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Enhanced hypotensive effect of nimodipine solid dispersions produced by supercritical CO2 drying
Powder Technology 278 (2015) 204–210
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Enhanced hypotensive effect of nimodipine solid dispersions produced by supercritical CO2 drying Manoela Klüppel Riekes a, Thiago Caon a, José da Silva Jr. b, Regina Sordi c, Gislaine Kuminek a, Larissa Sakis Bernardi d, Carlos Renato Rambo b, Carlos Eduardo Maduro de Campos e, Daniel Fernandes f, Hellen Karine Stulzer a,⁎ a
Programa de Pós-Graduação em Farmácia, Universidade Federal de Santa Catarina, Florianópolis 88040-970, Brazil Programa de Pós-Graduação em Engenharia Elétrica, Universidade Federal de Santa Catarina, Florianópolis 88040-970, Brazil c Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Catarina, Florianópolis 88040-970, Brazil d Departamento de Ciências Farmacêuticas, Universidade Estadual do Centro-Oeste, Guarapuava 85040-080, Brazil e Programa de Pós-Graduação em Física, Universidade Federal de Santa Catarina, Florianópolis 88040-970, Brazil f Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Ponta Grossa 84030-900, Brazil b
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Article history: Received 4 December 2014 Received in revised form 4 March 2015 Accepted 20 March 2015 Available online 28 March 2015 Keywords: Nimodipine PVP K-30 Solid dispersions Supercritical fluid technology
a b s t r a c t Solid dispersions of nimodipine and PVP K-30 were prepared by supercritical fluid technology at three drug:carrier ratios (1:9, 2:8, 3:7, w/w). The samples were characterized by X-ray powder diffraction, infrared spectroscopy and scanning electron microscopy, and evaluated by means of their solubility, dissolution rate and hypotensive effect. The solid dispersion with the highest amount of PVP K-30 (SD 1:9) presented an amorphous state, a porous surface and hydrogen bonds between nimodipine and the carrier. On the other hand, the other solid dispersions were semicrystalline. All formulations enhanced the solubility and dissolution rate of nimodipine and the best results were found for SD 1:9. This formulation promoted an increase of more than 1300% in the solubility of nimodipine, besides releasing 100% of the drug within 5 min. When submitted to in vivo studies SD 1:9 decreased significantly the mean arterial pressure and also reduced its phenylephrineinduced increase. © 2015 Elsevier B.V. All rights reserved.
1. Introduction As a consequence of the synthesis tools employed, the number of new drug candidates with poor aqueous solubility and dissolution rate has grown constantly over the past two decades [1]. Since the poor solubility of active pharmaceutical ingredients (APIs) is considered a limiting step for their oral bioavailability [2], this fact demonstrates an urgent need for new approaches regarding poorly water-soluble APIs. Amongst the available technological tools which have been developed to increase the solubility of poorly water-soluble compounds, converting a crystalline API into their amorphous counterpart is one of the most promising strategies [1–3]. As a result of their high internal energy, amorphous materials usually exhibit greater molecular mobility and enhanced thermodynamic properties compared to the crystalline structures, which generally lead to increased apparent solubility and dissolution rate [3–5]. In this regard, solid dispersions (SDs) have been widely explored [2,6,7] and can be defined as glass solutions of poorly soluble compounds with hydrophilic carriers [1]. However, the high internal energy of amorphous materials is also responsible for their ⁎ Corresponding author. Tel.: +55 48 3721 4585; fax: +55 48 3721 9542. E-mail address: [email protected] (H.K. Stulzer).
http://dx.doi.org/10.1016/j.powtec.2015.03.029 0032-5910/© 2015 Elsevier B.V. All rights reserved.
thermodynamic instability leading to relaxation, nucleation and crystal growth phenomena during storage, processing or dissolution in the gastrointestinal tract [1]. In this context, SDs can also be considered an efficient strategy, since the carrier can inhibit crystallization through the raise of the overall glass transition temperature of the dispersion, which reduces API molecular mobility, or through the interaction with the API via hydrogen bonding [6]. As a more recent approach to obtain SDs, the supercritical fluid technology is based on a compound existing as a single fluid phase above its critical temperature and critical pressure [8]. In pharmaceutical applications, carbon dioxide (CO2) is the most widely used supercritical fluid because it has a low critical point and is non-toxic [9]. The most typical methodology based on supercritical fluid extraction involves the use of CO2 as a solvent and is called as ‘rapid expansion of a supercritical solution’ (RESS). This procedure is characterized by firstly solubilizing the solute in a supercritical fluid, which is then rapidly expanded by sudden decompression, typically by passing through an orifice at low pressure [10]. As an advantage of RESS when compared to other supercritical fluid methodologies, it does not require the use of organic solvents, which generates particles of high purity and with reduced particle diameter [11]. Moreover, RESS is a very attractive process as it is simple and relatively easy to implement [12].
