Molecular dynamics of amorphous pharmaceutical fenofibrate studied by broadband dielectric spectroscopy.

Fenofibrate is mainly used to reduce cholesterol level in patients at risk of cardiovascular disease. Thermal transition study with the help of differential scanning calorimetry (DSC) shows that the aforesaid active pharmaceutical ingredient (API) is a good glass former. Based on our DSC study, the molecular dynamics of this API has been carried out by broadband dielectric spectroscopy (BDS) covering wide temperature and frequency ranges. Dielectric measurements of amorphous fenofibrate were performed after its vitrification by fast cooling from a few degrees above the melting point (Tm=354.11 K) to deep glassy state. The sample does not show any crystallization tendency during cooling and reaches the glassy state. The temperature dependence of the structural relaxation has been fitted by single Vogel-Fulcher-Tamman (VFT) equation. From VFT fit, glass transition temperature (Tg) was estimated as 250.56 K and fragility (m) was determined as 94.02. This drug is classified as a fragile glass former. Deviations of experimental data from Kohlrausch-Williams-Watts (KWW) fits on high-frequency flank of α-peak indicate the presence of an excess wing in fenofibrate. Based on Ngai׳s coupling model, we identified the excess wing as true Johari-Goldstein (JG) process. Below the glass transition temperature one can clearly see a secondary relaxation (γ) with an activation energy of 32.67 kJ/mol.

Introduction

To increase the solubility and bioavailability of the poorly water soluble crystalline drugs is a challenge to modern pharmaceutical scientists. One of the promising methods for improving solubility and bioavailability of poorly water soluble crystalline drugs is to prepare them in the amorphous form. Amorphous form of pharmacologically active materials is very important because this form has enhanced thermodynamic properties, having high internal energy, solubility, bioavailability, dissolution rate, better compression characteristics and greater therapeutic activity than the corresponding crystalline counterpart [1], [2], [3], [4]. However, amorphous systems are physically and chemically unstable and therefore show crystallization tendencies.

Recent studies on various amorphous pharmaceutical drugs show that molecular mobility above and below the glass transition temperature (Tg) plays an important role in the devitrification of the drugs. The knowledge about the molecular dynamics of the drugs in the amorphous phase is very important for safety storage in order to obtain the maximum shelf-life and stability [5], [6], [7], [8].

Fenofibrate had been chosen as a model drug for the current study. It is a fibric acid derivative which has greater high density lipoprotein cholesterol (HDL-CH) rising and greater low density lipoprotein cholesterol (LDL-CH) lowering action than other fibrates [9]. To the best of our knowledge, this will be the first attempt to describe the dielectric relaxation study of amorphous fenofibrate.

For this it is essential to have a better understanding of molecular dynamics in supercooled and glassy state of this API. In the present study, we used differential scanning calorimetry (DSC) for thermal transition analysis and observed that fenofibrate is a very good glass former. Based on DSC study, dielectric measurements of this API were carried out by broadband dielectric spectroscopy (BDS) as an investigative tool to study the molecular dynamics of amorphous systems in a wide range of frequencies (f=109–10−2 Hz) at different thermodynamic conditions (P, T). Recently this technique has been successfully applied to study the molecular dynamics of various amorphous pharmaceuticals [10], [11], [12]. To get a clear picture of hydrogen bonding in the crystalline and amorphous state of fenofibrate, infrared (IR) spectroscopy was used.

Material and methods

Material

Fenofibrate, a white crystalline powder CAS no. 6271-86-2, was supplied by Sigma Aldrich with purity ≥99%. The drug was crystalline in nature as confirmed by the sharp diffraction patterns of the PXRD result, which is depicted in Fig. 1. Its chemical structure is presented in Fig. 2. Obtained material was used as received without further purification.

Fig. 1

XRPD pattern of crystalline fenofibrate.

Fig. 2

The chemical structure of fenofibrate.

Methods

Image analysis system (IAS)

Image analysis was used to investigate micro- and macro-spe-cimens objectively to provide information regarding the microstructure, quantity, size, area, shape and phase analysis. Images were captured using light optical microscope and analyzed using image analysis software. This technique can be applied in the field of material science, biological science, pharmaceutical science, etc. Particle size of fenofibrate was taken by dispersing the sample in water.

DSC

A DSC instrument (821e Mettler-Toledo GmbH) operated with STARe software version 9.1 and equipped with an intracooler was used for the thermal transition studies. The instrument was calibrated by using indium for temperature and specific heat. Fenofibrate of 3.1540 mg was analyzed under dry nitrogen purge (50 mL/min) in a sealed pinhole aluminum pan. The sample was heated from room temperature to 91 °C and held for 3 min; it was cooled to −50 °C and held for 5 min; and again it was heated to 91 °C. During the entire process a constant heating and cooling rate of 10 °C/min was used. Thermograms were collected during heating. Melting point (Tm) was determined as the onset of the endothermic peak, whereas glass transition temperature (Tg) was measured as the onset of the glass transition.

