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Stable nimodipine tablets with high bioavailability containing NM-SD prepared by hot-melt extrusion
Powder Technology 204 (2010) 214–221
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Powder Technology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p o w t e c
Stable nimodipine tablets with high bioavailability containing NM-SD prepared by hot-melt extrusion Fu Jijun, Zhang Lili, Guan Tingting, Tang Xing ⁎, He Haibing Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, Liaoning, PR China
a r t i c l e
i n f o
Article history: Received 26 February 2010 Received in revised form 9 July 2010 Accepted 4 August 2010 Available online 13 August 2010 Keywords: Nimodipine Solid dispersions Hot-melt extrusion Direct compression Stability Bioavailability
a b s t r a c t In the present study, using PVP/VA (Kollidon VA64) as a carrier, a nimodipine (NM) solid dispersion (SD) was prepared by hot-melt extrusion (HME). The effect of temperature during HME, the ratio of NM to Kollidon VA64 and the particle size of the SD after milling on the dissolution behavior were investigated. The temperatures of the extruder barrel zones and die were set as follows: Zone1 = 140 °C, Zone2 = 150 °C, Zone3 = 150 °C, Zone4 = 150 °C and Die = 100 °C. The drug content in SD was set at 15% and SD was milled to pass through a no. 40 mesh sieve. In combination with HME, nimodipine tablets (NM-T-SD (F13)) were produced by direct compression, and the stability and related compounds of NM-T-SD (F13) were studied. The results revealed that NM-T-SD (F13) was stable during storage (40 °C, RH 75%) for six months and related compounds of NM-T-SD (F13) accounted for less than 0.5% and no new related compounds were produced during HME, indicating that NM was able to tolerate the high temperature during HME. Finally, the bioavailability of NM-T-SD (F11, F13) was evaluated in beagle dogs with Nimotop® (Bayer Health Company LTD) and nimodipine tablets (NM-T-C) produced by The Central Pharmaceutical Co., Ltd (Tianjin, China) as references; the results showed similar Cmax and AUC0 → 24 values for NM-T-SD (F13) and Nimotop®, while the Cmax of NM-T-SD (F11) was much lower than that of Nimotop® and provided evidence that Eudragit® EPO should not be used during HME because of its highly pH-dependent nature. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Because of its well plastic property, Eudragit® EPO is widely used in the preparation of SD through HME [1–3] and NM-SD was developed successfully at around the melting point of NM (130 °C) instead of a higher temperature which ensures the stability of NM during HME in a previous study with Eudragit® EPO as the main carrier (the ratio of Eudragit® EPO to Kollidon VA64 is 65:10). As the softening point of Kollidon VA64 is about 180 °C, at the temperature used in HME (120–130 °C), it did not melt completely and exhibited a high viscosity. Thus, it could not mix uniformly with other ingredients and, hence, a lower drug release was obtained when its content in the formulation of SD exceeded 10%. As a result, the content of Kollidon VA64 in SD was finally fixed at 10%. However, there is concern that the highly pH-dependent nature of NM-T-SD (F11) containing Eudragit® EPO, as one of the carriers during HME, will affect its bioavailability in vivo. From another point of view, since the glass transition temperature (Tg) of Kollidon VA64 (102 °C) is much higher than that of Eudragit® EPO (43.7 °C) and, if Eudragit® EPO is completely replaced by Kollidon VA64 in the formulation of NM-SD, the Tg of the
⁎ Corresponding author. Tel.: + 86 24 23986343; fax: +86 24 23911736. E-mail address: [email protected] (T. Xing). 0032-5910/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2010.08.003
extrudate will increase markedly, which could further enhance the stability of NM-T-SD. The purpose of the present study is to explore formulations of NMSD with Kollidon VA64 as the only carrier to ensure that the NM-T-SD developed does not exhibit pH-dependence and to enhance the stability of NM-T-SD further by increasing the Tg of the extrudate. In order to overcome the problem of the high viscosity of Kollidon VA64 during HME and the limited solubility of NM in Kollidon VA64 as a result, NM-SD with a different amount of drug and higher temperatures during HME were investigated in this study. Finally, NM-T-SD without a high pH-dependence was developed and its behavior in vivo was compared with Nimotop®, NM-T-SD (F11) and NM-T-C in beagle dogs. 2. Materials and methods 2.1. Materials NM and nitrendipine (internal standard) were obtained from Zhengzhou Ruikang Pharmaceutical Company (Zhengzhou, Henan, China). Kollidon VA64 (polyvinylpyrrolidone/vinyl acetate copolymer, PVP/VA) and Ludipress® were a gift from BASF Chemical Company (Germany). CMS-Na (sodium starch glycolate) was purchased from Huzhou Zhanwang Chemical Company (Huzhou, China). Talc was purchased from Guangxi Huashi Chemical Company
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(Guangxi, China). Opardry AMB was supplied by Colorcon Coating Technology Ltd. (Shanghai, China). Commercially available tablets Nimotop® (30 mg), used as a reference, were provided by Bayer Healthcare Company Ltd. (H200030010 Beijing, China). Nimodipine tablets, used as a reference, were purchased from The Central Pharmaceutical Co., Ltd. (H10910040 Tianjin, China). All the other regents were either of analytical or chromatographic grade. 2.2. Miscibility study of NM with Kollidon VA64 using thermal analysis Experimental determination of the miscibility by DSC was used to evaluate the processability of NM with the carrier. Differential Scanning Calorimeter-60 and Thermal Analyzer-60 WS (Shimadzu, Japan) instruments were used. Nitrogen was used as the purge gas at a flow rate of 40 mL/min. Samples were crimped in hermetic aluminum pans fitted with lids and then analyzed using a heating rate of 10 °C/ min from 30 °C to 200 °C. 2.3. Preparation of NM-SD NM and PVP/VA (Kollidon VA64) were accurately weighed and mixed by hand in a polyethylene bag for 10 min to obtain a homogeneous physical mixture. This was then extruded using a Coperion KEYATE-20 (Nanjing, China) twin-screw extruder. The extruder consisted of a hopper, barrel, die, kneading screw, and heaters distributed over the entire length of the barrel. Materials introduced into the hopper were carried forward by the feed screw, kneaded under high pressure by the kneading screw and then extruded from the die. The temperatures of the extruder barrel zones and die were set using external temperature controllers. The feed rate and screw rate were both set at 3.5 Hz. The extrudate was collected and allowed to cool at room temperature, and then milled using a laboratory cutting mill. 2.3.1. Choice of operating temperature during HME In present study, two series of temperatures were investigated. The temperatures of the extruder barrel zones and die were set as follows: series 1: Zone1 = 120 °C, Zone2 = 130 °C, Zone3 = 130 °C, Zone4 = 130 °C and Die = 80 °C, series 2: Zone1 = 140 °C, Zone2 = 150 °C, Zone3 = 150 °C, Zone4 = 150 °C and Die = 100 °C. The ratio of NM to Kollidon VA64 was set at 20:80 and the extrudate was milled to pass through a no. 80 mesh sieve after cooling at room temperature. 2.3.2. Selection of formulations of NM-SD The temperatures of the extruder barrel zones and die were set as follows: Zone1 = 140 °C, Zone2 = 150 °C, Zone3 = 150 °C, Zone4 = 150 °C and Die = 100 °C, and the extrudate was milled and passed through a no. 80 mesh sieve after cooling at room temperature. The formulations of NM-SD are shown in Table 1. 2.3.3. Optimization of the particle size of NM-SD The extrudate was milled and passed through no. 80 and no. 40 mesh sieves. The SD drug loading was 15% and the temperatures of Zone1, Zone2, Zone3, Zone4 and the Die were 140, 150, 150, 150 and 100 °C, respectively. Table 1 Formulations of NM-SD. Formulation no.
NM (%)
Kollidon VA64 (%)
SD1 SD2
20 15
80 85
215
2.4. Preparation of NM-T-SD (F13) The formulation of NM-T-SD (F13) is shown in Table 2 and the tablet weight was set at 500 mg. Firstly, the extrudate was pulverized and passed through a no. 40 mesh sieve, and then the ingredients (including lubricants) were weighed accurately and blended evenly and, finally, the powder blends were loaded into feeders and compressed into tablets using a TDP-5B type single punch tablet press (Shanghai, China). To prevent the tablets becoming moist, coating was performed with Opardry AMB in 40% alcohol as the coating material. The coating level of the tablets was 3% (w/w) and coating was carried out using a type B200/400 coating pan (Baoji, Shanxi, China). The coating temperature was 40 °C.
2.5. Dissolution testing The dissolution rate of NM under study was determined at 37 °C using a ZRS-8G dissolution apparatus. Phosphate buffer at pH 6.8 (900 mL) was chosen as the dissolution medium for the formulation and technology screening. Dissolution profiles of NM-T-SD (F13) were obtained in 0.1 mol/L HCl, in acetate buffer at pH 4.5, in purified water and in phosphate buffer at pH 6.8 to examine whether the dissolution was significantly affected by pH. The test was performed according to dissolution test method 2 as described in the China Pharmacopeia (2005) [4] with a paddle rotation speed of 75 rpm. Samples equivalent to 30 mg drug were added to the dissolution apparatus, and test fluid was withdrawn after 5, 10, 20, 30, 45 and 60 min. Dissolution samples were subsequently passed through a 0.45 μm Millipore filter and then the NM content was assayed immediately by UV spectrophotometry at 356 nm to avoid recrystallization of NM from the test fluid when the temperature became lower. In all experiments, the absorbance of the adjuvants at 356 nm was negligible.