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In addition, the selection of an ideal carrier is also crucial for successful SDs [1]. Polyvinylpyrrolidone (PVP) (Fig. 1A) is a hydrophilic polymer generated by polymerization of vinylpyrrolidone [2]. Due to its good aqueous solubility, biocompatibility, lack of toxicity, temperature-resistance and stability under different pH values [13], this second generation carrier has been often used to produce SDs of poorly water APIs [1,2,14]. More recently, PVP has been employed on the obtainment of several API solid solutions through supercritical fluid approaches [13,15,16]. Nimodipine (NMP) (Fig. 1B), a classical BCS II drug, is a dihydropyridine calcium channel blocker originally developed for the treatment of hypertension, although nowadays it has also been used to prevent subarachnoid hemorrhage complications [17–19]. Moreover, this compound presents limited bioavailability (only 13%), low aqueous solubility [20] and an extensive firstpass metabolism, which typically requires frequent doses (60 mg each 4 h) during the therapeutic treatment. For the patient, lower drug dosage administration intervals imply an inconvenient and non-compliant treatment [17–19]. Some SDs of NMP have been reported in literature during the past years. However, these reports are related to SDs obtained via conventional methods, such as solvent evaporation under vacuum, hot melt extrusion, melting and more recently, ball milling [21–37]. In addition, few studies have considered the in vivo impact of these preparations. In this context, the evaluation of a novel technique to obtain SDs of an API model is extremely relevant in order to optimize its characteristics and comprehend its in vivo behavior. Considering the limited biopharmaceutical properties of NMP and the need to explore novel manufacturing techniques, this study reported the production and physicochemical characterization of SDs composed of NMP and PVP K-30 through supercritical fluid drying technique. The formulation with the most promising results, regarding the enhancement of the solubility and dissolution rate of the API was also submitted to in vivo studies, in order to evaluate its hypotensive effect. 2. Material and methods 2.1. Materials NMP was purchased from Chang Zhow ComWin Fine Chemicals (batch 20110305; Jiangsu, China) and PVP K-30 was derived from Via Farma LTDA (batch 10713659/1; São Paulo, Brazil). High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were obtained from J.T. Baker® (Phillipsburg, NJ). Phenylephrine chloride was purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals used were of pharmaceutical grade. 2.2. Preparation of SDs SDs were composed of three different drug:carrier (w/w) ratios (1:9, 2:8 and 3:7) and denoted as SD 1:9, SD 2:8 and SD 3:7, respectively. These compositions were chosen based on previous reports of SDs of NMP and PVP K-30 [30]. An E3100 Critical Point Dryer equipment
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(Quorum Technologies®,West Sussex, United Kingdom) was employed and the RESS methodology applied was adapted according to the experimental procedures described by Guan and coworkers [11]. Within this context, amounts corresponding to 2.5 g of each sample were sealed in a porous cellulose pouch (22 μm pore size) and kept inside the vessel. The liquefied CO2 filled the vessel and was maintained in contact with the sample during 3 days, at room temperature and pressure. After this period, the CO2 was compressed and heated until achieving the controlled operating conditions of 100 bar and 40 °C. Under these conditions, the system was allowed to equilibrate for 3 days. In the end of the sixth day, the vessel was depressurized and the samples were collected from the cellulose pouch. The obtained SDs were passed through a 60 mesh sieve and stored under vacuum, at room temperature. Physical mixtures were prepared through simple spatulation of drug and carrier, accurately weighed at different proportions. No processing with supercritical CO2 drying was performed in these samples. The physical mixtures were named as PM 1:9, PM 2:8 and PM 3:7, corresponding to proportions of NMP:PVP K-30 (w/w) of 1:9, 2:8 and 3:7, respectively. 2.3. HPLC analysis The HPLC analyses were carried out through a stability-indicating method already described [38]. A Shimadzu® LC-10A system (Kyoto, Japan) equipped with an LC-10AD pump, DGU-14A degasser, SPD10AV variable-wavelength detector (set at 235 nm) and an SCL-10AVP system controller unit was used. The experiments were performed with a reversed-phase Phenomenex® (Torrance, CA) Luna C18 column (250 × 4.6 mm i.d., 5 μm), including a security guard holder (C18, 4.0 × 3.0 μm i.d.). The mobile phase consisted of an isocratic system composed of acetonitrile:methanol:water (55:11:34 v/v/v), with a flow rate of 0.5 mL min−1, at 40 °C. After the filtration in 45 μm membranes, 20 μL of sample (at theoretical concentration of 20 μg mL−1) was injected. Data acquisition was performed using the CLASS-VP software. This method was revalidated according to ICH Guidelines [39] and it was considered specific, linear (r N 0.999), sensible (limits of quantification and detection of 1.22 μg mL−1 and 0.40 μg mL−1, respectively), precise (intra- and interday relative standard deviations of 0.9 and 1.2%, respectively) and accurate (recoveries ranged from 98.2 to 100.8%), for measurements with SDs. 2.4. Measurement of solubility In order to determine the NMP solubility, an excess of drug in SDs was weighed and added to 30 mL of acetate buffer (pH 4.5) containing 0.3% of sodium lauryl sulfate (SLS), giving a final concentration of 2.0 mg mL−1. Samples were kept under stirring at 150 rpm in a thermostatically controlled water bath (37 ± 2 °C) (Dist®, model DI-06) until a constant concentration of NMP was reached. At pre-determined intervals, aliquots were withdrawn (with immediate replacement of fresh solution) and filtered through 22 μm filters before taking the readings on a UV/VIS spectrophotometer (Cary 50 BIO, Varian®) at 340 nm.
Fig. 1. (A) Monomeric unit of PVP K-30 and (B) chemical structure of NMP.
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2.5. Solid state characterization 2.5.1. X-ray powder diffraction (XRPD) X-ray diffraction patterns were obtained using the diffractometers PANAlytical® Xpert Pro Multi-Purpose, equipped with a Real Time Multiple Strip (RTMS) detector, and Bruker® D2 Phaser, equipped with a scintillation counter one dimensional LYNXEYE detector. Both equipments operated using Kα copper radiation (λ = 1.5418 Å), with 40 mA current and 45 kV voltage. The measurements were performed at room temperature, scanning at 2θ from 5° to 50°.
SD 1:9, both at 5 mg kg−1, loaded into size 9 gelatin capsules administered by gastric gavage using a dosing syringe for drug delivery capsule (Torpac®, Fairfield, NJ). Finally, group 3 (control) received empty capsules. Immediately after treatment, animals were instrumented for mean arterial pressure (MAP) recording as described below. At different time periods (15, 30, 45, 60 and 120 min) the blood pressure (mm Hg) and the change in response to the vasoconstrictor phenylephrine were monitored.
2.5.3. Scanning electron microscopy (SEM) The samples were mounted on aluminum stubs, sputtered with gold (IC-50 Ion Coater, Shimadzu®, Kyoto, Japan), and analyzed using a scanning electron microscope (SSX-550 Superscan, Shimadzu®, Kyoto, Japan) at an accelerating voltage of 10 or 15 kV.
2.7.3. MAP measurement The animals were anesthetized intramuscularly with ketamine and xylazine (90 and 15 mg/kg, respectively), and heparinized PE-20 and PE-50 polyethylene catheters were inserted into the left femoral vein for phenylephrine injections and into the right carotid artery for recording of MAP (mm Hg), respectively. Animals were allowed to breathe spontaneously via a tracheal cannula and body temperature was maintained at 36 ± 1 °C. MAP was recorded with a catheter pressure transducer (Mikro-Tip®, Millar Instruments, Inc., Houston, Texas, USA) coupled to a Powerlab 8/30 acquisition system (AD Instruments Pty Ltd., Castle Hill, Australia). Results were expressed as mean ± SEM of the peak changes in MAP (as mm Hg) following administration of phenylephrine (30 nmol kg−1, i.v.) relative to baseline. The rats were then sacrificed with a pentobarbitone overdose.
