- Email: [email protected]
Characterization of CoQ 10-lauric acid eutectic system
Accepted Manuscript Title: Characterization of CoQ 10-Lauric Acid Eutectic System Author: Bapurao Tarate Arvind K. Bansal PII: DOI: Reference:
S0040-6031(15)00026-X http://dx.doi.org/doi:10.1016/j.tca.2015.01.018 TCA 77129
To appear in:
Thermochimica Acta
Received date: Revised date: Accepted date:
22-9-2014 29-12-2014 25-1-2015
Please cite this article as: Bapurao Tarate, Arvind K.Bansal, Characterization of CoQ 10-Lauric Acid Eutectic System, Thermochimica Acta http://dx.doi.org/10.1016/j.tca.2015.01.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of CoQ 10-Lauric Acid Eutectic System Bapurao Tarate and Arvind K. Bansal* Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali), Punjab- 160062 (INDIA)
*Address correspondence to this author at the Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar (Mohali), Punjab- 160 062 (India). Tel:
+91
172
2214682;
Fax:
+91
172
2214692.
E-mail
address:
[email protected]
and
[email protected]
Highlights
Two polymorphic forms of CoQ 10.
CoQ 10 forms eutectic mixture with LA.
Experimental and predicted values of eutectic point are 70% CoQ 10 at 37.93 C ̊ and 87.7% CoQ 10 at 38.98 C ̊ , respectively.
Attraction exists between CoQ 10 and LA molecules.
Abstract Solid state characterization of Coenzyme Q10 (CoQ 10) was carried out using differential scanning calorimetry (DSC), variable temperature X-ray diffractometry (VT-XRD) and hot/cold stage microscopy (H/CSM). It revealed that CoQ 10 exists in two polymorphic forms. The recrystallized samples of CoQ 10 melted at different temperatures either due to the wide crystal size variation or change in crystallinity. Further, the binary mixture of CoQ 10 and lauric acid (LA) formed eutectic mixture in the ratio 70:30 melting at 37.93 ̊C, which was close to the predicted eutectic composition of 87.7:12.3 melting at 38.98 C ̊ . The values of actual liquidus temperatures for CoQ 10 are higher than the predicted liquidus temperatures. The experimental heat of fusion at eutectic point was less than the calculated heat of fusion. Activity coefficient of CoQ 10 in the binary mixture was less than unity, which indicates the attraction between the components of eutectic mixture. Keywords: Coenzyme Q10 (CoQ 10), DSC, enthalpy, eutectic, lauric acid, polymorphic forms.
1
Introduction
Coenzyme (CoQ 10) is a poorly water soluble [1] vitamin like substance. It is a lipophilic, naturally occurring substance having a role as an intermediate of electron transport system, an antioxidant, and is involved in cellular metabolism [2]. Apart from poor water solubility, bioavailability of CoQ 10 is limited by its high molecular weight [1] and efflux by p-glycoprotein [3]. Various methods for enhancement of bioavailability of CoQ 10 are reported in literature. Self nanoemulsifying drug delivery system (SNEDDS) of CoQ 10 with Witepsol® H35, Solutol® HS15, and Lauroglycol® FCC has been reported. The formulation provided 90% release of CoQ 10 in pH 6.8 phosphate buffer and displayed 4.6 and 5.2 fold increase in AUC and Cmax, respectively, over powder formulation [4]. Spray drying of CoQ 10 with polymeric excipients such as polyvinylpyrrolidone
(PVP),
hydroxypropylmethylcellulose
acetate
succinate
(HPMCAS)
or
hydroxypropylmethyl cellulose pthalate (HPMCP) generated amorphous CoQ 10, resulting in enhanced bioavailability by reducing the time for dissolution of 50% CoQ 10 by 100% or more [5]. CoQ 10 has a dose of 100–3000 mg per day [6], a melting point of around 50 C ̊ [7], and a log P of about 15 [8]. This makes it a good candidate for oral delivery using lipidic formulations. For oral delivery using lipidic formulation, we carried preliminary investigations for in-situ crystallization of binary mixture of CoQ 10 and Lauric acid (LA) using DSC. Binary lipidic mixture of CoQ 10-L-menthol investigated by DSC is reported in literature [9]. Few examples of eutectic mixtures having applications in drug delivery are lidocaine-prilocaine [10], aspirin-acetaminophen-urea [11], and curcumin with conformers such as nicotinamide, ferulic acid, hydroquinone, p-hydroxybenzoic acid, and L-tartaric acid [12]. Thermal characterization of various compositions of CoQ 10-LA products revealed generation of eutectic mixture of CoQ 10 with LA. This work covers the solid state characterization of CoQ 10 and construction of binary phase diagram for eutectic mixture of CoQ 10 and LA. 2
Materials and methods
1.2 Materials All the experiments were carried out on commercial reduced CoQ 10 samples procured from Hangzhou Joymore Technology Co., Ltd., China. At the time of experimentation, the samples contained less than 29.4% of reduced CoQ 10 due to its oxidation during storage. Oxidized CoQ 10 from Hangzhou Joymore Technology Co., Ltd., China was used as a reference sample. Sample of LA was obtained from Loba Chemie Pvt. Ltd., India. 2.3 Methods 2.3.1
CoQ 10:LA mixtures for generation of eutectic system
Eutectic mixtures of CoQ 10 with LA were prepared by weighing the different proportions of CoQ 10:LA in the range between 5:95 to 95:5 in the DSC pans. It was then subjected to heat-cool-heat cycle protocol mentioned in the proceeding section. 2.3.2
Differential scanning calorimetry (DSC)
Heat-flux DSC measurements were performed with the DSC instrument (TA Q2000, New Castle, Delaware, USA equipped with TA Universal Analysis software). It was calibrated for temperature and heat flow by using high purity indium standard. Accurately weighed amount 2.5 to 5 mg of CoQ 10, LA, and CoQ10:LA binary mixture were weighed in aluminium pan, cripped, and heated to 80 ̊C, at the heating rate of 10 ̊C/minutes, and then held isothermally for 5 minutes. The sample was then cooled to -80 C ̊ , held isothermally for 5 minutes, followed by heating at the same rate as mentioned previously. Nitrogen was purged during the DSC operation at the rate 50 ml/min. 2.3.3
Hot and cold stage microscopy (H/CSM)