2.6. Related compounds HPLC was used to assay related compounds of NM-T-SD (F13). A Hitachi L-2130 Intelligent Pump (Hitachi, Japan) was used. An AllsphereTM C18 column (250 mm × 4.6 mm, 5 μm, Alltech) was used with a mobile phase consisting of methanol–water (65:35, v/v). A Hitachi UV Detector L-2400 (Hitachi, Japan) was set at 235 nm and the chromatographic analyses were performed at 35 °C at a flow rate of 1.0 mL/min. The formulation of NM-T-SD (F13) has been described before. Temperatures were set at 140, 150, 150, 150 and 100 °C for Zone1, Zone2, Zone3, Zone4 and the die during HME. The final tablets were milled to a powder, and a sample equivalent to 10 mg drug was transferred to a 50 mL volumetric flask and then made up to the mark with mobile phase. After sonication, NM dissolved in the liquid completely. Then the liquid was passed through a 0.45 μm Millipore filter and 10 μL of the solution at a concentration of 200 μg/mL was injected into the HPLC system. Physical mixtures equivalent to the formulation of NM-T-SD (F13) were also processed and then assayed as described above.
Table 2 Formulation of NM-T-SD (F13) (mg per tablet). Component
Dose
NM in SD2 Ludipress® CMS-Na Talc
30 255 20 25
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2.7. Characterization of the state of NM 2.7.1. Differential scanning calorimetry (DSC) Differential Scanning Calorimeter-60 and Thermal Analyzer-60 WS (Shimadzu, Japan) instruments were used to characterize the thermal properties of NM-T-SD (F13). The experimental conditions were the same as described above. 2.7.2. Powder X-ray diffraction (PXRD) PXRD was performed using a D/Max-2400 X-ray Fluorescence Spectrometer (Rigaku, Japan) with a CuKa line as the source of radiation. Standard runs were carried out using a voltage of 56 kV, a current of 182 mA and a scanning rate of 2° min− 1 over a 2-theta range of 3–45°. The samples were NM, NM-T-SD (F13) and its adjuvants. 2.8. Determination of the glass transition temperature of the extrudate Temperature modulated DSC was used to determine the Tg of the NM-SD2, using a DSC 1 differential scanning calorimeter (Mettler Toledo, Switzerland) and applying TOPEM® with a modulation amplitude of ±0.5 °C and a heating rate of 1 °C/min. Nitrogen was used as the purge gas at a flow rate of 40 mL/min. Samples were crimped in aluminum pans with a pin hole. 2.9. Infrared spectroscopy Fourier-transform infrared (FT-IR) spectra were obtained on a BRUKER IFS 55 FT-IR system using the KBr disk method. The scanning range was 4000–400 cm− 1 and the resolution was 1 cm− 1. 2.10. Stability testing The stability of NM-T-SD (F13) was tested during storage (40 °C, RH 75%). NM-T-SD (F13) was sealed tightly in commercial packing. The stability was evaluated using four methods: dissolution testing, DSC, PXRD and FT-IR. The parameters were the same as those described above. 2.11. Bioavailability study 2.11.1. Administration program The study protocol was approved by the Ethics Committee of Shenyang Pharmaceutical University. The study design involved fasting, a single dose of four treatments and four periods. Each dog was given four preparations with a oneweek washout period between each. Six beagle dogs were used for each treatment group. After fasting overnight, preparations containing 60 mg NM (Nimotop®, NM-T-C, NM-T-SD (F11) and NM-T-SD (F13)) were given to the beagle dogs with 200 mL water. Four hours after dosing, the dogs were provided with standard food. On each dosing day, blood samples were taken before and then 0.1667, 0.333, 0.6667, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 16, and 24 h after dosing. Plasma was separated from samples by centrifugation (4000 rpm for 10 min) and stored at − 20 °C until analysis within one month. 2.11.2. Plasma sample preparation To prevent the photodegradation of nimodipine, all the experiments, including standard and QC preparation, plasma collection, sample preparation and instrumental analysis, were performed under dim light. For this, 0.5 mL plasma was spiked with 50 μL internal standard solution (nitrendipine, 4 μg/mL dissolved in methanol) and 50 μL methanol. The sample was subsequently made alkaline with 100 μL 0.1 M NaOH solution and extracted with 3 mL extraction solvent (n-hexane/diethylether = 1:1, v/v) by vortexing for 10 min. After centrifugation at 4000 rpm for 10 min, 2 mL of the supernatant
was transferred to a conical tube. The separated organic phase was then evaporated at 50 °C using a Centrifugal Concentrator (Centrivap® 78120-03, Labconco, Corp. USA). The residue was reconstituted with 1 mL methanol and vortexed for 5 min. After centrifugation at 12,000 rpm for 10 min, a 5 μL aliquot was injected into the UPLC-MS/ MS system. 2.11.3. Analysis conditions and method validation The analysis was carried out on an ACQUITYTM UPLC system (Waters Corp., Milford, MA, USA) with a cooling autosampler and column oven. An ACQUITY UPLCTM BEH C18 column (50 mm × 2.1 mm, 1.7 μm; Waters Corp., Milford, MA, USA) was employed for separation with the column temperature maintained at 35 °C. The chromatographic separation was achieved with gradient elution using a mobile phase composed of water containing 0.1% (v/v) formic acid and acetonitrile. The gradient elution started at 50% acetonitrile, increased linearly to 80% acetonitrile over 1 min, was maintained at 80% for 1.3 min and then returned to the initial percentage over 0.2 min and was maintained there for another 0.5 min. The flow rate was set at 0.2 mL/min. The autosampler temperature was kept at 4 °C and a sample solution of 5 μL was injected. A Waters ACQUITYTM TQD triple quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) with an electrospray ionization (ESI) interface was employed for mass analysis. The ESI source was operated in positive ionization model with optimal operation parameters as follows: capillary voltage 3.70 kV, cone voltage 20 V, extractor 3.0 V and RF 0.1 V with a source temperature of 100 °C and a desolvation temperature of 400 °C. Nitrogen was used as the desolvation and cone gas at a flow rate of 450 L/h and 50 L/h, respectively. For collision-induced dissociation (CID), argon was used as the collision gas at a flow rate of 0.17 mL/min. The quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 419 → 343 for nimodipine and m/z 361 → 315 for nitrendipine (I.S.), respectively. All data were collected in centroid mode and processed using MassLynxTM NT 4.1 software with a QuanLynxTM program (Waters Corp., Milford, MA, USA). The linear range of this method was 1–1000 ng/mL with an r (correlation coefficient) value of no less than 0.99. The LLOQ was 1 ng/ mL. The R.S.D. which reflected the intra-day and the inter-day precision of the QC samples were both not more than 5.4%. The R.E. of the QC samples was less than ±8.4%. The extraction recoveries of NM from the beagle dog QC plasma samples were 92.7±7.0%, 100.9±4.6% and 105.2 ±4.8%, respectively, and the mean extraction recovery of nitrendipine was 96.8±10.5%. 2.11.4. Calculation parameters The area under the plasma concentration–time curve (AUC0 → t) up to the last measurable plasma concentration (Ct) was calculated by the linear trapezoidal rule. 3. Results and discussion 3.1. Miscibility study by DSC In order to assess the behavior under thermal processing conditions, differential scanning calorimetry was applied to assess the processability of the drug: carrier physical mixtures. Interestingly, the endothermic peak of NM in the pure drug and the physical mixture (PM) is significantly different, as shown in Fig. 1. The melting peak of the pure drug is typically sharp, whereas the endothermic peak in the physical mixture becomes much broader. A significant reduction in melting temperature was also observed, with a peak temperature of 112 °C in the PM compared with 129 °C in the pure drug. The above phenomena were both caused by the gradual dissolution of NM in the carriers during the DSC heating ramp [3,5–6] and provided strong
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the carrier could not dissolve all the drug and much of the drug was in the crystalline form instead of the amorphous form, with the result that the drug release was only about 35% in one hour for NM-SD1, showing that the drug loading should be reduced. However, as SD is difficult to handle due to its high viscosity and sensitivity to moisture, the amount of SD contained in tablets should be as low as possible. If the drug loading was 10%, over half the tablet weight would be SD, which is not acceptable as far as flowability is concerned and the amount of dispersion required to administer the usual dose of the drug may be too high to produce a tablet or capsule that can be easily swallowed. Finally, the ratio of NM to Kollidon VA64 was set at 15:85. Fig. 1. DSC thermograms of the pure NM (a) and the mixture of NM and Kollidon VA64 (15:85) (b).
evidence for the miscibility of NM with Kollidon VA64, which suggested that Kollidon VA64 would be an optimum carrier for NM-SD. 3.2. Dissolution profiles of NM-SD and NM-T-SD (F13) 3.2.1. Choice of temperatures used during HME It was clear from Fig. 2 that increasing the temperature during HME led to an increased dissolution. As the softening point of Kollidon VA64 is quite high (about 180 °C) while the melting point of NM is only about 130 °C, when the temperatures used during HME were set around 130 °C, Kollidon VA64 was not softened effectively and exhibited a high viscosity, so, the drug and carrier could not be mixed to obtain a uniform state. When extrusion was performed at low temperatures, much of the NM was present in the crystalline form instead of the amorphous form, and the resulting dissolution was less than 15% in one hour. However, if the temperature exceeded 160 °C, the color of the extrudate changed from gold to brown which indicated that degradation of NM occurred at that temperature, so the temperature should not exceed 150 °C during HME. As a result, the temperatures used during HME were finally set at 140, 150, 150, 150 and 100 °C for Zone1, Zone2, Zone3, Zone4 and the die, respectively. 3.2.2. Choice of drug loading in NM-SD In a previous study, it was found that the drug loading affected the dissolution behavior in vitro. So, in the present study, the ratio of NM to Kollidon VA64 was also investigated. It can be seen from Fig. 2 that the drug release increased from about 35% to 70% when the drug loading fell from 20% to 15%. So, it was concluded that the drug loading markedly affected the dissolution of NM-SD. This is because there is a limited ability of the carrier to dissolve NM. When the ratio of NM to Kollidon VA64 was 20:80 in SD,
Fig. 2. Dissolution profiles of NM-SD in phosphate buffer at pH 6.8 (n = 3). (♦) temperature: 120, 130, 130, 130 and 80 °C, drug content 20%, particle size no. 80 mesh; (■) temperature: 140, 150, 150, 150 and 100 °C, drug content 20%, particle size no. 80 mesh; (▲) temperature: 140, 150, 150, 150 and 100 °C, drug content 15%, particle size no. 80 mesh; (△) temperature: 140, 150, 150, 150 and 100 °C, drug content 15%, particle size no. 40 mesh.