2.6. “In vitro” dissolution studies
3. Results and discussion
Dissolution studies of pure drug and SDs were performed at a Varian® VK 7000 dissolutor, using dissolution apparatus II (paddle), according to the specifications of British Pharmacopoeia [40], which describes the in vitro method for evaluating the dissolution of NMP tablets. Amounts corresponding to 30 mg of NMP, in pure drug and SDs, were weighed and powdered onto 900 mL of previously-deaerated acetate buffer pH 4.5 containing 0.3% of SLS, kept at 37 ± 0.5 °C and rotated at 75 rpm. At predetermined intervals of time (5, 10, 15, 20, 25, 30, 45 and 60 min), aliquots of 4 mL were withdrawn (with immediate replacement of fresh dissolution medium) and passed through 22 μm filters before measurements at UV/VIS spectrophotometer (Varian® UV/VIS Cary), at 340 nm. The correction for the cumulative dilution was calculated. Dissolution experiments were carried out in triplicate and protected from light. Dissolution profiles of NMP and SDs were evaluated by means of the dissolution efficiency (DE), a model-independent analysis. Through this analysis, the area under the dissolution curve at time t was calculated through the trapezoidal rule and expressed as the percentage of the rectangle area described by 100% of dissolution within the same period. The area under the curve was determined using the GraphPad PRISM® software. In addition, the dissolution profiles were compared point by point by means of the Tukey's test, in which significant results presented a probability lower than 5% (p ≤ 0.05 with a 95% confidence interval), through the GraphPad PRISM® software.
3.1. HPLC analysis
2.5.2. Fourier transform infrared (FTIR) spectroscopy FTIR spectra were acquired on a Shimadzu® spectrophotometer (FTIR Prestige) at room temperature from 4000 to 400 cm−1, with a collection of 20 scans at a resolution of 4 cm−1. Samples were prepared by gently mixing 2% (w/w) of drug, physical mixtures and SDs in potassium bromide (KBr).
2.7. “In vivo” studies 2.7.1. Animals Female Wistar rats (weighing 200–300 g) were housed in a temperature- and light-controlled room (23 ± 2 °C; 12 h light/ dark cycle), with free access to water and food. All procedures are in agreement with the Federal University of Santa Catarina Ethics Committee (project number PP00790) and the investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). 2.7.2. Experimental protocol Rats were randomly divided into 3 groups of 4 animals each. Group 1 received the crystalline raw material of NMP and group 2 received the
The experimental and relative values regarding the contents of NMP in SDs were 100.1% ± 0.12, 99.9% ± 0.36 and 100.0% ± 0.84 for SD 1:9, SD 2:8 and SD 3:7, respectively. In addition, no degradation peak was observed in the chromatograms analyzed (data not shown), indicating the stability of the drug on employing this technique. 3.2. Measurement of solubility Increases regarding the solubility of SDs in comparison with the crystalline API (0.13 mg/mL) were of 1304.5% ± 0.30 (1.66 mg/mL), 363.0% ± 1.80 (0.46 mg/mL) and 272.4% ± 0.80 (0.36 mg/mL) for SD 1:9, SD 2:8 and SD 3:7, respectively. These data demonstrate that the solubility of the API in these SDs was directly dependent on the carrier concentration, in agreement with literature reports [2]. 3.3. Solid state characterization 3.3.1. XRPD The X-ray diffractograms of NMP, PVP K-30, SDs and physical mixture are shown in Fig. 2. NMP presents two polymorphs; Modification I (Mod I), a racemic compound that exhibits higher solubility in water (0.36 ± 0.07 mg mL − 1 ), and Modification II (Mod II), the thermodynamically stable polymorph, which is chemically a conglomerate [41]. Polymorphism investigations are particularly important in drug and product development in the pharmaceutical industry, since they may have an important implication in the bioavailability and stability of a drug [42]. Particularly for NMP, solid-state transitions have already been reported in SD systems [23,29,30] and then they were also monitored in proposed formulations. The crystalline phases of NMP, Mod I and Mod II, can be distinguished through various solid state techniques. XRPD analysis can be considered appropriate since it distinguishes the diffractograms of these polymorphs especially at low 2θ angles. The reflection at 6.6° is only observed for Mod I, while that at 9.3° is present exclusively in Mod II [23, 43-47]. In this regard, XRPD analysis demonstrates that the raw material used for the development of the solid dispersions is composed of the metastable polymorph Mod I.
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3:7, which is characteristic of semi-crystalline materials. In addition to that, peaks relative to the less soluble polymorph Mod II are observed for these samples indicating a phase transition during the manufacturing process. These results demonstrate that the drug:carrier ratio plays an important role on the physical state of the obtained SDs.
Fig. 2. Calculated X-ray patterns of Mod I and Mod II originated from the Cambridge Structural Database and X-ray diffractograms of raw materials, physical mixture and SDs. The arrows demonstrate the presence of crystalline peaks respective to polymorph Mod II.
PVP K-30 was found to be completely amorphous, in agreement with previous data reported in literature [13,30,48–50]. The physical mixture PM 1:9 composed of the highest amount of the carrier presented characteristic reflections of NMP Mod I overlapped with the amorphous halo of the polymer, which indicates the absence of physical interactions between NMP and PVP K-30. The sample SD 1:9 presented an amorphous halo, while crystalline peaks of NMP overlapped with an amorphous halo for SD 2:8 and SD
3.3.2. FTIR FTIR studies were carried out to identify possible interactions between the NMP and PVP (Fig. 3). FTIR characteristic bands related to NMP appear at 3293, 1695 and 1523 cm− 1 (relative to N–H stretching for the polymorph Mod I, carbonyl bond and –NO2 group, respectively) [31,51–54]. The FTIR spectrum of the PVP K-30 presented characteristic bands at 2955 cm−1 (C–H stretch) and at 1654 cm−1 (C_O), as already reported [55]. Also, the spectrum relative to PM 1:9 is composed of an overlap of the characteristic bands of NMP and PVP K-30, without shifts, which indicates the absence of chemical interactions and/or polymorphic transitions. According to Papageorgiou and coworkers, the amine group of NMP is able to form hydrogen bonds with the carbonyl groups of PVP K-30 [30]. The interaction by means of hydrogen bonds shifts the respective bands to lower wavenumbers, usually followed by an increase of their intensity [37,49,56]. In this way, intermolecular interactions between NMP and PVP K-30 may shift the absorbance of the amine and carbonyl groups. In the spectrum of the sample SD 1:9, the band corresponding to the amine group of NMP disappeared. Moreover, a displacement of the carbonyl group of PVP K-30 from 1654 to 1649 cm− 1 was observed, indicating the formation of hydrogen bond between drug and carrier. This interaction between the API and the hydrophilic carrier provides an additional benefit for SDs, since it is able to increase the solid solubility of the drug into the hydrophilic carrier besides acting on inhibiting the crystallization of the API from a glass solution [2,57]. Similar results have already been found by Papageorgiou and coworkers [30] for SDs of NMP and PVP K-30 obtained through solvent evaporation under vacuum. On the other hand, SD 2:8 and SD 3:7 presented the amine band, but shifted to 3272 cm−1 and to 3269 cm−1, respectively, contributing to a higher molecular mobility.