Hot stage microscopy (HSM) and cold stage microscopy (CSM) of CoQ 10 samples were performed on Leica DMLP polarized microscope (Leica Microsystems Wetzalar GmBH, Germany) equipped with Linkam LTS 350 hot/cold stage. Small quantity of sample was placed on a glass slide, mixed with silicon oil, and covered with a glass cover slip. It was heated and cooled using a protocol similar to DSC operation. 2.3.4
Variable temperature X-ray diffractometry (VT-XRD)
Powder X-ray diffraction patterns were recorded on diffractometer using Cu-Kα X- ray radiation at 40 Kv power and 40 mA current. The wavelength of radiation was 2.54 Å. X-ray diffraction patterns were collected over 2θ range 3-40 at the scan rate of 0.01 and scan speed of 0.1. The measurements were carried out on the variable temperature stage to confirm the presence of pure components at different temperature. The heating and cooling protocol was similar to DSC. 3
Results and discussion
3.1 Solid state characterization of CoQ 10 3.1.1
DSC and H/CSM
All the experiments were conducted using reduced CoQ 10. However, due to its low atmospheric stability, it converted into the oxidized CoQ 10, and very small quantity of reduced CoQ 10 remained in the sample. This was evident from the orange colour of sample, since oxidized CoQ 10 is orange in colour while reduced CoQ 10 is of white colour [13]. Thus, all the studies were carried out on the CoQ 10 which was predominantly in the oxidized form. The DSC analysis of CoQ 10 was performed under different experimental protocols and the same is represented in shows Fig. 1 and Fig. 2. Fig. 1 shows the heat-cool-heat cycle of CoQ 10 at a fast cooling rate of 16 ̊C/min. Sample of the pure oxidized CoQ 10 was also characterized using similar protocol. First heating cycle of CoQ 10 and oxidized CoQ 10 indicated melting endotherm at 50.80 and 51.28 C ̊ , respectively. This is in conformity with reported literature, wherein melting points of 49.5 [14] and 49 C ̊ [15] have been reported for reduced and oxidized forms of CoQ 10, respectively. Thereafter, molten samples of CoQ 10 were cooled to -80 ̊C at a fast cooling rate of 16 C ̊ /min. Commercial and pure oxidized samples of CoQ 10 exhibited a recrystallization exotherm at around -14.58 and -8.98 C ̊ , respectively. This event was much sharper in case of oxidized CoQ 10.
Second heating cycle of commercial CoQ 10 indicated a subtle event at -72.60 C ̊ , which can be attributed to amorphous phase. It shows another exothermic event at -33.4 ̊C, which can be attributed to crystalization of amorphous CoQ 10. A combination of exothermic-endothermic-exothermic-endothermic events was observed at 16.11, 23.79, 26.34, and 45.52 C ̊ . A similar set of events was observed for oxidized CoQ 10 at 16.11, 23.31, 25.06, and 48.72 C ̊ .The oxidized CoQ 10 did not exhibit the thermal event corresponding to amorphous phase at any temperature. Therefore, the exothermic event at around 33 C ̊ was also absent in oxidized CoQ 10. A heat-cool-heat cycle at a heating and cooling rate (slow) of 10 C ̊ /min was carried out in an attempt to further resolve these events. Fig. 2 captures the heating curve, along with photomicrographs taken using hot stage microscope, at slow cooling rate. In the first cooling cycle, it shows recrystallization exotherm at -14.2 C ̊ . In slow and fast cooling, recrystallization of CoQ 10 occurred nearly at the same temperature. It was reported for CoQ 10 that lower the rate of cooling of molten sample, lower is the tempearature of recrystallization [16]. This was not observed in this study because difference in two cooling rates may not be too high. In the second heating cycle, a glass transition event at approximately -72.6 C ̊ indicated that only partial recrystallization occurred during cooling curve. Glass transition was followed by an exothermic event at 34.88 C ̊ . This event was observed at fast cooling rate also. This was followed by two exothermic events at 15.47 and 26.20 C ̊ . Additionally, two endothermic events were observed at 23.31 and 45.20 C ̊ . This is in good agreement with the previously reported study, in which CoQ 10 was found to exist in two crystalline phases. The metastable phase gets converted into the stable crysalline phase through such exothermic-endothemicexothermic events observed between 15 to 25 C ̊ [16]. The molten portion at -80 C ̊ is ‘undercooled liquidus’ below the thermodynamic equilibrium which can crystallize on the surface of existing crystals. Due to high temperature the formation of new nuclei and their growth is not possible, giving rise to the exotherm at 15.47 C ̊ [17]. This is followed by melting of metastable phase, crystallization of stable phase, and finally its melting.
3.1.2
VT- XRD
The powder XRD pattern of CoQ 10 at different tempearures was compared with its XRD spectra at 25 C ̊ in Fig. 3. The powder XRD of commercial sample, labelled as 25 C ̊ in Fig. 3, of CoQ 10 shows two major peaks at 2θ values around 18.5 and 23̊. Apart from this, it showed a small peak at 2θ value of 20.5 and many small peaks in between 25 to 40̊. The XRD spectra of oxidized CoQ 10 shows many peaks apart from sharp peak at 2θ value of 18º [18]. The sample of commercial CoQ 10 at the tempearure -10, -80, -33 and 16 C ̊ exhibited similar XRD pattern, wherein only two peaks at 2θ values of 18.5 and 23 were observed. Here, the small peaks observed in the range of 25 to 40 C ̊ were completely absent. Powder XRD pattern of CoQ 10 at 27 C ̊ was found to be similar to that of 25 C ̊ , except change in intensity, which may be due to reduced crystallinity of stable crystalline phase. Similar observations were obtained when CoQ 10 was studied with wide angle X-ray diffraction methods [16]. This indicates the existance of two crystalline phases viz., metastable and stable forms of CoQ 10, which is consistant with the DSC data discussed previously. Thus, it can be concluded from DSC, H/CSM, and VT-XRD studies that CoQ 10 exists as two crystalline forms viz., metastable form existing below 25 C ̊ and stable form above room temperature.