Fourier transform infrared (FTIR) spectroscopy

The IR spectra of crystalline and amorphous samples were collected on a Perkin Elmer (model: Synthesis Monitoring System) and Nicolet instruments corporation USA (Model MAGNA), respectively.

BDS

Isobaric dielectric measurements at ambient pressure were carried out using Novo-Control GMBH alpha dielectric spectrometer covering a frequency range from 10−2 to 107 Hz. Temperature was controlled by using a nitrogen gas cryostat with temperature stability better than 0.1 K. The tested sample was placed in a measurement capacitor made of stainless steel (diameter: 30 mm, gap: 0.20 mm). Teflon was used as the spacer. Dielectric measurements of fenofibrate were performed after its vitrification by fast cooling (10 K/min) from a few degrees above the melting point (Tm=354.11 K). The temperature measurements were carried out from 123.15 K to 305.15 K in different steps. However, the sample did not crystallize during cooling from the melting temperature.

Results and discussion

Particle size

Particle size of fenofibrate was analyzed by IAS and the maximum size was found to be 9.95 µm and the minimum was found to be 1.91 µm. The details of analysis are presented in Table 1.

Table 1

Class interval and number of particles of fenofibrate.

ID class	Class interval (μm)	No. of particles
1	0–2	280
2	2–4	777
3	4–6	371
4	6–8	163
5	8–10	79
6	10–12	0

Thermal studies

The onset glass transition was observed at 254.15 K, which is considered as the glass transition temperature (Tg) of fenofibrate. The onset melting of the sample was observed at 354.11 K, which is melting temperature (Tm) of the sample. No crystallization was observed either cooling below Tg or subsequent heating up to melting point. These results were in good agreement with those reported by Baird et al. [13] and Zhou et al. [7]. Thermodynamic quantities obtained from the current work and from the literature are listed in Table 2. The obtained thermogram of fenofibrate is presented in Fig. 3.

Table 2

Thermodynamic quantities during melting and glass transition of fenofibrate.

Compound	MW (g/mol)	Tm (K)	Tg (K)	Ref.
Fenofibrate	360.83	354.11	254.15	This work
		354.15	254.15	[13]
		353.65	253.15	[7]

Fig. 3

DSC thermogram of fenofibrate obtained during heating/cooling with 10 °C/min. Heating of the sample yields Tm=354.11 K as the onset melting temperature. Subsequent cooling of the melt from 364.15 K down to 223.15 K. Following heating of the vitrified fenofibrate to a temperature higher than Tm (364.15 K). The onset glass transition temperature (Tg) obtained on heating is 254.15 K: at higher temperature there is no evidence of crystallization.

Spectroscopic investigation of hydrogen bonding

The spectra of fenofibrate showed different strengths of hydrogen bonding (Fig. 4A and B). Two carbonyl peaks were seen in the crystalline and amorphous fenofibrate, one from the CO, and the other from the methyl ester carbonyl group. Crystalline fenofibrate showed two peaks at 1729.53 cm−1 and 1651.48 cm−1, respectively. But in the amorphous form two peaks were seen at 1728.25 cm−1 and 1654.22 cm−1, respectively. Because of the hydrogen bond formation the carbonyl peak position shifted to a lower wave number. In the amorphous phase, only one carbonyl group took part in the hydrogen bond formation by a downward shift of 1.28 cm−1. The other peak shifted by value of 2.74 cm−1 to a higher peak position. The upward shifted in the position of the CO group suggests that the hydrogen bond in the crystalline phase is stronger than that in the amorphous phase. In addition to the above groups capable of classical hydrogen bond formation, the stretching frequencies of the CH3 were investigated in both the phases [14], [15].

Fig. 4

Spectral variations of fenofibrate in CO stretching vibration region. (A) crystalline fenofibrate and (B) amorphous fenofibrate.