3.2.3. Selection of the particle size of SD As the drug loading of SD decreased, the amount of SD contained in the final dosage form increased. If the extrudate was still pulverized to pass through a no. 80 mesh sieve, good flowability ensuring automatic and continuous tabletting could not be achieved. So, the extrudate was pulverized to obtain a larger particle size. It can be seen from Fig. 2 that, after milling to no. 40 mesh instead of no. 80 mesh, the drug release increased from about 70% to 90%. During the dissolution testing, it was found that SD powders aggregated and floated on the dissolution medium within about 20 min and gradually dissolved when the particle size was no. 80 mesh, while it dispersed and dissolved in the dissolution medium immediately when the particle size was no. 40 mesh. It is likely that this phenomenon was due to the poorer wettability of SD powders with a particle size of no. 80 mesh compared with no. 40 mesh. Although the specific surface area increased as the particle size decreased, due to aggregation of SD powders because of the poor wettability, the effective specific surface area was reduced, which had an adverse effect on the dissolution of NM from SD. Finally, the flowability of the powder blends for direct compression increased when the particle size of NM-SD increased. The above factors were both favorable for direct compression and dissolution. As the particle size of Ludipress® is about no. 40 mesh, taking content uniformity into account, the particle size of NM-SD should not be greater, so, finally, the NM-SD2 produced by HME was pulverized and passed through a no. 40 mesh sieve in the present study. 3.2.4. Dissolution profiles of NM-T-SD (F13) in different media The dissolution behaviors of NM-T-SD (F13) obtained by HME combined with direct compression as described above in dissolution medium at different pH values are illustrated in Fig. 3. It can be seen from the figure that the drug release in different media exceeded 80% in every case. It was concluded that NM-T-SD (F13) did not exhibit a high pH-dependence. Because of this pHindependence, NM could be released from tablets in any part of gastrointestinal tract and, as a result, the in vivo bioavailability might be less affected by physiological factors compared with NM-T-SD (F11) in the previous study. It also can be inferred from the dissolution profiles that, after 20–30 min, dissolution profiles in different media were reduced. This is because after more than 80% of NM was
Fig. 3. Dissolution profiles of NM-T-SD (F13) in different media (n = 3). (♦) 0.1 mol/L HCl; (■) acetate buffer at pH 4.5; (▲) phosphate buffer at pH 6.8; (△) purified water.
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released, the drug was present in supersaturated form in the dissolution medium, which means that NM is not stable in the dissolution medium. So it easily recrystallizes when stirred. Generally speaking, compared with the dissolution behavior of Nimotop® in different media in our previous study, the dissolution rate of NM-T-SD (F13) was faster and the drug release was higher than that of Nimotop®. However, recrystallization was effectively inhibited in the case of Nimotop® as indicated by the finding that the dissolution curves did not decline in one hour, while drug crystals were formed after the maximum dissolution of NM-T-SD (F13) had occurred. Although, in the present study, this could not be achieved, it might not affect the bioavailability greatly as long as the uptake of NM was complete in about 20 min since NM was released from NM-T-SD (F13).
Fig. 5. DSC thermograms of NM (a) and NM-T-SD (F13) (b).
3.3. Related compounds Related compounds of NM-T-SD produced by HME combined with direct compression assayed by HPLC are illustrated in Fig. 4. From Fig. 4, it can be seen that no new related compounds were produced during HME although the area of the impurities became larger. The area of any individual impurity was not more than 0.2% and no more than 0.5% total impurities were found after HME. This result met the requirement of the China Pharmacopeia (2005) [4] and indicated that 150 °C used during HME was acceptable and no obvious degradation was observed at this temperature. 3.4. Physical characterization 3.4.1. Physical characterization by DSC Fig. 5 shows the DSC thermograms over the temperature range 30– 140 °C. The DSC recording of the pure NM exhibits a sharp endothermic peak around 129 °C, while NM-T-SD (F13) resulted in a complete suppression of the drug fusion peak. The DSC data showed that, using HME, NM was completely converted into an amorphous state in the carrier and this state was not altered by compression and coating. So, it could be concluded that the temperatures chosen and the ratio of NM to Kollidon VA64 selected were suitable to produce NM in an amorphous state. Another important conclusion was that Kollidon VA64 was still an excellent carrier for NM when the parameters of HME and formulation of SD were optimized, although the softening point of Kollidon VA64 (180 °C) was much higher than the melting point of NM (130 °C).
Fig. 6. PXRD patterns. (a) Kollidon VA64; (b) NM-SD2; (c) adjuncts of NM-T-SD (F13); (d) NM-T-SD (F13); (e) pure NM.
3.4.2. Physical characterization by PXRD PXRD was used to confirm the loss of drug crystals, and the results are shown in Fig. 6. Pure NM has several major peaks at 2-theta angles within 30° (2-theta angles of 6.5, 12.3, 12.8, 17.3, 19.7, 20.3, 20.8, 23.9, 24.7 and 26.3°). For NM-SD2 and NM-T-SD (F13), no detectable diffraction peak of NM was observed, suggesting that NM was in an amorphous state in NM-SD2 and NM-T-SD (F13). These results indicated that NM was transformed from the crystalline form to the amorphous form by HME and the amorphous state was not destroyed by compression and coating. Furthermore, this offered the possibility of high dissolution and, as a result, a high bioavailability in vivo. 3.5. Determination of glass transition temperature (Tg) It can be seen from Fig. 7 that the measured Tg of NM-SD2 is 89 °C and, as a result, it is estimated that the NM-T-SD prepared containing NM-SD2 will be stable during long-term storage (20–30 °C) [7,8]. 3.6. FT-IR In order to study the possibility of an interaction of NM with Kollidon VA64 in the solid state, information was gathered using FT-IR spectroscopy. From the structures of NM and Kollidon VA64 (Fig. 8), it can be assumed that a possible interaction could occur between the secondary amine hydrogen atom of NM and the amide function or ester function of Kollidon VA64. Thus, in this case any sign of interaction would be reflected by shifts in the C O vibration, depending on its extent. H-bonding leads to a shift of the peak maxima towards a lower wave number (bathochromic shift) and very often the peak width is increased [9,10]. The infrared spectra of NM, Kollidon VA64 and the SD are shown in Fig. 9. The position of the absorption bands of the ester function and amide function of Kollidon
Fig. 4. HPLC chromatogram of NM-T-SD (F13) before (1) and after (2) HME. (a) Impurity 1; (b) impurity 2; (c) NM; (d) impurity 3; (e) impurity 4.
Fig. 7. The revering heat flow profile of the NM-SD2.
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Fig. 8. Molecular structures of the compounds and polymers.
VA64 remained unchanged at 1739 and 1678 cm− 1 in SD, respectively. These results indicated the absence of a well-defined interaction between NM and Kollidon VA64. 3.7. Stability testing 3.7.1. Dissolution testing After storage for six months (40 °C, RH 75%), dissolution testing of NM-T-SD (F13) at several time intervals was carried out in different media. The results are shown in Fig. 10. It can be seen clearly from the figures that after storage (40 °C, RH 75%), the dissolution profiles were similar to the initial state, especially in the case of purified water, so it can be concluded that the amorphous state of NM in NM-T-SD (F13) was not destroyed and ageing did not occur during storage, which indicated that NM-T-SD (F13) made by HME combined with direct compression was stable. Greater variations were shown in 0.1 M HCl, acetate buffer at pH 4.5 and phosphate buffer at pH 6.8 because of the differences in the ionic strength in the media at different time intervals.
the polymer or interactions between the drug and the polymer, or a combination of both [12,13]. An anti-plasticizing effect of the polymer may contribute significantly by increasing the temperature at which the molecular mobility becomes significant with respect to recrystallization. In addition, the resulting system will exhibit high viscosity at room temperature, hence impairing crystallization [13]. As described above, although there were no interactions between NM and Kollidon VA64, the Tg of NM-SD2 was nearly 50 °C above storage temperature (40 °C) and, according to Hancock and Zografi [7,8], NM-SD2 would be
3.7.2. Physical characterization DSC and PXRD recordings of NM-T-SD (F13) before and after storage for one and two months (40 °C, RH 75%) are illustrated in Figs. 11 and 12. It can be seen from Fig. 11 that there was no endothermic peak around 130 °C and no detectable diffraction peak of NM in Fig. 12 after storage for two months, these results showed that NM remained in an amorphous state during storage. 3.7.3. FT-IR FT-IR profiles of NM-T-SD (F13) before and after storage for six months (40 °C, RH 75%) are illustrated in Fig. 13. No differences were seen among the profiles at different time intervals, which indicated that no changes have occurred to NM-T-SD (F13) and it was stable during storage for six months (40 °C, RH 75%). With respect to the physical stability of the amorphous state, it is generally accepted that recrystallization is dependent on the molecular mobility and the degree of supersaturation of the solute in the matrix [11]. As the molecular mobility increases, the risk of crystallization also increases. Usually, the addition of a polymer with a high Tg is sufficient to prevent crystallization. The protective effect of the polymer can be caused by two factors: an anti-plasticizing effect of
Fig. 9. FT-IR spectra: (a) NM; (b) Kollidon VA64; (c) NM-SD2.
Fig. 10. Dissolution profiles of NM-T-SD (F13) in different media after storage (n = 3). (♦) initial state; (■) one month; (▲) two months; (♢) three months; (□) four months; (△) five months; (×) six months.
