Pharm. 420, 1–10.

M. Mizoguchi et al. / European Journal of Pharmaceutical Sciences 74 (2015) 77–85 85

Ku, M.S., Dulin, W., 2012. A biopharmaceutical classification-based Right-First-Timeformulation approach to reduce human pharmacokinetic variability and projectcycle time from First-In-Human to clinical Proof-Of-Concept. Pharm. Dev.Technol. 17, 285–302.

Kume, S., Lee, Y.H., Miyamoto, Y., Fukada, H., Goto, Y., Inui, T., 2012. Systematicinteraction analysis of human lipocalin-type prostaglandin D synthase withsmall lipophilic ligands. Biochem. J. 446, 279–289.

Kume, S., Lee, Y.H., Nakatsuji, M., Teraoka, Y., Yamaguchi, K., Goto, Y., Inui, T., 2014.Fine-tuned broad binding capability of human lipocalin-type prostaglandin Dsynthase for various small lipophilic ligands. FEBS Lett. 588, 962–969.

Lipinski, C.A., 2000. Drug-like properties and the causes of poor solubility and poorpermeability. J. Pharmacol. Toxicol. Methods 44, 235–249.

Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J., 2001. Experimental andcomputational approaches to estimate solubility and permeability in drugdiscovery and development settings. Adv. Drug Deliv. Rev. 46, 3–26.

Loftsson, T., Brewster, M.E., 2012. Cyclodextrins as functional excipients: methodsto enhance complexation efficiency. J. Pharm. Sci. 101, 3019–3032.

Mahler, H.C., Muller, R., Friess, W., Delille, A., Matheus, S., 2005. Induction andanalysis of aggregates in a liquid IgG1-antibody formulation. Eur. J. Pharm.Biopharm. 59, 407–417.

Marasini, N., Tran, T.H., Poudel, B.K., Cho, H.J., Choi, Y.K., Chi, S.C., Choi, H.G., Yong,C.S., Kim, J.O., 2013. Fabrication and evaluation of pH-modulated soliddispersion for telmisartan by spray-drying technique. Int. J. Pharm. 441, 424–432.

Miyamoto, Y., Nishimura, S., Inoue, K., Shimamoto, S., Yoshida, T., Fukuhara, A.,Yamada, M., Urade, Y., Yagi, N., Ohkubo, T., Inui, T., 2010. Structural analysis oflipocalin-type prostaglandin D synthase complexed with biliverdin by small-angle X-ray scattering and multi-dimensional NMR. J. Struct. Biol. 169,209–218.

Morihara, M., Aoyagi, N., Kaniwa, N., Kojima, S., Ogata, H., 2001. Assessment ofgastric acidity of Japanese subjects over the last 15 years. Biol. Pharm. Bull. 24,313–315.

Nagata, A., Suzuki, Y., Igarashi, M., Eguchi, N., Toh, H., Urade, Y., Hayaishi, O., 1991.Human brain prostaglandin D synthase has been evolutionarily differentiatedfrom lipophilic-ligand carrier proteins. Proc. Natl. Acad. Sci. USA 88,4020–4024.

Nakatani, M., Sawada, T., Ohki, T., Toyoshima, K., 2004. Solid pharmaceuticalformulations comprising telmisartan. US Patent 2004/0110813 A1.

Reagan-Shaw, S., Nihal, M., Ahmad, N., 2008. Dose translation from animal tohuman studies revisited. FASEB J. 22, 659–661.

Sangwai, M., Vavia, P., 2013. Amorphous ternary cyclodextrin nanocomposites oftelmisartan for oral drug delivery: improved solubility and reducedpharmacokinetic variability. Int. J. Pharm. 453, 423–432.

Schule, S., Schulz-Fademrecht, T., Garidel, P., Bechtold-Peters, K., Frieb, W., 2008.Stabilization of IgG1 in spray-dried powders for inhalation. Eur. J. Pharm.Biopharm. 69, 793–807.

Shimamoto, S., Yoshida, T., Inui, T., Gohda, K., Kobayashi, Y., Fujimori, K., Tsurumura,T., Aritake, K., Urade, Y., Ohkubo, T., 2007. NMR solution structure of lipocalin-type prostaglandin D synthase: evidence for partial overlapping of catalyticpocket and retinoic acid-binding pocket within the central cavity. J. Biol. Chem.282, 31373–31379.

Shoyele, S.A., Sivadas, N., Cryan, S.A., 2011. The effects of excipients and particleengineering on the biophysical stability and aerosol performance of parathyroidhormone (1-34) prepared as a dry powder for inhalation. AAPS PharmSciTech12, 304–311.

Strickley, R.G., 2004. Solubilizing excipients in oral and injectable formulations.Pharm. Res. 21, 201–230.

Taniguchi, C., Kawabata, Y., Wada, K., Yamada, S., Onoue, S., 2014.Microenvironmental pH-modification to improve dissolution behavior andoral absorption for drugs with pH-dependent solubility. Expert Opin. DrugDeliv. 11, 505–516.

Toh, H., Kubodera, H., Nakajima, N., Sekiya, T., Eguchi, N., Tanaka, T., Urade, Y.,Hayaishi, O., 1996. Glutathione-independent prostaglandin D synthase as a leadmolecule for designing new functional proteins. Protein Eng. 9, 1067–1082.

Tran, P.H., Tran, H.T., Lee, B.J., 2008. Modulation of microenvironmental pH andcrystallinity of ionizable telmisartan using alkalizers in solid dispersions forcontrolled release. J. Control. Release 129, 59–65.

Vehring, R., 2008. Pharmaceutical particle engineering via spray drying. Pharm. Res.25, 999–1022.

Vertzoni, M., Dressman, J., Butler, J., Hempenstall, J., Reppas, C., 2005. Simulation offasting gastric conditions and its importance for the in vivo dissolution oflipophilic compounds. Eur. J. Pharm. Biopharm. 60, 413–417.

Wang, W., 1999. Instability, stabilization, and formulation of liquid proteinpharmaceuticals. Int. J. Pharm. 185, 129–188.

Wang, W., 2000. Lyophilization and development of solid protein pharmaceuticals.Int. J. Pharm. 203, 1–60.

Wienen, W., Schierok, H.J., 2001. Effects of telmisartan, hydrochlorothiazide andtheir combination on blood pressure and renal excretory parameters inspontaneously hypertensive rats. J. Renin Angiotensin Aldosterone Syst. 2,123–128.

Wienen, W., Entzeroth, M., van Meel, J.C.A., Stangier, J., Busch, U., Ebner, T., Schmid,J., Lehmann, H., Matzek, K., Kempthorne-Rawson, J., Gladigau, V., Hauel, N.H.,2000. A review on telmisartan: a novel, long-acting angiotensin II-receptorantagonist. Cardiovasc. Drug Rev. 18, 127–154.

Xu, S., Dai, W.G., 2013. Drug precipitation inhibitors in supersaturable formulations.Int. J. Pharm. 453, 36–43.

Zhong, L., Zhu, X., Luo, X., Su, W., 2013. Dissolution properties and physicalcharacterization of telmisartan–chitosan solid dispersions prepared bymechanochemical activation. AAPS PharmSciTech 14, 541–550.

Zhou, Y., Shaw, N., Li, Y., Zhao, Y., Zhang, R., Liu, Z.J., 2010. Structure-functionanalysis of human l-prostaglandin D synthase bound with fatty acid molecules.FASEB J. 24, 4668–4677.