3.2 Eutectic phase diagram of CoQ 10:LA mixture As shown in Fig. 4, many thermal events were observed in the binary mixture of CoQ 10 and LA. These mixtures showed disappearance of melting endotherm at around 45 C ̊ of pure CoQ 10 treated similar to binary mixtures. Many thermal events observed before melting of stable crystalline form of CoQ 10 in the binary mixture are indicative of phase transformation of metastable to stable form, as discussed in the preceding section. No such behaviour is reported for LA. In the thermogram of binary mixtures, additional melting endotherm at 18 ̊C was observed and the exotherm of CoQ 10 at 17 ̊C shifted to slightly higher tempearature i.e., at around 20 C ̊ . This can be attributed to the presence of crystals of different sizes [17] or due to difference in crystallinity. In these mixtures, the melting endotherms appearing in pure CoQ 10 and LA disappeared. This indicates CoQ 10 is forming eutectic with LA in the binary mixture. In the binary mixtures, the endotherm at around 42 C ̊ is due to the unreacted excess LA. Similar observations have been reported in literature for the binary eutectic mixture of acyclovir and flucinolone acetonoid which showed two melting events viz., the lower eutectic melting endotherm and higher excess unreacted flucinolone acetonoid [19]. There is slight change in the melting temperature of LA to that of the reported value which may be due to the change in particle size. All the other thermal events occurring at the temperature between 35 to 40 ̊C can be attributed to the eutectic mixture of CoQ 10 and LA.
The binary phase diagram of CoQ 10 and LA is shown in Fig. 5. The melting points of pure LA and CoQ 10, after treating similar to binary mixture, were 44.9 and 45.3 C ̊ , respectively. It is evident from Fig. 5 that there is decrease in the melting point of LA and CoQ 10 in the binary mixture with change in the composition. It reaches a minimum value of 37.93 C ̊ which is called as eutectic point. It occurs when the binary mixture contains 70% of CoQ 10. Three different phases occurring at eutectic point are solid CoQ 10, solid LA, and conjugate liquid phase. There are four different regions in the binary phase diagram of CoQ 10 and LA viz., liquid phase containing CoQ 10 and LA above eutectic point (I), solid phase containing CoQ 10 and LA below eutectic point (II), solid CoQ 10 and conjugate liquid phase to the right of eutectic point (III), and solid LA and conjugate liquid phase to the left of eutectic point (IV).
The depression in the melting point of CoQ 10 in presence of LA is a colligative property. The mole fraction of liquid component across the liquidus curve in Fig. 6 can be predicted by the following equation [20].
Where, x is the mole fraction of drug into the eutectic mixture,
is the enthalpy of fusion of pure component,
R is the gas constant, T0 is the fusion temperature of pure component, and T is the melting point of eutectic mixture.
The point where the two melting curves intersect each other, can be taken as the predicted eutectic point. The predicted eutectic point of CoQ 10 and LA binary mixture was found to be 38.98 C ̊ at 87.7% CoQ 10. This is shown in Fig. 6. The predicted and experimental eutectic points were close to each other.
3.3 Reasons for deviations from ideality The predicted eutectic point of CoQ 10 and LA binary mixture was found to be 38.98 C ̊ and the eutectic composition at this point was 87.7% of CoQ 10. The experimental liquidus curve of CoQ 10 was located above the theoretical curve. Since activity coefficient of CoQ10 in the binary mixture was less than unity, attractive forces exist between the CoQ 10 and LA molecules. These attractions may be evidenced by calculating the fusion excess enthalpy (
) at eutectic point as
follows.
is obtained by the following equation.
In the above equation xi is mass fraction and ΔHf,i is the fusion enthalpy. Thus, calculated value of ΔHexcess at eutectic point is -8.319 J/g. The negative values of excess enthalpy of predicted eutectic mixture explains the attractive forces between CoQ 10 and LA molecules. There exists a correlation between the enthalpy and composition of eutectic mixture. The typical relationship between the enthalpy of melting and percent composition can be represented by the Fig. 7 [21].
The actual values of eutectic enthalpy were plotted against the % CoQ 10 in Fig. 8. The shape was found to deviate from the typical ‘triangular shape’ shown in Fig. 8. This may be due to the overlap between eutectic and excess component peak. The enthalpy of excess component is plotted against % composition in the Fig. 9 and Fig. 10. The enthalpy values were found to decrease with increase in % CoQ 10 and increase with increase in % LA. The two curves are mirror images of each other.