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Fig. 13. FT-IR spectra of NM-T-SD (F13) after storage: (a) initial state; (b) one month; (c) two months; (d) three months; (e) four months; (f) five months; (g) six months. Fig. 11. DSC thermograms of NM-T-SD (F13) after storage. (a) The pure NM; (b) NM-TSD (F13) before storage; (c) NM-T-SD (F13) after storage for one month; (d) NM-T-SD (F13) after storage for two months.
stable during storage (40 °C, RH 75%). The good stability of NM-T-SD (F13) was also proved in this study and it was demonstrated that the anti-plasticizing effect of the polymer which increased the viscosity of the binary system and decreased the diffusion of drug molecules necessary to form a lattice was the only stabilizing factor in the dispersions of NM and Kollidon VA64. 3.8. Bioavailability in vivo NM-T-SD produced by direct compression tabletting (F11, F13) was used to study the bioavailability in vivo, with Nimotop® (Bayer, H200030010 Beijing, China) and NM-T-C as reference agents. The plasma concentration versus time curves for the four preparations are shown in Fig. 14 and the bioavailability parameters are given in Table 3. The Cmax of Nimotop® and NM-T-C was 409.2 ± 200.1 and 302.0 ± 167.4 ng/mL respectively, while the Cmax of NM-T-SD (F11) was only 115.5 ± 65.0 ng/mL, much lower than that of the references, although the AUC0 → 24 of Nimotop®, NM-T-C and NM-T-SD (F11) were similar (861.5 ± 296.3, 705.0 ± 177.7 and 720.2 ± 625.8 ng h/mL). The low Cmax of NM-T-SD (F11) might be caused by Eudragit® EPO used as a carrier during HME. Eudragit® EPO is a highly pH-dependent material which dissolves only when the pH of the medium is below 5.0. As indicated by the dissolution profiles of NM-T-SD (F11) in different media in vitro, NM could not be released from NM-T-SD (F11) in phosphate buffer at pH 6.8 and purified water. Perhaps NM-T-SD (F11) entered the small intestine before NM was released completely and, as the pH of the intestinal fluid was higher than 5.0, NM still in the tablets could not be released at all as indicated by the dissolution in vitro. So, from the moment NM-T-SD (F11) entered the small intestine or slightly later, uptake of NM ceased and the increasing plasma concentration due to the release of NM from NM-T-SD (F11) in the stomach could not continue and, as a result, the Cmax of the test was quite low. The pharmacokinetic behaviors of Nimotop® and NM-T-SD (F13) were similar as indicated by the Cmax and AUC0 → 24. After excluding Eudragit® EPO from formulations of SD, the Cmax increased markedly from 115.5 ± 65.0 ng/mL to 334.3 ± 149.9 ng/mL. Without Eudragit® EPO, NM could be released from the dosage form in any part of the
Fig. 12. PXRD recordings of NM-T-SD (F13) after storage. (a) NM-T-SD (F13) before storage; (b) NM-T-SD (F13) after storage for one month; (c) NM-T-SD (F13) after storage for two months; (d) pure NM; (e) adjuncts of NM-T-SD (F13).
gastrointestinal tract after oral administration and, as a result, the influence of physiological factors on the in vivo behavior of NM-T-SD (F13) was reduced, as indicated by the higher Cmax. This proved that the hypothesis above was correct and Eudragit® EPO was not an ideal carrier for HME because of its high pH-dependence although an amorphous state of NM could be achieved at a lower temperature and with increased drug loading. It was reported by other authors that the Cmax and the AUC0 → 12 of the pure NM were only a bit more than 30% of those of Nimotop® [14]. As the Cmax and the AUC0 → 24 were similar between the NM-T-SD (F13) and Nimotop® in the present study, it can be concluded that HME was quite an efficient technology in improving the oral bioavailability of NM. It can be seen from the dissolution profiles in vitro that the dissolution curve of NM-T-SD (F11) was higher than that of the references for most of the time in 0.1 mol/L HCl. However, taking the high pH-dependence of NM-T-SD (F11) into account, NM released from NM-T-SD (F11) differed significantly among beagle dogs as the physiological state of the beagle dogs was different and it may be that NM was released almost completely from NM-T-SD (F11) in some beagle dogs while only a little was released in others, as indicated by the much higher R.S.D. of the AUC0 → 24 of NM-T-SD (F11) (86.9%) compared with Nimotop® (34.4%) and NM-T-C (25.2%). For these two reasons, the AUC0 → 24 of NM-T-SD (F11) was slightly lower than that of Nimotop® and similar to that of NM-T-C. Another interesting phenomenon was observed in Fig. 14. Double peaks were observed in the plasma concentration versus time curves of NM-T-SD (F11) and commercially available NM-T-C and there was a shoulder peak in the plasma concentration versus time curves of NMT-SD (F13), while this was not the case for Nimotop®. This phenomenon resulted in a higher bioavailability of NM-T-SD (F13) compared with Nimotop®, although the Cmax was lower. It seems that the plasma concentration versus time curves for NM-T-SD (F11, F13) and NM-T-C were not classic. Perhaps this phenomenon was caused by recrystallization of NM in the gastrointestinal tract after release from NM-T-SD (F11, F13) as indicated by the dissolution profiles in vitro. If recrystallization occurred, although NM in crystalline form was carried down to the effective absorption region, it could not be
Fig. 14. Mean NM plasma profiles from a single dose (60 mg) bioavailability study compared with Nimotop® (n = 6). (♦) Nimotop®; (■) NM-T-C; (▲) NM-T-SD (F11); (×) NM-T-SD (F13).
F. Jijun et al. / Powder Technology 204 (2010) 214–221 Table 3 Bioavailability parameters of NM for different tablets in beagle dogs (n = 6). Sample
Cmax (ng/mL)
tmax (h)
AUC0 → 24 (ng h/mL)
Nimotop® NM-T-C NM-T-SD (F11) NM-T-SD (F13)
409.2 ± 200.1 302.0 ± 167.4 115.5 ± 65.0 334.3 ± 149.9
0.8 ± 0.3 1.5 ± 1.6 1.6 ± 1.1 1.8 ± 2.3
861.5 ± 296.3 705.0 ± 177.7 720.2 ± 625.8 1060.9 ± 430.7
Each value represents the mean ± S.D.
taken up immediately while, in the amorphous state, it was taken up as soon as it reached the effective absorption region. The uptake of the amorphous drug resulted in the first peak in the plasma concentration versus time curve. Although the drug release of NM-T-SD (F11, F13) was higher than Nimotop®, some of the NM released from NM-T-SD (F11, F13) recrystallized and could not be taken up immediately while nearly all the NM released from Nimotop® was present in the amorphous form and was taken up rapidly, so the Cmax of NM-T-SD (F11, F13) was lower. Only after NM in crystalline form was emulsified by endogenous surfactants such as bile, could the drug be taken up later and, as a result, shoulder or double peaks appeared. Investigation of the dissolution behavior in vitro showed that the rate of recrystallization was much slower for Nimotop® compared with all the other dosage forms, so only a single peak appeared in the plasma concentration versus time curve of Nimotop®. It can be seen from the dissolution profiles in vitro that the dissolution curve of NM-T-SD (F13) was higher than that of Nimotop® for most of the one hour period and NM could be released from NM-T-SD (F13) in media with different pH values as Nimotop®, so the AUC0 → 24 of NM-T-SD (F13) was higher than that of Nimotop®. For the above reasons, the AUC0 → 24 of NM-T-SD (F13) was higher than that of Nimotop® while the Cmax was lower. It can be seen from the above curve that the peak in the plasma concentration versus time curve of NM-T-SD (F11, F13) was earlier than that of Nimotop®, which was attributed to the faster dissolution of NM-T-SD (F11, F13) compared with Nimotop®, as indicated by the dissolution in vitro. However, as there were double peaks or the shoulder peak of NM-T-SD (F11, F13) in the plasma concentration versus time curve, the tmax of the tests (1.6, 1.8 h) were later than that of Nimotop® (0.8 h). 4. Conclusions By increasing the temperature during HME and reducing the drug loading in SD, NM could be obtained in an amorphous state in the present study and this amorphous state of NM was demonstrated by DSC and PXRD. Related compounds assayed by HPLC showed that no dramatic degradation of NM occurred during HME and indicated that
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the temperature used during HME was acceptable. The suitable particle size of NM-SD was also studied. NM-T-SD (F13) was made by HME combined with direct compression and its dissolution behavior in different media proved that it was pH-independent. The results of the stability study showed that the amorphous state of NM in NM-TSD (F13) was not destroyed during two months of storage (40 °C, RH 75%) and further studies are in progress. The bioavailability study showed that NM-T-SD (F13) exhibited a similar behavior in vivo to Nimotop®, while the Cmax of NM-T-SD (F11) was much lower than that of Nimotop®, which confirmed the belief that Eudragit® EPO should not be included in formulations of SD because of its highly pHdependent nature.
