Application of hydrophilic polymers for the preparation of tadalafil solid dispersions: micromeritics properties, release and erectile dysfunction studies in male rats

Preparation of TFL- SDs by solvent evaporation

Three batches of drug-polymers SDs in the weight ratio (1:1w/w) were prepared by dissolving TFL and hydrophilic polymers; Kolliphor^® P188, Kollidon^®-VA64 and Kollidon^® 30, separately in 50 mL of 1:1v/v water and ethanol solvent mixture. The hydroalcoholic solution containing the drug and polymer was then placed in ultrasonication for 5 min. Thereafter, it was transferred to a round bottom flask, and the solvent was evaporated for 5 h on rota-evaporator, Buchi Rotavapor R-215 at 60 °C with 50 rpm flask rotation. The collected SDs were dried under vacuum to remove the residual solvent and pulverized. The dried product was packed in vials and labeled as TFL: Kolliphor^® P188 (TK188), TFL: Kollidon^®-VA64 (TK64), and TFL: Kollidon^® 30 (TK30), respectively (Table 1). SD batches were stored at room temperature (20  ± 2 °C) until further testing and analysis (Mesallati, Umerska & Tajber, 2019).

Practical yield percentage calculations

Practical yield (%) results reflect the efficiency of the production method and technology parameters. It’s an essential tool for evaluating the cost-effectiveness of the batch. Practical yield (%) can be calculated by using the weight ratio of the precursors and product as indicated in the below equation. ${\displaystyle \text{Practical yield}\left(\text{\%}\right)=\frac{\text{Weight of the precusors}}{\text{Weight of product}}\times 100.}$

Drug content estimation

SD equivalent to 5 mg of TFL was dissolved in ethanol (10 mL), vortexed for one minute. The solution is then filtered through a 0.2 µm syringe filter (Millex™-LG, Hydrophilic PTFE) and diluted suitably. TFL content was estimated by UV-spectroscopy at λ_max 285 nm using UV-Visible Spectrophotometer. (Jasco, V630). All the samples were analyzed in triplicate to get the standard deviation (Mesallati, Umerska & Tajber, 2019). ${\displaystyle \text{Drug content}\left(\text{\%}\right)=\frac{\text{The absorbance of the SD sample}}{\text{Absorbance of TDL}}\times 100.}$

Micromeritic-powder characterization

Fourier Transform Infrared (FTIR) Spectroscopy

FTIR spectrums of polymers; K30, K64, K P 188, TFL, and their respective SDs were acquired by triturating them in KBr individually and making the thin-translucent pellet after compression. A Prepared KBr-sample pellet was placed into the pre-validated FTIR spectrometer (FT/IR-4000; Jasco, Japan). Infrared spectra from the interferometer were obtained by processing the sample signals. IR radiation of wavenumber region from 400–4,000 cm⁻¹ was allowed to pass through the sample analyte, and the resulting signals were recorded, collaged, and interpreted for the possible polymer-drug interactions.

Differential scanning calorimetric analysis

Pure drug (TLF) and prepared three SDs were sealed into the hemispherical aluminum pan by compression. Then the sample (5 mg) loaded pan was placed inside the DSC sample unit, the empty pan was also placed beside it, which acted as a reference. The samples were exposed to controlled temperature from 25–305 °C scan rate 20 °C/min, the differential scanning calorimeter (DSC N-650, SCINCO, Seoul, Korea) was connected with nitrogen supply (10 mL min⁻¹) (Khuroo et al., 2018). Thermal analysis was performed to confirm the crystalline state of the TLF inside hydrophilic polymers’ SDs.

X-ray Powder Diffraction (XRD) analysis

Diffraction pattern of polymers (K30, K64, and K P 188), pure drug TFL and SDs were obtained using diffractometer Ultima-IV (Tokyo, Japan) equipped with Ni-filtered Cu-K α as an ionized radiation X-ray source. Instrument was run using a Cu tube (Anode) at a generator of 40 kV using current 40 mA, 2-theta peak patterns were collected for each sample with scanned rate of 0.02o /min. X-ray beam reflections from the samples are monitored in Counts/sec as a function of the scattered angle.

Scanning electron microscopy

The SEM Zeiss EVO LS10 (Cambridge, United Kingdom) was operated under vacuum. The samples under study (K30, K64 and K P 188) and their respective SD were mounted on the stubs held by two-sided adhesive carbon tape. Current conduct was achieved by a gold (Au) coat on the mounted sample at 20 mA using argon gas atmosphere. The beam of electron passed through the sample detected, converted to voltage, and amplified. The images were captured by tuning the desired zone at 15 kV current voltage (Khuroo et al., 2014).