Fig. 3. FTIR spectra of raw materials, physical mixture and SDs. The dotted rectangle indicates the area of appearance of the NMP amine bands.
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3.3.3. SEM SEM analyses (Fig. 4) were carried out to evaluate the impact of the preparation on the morphology of the SDs, as well as the impact of the different drug:carrier ratios. Small tabular crystals with particle size ranging from 1 to 10 μm were observed for the NMP raw material. On the other hand, spheroid particles with indentations and multimodal particle size distribution were observed for PVP K-30. This spherical cage-like shape of the polymer particles is a typical result of spray drying process (Fig. 4B) [58]. Concerning the SDs, the drug:carrier ratios also seem to have an influence on SDs morphology. SD 1:9 exhibited a very porous surface, with a large available surface area, which in combination with the amorphous structure of the drug and the majority composition of PVP K-30, represents a promising result regarding the dissolution rate of the API. In contrast, as the content of the drug increased, the porous aspect of SD 2:8 and SD 3:7 surfaces vanished, and NMP crystals were observed on their surface. These results also support the hypothesis on a semicrystalline state for these samples. The porous aspect observed in SD 1:9 (Fig. 4C and D) can be attributed to the presence of PVP K-30 and its porous-ability, as previously described by Caon et al. [59]. As the content of the carrier decreased in SD
2:8 and SD 3:7 (Fig. 4E and F), the amount of PVP K-30 was not enough to produce porous and amorphous materials. 3.4. “In vitro” dissolution studies All the SDs increased the dissolution rate of NMP (Fig. 5) since only 33.4 ± 1.9% of the crystalline API was released within 60 min. The samples were considered statistically different (p b 0.05) of the pure API. However, differences were not observed between SD 2:8 and SD 3:7. Additionally, all of the dissolution rates were influenced by the drug:carrier ratios and the best result was attributed to SD 1:9, which was able to release 100.73% ± 0.41 of the API within 5 min. This data can be directly related to highly porous area surface of this SD, as qualitatively observed by SEM, and to the amorphous state of the drug, identified by XRPD. In contrast, and as expected, the semi-crystalline SDs released 73.18% ± .14 (SD 2:8) and 68.78% ± 0.20 (SD 3:7) at 60 min. Concerning the DE values, SDs also presented improved results; 95.81% for SD 1:9, 61.95% for SD 2:8 and 59.7% for SD 3:7, in comparison with pure NMP (19.4%). Different factors can contribute to the enhancement of the dissolution rate of a poorly water-soluble API by SDs. Based on the obtained
Fig. 4. Photomicrographs of (A) NMP (3000×), (B) PVP K-30 (500×), (C) SD 1:9 (50×), (D) SD 1:9 (500×), (E) SD 2:8 (100×) and (F) SD 3:7 (100×).
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crystalline NMP did not promote any decrease in the MAP compared with the control group at the tested dose (5 mg kg−1, p.o.) during the evaluated time (120 min). Regarding to phenylephrine-induced increase in blood pressure, more expressive results were obtained. The sample SD 1:9 reduced phenylephrine response for all the time periods considered. After 15 min of oral administration, SD 1:9 reduced the phenylephrine response by 86.4% and 82.4% compared to control and to crystalline NMP group, respectively. On the other hand, crystalline NMP reduced phenylephrine response (38.6% compared to control) only 2 h after oral administration. In summary, this data also reinforces the advantage of SD 1:9 since it is able to maintain a low MAP even against more drastic conditions, such the administration of a vasoconstrictor agent. Fig. 5. In vitro dissolution profiles of crystalline NMP (●), SD 1:9 (♦), SD 2:8 (■) and SD 3:7 (▲).
4. Conclusions results, it is possible to state that the SDs composed of NMP and PVP K30 enhanced the physicochemical properties of the API due to the decrease of crystallinity of NMP, the increased wettability, the increased surface area, characteristic porous morphology, and the establishment of hydrogen bonds between the API drug and its carrier. As SD 1:9 presented the highest solubility and faster drug release it was selected for in vivo efficacy studies. 3.5. “In vivo” studies Fig. 6 shows the in vivo assays results (MAP and MAP change in response to phenylephrine) in function of time for animals treated with SD 1:9 and NMP. Animals treated with sterile water were used as controls. The treatment with SD 1:9 promoted a significant reduction in the animals MAP compared to the crystalline NMP and control groups. At 15 min, for example, SD 1:9 reduced blood pressure by 36% and 32.9% compared to control group and crystalline NMP, respectively. The hypotensive effect of the SD 1:9 remains during all analyzed time. The
SDs of NMP and PVP K-30 were successfully obtained by supercritical fluid extraction technology. Different drug:carrier ratios impacted on the physicochemical properties of the samples. While SD 1:9 presented an amorphous nature, remaining drug crystallinity was observed for SD 2:8 and SD 3:7. Nevertheless, all the SDs exhibited higher solubility and dissolution rates (although in different grades) compared with the pure drug. The best result was attributed to SD 1:9, which provided an increase of more than 1300% in the solubility of NMP and a 5-fold increase in the DE value, besides releasing 100% of the API within 5 min. The performance of this SD was attributed to its amorphous state, the establishment of hydrogen bond between NMP and PVP K30 and the increased surface area, due to its highly porous morphology. SD 1:9 was able to decrease the MAP and reduced the phenylephrineinduced increase in blood pressure. Similar data were not observed for the crystalline drug in the dosage tested, verifying the advantage of this SD versus the crystalline API. Acknowledgments The authors acknowledge CNPq (MCT/CNPq 014/2010) and FAPESC (04/2011) for the financial support and CAPES for the student scholarships. Some of the XRPD analyses were carried out at multiuser X-ray diffraction laboratory (LDRX) of the Federal University of Santa Catarina (UFSC). The authors also wish to thank Dr. Milton Domingues Michel, from Universidade Estadual de Ponta Grossa (UEPG), for the SEM analyses. Thanks are also due to the Consejo Superior de Investigaciones Científicas (CSIC) in Spain, for awarding a license for the use of the Cambridge Structural Database (CSD). References
Fig. 6. Graphs respective to (A) MAP and (B) its change in response to phenylephrine, for animals that received SD 1:9, NMP and empty capsules (control group). Each point of bar represents the mean of 4 animals and vertical lines are the S.E.M. ⁎p b 0.05 compared with the control group and #p b 0.05 compared with the group that received NMP. Statistical analysis was performed using ANOVA test followed by Bonferroni's posthoc test.