4
Conclusions
The DSC heat-cool-heat cycles of CoQ 10 revealed the presence of metastable and stable polymorphs of CoQ 10, which was in compliance with previously published findings. CoQ 10 was found to form eutectic with LA at a composition of 70 %w/w. Individual melting point of CoQ 10 and LA were 50 and 42 ̊C, respectively. Eutectic mixture showed a melting point of 37.93 C ̊ . The predicted eutectic point was near to actual eutectic point. The predicted liquidus curve for CoQ 10 was below the actual one. The experimental heat of fusion at eutectic point was less than the calculated heat of fusion. Activity coefficient of CoQ 10 in the binary mixture was found to be less than unity, which indicates the attractive forces between CoQ 10 and LA. The shape of eutectic enthalpy verses % composition was consistent with the theoretical curve. The enthalpy of excess component verses % CoQ 10, and that of verses % LA are mirror images of each other. Acknowledgement The work was performed with the financial support from NIPER, S.A.S. Nagar (India). References [1] T. Kommuru, B. Gurley, M. Khan, I. Reddy, Self-emulsifying drug delivery systems (SEDDS) of coenzyme Q10: formulation development and bioavailability assessment, Int. J. Pharm. 212 (2001) 233-246. [2] S. Onoue, N. Terasawa, T. Nakamura, K. Yuminoki, N. Hashimoto, S. Yamada, Biopharmaceutical characterization of nanocrystalline solid dispersion of coenzyme Q10 prepared with cold wet-milling system, Eur. J. Pharm. Sci. 53 (2014) 118-125. [3] S. Itagaki, A. Ochiai, M. Kobayashi, M. Sugawara, T. Hirano, K. Iseki, Grapefruit juice enhance the uptake of coenzyme Q10 in the human intestinal cell-linͦ e Caco-2, Food Chem. 120 (2010) 552-555. [4] P.R. Nepal, H-K. Han, H-K. Choi, Preparation and in vitro in vivo evaluation of Witepsol H35 based selfnanoemulsifying drug delivery systems (SNEDDS) of coenzyme Q10, Eur. J. Pharm. Sci. 39 (2010) 224-232. [5] S. Olsen, J. Doney, C. Shores, Benzoquinones of enhanced bioavailability, US20070026072A1 (2006). [6] Available from: http://www.rxlist.com/coenzyme_q-10-page3/supplements.htm (accessed 08.01.14). [7] P.R. Nepal, H-K. Han, H-K. Choi, Enhancement of solubility and dissolution of coenzyme Q10 using solid dispersion formulation, Int. J. Pharm. 383 (2010) 147-153. [8] C.A. Bonda, A. Pavlovic, J. Zhang, Photostabilization of coenzyme Q compounds with alkoxycrylene compounds, EP2436272A1 (2012). [9] S. Nazzal, I. Smalyukh, O. Lavrentovich, M.A. Khan, Preparation and in vitro characterization of a eutectic based semisolid self-nanoemulsified drug delivery system (SNEDDS) of ubiquinone: mechanism and progress of emulsion formation, Int. J. Pharm. 235 (2002) 247-265. [10] M.M. Buckley, P. Benfield, Eutectic lidocaine/prilocaine cream. A review of the topical anaesthetic/analgesic efficacy of a eutectic mixture of local anaesthetics (EMLA), Drugs 46 (1993) 126-51. [11] H.M. El‐Banna, Solid dispersion of pharmaceutical ternary systems I: Phase diagram of aspirin‐acetaminophen‐urea system, J. Pharm. Sci. 67 (1978) 1109-1111. [12] N.R. Goud, K. Suresh, P. Sanphui, A. Nangia, Fast dissolving eutectic compositions of curcumin, Int. J. Pharm. 439 (2012) 63-72. [13] P. Lambrechts, S. Siebrecht, Coenzyme Q10 and ubiquinol as adjunctive therapy for heart failure, Agro Food Indust. Hi-Tech. 24 (2013) 60-62. [14] Compositional guideline for ubiquinol-10 Available from: http://www.tga.gov.au/pdf/cm-cg-ubiquinol10.pdf (accessed 14.04.14). [15] Available from: http://lipidbank.jp/cgi-bin/detail.cgi?id=VCQ0001 (accessed 14.04.14). [16] H. Katsikas, P.J. Quinn, The thermotropic properties of coenzyme Q10 and its lower homologues, J. Bioenerg. Biomembr. 15 (1983) 67-79. [17] D. Fabri, J. Guan, A. Cesàro, Crystallisation and melting behaviour of poly (3-hydroxybutyrate) in dilute solution: towards an understanding of physical gels, Thermochim. Acta 321 (1998) 3-16. [18] J. Hatanaka, Y. Kimura, Z. Lai-Fu, S. Onoue, S. Yamada, Physicochemical and pharmacokinetic characterization of water-soluble coenzyme Q10 formulations, Int. J. Pharm. 363 (2008) 112-117.
[19] D-C. Marinescu, E. Pincu, I. Stanculescu, V. Meltzer, Thermal and spectral characterization of a binary mixture (acyclovir and fluocinolone acetonide): eutectic reaction and inclusion complexes with β-cyclodextrin, Thermochim. Acta 560 (2013) 104-111. [20] Z. Naima, T. Siro, G-D. Juan-Manuel, C. Chantal, C. René, D. Jerome, Interactions between carbamazepine and polyethylene glycol (PEG) 6000: characterisations of the physical, solid dispersed and eutectic mixtures, Eur. J. Pharm. Sci. 12 (2001) 395-404. [21] S. Lerdkanchanaporn, D. Dollimore, S.J. Evans, Phase diagram for the mixtures of ibuprofen and stearic acid, Thermochim. Acta 367 (2001) 1-8.
Figure captions Fig. 1. Heat-cool-heat curves ofcommercial CoQ 10 and oxidized CoQ 10 sample in which cooling was carried out at a fast rate of 16 ̊C/min. Fig. 2. Heat-cool-heat curves and H/CSM of CoQ 10 sample in which cooling is carried out at at a slow rate of 10 C ̊ /min. Fig. 3. VT-XRD of CoQ 10 obtained at different temperatures (25 C ̊ : Room temperature, -10 and -80 ̊C: Recrystallised during first cooling run, -33, 16, and 27 C ̊ : Recrystallised during second heating run). Fig. 4. DSC heating curves of binary mixture of CoQ 10 and LA. Fig. 5. Eutectic behaviour of CoQ 10 in presence of LA. Fig. 6. Predicted and actual eutectic behaviour of binary mixture of CoQ 10 and LA. Fig. 7. Schematic diagram representing the relationship between the enthalpy of eutectic and composition of binary mixture. Fig. 8. Enthalpy of melting of eutectic mixture verses composition of CoQ 10 and LA binary mixture. Fig. 9. Enthalpy of excess solid phase verses mole fraction of CoQ 10 in the binary mixture of CoQ 10 and LA. Fig. 10. Enthalpy of excess solid phase verses mole fraction of LA in the binary mixture of CoQ 10 and LA.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
S0040-6031(15)00026-X http://dx.doi.org/doi:10.1016/j.tca.2015.01.018 TCA 77129
To appear in:
Thermochimica Acta
Received date: Revised date: Accepted date:
22-9-2014 29-12-2014 25-1-2015
Please cite this article as: Bapurao Tarate, Arvind K.Bansal, Characterization of CoQ 10-Lauric Acid Eutectic System, Thermochimica Acta http://dx.doi.org/10.1016/j.tca.2015.01.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of CoQ 10-Lauric Acid Eutectic System Bapurao Tarate and Arvind K. Bansal* Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali), Punjab- 160062 (INDIA)
*Address correspondence to this author at the Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar (Mohali), Punjab- 160 062 (India). Tel:
+91
172
2214682;
Fax:
+91
172
2214692.