References [1] R.J. Chokshi, H. Sandhu, R.M. Iyer, N.H. Shah, A.W. Malick, H. Zia, Characterization of physical-mechanical properties of indomethacin and polymers to assess their suitability for hot-melt extrusion process as a means to manufacture solid dispersion/solution, J. Pharm. Sci. 11 (2005) 2463–2474. [2] H.J. Liu, P. W, X.Y. Zh, F. Sh, C.G. Gogos, Effects of extrusion process parameters on the dissolution behavior of indomethacin in Eudragit® EPO solid dispersions, Int. J. Pharm. 383 (2010) 161–169. [3] S. Qi, A. Gryczke, P. Belton, D.Q.M. Craig, Characterization of solid dispersions paracetamol and EUDRAGIT E prepared by hot-melt extrusion using thermal, microthermal and spectroscopic analysis, Int. J. Pharm. 354 (2008) 158–167. [4] China Pharmacopeia, Beijing, 2005, pp. 166–167. [5] A. Forster, J. Hempenstall, I. Tucker, T. Rades, Selection of excipients for melt extrusion with two poorly water-soluble drugs by solubility parameter calculation and thermal analysis, Int. J. Pharm. 226 (2001) 147–161. [6] D.J. Greenhalgh, A.C. Williams, P. Timmins, P. York, Solubility parameters as predictors of miscibility in solid dispersions, J. Pharm. Sci. 88 (1999) 1182–1190. [7] B.C. Hancock, S.L. Shamblin, G. Zografi, Molecular mobility of amorphous pharmaceutical solids below their glass transition temperature, Pharm. Res. 12 (1995) 799–806. [8] M. Yoshioka, B.C. Hancock, G. Zografi, Inhibition of indomethacin crystallization in poly(vinylpyrrolidone) coprecipitates, J. Pharm. Sci. 84 (1995) 983–986. [9] I. Weuts, D. Kempen, A. Decorte, G. Verreck, J. Peeters, M. Brewster, G. Van den Mooter, Phase behaviour analysis of solid dispersions of loperamide and two structurally related compounds with the polymers PVP-K30 and PVP-VA64, Eur. J. Pharm. Sci. 22 (2004) 375–385. [10] X. Zheng, R. Yang, X. Tang, L.Y. Zheng, Characterization of solid dispersions of nimodipine prepared by hot-melt extrusion, Drug Dev. Ind. Pharm. 33 (2007) 791–802. [11] I. Weuts, D. Kempen, A. Decorte, G. Verreck, J. Peeters, M. Brewster, G. Van den Mooter, Physical stability of the amorphous state of loperamide and two fragment molecules in solid dispersions with the polymers PVP-K30 and PVP-VA64, Eur. J. Pharm. Sci. 25 (2005) 313–320. [12] T. Matsumoto, G. Zografi, Physical properties of solid molecular dispersions of Indomethacin with poly(vinylpyrrolidone) and poly(vinylpyrrolidone-co-vinylacetate) in relation to Indomethacin crystallization, Pharm. Res. 16 (1999) 1722–1728. [13] G. Van den Mooter, M. Wuyts, N. Blaton, R. Busson, P. Grobet, P. Augustijns, R. Kinget, Physical stabilization of amorphous ketoconazole in solid dispersions with polyvinylpyrrolidone K25, Eur. J. Pharm. Sci. 12 (2001) 261–269. [14] X. Zheng, R. Yang, Y. Zhang, Zh.J Wang,, X. Tang, L.Y. Zheng, Bioavailability in beagle dogs of nimodipine solid dispersions prepared by hot-melt extrusion, Drug Dev. Ind. Pharm. 33 (2007) 783–789.
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Powder Technology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p o w t e c
Stable nimodipine tablets with high bioavailability containing NM-SD prepared by hot-melt extrusion Fu Jijun, Zhang Lili, Guan Tingting, Tang Xing ⁎, He Haibing Department of Pharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, Liaoning, PR China
a r t i c l e
i n f o
Article history: Received 26 February 2010 Received in revised form 9 July 2010 Accepted 4 August 2010 Available online 13 August 2010 Keywords: Nimodipine Solid dispersions Hot-melt extrusion Direct compression Stability Bioavailability
a b s t r a c t In the present study, using PVP/VA (Kollidon VA64) as a carrier, a nimodipine (NM) solid dispersion (SD) was prepared by hot-melt extrusion (HME). The effect of temperature during HME, the ratio of NM to Kollidon VA64 and the particle size of the SD after milling on the dissolution behavior were investigated. The temperatures of the extruder barrel zones and die were set as follows: Zone1 = 140 °C, Zone2 = 150 °C, Zone3 = 150 °C, Zone4 = 150 °C and Die = 100 °C. The drug content in SD was set at 15% and SD was milled to pass through a no. 40 mesh sieve. In combination with HME, nimodipine tablets (NM-T-SD (F13)) were produced by direct compression, and the stability and related compounds of NM-T-SD (F13) were studied. The results revealed that NM-T-SD (F13) was stable during storage (40 °C, RH 75%) for six months and related compounds of NM-T-SD (F13) accounted for less than 0.5% and no new related compounds were produced during HME, indicating that NM was able to tolerate the high temperature during HME. Finally, the bioavailability of NM-T-SD (F11, F13) was evaluated in beagle dogs with Nimotop® (Bayer Health Company LTD) and nimodipine tablets (NM-T-C) produced by The Central Pharmaceutical Co., Ltd (Tianjin, China) as references; the results showed similar Cmax and AUC0 → 24 values for NM-T-SD (F13) and Nimotop®, while the Cmax of NM-T-SD (F11) was much lower than that of Nimotop® and provided evidence that Eudragit® EPO should not be used during HME because of its highly pH-dependent nature. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Because of its well plastic property, Eudragit® EPO is widely used in the preparation of SD through HME [1–3] and NM-SD was developed successfully at around the melting point of NM (130 °C) instead of a higher temperature which ensures the stability of NM during HME in a previous study with Eudragit® EPO as the main carrier (the ratio of Eudragit® EPO to Kollidon VA64 is 65:10). As the softening point of Kollidon VA64 is about 180 °C, at the temperature used in HME (120–130 °C), it did not melt completely and exhibited a high viscosity. Thus, it could not mix uniformly with other ingredients and, hence, a lower drug release was obtained when its content in the formulation of SD exceeded 10%. As a result, the content of Kollidon VA64 in SD was finally fixed at 10%. However, there is concern that the highly pH-dependent nature of NM-T-SD (F11) containing Eudragit® EPO, as one of the carriers during HME, will affect its bioavailability in vivo. From another point of view, since the glass transition temperature (Tg) of Kollidon VA64 (102 °C) is much higher than that of Eudragit® EPO (43.7 °C) and, if Eudragit® EPO is completely replaced by Kollidon VA64 in the formulation of NM-SD, the Tg of the
⁎ Corresponding author. Tel.: + 86 24 23986343; fax: +86 24 23911736. E-mail address: [email protected] (T. Xing). 0032-5910/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2010.08.003
extrudate will increase markedly, which could further enhance the stability of NM-T-SD. The purpose of the present study is to explore formulations of NMSD with Kollidon VA64 as the only carrier to ensure that the NM-T-SD developed does not exhibit pH-dependence and to enhance the stability of NM-T-SD further by increasing the Tg of the extrudate. In order to overcome the problem of the high viscosity of Kollidon VA64 during HME and the limited solubility of NM in Kollidon VA64 as a result, NM-SD with a different amount of drug and higher temperatures during HME were investigated in this study. Finally, NM-T-SD without a high pH-dependence was developed and its behavior in vivo was compared with Nimotop®, NM-T-SD (F11) and NM-T-C in beagle dogs. 2. Materials and methods 2.1. Materials NM and nitrendipine (internal standard) were obtained from Zhengzhou Ruikang Pharmaceutical Company (Zhengzhou, Henan, China). Kollidon VA64 (polyvinylpyrrolidone/vinyl acetate copolymer, PVP/VA) and Ludipress® were a gift from BASF Chemical Company (Germany). CMS-Na (sodium starch glycolate) was purchased from Huzhou Zhanwang Chemical Company (Huzhou, China). Talc was purchased from Guangxi Huashi Chemical Company
F. Jijun et al. / Powder Technology 204 (2010) 214–221
(Guangxi, China). Opardry AMB was supplied by Colorcon Coating Technology Ltd. (Shanghai, China). Commercially available tablets Nimotop® (30 mg), used as a reference, were provided by Bayer Healthcare Company Ltd. (H200030010 Beijing, China). Nimodipine tablets, used as a reference, were purchased from The Central Pharmaceutical Co., Ltd. (H10910040 Tianjin, China). All the other regents were either of analytical or chromatographic grade. 2.2. Miscibility study of NM with Kollidon VA64 using thermal analysis Experimental determination of the miscibility by DSC was used to evaluate the processability of NM with the carrier. Differential Scanning Calorimeter-60 and Thermal Analyzer-60 WS (Shimadzu, Japan) instruments were used. Nitrogen was used as the purge gas at a flow rate of 40 mL/min. Samples were crimped in hermetic aluminum pans fitted with lids and then analyzed using a heating rate of 10 °C/ min from 30 °C to 200 °C. 2.3. Preparation of NM-SD NM and PVP/VA (Kollidon VA64) were accurately weighed and mixed by hand in a polyethylene bag for 10 min to obtain a homogeneous physical mixture. This was then extruded using a Coperion KEYATE-20 (Nanjing, China) twin-screw extruder. The extruder consisted of a hopper, barrel, die, kneading screw, and heaters distributed over the entire length of the barrel. Materials introduced into the hopper were carried forward by the feed screw, kneaded under high pressure by the kneading screw and then extruded from the die. The temperatures of the extruder barrel zones and die were set using external temperature controllers. The feed rate and screw rate were both set at 3.5 Hz. The extrudate was collected and allowed to cool at room temperature, and then milled using a laboratory cutting mill. 2.3.1. Choice of operating temperature during HME In present study, two series of temperatures were investigated. The temperatures of the extruder barrel zones and die were set as follows: series 1: Zone1 = 120 °C, Zone2 = 130 °C, Zone3 = 130 °C, Zone4 = 130 °C and Die = 80 °C, series 2: Zone1 = 140 °C, Zone2 = 150 °C, Zone3 = 150 °C, Zone4 = 150 °C and Die = 100 °C. The ratio of NM to Kollidon VA64 was set at 20:80 and the extrudate was milled to pass through a no. 80 mesh sieve after cooling at room temperature. 2.3.2. Selection of formulations of NM-SD The temperatures of the extruder barrel zones and die were set as follows: Zone1 = 140 °C, Zone2 = 150 °C, Zone3 = 150 °C, Zone4 = 150 °C and Die = 100 °C, and the extrudate was milled and passed through a no. 80 mesh sieve after cooling at room temperature. The formulations of NM-SD are shown in Table 1. 2.3.3. Optimization of the particle size of NM-SD The extrudate was milled and passed through no. 80 and no. 40 mesh sieves. The SD drug loading was 15% and the temperatures of Zone1, Zone2, Zone3, Zone4 and the Die were 140, 150, 150, 150 and 100 °C, respectively. Table 1 Formulations of NM-SD. Formulation no.
NM (%)
Kollidon VA64 (%)
SD1 SD2
20 15
80 85
215
2.4. Preparation of NM-T-SD (F13) The formulation of NM-T-SD (F13) is shown in Table 2 and the tablet weight was set at 500 mg. Firstly, the extrudate was pulverized and passed through a no. 40 mesh sieve, and then the ingredients (including lubricants) were weighed accurately and blended evenly and, finally, the powder blends were loaded into feeders and compressed into tablets using a TDP-5B type single punch tablet press (Shanghai, China). To prevent the tablets becoming moist, coating was performed with Opardry AMB in 40% alcohol as the coating material. The coating level of the tablets was 3% (w/w) and coating was carried out using a type B200/400 coating pan (Baoji, Shanxi, China). The coating temperature was 40 °C.