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Enhanced hypotensive effect of nimodipine solid dispersions produced by supercritical CO2 drying Manoela Klüppel Riekes a, Thiago Caon a, José da Silva Jr. b, Regina Sordi c, Gislaine Kuminek a, Larissa Sakis Bernardi d, Carlos Renato Rambo b, Carlos Eduardo Maduro de Campos e, Daniel Fernandes f, Hellen Karine Stulzer a,⁎ a
Programa de Pós-Graduação em Farmácia, Universidade Federal de Santa Catarina, Florianópolis 88040-970, Brazil Programa de Pós-Graduação em Engenharia Elétrica, Universidade Federal de Santa Catarina, Florianópolis 88040-970, Brazil c Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Catarina, Florianópolis 88040-970, Brazil d Departamento de Ciências Farmacêuticas, Universidade Estadual do Centro-Oeste, Guarapuava 85040-080, Brazil e Programa de Pós-Graduação em Física, Universidade Federal de Santa Catarina, Florianópolis 88040-970, Brazil f Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Ponta Grossa 84030-900, Brazil b
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Article history: Received 4 December 2014 Received in revised form 4 March 2015 Accepted 20 March 2015 Available online 28 March 2015 Keywords: Nimodipine PVP K-30 Solid dispersions Supercritical fluid technology
a b s t r a c t Solid dispersions of nimodipine and PVP K-30 were prepared by supercritical fluid technology at three drug:carrier ratios (1:9, 2:8, 3:7, w/w). The samples were characterized by X-ray powder diffraction, infrared spectroscopy and scanning electron microscopy, and evaluated by means of their solubility, dissolution rate and hypotensive effect. The solid dispersion with the highest amount of PVP K-30 (SD 1:9) presented an amorphous state, a porous surface and hydrogen bonds between nimodipine and the carrier. On the other hand, the other solid dispersions were semicrystalline. All formulations enhanced the solubility and dissolution rate of nimodipine and the best results were found for SD 1:9. This formulation promoted an increase of more than 1300% in the solubility of nimodipine, besides releasing 100% of the drug within 5 min. When submitted to in vivo studies SD 1:9 decreased significantly the mean arterial pressure and also reduced its phenylephrineinduced increase. © 2015 Elsevier B.V. All rights reserved.
1. Introduction As a consequence of the synthesis tools employed, the number of new drug candidates with poor aqueous solubility and dissolution rate has grown constantly over the past two decades [1]. Since the poor solubility of active pharmaceutical ingredients (APIs) is considered a limiting step for their oral bioavailability [2], this fact demonstrates an urgent need for new approaches regarding poorly water-soluble APIs. Amongst the available technological tools which have been developed to increase the solubility of poorly water-soluble compounds, converting a crystalline API into their amorphous counterpart is one of the most promising strategies [1–3]. As a result of their high internal energy, amorphous materials usually exhibit greater molecular mobility and enhanced thermodynamic properties compared to the crystalline structures, which generally lead to increased apparent solubility and dissolution rate [3–5]. In this regard, solid dispersions (SDs) have been widely explored [2,6,7] and can be defined as glass solutions of poorly soluble compounds with hydrophilic carriers [1]. However, the high internal energy of amorphous materials is also responsible for their ⁎ Corresponding author. Tel.: +55 48 3721 4585; fax: +55 48 3721 9542. E-mail address: [email protected] (H.K. Stulzer).
http://dx.doi.org/10.1016/j.powtec.2015.03.029 0032-5910/© 2015 Elsevier B.V. All rights reserved.
thermodynamic instability leading to relaxation, nucleation and crystal growth phenomena during storage, processing or dissolution in the gastrointestinal tract [1]. In this context, SDs can also be considered an efficient strategy, since the carrier can inhibit crystallization through the raise of the overall glass transition temperature of the dispersion, which reduces API molecular mobility, or through the interaction with the API via hydrogen bonding [6]. As a more recent approach to obtain SDs, the supercritical fluid technology is based on a compound existing as a single fluid phase above its critical temperature and critical pressure [8]. In pharmaceutical applications, carbon dioxide (CO2) is the most widely used supercritical fluid because it has a low critical point and is non-toxic [9]. The most typical methodology based on supercritical fluid extraction involves the use of CO2 as a solvent and is called as ‘rapid expansion of a supercritical solution’ (RESS). This procedure is characterized by firstly solubilizing the solute in a supercritical fluid, which is then rapidly expanded by sudden decompression, typically by passing through an orifice at low pressure [10]. As an advantage of RESS when compared to other supercritical fluid methodologies, it does not require the use of organic solvents, which generates particles of high purity and with reduced particle diameter [11]. Moreover, RESS is a very attractive process as it is simple and relatively easy to implement [12].