Molecular dynamics

Dielectric loss spectra (i.e., imaginary part of dielectric permittivity ε″ plotted as a function of frequency f) of fenofibrate is shown in Fig. 5. Fig. 5A shows the primitive relaxation process measured above Tg. Tg is defined as the temperature at which the structural relaxation time (τα) approaches a value of 100 s. Dielectric loss spectra collected above Tg exhibited the most intense relaxation i.e., α-relaxation (originating from cooperative reorientational motion of molecules) along with conductivity. The strength of the dielectric spectra started decreasing from 289.15 K onwards, indicating that fenofibrate started crystallizing. Fig. 5B shows the secondary relaxation present below Tg of this sample. The decrease in dipoles due to crystallization was also observed from the real part of the permittivity (Fig. 6). Both ε′ and ε″ were analyzed by using the Win FIT V (3.2) software. The dielectric spectra were fitted by using the Havriliak–Negami (HN) function as given belowwhere k sums over different relaxation processes, ω is the angular frequency, τHN is the characteristic relaxation time which is related to the frequency of maximal loss fmax, Δε is the dielectric strength, ε∞ is the high frequency limit of the real part ε′(ω); αHN and βHN are the symmetric and asymmetric broadenings of loss curve. For the secondary relaxation process, βHN=1, then HN function becomes the Cole–Cole function.

Fig. 5

Dielectric loss curves obtained for fenofibrate during heating. (A) the primary relaxation above Tg and (B) the secondary relaxation below Tg.

Fig. 6

Variation of real part of the complex permittivity for fenofibrate with temperature at different frequencies.

In order to check the presence of true secondary relaxation coming from the intermolecular motion called Johari–Goldstein (JG) or (β) relaxation in fenofibrate, coupling model (CM) prediction was applied [16], [17]. According to this theoretical tool there is a good agreement between the JG-relaxation time (τJG) and the primitive relaxation time (τ0) calculated from Eq. (2)where n=(1−βKWW) is the coupling parameter, tc is the crossover time which represents the crossover from independent to cooperative motions and equal to 2 ps for small molecular glass formers.

To describe the non-exponential behavior of α-relaxation the one-sided Fourier transform of KWW function was also used as given below

In the frequency domain, the parameter βKWW describes the shape of the spectra. If βKWW=1, it denotes symmetric and narrow relaxation peak, value of βKWW=0 indicates very broad and asymmetric peak. For fenofibrate the procedure of fitting the α-peak by one-side Fourier transform of stretched exponential function at 257.15 K gives the value of βKWW=0.70. This means that experimentally observed structural relaxation peak is asymmetric and broader than that of classical Debye response. This indicates the presence of an excess wing (EW). EW is a submerged process in which true secondary relaxation coming from the intermolecular motion is hidden under the prominent α-peak.

For fenofibrate, the value of τ0=2.643×10−4 s at T=257.15 K, corresponds to a frequency of f0=602.55 Hz. Where f0 is the primitive relaxation frequency and it was found that f0 was far from the maximum of secondary γ-relaxation peak and lay within the prominent structural relaxation peak. It clearly indicates that the secondary relaxation β (JG) which is a precursor of the α-relaxation and originating from the intermolecular motions is hidden under the prominent structural α-peak, which means that the well resolved γ-relaxation is non-JG secondary relaxation. Many investigations showed that high pressure measurements [18], [19], [20], [21], [22] as well as physical ageing [22], [23] can separate the α-peak and EW.

The master curve was constructed in the following way. Several spectra above Tg were shifted horizontally to superimpose with that obtained at 257.15 K as shown in Fig. 7. The exponents αHN and βHN show constant value, i.e., αHN≈0.98; βHN≈0.57, which indicates that the shape of the α-peak of fenofibrate does not change in that region.

Fig. 7

Super imposed spectra of fenofibrate formed by shifting several spectra [255.15–281.15 K, ΔT=2 K] above Tg to overlap the spectrum at 257.15 K. The KWW fit of the α-peak at 257.15 K yields βKWW=0.70. The black arrow denotes the position of primitive relaxation frequency (f0) calculated at 257.15 K using CM predictions.

Shamblin et al. [24] have suggested that the stretching parameter βKWW has a very important role in predicting the stability of amorphous pharmaceuticals against crystallization. As βKWW decreases (the asymmetric distribution of relaxation times becomes broader) stability would also decrease. By comparing the value of βKWW of fenofibrate with other APIs (acetaminophen βKWW=0.79 [12], Nonivamide βKWW=0.79 [25], Glibenclamide βKWW=0.74 [26], Indapamide βKWW=0.72 [27], and Ketoprofen βKWW=0.71 [10]) according to the Shamblin criterion, it was found that stability of amorphous fenofibrate was lower than that of ketoprofen.

The observed secondary (γ-) relaxation probably originated from the intramolecular motion of small part of the fenofibrate molecule (local motions originating from the molecular fluctuations of methyl group). In FTIR spectrum (Fig. 5B) two distinct bands occurred at 2983.26 and 2939.18 cm−1. The first band corresponded to asymmetrical stretching mode of C—H bonds of methyl group and the second band arose from the symmetrical stretching of C—H bonds.