address:
[email protected]
and
[email protected]
Highlights
Two polymorphic forms of CoQ 10.
CoQ 10 forms eutectic mixture with LA.
Experimental and predicted values of eutectic point are 70% CoQ 10 at 37.93 C ̊ and 87.7% CoQ 10 at 38.98 C ̊ , respectively.
Attraction exists between CoQ 10 and LA molecules.
Abstract Solid state characterization of Coenzyme Q10 (CoQ 10) was carried out using differential scanning calorimetry (DSC), variable temperature X-ray diffractometry (VT-XRD) and hot/cold stage microscopy (H/CSM). It revealed that CoQ 10 exists in two polymorphic forms. The recrystallized samples of CoQ 10 melted at different temperatures either due to the wide crystal size variation or change in crystallinity. Further, the binary mixture of CoQ 10 and lauric acid (LA) formed eutectic mixture in the ratio 70:30 melting at 37.93 ̊C, which was close to the predicted eutectic composition of 87.7:12.3 melting at 38.98 C ̊ . The values of actual liquidus temperatures for CoQ 10 are higher than the predicted liquidus temperatures. The experimental heat of fusion at eutectic point was less than the calculated heat of fusion. Activity coefficient of CoQ 10 in the binary mixture was less than unity, which indicates the attraction between the components of eutectic mixture. Keywords: Coenzyme Q10 (CoQ 10), DSC, enthalpy, eutectic, lauric acid, polymorphic forms.
1
Introduction
Coenzyme (CoQ 10) is a poorly water soluble [1] vitamin like substance. It is a lipophilic, naturally occurring substance having a role as an intermediate of electron transport system, an antioxidant, and is involved in cellular metabolism [2]. Apart from poor water solubility, bioavailability of CoQ 10 is limited by its high molecular weight [1] and efflux by p-glycoprotein [3]. Various methods for enhancement of bioavailability of CoQ 10 are reported in literature. Self nanoemulsifying drug delivery system (SNEDDS) of CoQ 10 with Witepsol® H35, Solutol® HS15, and Lauroglycol® FCC has been reported. The formulation provided 90% release of CoQ 10 in pH 6.8 phosphate buffer and displayed 4.6 and 5.2 fold increase in AUC and Cmax, respectively, over powder formulation [4]. Spray drying of CoQ 10 with polymeric excipients such as polyvinylpyrrolidone
(PVP),
hydroxypropylmethylcellulose
acetate
succinate
(HPMCAS)
or
hydroxypropylmethyl cellulose pthalate (HPMCP) generated amorphous CoQ 10, resulting in enhanced bioavailability by reducing the time for dissolution of 50% CoQ 10 by 100% or more [5]. CoQ 10 has a dose of 100–3000 mg per day [6], a melting point of around 50 C ̊ [7], and a log P of about 15 [8]. This makes it a good candidate for oral delivery using lipidic formulations. For oral delivery using lipidic formulation, we carried preliminary investigations for in-situ crystallization of binary mixture of CoQ 10 and Lauric acid (LA) using DSC. Binary lipidic mixture of CoQ 10-L-menthol investigated by DSC is reported in literature [9]. Few examples of eutectic mixtures having applications in drug delivery are lidocaine-prilocaine [10], aspirin-acetaminophen-urea [11], and curcumin with conformers such as nicotinamide, ferulic acid, hydroquinone, p-hydroxybenzoic acid, and L-tartaric acid [12]. Thermal characterization of various compositions of CoQ 10-LA products revealed generation of eutectic mixture of CoQ 10 with LA. This work covers the solid state characterization of CoQ 10 and construction of binary phase diagram for eutectic mixture of CoQ 10 and LA. 2
Materials and methods
1.2 Materials All the experiments were carried out on commercial reduced CoQ 10 samples procured from Hangzhou Joymore Technology Co., Ltd., China. At the time of experimentation, the samples contained less than 29.4% of reduced CoQ 10 due to its oxidation during storage. Oxidized CoQ 10 from Hangzhou Joymore Technology Co., Ltd., China was used as a reference sample. Sample of LA was obtained from Loba Chemie Pvt. Ltd., India. 2.3 Methods 2.3.1
CoQ 10:LA mixtures for generation of eutectic system
Eutectic mixtures of CoQ 10 with LA were prepared by weighing the different proportions of CoQ 10:LA in the range between 5:95 to 95:5 in the DSC pans. It was then subjected to heat-cool-heat cycle protocol mentioned in the proceeding section. 2.3.2
Differential scanning calorimetry (DSC)
Heat-flux DSC measurements were performed with the DSC instrument (TA Q2000, New Castle, Delaware, USA equipped with TA Universal Analysis software). It was calibrated for temperature and heat flow by using high purity indium standard. Accurately weighed amount 2.5 to 5 mg of CoQ 10, LA, and CoQ10:LA binary mixture were weighed in aluminium pan, cripped, and heated to 80 ̊C, at the heating rate of 10 ̊C/minutes, and then held isothermally for 5 minutes. The sample was then cooled to -80 C ̊ , held isothermally for 5 minutes, followed by heating at the same rate as mentioned previously. Nitrogen was purged during the DSC operation at the rate 50 ml/min. 2.3.3
Hot and cold stage microscopy (H/CSM)
Hot stage microscopy (HSM) and cold stage microscopy (CSM) of CoQ 10 samples were performed on Leica DMLP polarized microscope (Leica Microsystems Wetzalar GmBH, Germany) equipped with Linkam LTS 350 hot/cold stage. Small quantity of sample was placed on a glass slide, mixed with silicon oil, and covered with a glass cover slip. It was heated and cooled using a protocol similar to DSC operation. 2.3.4
Variable temperature X-ray diffractometry (VT-XRD)