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In addition, the selection of an ideal carrier is also crucial for successful SDs [1]. Polyvinylpyrrolidone (PVP) (Fig. 1A) is a hydrophilic polymer generated by polymerization of vinylpyrrolidone [2]. Due to its good aqueous solubility, biocompatibility, lack of toxicity, temperature-resistance and stability under different pH values [13], this second generation carrier has been often used to produce SDs of poorly water APIs [1,2,14]. More recently, PVP has been employed on the obtainment of several API solid solutions through supercritical fluid approaches [13,15,16]. Nimodipine (NMP) (Fig. 1B), a classical BCS II drug, is a dihydropyridine calcium channel blocker originally developed for the treatment of hypertension, although nowadays it has also been used to prevent subarachnoid hemorrhage complications [17–19]. Moreover, this compound presents limited bioavailability (only 13%), low aqueous solubility [20] and an extensive firstpass metabolism, which typically requires frequent doses (60 mg each 4 h) during the therapeutic treatment. For the patient, lower drug dosage administration intervals imply an inconvenient and non-compliant treatment [17–19]. Some SDs of NMP have been reported in literature during the past years. However, these reports are related to SDs obtained via conventional methods, such as solvent evaporation under vacuum, hot melt extrusion, melting and more recently, ball milling [21–37]. In addition, few studies have considered the in vivo impact of these preparations. In this context, the evaluation of a novel technique to obtain SDs of an API model is extremely relevant in order to optimize its characteristics and comprehend its in vivo behavior. Considering the limited biopharmaceutical properties of NMP and the need to explore novel manufacturing techniques, this study reported the production and physicochemical characterization of SDs composed of NMP and PVP K-30 through supercritical fluid drying technique. The formulation with the most promising results, regarding the enhancement of the solubility and dissolution rate of the API was also submitted to in vivo studies, in order to evaluate its hypotensive effect. 2. Material and methods 2.1. Materials NMP was purchased from Chang Zhow ComWin Fine Chemicals (batch 20110305; Jiangsu, China) and PVP K-30 was derived from Via Farma LTDA (batch 10713659/1; São Paulo, Brazil). High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were obtained from J.T. Baker® (Phillipsburg, NJ). Phenylephrine chloride was purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals used were of pharmaceutical grade. 2.2. Preparation of SDs SDs were composed of three different drug:carrier (w/w) ratios (1:9, 2:8 and 3:7) and denoted as SD 1:9, SD 2:8 and SD 3:7, respectively. These compositions were chosen based on previous reports of SDs of NMP and PVP K-30 [30]. An E3100 Critical Point Dryer equipment
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(Quorum Technologies®,West Sussex, United Kingdom) was employed and the RESS methodology applied was adapted according to the experimental procedures described by Guan and coworkers [11]. Within this context, amounts corresponding to 2.5 g of each sample were sealed in a porous cellulose pouch (22 μm pore size) and kept inside the vessel. The liquefied CO2 filled the vessel and was maintained in contact with the sample during 3 days, at room temperature and pressure. After this period, the CO2 was compressed and heated until achieving the controlled operating conditions of 100 bar and 40 °C. Under these conditions, the system was allowed to equilibrate for 3 days. In the end of the sixth day, the vessel was depressurized and the samples were collected from the cellulose pouch. The obtained SDs were passed through a 60 mesh sieve and stored under vacuum, at room temperature. Physical mixtures were prepared through simple spatulation of drug and carrier, accurately weighed at different proportions. No processing with supercritical CO2 drying was performed in these samples. The physical mixtures were named as PM 1:9, PM 2:8 and PM 3:7, corresponding to proportions of NMP:PVP K-30 (w/w) of 1:9, 2:8 and 3:7, respectively. 2.3. HPLC analysis The HPLC analyses were carried out through a stability-indicating method already described [38]. A Shimadzu® LC-10A system (Kyoto, Japan) equipped with an LC-10AD pump, DGU-14A degasser, SPD10AV variable-wavelength detector (set at 235 nm) and an SCL-10AVP system controller unit was used. The experiments were performed with a reversed-phase Phenomenex® (Torrance, CA) Luna C18 column (250 × 4.6 mm i.d., 5 μm), including a security guard holder (C18, 4.0 × 3.0 μm i.d.). The mobile phase consisted of an isocratic system composed of acetonitrile:methanol:water (55:11:34 v/v/v), with a flow rate of 0.5 mL min−1, at 40 °C. After the filtration in 45 μm membranes, 20 μL of sample (at theoretical concentration of 20 μg mL−1) was injected. Data acquisition was performed using the CLASS-VP software. This method was revalidated according to ICH Guidelines [39] and it was considered specific, linear (r N 0.999), sensible (limits of quantification and detection of 1.22 μg mL−1 and 0.40 μg mL−1, respectively), precise (intra- and interday relative standard deviations of 0.9 and 1.2%, respectively) and accurate (recoveries ranged from 98.2 to 100.8%), for measurements with SDs. 2.4. Measurement of solubility In order to determine the NMP solubility, an excess of drug in SDs was weighed and added to 30 mL of acetate buffer (pH 4.5) containing 0.3% of sodium lauryl sulfate (SLS), giving a final concentration of 2.0 mg mL−1. Samples were kept under stirring at 150 rpm in a thermostatically controlled water bath (37 ± 2 °C) (Dist®, model DI-06) until a constant concentration of NMP was reached. At pre-determined intervals, aliquots were withdrawn (with immediate replacement of fresh solution) and filtered through 22 μm filters before taking the readings on a UV/VIS spectrophotometer (Cary 50 BIO, Varian®) at 340 nm.
Fig. 1. (A) Monomeric unit of PVP K-30 and (B) chemical structure of NMP.
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2.5. Solid state characterization 2.5.1. X-ray powder diffraction (XRPD) X-ray diffraction patterns were obtained using the diffractometers PANAlytical® Xpert Pro Multi-Purpose, equipped with a Real Time Multiple Strip (RTMS) detector, and Bruker® D2 Phaser, equipped with a scintillation counter one dimensional LYNXEYE detector. Both equipments operated using Kα copper radiation (λ = 1.5418 Å), with 40 mA current and 45 kV voltage. The measurements were performed at room temperature, scanning at 2θ from 5° to 50°.
SD 1:9, both at 5 mg kg−1, loaded into size 9 gelatin capsules administered by gastric gavage using a dosing syringe for drug delivery capsule (Torpac®, Fairfield, NJ). Finally, group 3 (control) received empty capsules. Immediately after treatment, animals were instrumented for mean arterial pressure (MAP) recording as described below. At different time periods (15, 30, 45, 60 and 120 min) the blood pressure (mm Hg) and the change in response to the vasoconstrictor phenylephrine were monitored.
2.5.3. Scanning electron microscopy (SEM) The samples were mounted on aluminum stubs, sputtered with gold (IC-50 Ion Coater, Shimadzu®, Kyoto, Japan), and analyzed using a scanning electron microscope (SSX-550 Superscan, Shimadzu®, Kyoto, Japan) at an accelerating voltage of 10 or 15 kV.
2.7.3. MAP measurement The animals were anesthetized intramuscularly with ketamine and xylazine (90 and 15 mg/kg, respectively), and heparinized PE-20 and PE-50 polyethylene catheters were inserted into the left femoral vein for phenylephrine injections and into the right carotid artery for recording of MAP (mm Hg), respectively. Animals were allowed to breathe spontaneously via a tracheal cannula and body temperature was maintained at 36 ± 1 °C. MAP was recorded with a catheter pressure transducer (Mikro-Tip®, Millar Instruments, Inc., Houston, Texas, USA) coupled to a Powerlab 8/30 acquisition system (AD Instruments Pty Ltd., Castle Hill, Australia). Results were expressed as mean ± SEM of the peak changes in MAP (as mm Hg) following administration of phenylephrine (30 nmol kg−1, i.v.) relative to baseline. The rats were then sacrificed with a pentobarbitone overdose.