The temperature evolution of the structural relaxation time τα very often follows the time honored Vogel–Fulcher–Tamman (VFT) expression, given as belowwith τVF, B, and T0 as parameters.

From VFT fit, we calculated the fragility “m” of fenofibrate by using Eq. (5):

VFT fit reveals the value of Tg as 250.56 K. Tg obtained from VFT fit is in close agreement with that of our DSC study as well as that of the DSC study done by Baird et al. [13] and Zhou et al. [7]. The non-Arrhenius behavior of the α-peak gives the measure of the fragility parameter [28], [29]. Strong systems have fragility less than 40 and fragile systems have fragility greater than 75 [1]. Fragile glass formers are physically unstable compared with the strong one. Fragility “m” in the case of fenofibrate was estimated as 94.02 and classified this API as a fragile glass former. Recent studies on ketoprofen by Shibata et al. [32] showed that there is a strong correlation between the fragility, boson peak frequency and shear modulus; large value of fragility is related to weak boson peak (or lower boson peak frequency which is caused by small shear modulus). So fragility is a very important parameter, which gives an insight to the theory of liquid–glass transition [30]. The strength parameter D was estimated as 7.04 proving fenofibrate as a fragile glass former. (Tg–To) was found to be 41.24, and Tm/Tg was found to be 1.4, and both the methods showed this API belongs to fragile group. The value of T0 was obtained as 209.32 K. The temperature T0 is often referred to as the Kauzmann temperature TK, a hypothetical temperature where configurational entropy of supercooled liquid would be equal to entropy of the crystal and molecular mobility would reduce to the same level as that of the crystal (strictly speaking the molecular mobility is not zero). For most of the glassy systems, it lies between 50 and 70 K, below Tg and it is sufficient to ensure safety storage [4]. Recently Pajula et al. [31], [32] have showed that the stabilization of amorphous drugs can be achieved by the addition of small molecular weight excipients.

Now we would like to discuss the secondary relaxation coming from the small part of the fenofibrate molecule which is the gamma relaxation. The temperature dependence of the relaxation time of the γ-process shows a linear dependence and can be described by the Arrhenius equationwhere τ is constant and Ea is the activation energy. The activation energy for fenofibrate was calculated and obtained as 32.67 kJ/mol. Fig. 8 shows the relaxation map of fenofibrate. The fitting parameters of VFT and Arrhenius equations are presented in Table 3.

Fig. 8

Arrhenius plot of fenofibrate. Filled circles denote α-relaxation, while filled squares are for γ-relaxation. The solid lines are fit to the dielectric data using VFT form for α-process, and Arrhenius equation for γ-process.

Table 3

The fitting parameters for the VFT and Arrhenius equations of fenofibrate.

α-Process, VFT			γ-Process, Arrhenius
T0 (K)	log τVF	D	log τ∞,γ	Eγ (kJ/mol)	m	Tg (K) (BDS)
209.32±1.02	−13.52±0.17	7.04±0.20	−14.63±0.09	32.67±0.01	94.02	250.56

Conclusions

The molecular dynamics of fenofibrate in the glassy and supercooled states was studied by different experimental techniques. Based on the study of the molecular dynamics of fenofibrate in the glassy and supercooled states by BDS, we are summarising the important results. Our measurements during cooling with a cooling rate of 10 K/min from the melt to deep glassy state do not show any crystallization tendency in fenofibrate, thereby showing their excellent glass forming ability. Fenofibrate shows structural (α) relaxation above Tg and one secondary relaxation process below Tg which is characterized as γ-relaxation.

The temperature dependence of α-relaxation times was described by single Vogel–Fulcher–Tamman equation. From VFT fits the glass transition temperature Tg was estimated as 250.56 K. Tg values calculated from dielectric measurements were found to be in good agreement with those measured from our DSC experiments as well as with the literature values.

Fragility represents the deviation from the Arrhenius behavior. It was obtained as 94.02 and we classified this API as a fragile glass former. T0 was obtained as 209.32 K. So it is suggested that this API can provide a shelf life of at least 3–5 years when it is stored in freezer at a temperature less than T0.

Application of the CM prediction indicated that the Johari–Goldstein (JG) relaxation is hidden under the prominent structural relaxation and manifested as an excess wing. The observed secondary relaxation is characterized as non-JG relaxation (γ relaxation) and assumed to be originated from the intramolecular degrees of freedom. The γ-relaxation has an Arrhenius temperature dependence and the activation energy is found to be Eγ=32.67 kJ/mol. This drug shows crystallization tendency from 289.15 K onwards during heating in the dielectric measurements by BDS. The existence of intermolecular hydrogen bonding was verified by IR spectroscopy.