2.5. Dissolution testing The dissolution rate of NM under study was determined at 37 °C using a ZRS-8G dissolution apparatus. Phosphate buffer at pH 6.8 (900 mL) was chosen as the dissolution medium for the formulation and technology screening. Dissolution profiles of NM-T-SD (F13) were obtained in 0.1 mol/L HCl, in acetate buffer at pH 4.5, in purified water and in phosphate buffer at pH 6.8 to examine whether the dissolution was significantly affected by pH. The test was performed according to dissolution test method 2 as described in the China Pharmacopeia (2005) [4] with a paddle rotation speed of 75 rpm. Samples equivalent to 30 mg drug were added to the dissolution apparatus, and test fluid was withdrawn after 5, 10, 20, 30, 45 and 60 min. Dissolution samples were subsequently passed through a 0.45 μm Millipore filter and then the NM content was assayed immediately by UV spectrophotometry at 356 nm to avoid recrystallization of NM from the test fluid when the temperature became lower. In all experiments, the absorbance of the adjuvants at 356 nm was negligible.
2.6. Related compounds HPLC was used to assay related compounds of NM-T-SD (F13). A Hitachi L-2130 Intelligent Pump (Hitachi, Japan) was used. An AllsphereTM C18 column (250 mm × 4.6 mm, 5 μm, Alltech) was used with a mobile phase consisting of methanol–water (65:35, v/v). A Hitachi UV Detector L-2400 (Hitachi, Japan) was set at 235 nm and the chromatographic analyses were performed at 35 °C at a flow rate of 1.0 mL/min. The formulation of NM-T-SD (F13) has been described before. Temperatures were set at 140, 150, 150, 150 and 100 °C for Zone1, Zone2, Zone3, Zone4 and the die during HME. The final tablets were milled to a powder, and a sample equivalent to 10 mg drug was transferred to a 50 mL volumetric flask and then made up to the mark with mobile phase. After sonication, NM dissolved in the liquid completely. Then the liquid was passed through a 0.45 μm Millipore filter and 10 μL of the solution at a concentration of 200 μg/mL was injected into the HPLC system. Physical mixtures equivalent to the formulation of NM-T-SD (F13) were also processed and then assayed as described above.
Table 2 Formulation of NM-T-SD (F13) (mg per tablet). Component
Dose
NM in SD2 Ludipress® CMS-Na Talc
30 255 20 25
216
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2.7. Characterization of the state of NM 2.7.1. Differential scanning calorimetry (DSC) Differential Scanning Calorimeter-60 and Thermal Analyzer-60 WS (Shimadzu, Japan) instruments were used to characterize the thermal properties of NM-T-SD (F13). The experimental conditions were the same as described above. 2.7.2. Powder X-ray diffraction (PXRD) PXRD was performed using a D/Max-2400 X-ray Fluorescence Spectrometer (Rigaku, Japan) with a CuKa line as the source of radiation. Standard runs were carried out using a voltage of 56 kV, a current of 182 mA and a scanning rate of 2° min− 1 over a 2-theta range of 3–45°. The samples were NM, NM-T-SD (F13) and its adjuvants. 2.8. Determination of the glass transition temperature of the extrudate Temperature modulated DSC was used to determine the Tg of the NM-SD2, using a DSC 1 differential scanning calorimeter (Mettler Toledo, Switzerland) and applying TOPEM® with a modulation amplitude of ±0.5 °C and a heating rate of 1 °C/min. Nitrogen was used as the purge gas at a flow rate of 40 mL/min. Samples were crimped in aluminum pans with a pin hole. 2.9. Infrared spectroscopy Fourier-transform infrared (FT-IR) spectra were obtained on a BRUKER IFS 55 FT-IR system using the KBr disk method. The scanning range was 4000–400 cm− 1 and the resolution was 1 cm− 1. 2.10. Stability testing The stability of NM-T-SD (F13) was tested during storage (40 °C, RH 75%). NM-T-SD (F13) was sealed tightly in commercial packing. The stability was evaluated using four methods: dissolution testing, DSC, PXRD and FT-IR. The parameters were the same as those described above. 2.11. Bioavailability study 2.11.1. Administration program The study protocol was approved by the Ethics Committee of Shenyang Pharmaceutical University. The study design involved fasting, a single dose of four treatments and four periods. Each dog was given four preparations with a oneweek washout period between each. Six beagle dogs were used for each treatment group. After fasting overnight, preparations containing 60 mg NM (Nimotop®, NM-T-C, NM-T-SD (F11) and NM-T-SD (F13)) were given to the beagle dogs with 200 mL water. Four hours after dosing, the dogs were provided with standard food. On each dosing day, blood samples were taken before and then 0.1667, 0.333, 0.6667, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 16, and 24 h after dosing. Plasma was separated from samples by centrifugation (4000 rpm for 10 min) and stored at − 20 °C until analysis within one month. 2.11.2. Plasma sample preparation To prevent the photodegradation of nimodipine, all the experiments, including standard and QC preparation, plasma collection, sample preparation and instrumental analysis, were performed under dim light. For this, 0.5 mL plasma was spiked with 50 μL internal standard solution (nitrendipine, 4 μg/mL dissolved in methanol) and 50 μL methanol. The sample was subsequently made alkaline with 100 μL 0.1 M NaOH solution and extracted with 3 mL extraction solvent (n-hexane/diethylether = 1:1, v/v) by vortexing for 10 min. After centrifugation at 4000 rpm for 10 min, 2 mL of the supernatant
was transferred to a conical tube. The separated organic phase was then evaporated at 50 °C using a Centrifugal Concentrator (Centrivap® 78120-03, Labconco, Corp. USA). The residue was reconstituted with 1 mL methanol and vortexed for 5 min. After centrifugation at 12,000 rpm for 10 min, a 5 μL aliquot was injected into the UPLC-MS/ MS system. 2.11.3. Analysis conditions and method validation The analysis was carried out on an ACQUITYTM UPLC system (Waters Corp., Milford, MA, USA) with a cooling autosampler and column oven. An ACQUITY UPLCTM BEH C18 column (50 mm × 2.1 mm, 1.7 μm; Waters Corp., Milford, MA, USA) was employed for separation with the column temperature maintained at 35 °C. The chromatographic separation was achieved with gradient elution using a mobile phase composed of water containing 0.1% (v/v) formic acid and acetonitrile. The gradient elution started at 50% acetonitrile, increased linearly to 80% acetonitrile over 1 min, was maintained at 80% for 1.3 min and then returned to the initial percentage over 0.2 min and was maintained there for another 0.5 min. The flow rate was set at 0.2 mL/min. The autosampler temperature was kept at 4 °C and a sample solution of 5 μL was injected. A Waters ACQUITYTM TQD triple quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) with an electrospray ionization (ESI) interface was employed for mass analysis. The ESI source was operated in positive ionization model with optimal operation parameters as follows: capillary voltage 3.70 kV, cone voltage 20 V, extractor 3.0 V and RF 0.1 V with a source temperature of 100 °C and a desolvation temperature of 400 °C. Nitrogen was used as the desolvation and cone gas at a flow rate of 450 L/h and 50 L/h, respectively. For collision-induced dissociation (CID), argon was used as the collision gas at a flow rate of 0.17 mL/min. The quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 419 → 343 for nimodipine and m/z 361 → 315 for nitrendipine (I.S.), respectively. All data were collected in centroid mode and processed using MassLynxTM NT 4.1 software with a QuanLynxTM program (Waters Corp., Milford, MA, USA). The linear range of this method was 1–1000 ng/mL with an r (correlation coefficient) value of no less than 0.99. The LLOQ was 1 ng/ mL. The R.S.D. which reflected the intra-day and the inter-day precision of the QC samples were both not more than 5.4%. The R.E. of the QC samples was less than ±8.4%. The extraction recoveries of NM from the beagle dog QC plasma samples were 92.7±7.0%, 100.9±4.6% and 105.2 ±4.8%, respectively, and the mean extraction recovery of nitrendipine was 96.8±10.5%. 2.11.4. Calculation parameters The area under the plasma concentration–time curve (AUC0 → t) up to the last measurable plasma concentration (Ct) was calculated by the linear trapezoidal rule. 3. Results and discussion 3.1. Miscibility study by DSC In order to assess the behavior under thermal processing conditions, differential scanning calorimetry was applied to assess the processability of the drug: carrier physical mixtures. Interestingly, the endothermic peak of NM in the pure drug and the physical mixture (PM) is significantly different, as shown in Fig. 1. The melting peak of the pure drug is typically sharp, whereas the endothermic peak in the physical mixture becomes much broader. A significant reduction in melting temperature was also observed, with a peak temperature of 112 °C in the PM compared with 129 °C in the pure drug. The above phenomena were both caused by the gradual dissolution of NM in the carriers during the DSC heating ramp [3,5–6] and provided strong
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the carrier could not dissolve all the drug and much of the drug was in the crystalline form instead of the amorphous form, with the result that the drug release was only about 35% in one hour for NM-SD1, showing that the drug loading should be reduced. However, as SD is difficult to handle due to its high viscosity and sensitivity to moisture, the amount of SD contained in tablets should be as low as possible. If the drug loading was 10%, over half the tablet weight would be SD, which is not acceptable as far as flowability is concerned and the amount of dispersion required to administer the usual dose of the drug may be too high to produce a tablet or capsule that can be easily swallowed. Finally, the ratio of NM to Kollidon VA64 was set at 15:85. Fig. 1. DSC thermograms of the pure NM (a) and the mixture of NM and Kollidon VA64 (15:85) (b).