2.6. “In vitro” dissolution studies
3. Results and discussion
Dissolution studies of pure drug and SDs were performed at a Varian® VK 7000 dissolutor, using dissolution apparatus II (paddle), according to the specifications of British Pharmacopoeia [40], which describes the in vitro method for evaluating the dissolution of NMP tablets. Amounts corresponding to 30 mg of NMP, in pure drug and SDs, were weighed and powdered onto 900 mL of previously-deaerated acetate buffer pH 4.5 containing 0.3% of SLS, kept at 37 ± 0.5 °C and rotated at 75 rpm. At predetermined intervals of time (5, 10, 15, 20, 25, 30, 45 and 60 min), aliquots of 4 mL were withdrawn (with immediate replacement of fresh dissolution medium) and passed through 22 μm filters before measurements at UV/VIS spectrophotometer (Varian® UV/VIS Cary), at 340 nm. The correction for the cumulative dilution was calculated. Dissolution experiments were carried out in triplicate and protected from light. Dissolution profiles of NMP and SDs were evaluated by means of the dissolution efficiency (DE), a model-independent analysis. Through this analysis, the area under the dissolution curve at time t was calculated through the trapezoidal rule and expressed as the percentage of the rectangle area described by 100% of dissolution within the same period. The area under the curve was determined using the GraphPad PRISM® software. In addition, the dissolution profiles were compared point by point by means of the Tukey's test, in which significant results presented a probability lower than 5% (p ≤ 0.05 with a 95% confidence interval), through the GraphPad PRISM® software.
3.1. HPLC analysis
2.5.2. Fourier transform infrared (FTIR) spectroscopy FTIR spectra were acquired on a Shimadzu® spectrophotometer (FTIR Prestige) at room temperature from 4000 to 400 cm−1, with a collection of 20 scans at a resolution of 4 cm−1. Samples were prepared by gently mixing 2% (w/w) of drug, physical mixtures and SDs in potassium bromide (KBr).
2.7. “In vivo” studies 2.7.1. Animals Female Wistar rats (weighing 200–300 g) were housed in a temperature- and light-controlled room (23 ± 2 °C; 12 h light/ dark cycle), with free access to water and food. All procedures are in agreement with the Federal University of Santa Catarina Ethics Committee (project number PP00790) and the investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). 2.7.2. Experimental protocol Rats were randomly divided into 3 groups of 4 animals each. Group 1 received the crystalline raw material of NMP and group 2 received the
The experimental and relative values regarding the contents of NMP in SDs were 100.1% ± 0.12, 99.9% ± 0.36 and 100.0% ± 0.84 for SD 1:9, SD 2:8 and SD 3:7, respectively. In addition, no degradation peak was observed in the chromatograms analyzed (data not shown), indicating the stability of the drug on employing this technique. 3.2. Measurement of solubility Increases regarding the solubility of SDs in comparison with the crystalline API (0.13 mg/mL) were of 1304.5% ± 0.30 (1.66 mg/mL), 363.0% ± 1.80 (0.46 mg/mL) and 272.4% ± 0.80 (0.36 mg/mL) for SD 1:9, SD 2:8 and SD 3:7, respectively. These data demonstrate that the solubility of the API in these SDs was directly dependent on the carrier concentration, in agreement with literature reports [2]. 3.3. Solid state characterization 3.3.1. XRPD The X-ray diffractograms of NMP, PVP K-30, SDs and physical mixture are shown in Fig. 2. NMP presents two polymorphs; Modification I (Mod I), a racemic compound that exhibits higher solubility in water (0.36 ± 0.07 mg mL − 1 ), and Modification II (Mod II), the thermodynamically stable polymorph, which is chemically a conglomerate [41]. Polymorphism investigations are particularly important in drug and product development in the pharmaceutical industry, since they may have an important implication in the bioavailability and stability of a drug [42]. Particularly for NMP, solid-state transitions have already been reported in SD systems [23,29,30] and then they were also monitored in proposed formulations. The crystalline phases of NMP, Mod I and Mod II, can be distinguished through various solid state techniques. XRPD analysis can be considered appropriate since it distinguishes the diffractograms of these polymorphs especially at low 2θ angles. The reflection at 6.6° is only observed for Mod I, while that at 9.3° is present exclusively in Mod II [23, 43-47]. In this regard, XRPD analysis demonstrates that the raw material used for the development of the solid dispersions is composed of the metastable polymorph Mod I.
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3:7, which is characteristic of semi-crystalline materials. In addition to that, peaks relative to the less soluble polymorph Mod II are observed for these samples indicating a phase transition during the manufacturing process. These results demonstrate that the drug:carrier ratio plays an important role on the physical state of the obtained SDs.
Fig. 2. Calculated X-ray patterns of Mod I and Mod II originated from the Cambridge Structural Database and X-ray diffractograms of raw materials, physical mixture and SDs. The arrows demonstrate the presence of crystalline peaks respective to polymorph Mod II.
PVP K-30 was found to be completely amorphous, in agreement with previous data reported in literature [13,30,48–50]. The physical mixture PM 1:9 composed of the highest amount of the carrier presented characteristic reflections of NMP Mod I overlapped with the amorphous halo of the polymer, which indicates the absence of physical interactions between NMP and PVP K-30. The sample SD 1:9 presented an amorphous halo, while crystalline peaks of NMP overlapped with an amorphous halo for SD 2:8 and SD
3.3.2. FTIR FTIR studies were carried out to identify possible interactions between the NMP and PVP (Fig. 3). FTIR characteristic bands related to NMP appear at 3293, 1695 and 1523 cm− 1 (relative to N–H stretching for the polymorph Mod I, carbonyl bond and –NO2 group, respectively) [31,51–54]. The FTIR spectrum of the PVP K-30 presented characteristic bands at 2955 cm−1 (C–H stretch) and at 1654 cm−1 (C_O), as already reported [55]. Also, the spectrum relative to PM 1:9 is composed of an overlap of the characteristic bands of NMP and PVP K-30, without shifts, which indicates the absence of chemical interactions and/or polymorphic transitions. According to Papageorgiou and coworkers, the amine group of NMP is able to form hydrogen bonds with the carbonyl groups of PVP K-30 [30]. The interaction by means of hydrogen bonds shifts the respective bands to lower wavenumbers, usually followed by an increase of their intensity [37,49,56]. In this way, intermolecular interactions between NMP and PVP K-30 may shift the absorbance of the amine and carbonyl groups. In the spectrum of the sample SD 1:9, the band corresponding to the amine group of NMP disappeared. Moreover, a displacement of the carbonyl group of PVP K-30 from 1654 to 1649 cm− 1 was observed, indicating the formation of hydrogen bond between drug and carrier. This interaction between the API and the hydrophilic carrier provides an additional benefit for SDs, since it is able to increase the solid solubility of the drug into the hydrophilic carrier besides acting on inhibiting the crystallization of the API from a glass solution [2,57]. Similar results have already been found by Papageorgiou and coworkers [30] for SDs of NMP and PVP K-30 obtained through solvent evaporation under vacuum. On the other hand, SD 2:8 and SD 3:7 presented the amine band, but shifted to 3272 cm−1 and to 3269 cm−1, respectively, contributing to a higher molecular mobility.