Powder X-ray diffraction patterns were recorded on diffractometer using Cu-Kα X- ray radiation at 40 Kv power and 40 mA current. The wavelength of radiation was 2.54 Å. X-ray diffraction patterns were collected over 2θ range 3-40 at the scan rate of 0.01 and scan speed of 0.1. The measurements were carried out on the variable temperature stage to confirm the presence of pure components at different temperature. The heating and cooling protocol was similar to DSC. 3
Results and discussion
3.1 Solid state characterization of CoQ 10 3.1.1
DSC and H/CSM
All the experiments were conducted using reduced CoQ 10. However, due to its low atmospheric stability, it converted into the oxidized CoQ 10, and very small quantity of reduced CoQ 10 remained in the sample. This was evident from the orange colour of sample, since oxidized CoQ 10 is orange in colour while reduced CoQ 10 is of white colour [13]. Thus, all the studies were carried out on the CoQ 10 which was predominantly in the oxidized form. The DSC analysis of CoQ 10 was performed under different experimental protocols and the same is represented in shows Fig. 1 and Fig. 2. Fig. 1 shows the heat-cool-heat cycle of CoQ 10 at a fast cooling rate of 16 ̊C/min. Sample of the pure oxidized CoQ 10 was also characterized using similar protocol. First heating cycle of CoQ 10 and oxidized CoQ 10 indicated melting endotherm at 50.80 and 51.28 C ̊ , respectively. This is in conformity with reported literature, wherein melting points of 49.5 [14] and 49 C ̊ [15] have been reported for reduced and oxidized forms of CoQ 10, respectively. Thereafter, molten samples of CoQ 10 were cooled to -80 ̊C at a fast cooling rate of 16 C ̊ /min. Commercial and pure oxidized samples of CoQ 10 exhibited a recrystallization exotherm at around -14.58 and -8.98 C ̊ , respectively. This event was much sharper in case of oxidized CoQ 10.
Second heating cycle of commercial CoQ 10 indicated a subtle event at -72.60 C ̊ , which can be attributed to amorphous phase. It shows another exothermic event at -33.4 ̊C, which can be attributed to crystalization of amorphous CoQ 10. A combination of exothermic-endothermic-exothermic-endothermic events was observed at 16.11, 23.79, 26.34, and 45.52 C ̊ . A similar set of events was observed for oxidized CoQ 10 at 16.11, 23.31, 25.06, and 48.72 C ̊ .The oxidized CoQ 10 did not exhibit the thermal event corresponding to amorphous phase at any temperature. Therefore, the exothermic event at around 33 C ̊ was also absent in oxidized CoQ 10. A heat-cool-heat cycle at a heating and cooling rate (slow) of 10 C ̊ /min was carried out in an attempt to further resolve these events. Fig. 2 captures the heating curve, along with photomicrographs taken using hot stage microscope, at slow cooling rate. In the first cooling cycle, it shows recrystallization exotherm at -14.2 C ̊ . In slow and fast cooling, recrystallization of CoQ 10 occurred nearly at the same temperature. It was reported for CoQ 10 that lower the rate of cooling of molten sample, lower is the tempearature of recrystallization [16]. This was not observed in this study because difference in two cooling rates may not be too high. In the second heating cycle, a glass transition event at approximately -72.6 C ̊ indicated that only partial recrystallization occurred during cooling curve. Glass transition was followed by an exothermic event at 34.88 C ̊ . This event was observed at fast cooling rate also. This was followed by two exothermic events at 15.47 and 26.20 C ̊ . Additionally, two endothermic events were observed at 23.31 and 45.20 C ̊ . This is in good agreement with the previously reported study, in which CoQ 10 was found to exist in two crystalline phases. The metastable phase gets converted into the stable crysalline phase through such exothermic-endothemicexothermic events observed between 15 to 25 C ̊ [16]. The molten portion at -80 C ̊ is ‘undercooled liquidus’ below the thermodynamic equilibrium which can crystallize on the surface of existing crystals. Due to high temperature the formation of new nuclei and their growth is not possible, giving rise to the exotherm at 15.47 C ̊ [17]. This is followed by melting of metastable phase, crystallization of stable phase, and finally its melting.
3.1.2
VT- XRD
The powder XRD pattern of CoQ 10 at different tempearures was compared with its XRD spectra at 25 C ̊ in Fig. 3. The powder XRD of commercial sample, labelled as 25 C ̊ in Fig. 3, of CoQ 10 shows two major peaks at 2θ values around 18.5 and 23̊. Apart from this, it showed a small peak at 2θ value of 20.5 and many small peaks in between 25 to 40̊. The XRD spectra of oxidized CoQ 10 shows many peaks apart from sharp peak at 2θ value of 18º [18]. The sample of commercial CoQ 10 at the tempearure -10, -80, -33 and 16 C ̊ exhibited similar XRD pattern, wherein only two peaks at 2θ values of 18.5 and 23 were observed. Here, the small peaks observed in the range of 25 to 40 C ̊ were completely absent. Powder XRD pattern of CoQ 10 at 27 C ̊ was found to be similar to that of 25 C ̊ , except change in intensity, which may be due to reduced crystallinity of stable crystalline phase. Similar observations were obtained when CoQ 10 was studied with wide angle X-ray diffraction methods [16]. This indicates the existance of two crystalline phases viz., metastable and stable forms of CoQ 10, which is consistant with the DSC data discussed previously. Thus, it can be concluded from DSC, H/CSM, and VT-XRD studies that CoQ 10 exists as two crystalline forms viz., metastable form existing below 25 C ̊ and stable form above room temperature.