evidence for the miscibility of NM with Kollidon VA64, which suggested that Kollidon VA64 would be an optimum carrier for NM-SD. 3.2. Dissolution profiles of NM-SD and NM-T-SD (F13) 3.2.1. Choice of temperatures used during HME It was clear from Fig. 2 that increasing the temperature during HME led to an increased dissolution. As the softening point of Kollidon VA64 is quite high (about 180 °C) while the melting point of NM is only about 130 °C, when the temperatures used during HME were set around 130 °C, Kollidon VA64 was not softened effectively and exhibited a high viscosity, so, the drug and carrier could not be mixed to obtain a uniform state. When extrusion was performed at low temperatures, much of the NM was present in the crystalline form instead of the amorphous form, and the resulting dissolution was less than 15% in one hour. However, if the temperature exceeded 160 °C, the color of the extrudate changed from gold to brown which indicated that degradation of NM occurred at that temperature, so the temperature should not exceed 150 °C during HME. As a result, the temperatures used during HME were finally set at 140, 150, 150, 150 and 100 °C for Zone1, Zone2, Zone3, Zone4 and the die, respectively. 3.2.2. Choice of drug loading in NM-SD In a previous study, it was found that the drug loading affected the dissolution behavior in vitro. So, in the present study, the ratio of NM to Kollidon VA64 was also investigated. It can be seen from Fig. 2 that the drug release increased from about 35% to 70% when the drug loading fell from 20% to 15%. So, it was concluded that the drug loading markedly affected the dissolution of NM-SD. This is because there is a limited ability of the carrier to dissolve NM. When the ratio of NM to Kollidon VA64 was 20:80 in SD,
Fig. 2. Dissolution profiles of NM-SD in phosphate buffer at pH 6.8 (n = 3). (♦) temperature: 120, 130, 130, 130 and 80 °C, drug content 20%, particle size no. 80 mesh; (■) temperature: 140, 150, 150, 150 and 100 °C, drug content 20%, particle size no. 80 mesh; (▲) temperature: 140, 150, 150, 150 and 100 °C, drug content 15%, particle size no. 80 mesh; (△) temperature: 140, 150, 150, 150 and 100 °C, drug content 15%, particle size no. 40 mesh.
3.2.3. Selection of the particle size of SD As the drug loading of SD decreased, the amount of SD contained in the final dosage form increased. If the extrudate was still pulverized to pass through a no. 80 mesh sieve, good flowability ensuring automatic and continuous tabletting could not be achieved. So, the extrudate was pulverized to obtain a larger particle size. It can be seen from Fig. 2 that, after milling to no. 40 mesh instead of no. 80 mesh, the drug release increased from about 70% to 90%. During the dissolution testing, it was found that SD powders aggregated and floated on the dissolution medium within about 20 min and gradually dissolved when the particle size was no. 80 mesh, while it dispersed and dissolved in the dissolution medium immediately when the particle size was no. 40 mesh. It is likely that this phenomenon was due to the poorer wettability of SD powders with a particle size of no. 80 mesh compared with no. 40 mesh. Although the specific surface area increased as the particle size decreased, due to aggregation of SD powders because of the poor wettability, the effective specific surface area was reduced, which had an adverse effect on the dissolution of NM from SD. Finally, the flowability of the powder blends for direct compression increased when the particle size of NM-SD increased. The above factors were both favorable for direct compression and dissolution. As the particle size of Ludipress® is about no. 40 mesh, taking content uniformity into account, the particle size of NM-SD should not be greater, so, finally, the NM-SD2 produced by HME was pulverized and passed through a no. 40 mesh sieve in the present study. 3.2.4. Dissolution profiles of NM-T-SD (F13) in different media The dissolution behaviors of NM-T-SD (F13) obtained by HME combined with direct compression as described above in dissolution medium at different pH values are illustrated in Fig. 3. It can be seen from the figure that the drug release in different media exceeded 80% in every case. It was concluded that NM-T-SD (F13) did not exhibit a high pH-dependence. Because of this pHindependence, NM could be released from tablets in any part of gastrointestinal tract and, as a result, the in vivo bioavailability might be less affected by physiological factors compared with NM-T-SD (F11) in the previous study. It also can be inferred from the dissolution profiles that, after 20–30 min, dissolution profiles in different media were reduced. This is because after more than 80% of NM was
Fig. 3. Dissolution profiles of NM-T-SD (F13) in different media (n = 3). (♦) 0.1 mol/L HCl; (■) acetate buffer at pH 4.5; (▲) phosphate buffer at pH 6.8; (△) purified water.
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released, the drug was present in supersaturated form in the dissolution medium, which means that NM is not stable in the dissolution medium. So it easily recrystallizes when stirred. Generally speaking, compared with the dissolution behavior of Nimotop® in different media in our previous study, the dissolution rate of NM-T-SD (F13) was faster and the drug release was higher than that of Nimotop®. However, recrystallization was effectively inhibited in the case of Nimotop® as indicated by the finding that the dissolution curves did not decline in one hour, while drug crystals were formed after the maximum dissolution of NM-T-SD (F13) had occurred. Although, in the present study, this could not be achieved, it might not affect the bioavailability greatly as long as the uptake of NM was complete in about 20 min since NM was released from NM-T-SD (F13).
Fig. 5. DSC thermograms of NM (a) and NM-T-SD (F13) (b).
3.3. Related compounds Related compounds of NM-T-SD produced by HME combined with direct compression assayed by HPLC are illustrated in Fig. 4. From Fig. 4, it can be seen that no new related compounds were produced during HME although the area of the impurities became larger. The area of any individual impurity was not more than 0.2% and no more than 0.5% total impurities were found after HME. This result met the requirement of the China Pharmacopeia (2005) [4] and indicated that 150 °C used during HME was acceptable and no obvious degradation was observed at this temperature. 3.4. Physical characterization 3.4.1. Physical characterization by DSC Fig. 5 shows the DSC thermograms over the temperature range 30– 140 °C. The DSC recording of the pure NM exhibits a sharp endothermic peak around 129 °C, while NM-T-SD (F13) resulted in a complete suppression of the drug fusion peak. The DSC data showed that, using HME, NM was completely converted into an amorphous state in the carrier and this state was not altered by compression and coating. So, it could be concluded that the temperatures chosen and the ratio of NM to Kollidon VA64 selected were suitable to produce NM in an amorphous state. Another important conclusion was that Kollidon VA64 was still an excellent carrier for NM when the parameters of HME and formulation of SD were optimized, although the softening point of Kollidon VA64 (180 °C) was much higher than the melting point of NM (130 °C).
Fig. 6. PXRD patterns. (a) Kollidon VA64; (b) NM-SD2; (c) adjuncts of NM-T-SD (F13); (d) NM-T-SD (F13); (e) pure NM.
3.4.2. Physical characterization by PXRD PXRD was used to confirm the loss of drug crystals, and the results are shown in Fig. 6. Pure NM has several major peaks at 2-theta angles within 30° (2-theta angles of 6.5, 12.3, 12.8, 17.3, 19.7, 20.3, 20.8, 23.9, 24.7 and 26.3°). For NM-SD2 and NM-T-SD (F13), no detectable diffraction peak of NM was observed, suggesting that NM was in an amorphous state in NM-SD2 and NM-T-SD (F13). These results indicated that NM was transformed from the crystalline form to the amorphous form by HME and the amorphous state was not destroyed by compression and coating. Furthermore, this offered the possibility of high dissolution and, as a result, a high bioavailability in vivo. 3.5. Determination of glass transition temperature (Tg) It can be seen from Fig. 7 that the measured Tg of NM-SD2 is 89 °C and, as a result, it is estimated that the NM-T-SD prepared containing NM-SD2 will be stable during long-term storage (20–30 °C) [7,8]. 3.6. FT-IR In order to study the possibility of an interaction of NM with Kollidon VA64 in the solid state, information was gathered using FT-IR spectroscopy. From the structures of NM and Kollidon VA64 (Fig. 8), it can be assumed that a possible interaction could occur between the secondary amine hydrogen atom of NM and the amide function or ester function of Kollidon VA64. Thus, in this case any sign of interaction would be reflected by shifts in the C O vibration, depending on its extent. H-bonding leads to a shift of the peak maxima towards a lower wave number (bathochromic shift) and very often the peak width is increased [9,10]. The infrared spectra of NM, Kollidon VA64 and the SD are shown in Fig. 9. The position of the absorption bands of the ester function and amide function of Kollidon
Fig. 4. HPLC chromatogram of NM-T-SD (F13) before (1) and after (2) HME. (a) Impurity 1; (b) impurity 2; (c) NM; (d) impurity 3; (e) impurity 4.
Fig. 7. The revering heat flow profile of the NM-SD2.
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Fig. 8. Molecular structures of the compounds and polymers.
VA64 remained unchanged at 1739 and 1678 cm− 1 in SD, respectively. These results indicated the absence of a well-defined interaction between NM and Kollidon VA64. 3.7. Stability testing 3.7.1. Dissolution testing After storage for six months (40 °C, RH 75%), dissolution testing of NM-T-SD (F13) at several time intervals was carried out in different media. The results are shown in Fig. 10. It can be seen clearly from the figures that after storage (40 °C, RH 75%), the dissolution profiles were similar to the initial state, especially in the case of purified water, so it can be concluded that the amorphous state of NM in NM-T-SD (F13) was not destroyed and ageing did not occur during storage, which indicated that NM-T-SD (F13) made by HME combined with direct compression was stable. Greater variations were shown in 0.1 M HCl, acetate buffer at pH 4.5 and phosphate buffer at pH 6.8 because of the differences in the ionic strength in the media at different time intervals.
the polymer or interactions between the drug and the polymer, or a combination of both [12,13]. An anti-plasticizing effect of the polymer may contribute significantly by increasing the temperature at which the molecular mobility becomes significant with respect to recrystallization. In addition, the resulting system will exhibit high viscosity at room temperature, hence impairing crystallization [13]. As described above, although there were no interactions between NM and Kollidon VA64, the Tg of NM-SD2 was nearly 50 °C above storage temperature (40 °C) and, according to Hancock and Zografi [7,8], NM-SD2 would be
3.7.2. Physical characterization DSC and PXRD recordings of NM-T-SD (F13) before and after storage for one and two months (40 °C, RH 75%) are illustrated in Figs. 11 and 12. It can be seen from Fig. 11 that there was no endothermic peak around 130 °C and no detectable diffraction peak of NM in Fig. 12 after storage for two months, these results showed that NM remained in an amorphous state during storage. 3.7.3. FT-IR FT-IR profiles of NM-T-SD (F13) before and after storage for six months (40 °C, RH 75%) are illustrated in Fig. 13. No differences were seen among the profiles at different time intervals, which indicated that no changes have occurred to NM-T-SD (F13) and it was stable during storage for six months (40 °C, RH 75%). With respect to the physical stability of the amorphous state, it is generally accepted that recrystallization is dependent on the molecular mobility and the degree of supersaturation of the solute in the matrix [11]. As the molecular mobility increases, the risk of crystallization also increases. Usually, the addition of a polymer with a high Tg is sufficient to prevent crystallization. The protective effect of the polymer can be caused by two factors: an anti-plasticizing effect of
Fig. 9. FT-IR spectra: (a) NM; (b) Kollidon VA64; (c) NM-SD2.