Fig. 3. FTIR spectra of raw materials, physical mixture and SDs. The dotted rectangle indicates the area of appearance of the NMP amine bands.
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3.3.3. SEM SEM analyses (Fig. 4) were carried out to evaluate the impact of the preparation on the morphology of the SDs, as well as the impact of the different drug:carrier ratios. Small tabular crystals with particle size ranging from 1 to 10 μm were observed for the NMP raw material. On the other hand, spheroid particles with indentations and multimodal particle size distribution were observed for PVP K-30. This spherical cage-like shape of the polymer particles is a typical result of spray drying process (Fig. 4B) [58]. Concerning the SDs, the drug:carrier ratios also seem to have an influence on SDs morphology. SD 1:9 exhibited a very porous surface, with a large available surface area, which in combination with the amorphous structure of the drug and the majority composition of PVP K-30, represents a promising result regarding the dissolution rate of the API. In contrast, as the content of the drug increased, the porous aspect of SD 2:8 and SD 3:7 surfaces vanished, and NMP crystals were observed on their surface. These results also support the hypothesis on a semicrystalline state for these samples. The porous aspect observed in SD 1:9 (Fig. 4C and D) can be attributed to the presence of PVP K-30 and its porous-ability, as previously described by Caon et al. [59]. As the content of the carrier decreased in SD
2:8 and SD 3:7 (Fig. 4E and F), the amount of PVP K-30 was not enough to produce porous and amorphous materials. 3.4. “In vitro” dissolution studies All the SDs increased the dissolution rate of NMP (Fig. 5) since only 33.4 ± 1.9% of the crystalline API was released within 60 min. The samples were considered statistically different (p b 0.05) of the pure API. However, differences were not observed between SD 2:8 and SD 3:7. Additionally, all of the dissolution rates were influenced by the drug:carrier ratios and the best result was attributed to SD 1:9, which was able to release 100.73% ± 0.41 of the API within 5 min. This data can be directly related to highly porous area surface of this SD, as qualitatively observed by SEM, and to the amorphous state of the drug, identified by XRPD. In contrast, and as expected, the semi-crystalline SDs released 73.18% ± .14 (SD 2:8) and 68.78% ± 0.20 (SD 3:7) at 60 min. Concerning the DE values, SDs also presented improved results; 95.81% for SD 1:9, 61.95% for SD 2:8 and 59.7% for SD 3:7, in comparison with pure NMP (19.4%). Different factors can contribute to the enhancement of the dissolution rate of a poorly water-soluble API by SDs. Based on the obtained
Fig. 4. Photomicrographs of (A) NMP (3000×), (B) PVP K-30 (500×), (C) SD 1:9 (50×), (D) SD 1:9 (500×), (E) SD 2:8 (100×) and (F) SD 3:7 (100×).
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crystalline NMP did not promote any decrease in the MAP compared with the control group at the tested dose (5 mg kg−1, p.o.) during the evaluated time (120 min). Regarding to phenylephrine-induced increase in blood pressure, more expressive results were obtained. The sample SD 1:9 reduced phenylephrine response for all the time periods considered. After 15 min of oral administration, SD 1:9 reduced the phenylephrine response by 86.4% and 82.4% compared to control and to crystalline NMP group, respectively. On the other hand, crystalline NMP reduced phenylephrine response (38.6% compared to control) only 2 h after oral administration. In summary, this data also reinforces the advantage of SD 1:9 since it is able to maintain a low MAP even against more drastic conditions, such the administration of a vasoconstrictor agent. Fig. 5. In vitro dissolution profiles of crystalline NMP (●), SD 1:9 (♦), SD 2:8 (■) and SD 3:7 (▲).
4. Conclusions results, it is possible to state that the SDs composed of NMP and PVP K30 enhanced the physicochemical properties of the API due to the decrease of crystallinity of NMP, the increased wettability, the increased surface area, characteristic porous morphology, and the establishment of hydrogen bonds between the API drug and its carrier. As SD 1:9 presented the highest solubility and faster drug release it was selected for in vivo efficacy studies. 3.5. “In vivo” studies Fig. 6 shows the in vivo assays results (MAP and MAP change in response to phenylephrine) in function of time for animals treated with SD 1:9 and NMP. Animals treated with sterile water were used as controls. The treatment with SD 1:9 promoted a significant reduction in the animals MAP compared to the crystalline NMP and control groups. At 15 min, for example, SD 1:9 reduced blood pressure by 36% and 32.9% compared to control group and crystalline NMP, respectively. The hypotensive effect of the SD 1:9 remains during all analyzed time. The
SDs of NMP and PVP K-30 were successfully obtained by supercritical fluid extraction technology. Different drug:carrier ratios impacted on the physicochemical properties of the samples. While SD 1:9 presented an amorphous nature, remaining drug crystallinity was observed for SD 2:8 and SD 3:7. Nevertheless, all the SDs exhibited higher solubility and dissolution rates (although in different grades) compared with the pure drug. The best result was attributed to SD 1:9, which provided an increase of more than 1300% in the solubility of NMP and a 5-fold increase in the DE value, besides releasing 100% of the API within 5 min. The performance of this SD was attributed to its amorphous state, the establishment of hydrogen bond between NMP and PVP K30 and the increased surface area, due to its highly porous morphology. SD 1:9 was able to decrease the MAP and reduced the phenylephrineinduced increase in blood pressure. Similar data were not observed for the crystalline drug in the dosage tested, verifying the advantage of this SD versus the crystalline API. Acknowledgments The authors acknowledge CNPq (MCT/CNPq 014/2010) and FAPESC (04/2011) for the financial support and CAPES for the student scholarships. Some of the XRPD analyses were carried out at multiuser X-ray diffraction laboratory (LDRX) of the Federal University of Santa Catarina (UFSC). The authors also wish to thank Dr. Milton Domingues Michel, from Universidade Estadual de Ponta Grossa (UEPG), for the SEM analyses. Thanks are also due to the Consejo Superior de Investigaciones Científicas (CSIC) in Spain, for awarding a license for the use of the Cambridge Structural Database (CSD). References
Fig. 6. Graphs respective to (A) MAP and (B) its change in response to phenylephrine, for animals that received SD 1:9, NMP and empty capsules (control group). Each point of bar represents the mean of 4 animals and vertical lines are the S.E.M. ⁎p b 0.05 compared with the control group and #p b 0.05 compared with the group that received NMP. Statistical analysis was performed using ANOVA test followed by Bonferroni's posthoc test.
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