3.2 Eutectic phase diagram of CoQ 10:LA mixture As shown in Fig. 4, many thermal events were observed in the binary mixture of CoQ 10 and LA. These mixtures showed disappearance of melting endotherm at around 45 C ̊ of pure CoQ 10 treated similar to binary mixtures. Many thermal events observed before melting of stable crystalline form of CoQ 10 in the binary mixture are indicative of phase transformation of metastable to stable form, as discussed in the preceding section. No such behaviour is reported for LA. In the thermogram of binary mixtures, additional melting endotherm at 18 ̊C was observed and the exotherm of CoQ 10 at 17 ̊C shifted to slightly higher tempearature i.e., at around 20 C ̊ . This can be attributed to the presence of crystals of different sizes [17] or due to difference in crystallinity. In these mixtures, the melting endotherms appearing in pure CoQ 10 and LA disappeared. This indicates CoQ 10 is forming eutectic with LA in the binary mixture. In the binary mixtures, the endotherm at around 42 C ̊ is due to the unreacted excess LA. Similar observations have been reported in literature for the binary eutectic mixture of acyclovir and flucinolone acetonoid which showed two melting events viz., the lower eutectic melting endotherm and higher excess unreacted flucinolone acetonoid [19]. There is slight change in the melting temperature of LA to that of the reported value which may be due to the change in particle size. All the other thermal events occurring at the temperature between 35 to 40 ̊C can be attributed to the eutectic mixture of CoQ 10 and LA.
The binary phase diagram of CoQ 10 and LA is shown in Fig. 5. The melting points of pure LA and CoQ 10, after treating similar to binary mixture, were 44.9 and 45.3 C ̊ , respectively. It is evident from Fig. 5 that there is decrease in the melting point of LA and CoQ 10 in the binary mixture with change in the composition. It reaches a minimum value of 37.93 C ̊ which is called as eutectic point. It occurs when the binary mixture contains 70% of CoQ 10. Three different phases occurring at eutectic point are solid CoQ 10, solid LA, and conjugate liquid phase. There are four different regions in the binary phase diagram of CoQ 10 and LA viz., liquid phase containing CoQ 10 and LA above eutectic point (I), solid phase containing CoQ 10 and LA below eutectic point (II), solid CoQ 10 and conjugate liquid phase to the right of eutectic point (III), and solid LA and conjugate liquid phase to the left of eutectic point (IV).
The depression in the melting point of CoQ 10 in presence of LA is a colligative property. The mole fraction of liquid component across the liquidus curve in Fig. 6 can be predicted by the following equation [20].
Where, x is the mole fraction of drug into the eutectic mixture,
is the enthalpy of fusion of pure component,
R is the gas constant, T0 is the fusion temperature of pure component, and T is the melting point of eutectic mixture.
The point where the two melting curves intersect each other, can be taken as the predicted eutectic point. The predicted eutectic point of CoQ 10 and LA binary mixture was found to be 38.98 C ̊ at 87.7% CoQ 10. This is shown in Fig. 6. The predicted and experimental eutectic points were close to each other.
3.3 Reasons for deviations from ideality The predicted eutectic point of CoQ 10 and LA binary mixture was found to be 38.98 C ̊ and the eutectic composition at this point was 87.7% of CoQ 10. The experimental liquidus curve of CoQ 10 was located above the theoretical curve. Since activity coefficient of CoQ10 in the binary mixture was less than unity, attractive forces exist between the CoQ 10 and LA molecules. These attractions may be evidenced by calculating the fusion excess enthalpy (
) at eutectic point as
follows.
is obtained by the following equation.
In the above equation xi is mass fraction and ΔHf,i is the fusion enthalpy. Thus, calculated value of ΔHexcess at eutectic point is -8.319 J/g. The negative values of excess enthalpy of predicted eutectic mixture explains the attractive forces between CoQ 10 and LA molecules. There exists a correlation between the enthalpy and composition of eutectic mixture. The typical relationship between the enthalpy of melting and percent composition can be represented by the Fig. 7 [21].
The actual values of eutectic enthalpy were plotted against the % CoQ 10 in Fig. 8. The shape was found to deviate from the typical ‘triangular shape’ shown in Fig. 8. This may be due to the overlap between eutectic and excess component peak. The enthalpy of excess component is plotted against % composition in the Fig. 9 and Fig. 10. The enthalpy values were found to decrease with increase in % CoQ 10 and increase with increase in % LA. The two curves are mirror images of each other.