Fig. 10. Dissolution profiles of NM-T-SD (F13) in different media after storage (n = 3). (♦) initial state; (■) one month; (▲) two months; (♢) three months; (□) four months; (△) five months; (×) six months.
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Fig. 13. FT-IR spectra of NM-T-SD (F13) after storage: (a) initial state; (b) one month; (c) two months; (d) three months; (e) four months; (f) five months; (g) six months. Fig. 11. DSC thermograms of NM-T-SD (F13) after storage. (a) The pure NM; (b) NM-TSD (F13) before storage; (c) NM-T-SD (F13) after storage for one month; (d) NM-T-SD (F13) after storage for two months.
stable during storage (40 °C, RH 75%). The good stability of NM-T-SD (F13) was also proved in this study and it was demonstrated that the anti-plasticizing effect of the polymer which increased the viscosity of the binary system and decreased the diffusion of drug molecules necessary to form a lattice was the only stabilizing factor in the dispersions of NM and Kollidon VA64. 3.8. Bioavailability in vivo NM-T-SD produced by direct compression tabletting (F11, F13) was used to study the bioavailability in vivo, with Nimotop® (Bayer, H200030010 Beijing, China) and NM-T-C as reference agents. The plasma concentration versus time curves for the four preparations are shown in Fig. 14 and the bioavailability parameters are given in Table 3. The Cmax of Nimotop® and NM-T-C was 409.2 ± 200.1 and 302.0 ± 167.4 ng/mL respectively, while the Cmax of NM-T-SD (F11) was only 115.5 ± 65.0 ng/mL, much lower than that of the references, although the AUC0 → 24 of Nimotop®, NM-T-C and NM-T-SD (F11) were similar (861.5 ± 296.3, 705.0 ± 177.7 and 720.2 ± 625.8 ng h/mL). The low Cmax of NM-T-SD (F11) might be caused by Eudragit® EPO used as a carrier during HME. Eudragit® EPO is a highly pH-dependent material which dissolves only when the pH of the medium is below 5.0. As indicated by the dissolution profiles of NM-T-SD (F11) in different media in vitro, NM could not be released from NM-T-SD (F11) in phosphate buffer at pH 6.8 and purified water. Perhaps NM-T-SD (F11) entered the small intestine before NM was released completely and, as the pH of the intestinal fluid was higher than 5.0, NM still in the tablets could not be released at all as indicated by the dissolution in vitro. So, from the moment NM-T-SD (F11) entered the small intestine or slightly later, uptake of NM ceased and the increasing plasma concentration due to the release of NM from NM-T-SD (F11) in the stomach could not continue and, as a result, the Cmax of the test was quite low. The pharmacokinetic behaviors of Nimotop® and NM-T-SD (F13) were similar as indicated by the Cmax and AUC0 → 24. After excluding Eudragit® EPO from formulations of SD, the Cmax increased markedly from 115.5 ± 65.0 ng/mL to 334.3 ± 149.9 ng/mL. Without Eudragit® EPO, NM could be released from the dosage form in any part of the
Fig. 12. PXRD recordings of NM-T-SD (F13) after storage. (a) NM-T-SD (F13) before storage; (b) NM-T-SD (F13) after storage for one month; (c) NM-T-SD (F13) after storage for two months; (d) pure NM; (e) adjuncts of NM-T-SD (F13).
gastrointestinal tract after oral administration and, as a result, the influence of physiological factors on the in vivo behavior of NM-T-SD (F13) was reduced, as indicated by the higher Cmax. This proved that the hypothesis above was correct and Eudragit® EPO was not an ideal carrier for HME because of its high pH-dependence although an amorphous state of NM could be achieved at a lower temperature and with increased drug loading. It was reported by other authors that the Cmax and the AUC0 → 12 of the pure NM were only a bit more than 30% of those of Nimotop® [14]. As the Cmax and the AUC0 → 24 were similar between the NM-T-SD (F13) and Nimotop® in the present study, it can be concluded that HME was quite an efficient technology in improving the oral bioavailability of NM. It can be seen from the dissolution profiles in vitro that the dissolution curve of NM-T-SD (F11) was higher than that of the references for most of the time in 0.1 mol/L HCl. However, taking the high pH-dependence of NM-T-SD (F11) into account, NM released from NM-T-SD (F11) differed significantly among beagle dogs as the physiological state of the beagle dogs was different and it may be that NM was released almost completely from NM-T-SD (F11) in some beagle dogs while only a little was released in others, as indicated by the much higher R.S.D. of the AUC0 → 24 of NM-T-SD (F11) (86.9%) compared with Nimotop® (34.4%) and NM-T-C (25.2%). For these two reasons, the AUC0 → 24 of NM-T-SD (F11) was slightly lower than that of Nimotop® and similar to that of NM-T-C. Another interesting phenomenon was observed in Fig. 14. Double peaks were observed in the plasma concentration versus time curves of NM-T-SD (F11) and commercially available NM-T-C and there was a shoulder peak in the plasma concentration versus time curves of NMT-SD (F13), while this was not the case for Nimotop®. This phenomenon resulted in a higher bioavailability of NM-T-SD (F13) compared with Nimotop®, although the Cmax was lower. It seems that the plasma concentration versus time curves for NM-T-SD (F11, F13) and NM-T-C were not classic. Perhaps this phenomenon was caused by recrystallization of NM in the gastrointestinal tract after release from NM-T-SD (F11, F13) as indicated by the dissolution profiles in vitro. If recrystallization occurred, although NM in crystalline form was carried down to the effective absorption region, it could not be
Fig. 14. Mean NM plasma profiles from a single dose (60 mg) bioavailability study compared with Nimotop® (n = 6). (♦) Nimotop®; (■) NM-T-C; (▲) NM-T-SD (F11); (×) NM-T-SD (F13).
F. Jijun et al. / Powder Technology 204 (2010) 214–221 Table 3 Bioavailability parameters of NM for different tablets in beagle dogs (n = 6). Sample
Cmax (ng/mL)
tmax (h)
AUC0 → 24 (ng h/mL)
Nimotop® NM-T-C NM-T-SD (F11) NM-T-SD (F13)
409.2 ± 200.1 302.0 ± 167.4 115.5 ± 65.0 334.3 ± 149.9
0.8 ± 0.3 1.5 ± 1.6 1.6 ± 1.1 1.8 ± 2.3
861.5 ± 296.3 705.0 ± 177.7 720.2 ± 625.8 1060.9 ± 430.7
Each value represents the mean ± S.D.
taken up immediately while, in the amorphous state, it was taken up as soon as it reached the effective absorption region. The uptake of the amorphous drug resulted in the first peak in the plasma concentration versus time curve. Although the drug release of NM-T-SD (F11, F13) was higher than Nimotop®, some of the NM released from NM-T-SD (F11, F13) recrystallized and could not be taken up immediately while nearly all the NM released from Nimotop® was present in the amorphous form and was taken up rapidly, so the Cmax of NM-T-SD (F11, F13) was lower. Only after NM in crystalline form was emulsified by endogenous surfactants such as bile, could the drug be taken up later and, as a result, shoulder or double peaks appeared. Investigation of the dissolution behavior in vitro showed that the rate of recrystallization was much slower for Nimotop® compared with all the other dosage forms, so only a single peak appeared in the plasma concentration versus time curve of Nimotop®. It can be seen from the dissolution profiles in vitro that the dissolution curve of NM-T-SD (F13) was higher than that of Nimotop® for most of the one hour period and NM could be released from NM-T-SD (F13) in media with different pH values as Nimotop®, so the AUC0 → 24 of NM-T-SD (F13) was higher than that of Nimotop®. For the above reasons, the AUC0 → 24 of NM-T-SD (F13) was higher than that of Nimotop® while the Cmax was lower. It can be seen from the above curve that the peak in the plasma concentration versus time curve of NM-T-SD (F11, F13) was earlier than that of Nimotop®, which was attributed to the faster dissolution of NM-T-SD (F11, F13) compared with Nimotop®, as indicated by the dissolution in vitro. However, as there were double peaks or the shoulder peak of NM-T-SD (F11, F13) in the plasma concentration versus time curve, the tmax of the tests (1.6, 1.8 h) were later than that of Nimotop® (0.8 h). 4. Conclusions By increasing the temperature during HME and reducing the drug loading in SD, NM could be obtained in an amorphous state in the present study and this amorphous state of NM was demonstrated by DSC and PXRD. Related compounds assayed by HPLC showed that no dramatic degradation of NM occurred during HME and indicated that
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the temperature used during HME was acceptable. The suitable particle size of NM-SD was also studied. NM-T-SD (F13) was made by HME combined with direct compression and its dissolution behavior in different media proved that it was pH-independent. The results of the stability study showed that the amorphous state of NM in NM-TSD (F13) was not destroyed during two months of storage (40 °C, RH 75%) and further studies are in progress. The bioavailability study showed that NM-T-SD (F13) exhibited a similar behavior in vivo to Nimotop®, while the Cmax of NM-T-SD (F11) was much lower than that of Nimotop®, which confirmed the belief that Eudragit® EPO should not be included in formulations of SD because of its highly pHdependent nature.
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