4
Conclusions
The DSC heat-cool-heat cycles of CoQ 10 revealed the presence of metastable and stable polymorphs of CoQ 10, which was in compliance with previously published findings. CoQ 10 was found to form eutectic with LA at a composition of 70 %w/w. Individual melting point of CoQ 10 and LA were 50 and 42 ̊C, respectively. Eutectic mixture showed a melting point of 37.93 C ̊ . The predicted eutectic point was near to actual eutectic point. The predicted liquidus curve for CoQ 10 was below the actual one. The experimental heat of fusion at eutectic point was less than the calculated heat of fusion. Activity coefficient of CoQ 10 in the binary mixture was found to be less than unity, which indicates the attractive forces between CoQ 10 and LA. The shape of eutectic enthalpy verses % composition was consistent with the theoretical curve. The enthalpy of excess component verses % CoQ 10, and that of verses % LA are mirror images of each other. Acknowledgement The work was performed with the financial support from NIPER, S.A.S. Nagar (India). References [1] T. Kommuru, B. Gurley, M. Khan, I. Reddy, Self-emulsifying drug delivery systems (SEDDS) of coenzyme Q10: formulation development and bioavailability assessment, Int. J. Pharm. 212 (2001) 233-246. [2] S. Onoue, N. Terasawa, T. Nakamura, K. Yuminoki, N. Hashimoto, S. Yamada, Biopharmaceutical characterization of nanocrystalline solid dispersion of coenzyme Q10 prepared with cold wet-milling system, Eur. J. Pharm. Sci. 53 (2014) 118-125. [3] S. Itagaki, A. Ochiai, M. Kobayashi, M. Sugawara, T. Hirano, K. Iseki, Grapefruit juice enhance the uptake of coenzyme Q10 in the human intestinal cell-linͦ e Caco-2, Food Chem. 120 (2010) 552-555. [4] P.R. Nepal, H-K. Han, H-K. Choi, Preparation and in vitro in vivo evaluation of Witepsol H35 based selfnanoemulsifying drug delivery systems (SNEDDS) of coenzyme Q10, Eur. J. Pharm. Sci. 39 (2010) 224-232. [5] S. Olsen, J. Doney, C. Shores, Benzoquinones of enhanced bioavailability, US20070026072A1 (2006). [6] Available from: http://www.rxlist.com/coenzyme_q-10-page3/supplements.htm (accessed 08.01.14). [7] P.R. Nepal, H-K. Han, H-K. Choi, Enhancement of solubility and dissolution of coenzyme Q10 using solid dispersion formulation, Int. J. Pharm. 383 (2010) 147-153. [8] C.A. Bonda, A. Pavlovic, J. Zhang, Photostabilization of coenzyme Q compounds with alkoxycrylene compounds, EP2436272A1 (2012). [9] S. Nazzal, I. Smalyukh, O. Lavrentovich, M.A. Khan, Preparation and in vitro characterization of a eutectic based semisolid self-nanoemulsified drug delivery system (SNEDDS) of ubiquinone: mechanism and progress of emulsion formation, Int. J. Pharm. 235 (2002) 247-265. [10] M.M. Buckley, P. Benfield, Eutectic lidocaine/prilocaine cream. A review of the topical anaesthetic/analgesic efficacy of a eutectic mixture of local anaesthetics (EMLA), Drugs 46 (1993) 126-51. [11] H.M. El‐Banna, Solid dispersion of pharmaceutical ternary systems I: Phase diagram of aspirin‐acetaminophen‐urea system, J. Pharm. Sci. 67 (1978) 1109-1111. [12] N.R. Goud, K. Suresh, P. Sanphui, A. Nangia, Fast dissolving eutectic compositions of curcumin, Int. J. Pharm. 439 (2012) 63-72. [13] P. Lambrechts, S. Siebrecht, Coenzyme Q10 and ubiquinol as adjunctive therapy for heart failure, Agro Food Indust. Hi-Tech. 24 (2013) 60-62. [14] Compositional guideline for ubiquinol-10 Available from: http://www.tga.gov.au/pdf/cm-cg-ubiquinol10.pdf (accessed 14.04.14). [15] Available from: http://lipidbank.jp/cgi-bin/detail.cgi?id=VCQ0001 (accessed 14.04.14). [16] H. Katsikas, P.J. Quinn, The thermotropic properties of coenzyme Q10 and its lower homologues, J. Bioenerg. Biomembr. 15 (1983) 67-79. [17] D. Fabri, J. Guan, A. Cesàro, Crystallisation and melting behaviour of poly (3-hydroxybutyrate) in dilute solution: towards an understanding of physical gels, Thermochim. Acta 321 (1998) 3-16. [18] J. Hatanaka, Y. Kimura, Z. Lai-Fu, S. Onoue, S. Yamada, Physicochemical and pharmacokinetic characterization of water-soluble coenzyme Q10 formulations, Int. J. Pharm. 363 (2008) 112-117.
[19] D-C. Marinescu, E. Pincu, I. Stanculescu, V. Meltzer, Thermal and spectral characterization of a binary mixture (acyclovir and fluocinolone acetonide): eutectic reaction and inclusion complexes with β-cyclodextrin, Thermochim. Acta 560 (2013) 104-111. [20] Z. Naima, T. Siro, G-D. Juan-Manuel, C. Chantal, C. René, D. Jerome, Interactions between carbamazepine and polyethylene glycol (PEG) 6000: characterisations of the physical, solid dispersed and eutectic mixtures, Eur. J. Pharm. Sci. 12 (2001) 395-404. [21] S. Lerdkanchanaporn, D. Dollimore, S.J. Evans, Phase diagram for the mixtures of ibuprofen and stearic acid, Thermochim. Acta 367 (2001) 1-8.
Figure captions Fig. 1. Heat-cool-heat curves ofcommercial CoQ 10 and oxidized CoQ 10 sample in which cooling was carried out at a fast rate of 16 ̊C/min. Fig. 2. Heat-cool-heat curves and H/CSM of CoQ 10 sample in which cooling is carried out at at a slow rate of 10 C ̊ /min. Fig. 3. VT-XRD of CoQ 10 obtained at different temperatures (25 C ̊ : Room temperature, -10 and -80 ̊C: Recrystallised during first cooling run, -33, 16, and 27 C ̊ : Recrystallised during second heating run). Fig. 4. DSC heating curves of binary mixture of CoQ 10 and LA. Fig. 5. Eutectic behaviour of CoQ 10 in presence of LA. Fig. 6. Predicted and actual eutectic behaviour of binary mixture of CoQ 10 and LA. Fig. 7. Schematic diagram representing the relationship between the enthalpy of eutectic and composition of binary mixture. Fig. 8. Enthalpy of melting of eutectic mixture verses composition of CoQ 10 and LA binary mixture. Fig. 9. Enthalpy of excess solid phase verses mole fraction of CoQ 10 in the binary mixture of CoQ 10 and LA. Fig. 10. Enthalpy of excess solid phase verses mole fraction of LA in the binary mixture of CoQ 10 and LA.
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Fig. 10