In-vitro dissolution studies

Prepared SDs were studied for in-vitro dissolution study to interpret the mass conversion rate of TFL from solid to the solution. The sample (SDs equivalent to 50 mg of TFL) was dispersed into 900 mL of 0.1 N HCl dissolution medium (DM) containing 0.25% of dodecyl sulfate sodium salt. USP-II paddle dissolution apparatus (Erweka DT 600, Heusnstamm, Germany) was used under controlled conditions of 37 ± 0.5 °C temperature and rotation speed 50 rpm. The run time was 90 min; at pre-determined time intervals, five mL samples were withdrawn and replaced with fresh DM. Then pre-filtered samples were suitably diluted and analyzed spectrophotometrically at λ_max 285 nm using UV-Visible Spectrophotometer (V630; Jasco, Japan). The mean of at least three aliquots readings was used to plot the drug dissolution-time profiles (Mesallati, Umerska & Tajber, 2019).

In-vivo aphrodisiac study in male-rats

Stability study

The stability study was performed by packing the sample under study into a tightly closed screw-lid glass (Glass laboratory 50 ml) vial. The study was conducted as suggested by MM. et al. by exposing the package for 40 °C or 40 °C/75 ± 0.5% relative humidity (RH) for four weeks, and 60 °C for two weeks respectively in a stability chamber. Thereafter, a drug content estimation and dissolution study was performed in the identical conditions of dissolution study and drug analysis (Ahmed et al., 2021). The dissolution profiles of the SD (TK188) before and after the stability study were compared and the similarity index was calculated by using the equation suggested by Moore and Flanner. ${\displaystyle {f_2}_{=}50_{\mathrm{X}}lo{g}_{10}⌈\frac{100}{\sqrt{1+\frac{\sum _{t=1}^n{\left(R_t-T_t\right)}^2}{n}}}⌉}$ where; R_t and T_t are the mean dissolution value for the reference and test product, respectively at time t, and n is the number of time points.

The f2 value is greater than 50 or (50–100) indicates the release profiles are similar, same or equivalent. A higher f2 value indicates similarity between the test and reference dissolution profiles.

Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) and followed by Tukey test post hoc analysis. Results were expressed as a means of three replicates ± standard deviation; data are presented as mean  ± standard error of the mean. The difference between two values considered as significant levels if P < 0.05. All the calculation and statistical analysis was performed by using Microsoft Office Excel 2016.

Characterization of SDs

It has been observed that the physicochemical properties of SDs chiefly dependent on hydrophilic polymers Kolliphor^® P188, Kollidon^® VA64, and Kollidon^® 30 used in the study.

Yield, drug content percentage, and micromeritics-powder characterizations

The percentage yield of prepared SDs was found to be in the range of (80.81 ± 2.21–87.27 ± 3.13%w/w). The loss of product could be due to the several unit operations involved during the preparations and sticking of SDs to the wall of equipment- glassware. The results were in agreement with Ahmed et al. (2020a) reported (>85%) practical yield of prepared spray-dried sildenafil amorphous SDs. Percentage yield could be improved with batch size and process optimization. The drug content of SDs was found to be between (96.17 ± 2.23 to 99.31 ± 1.00%). All three batches of SDs showed compendia limits of drug content, indicating uniform drug distribution in the polymeric dispersions. Higher drug content represents the presence of hydrophilic functional groups in the polymers. Macroscopic images are reflected in Fig. 1; particles size was measured, and the mean values are presented in Table 2. Particle analysis revealed the size ranges (2.175 ± 0.24 to 4.448 ± 0.11 µm) lower the particle size attributes to low bulk density and flowability. Results of micromeritics properties of all three batches of TFL SDs have been enlisted in Table 2. Bulk and true densities were found to be ranging between (0.496 ± 0.005 to 0.544 ± 0.002 g/cm³), (0.646 ± 0.003 to 0.618 ± 0.002 g/cm³), respectively. The bulk density of the SDs perhaps increases with an increase in the molecular weight of the polymers used. The higher true density value of TK188 represents the fluffy nature of Kolliphor^® P188 polymer due to inter-particulate voids. True density indicates the compactness of powders higher the value, the more compactness of the powder, which could be due to fewer inter-particle spaces. Carr’s Index and Hausner ratio show the propensity of powder under shear stress. Carr’s compressibility index and Hausner’s ratio range was found to be (23.25 ± 0.81 to 12.00 ± 0.23), (1.303 ± 0.003 to 1.136 ± 0.002), respectively. Inference of Carr’s Index and Hausner ratio governs TK188 batch has adequate flow whereas TK64 and TK30 have the good flow. The angle of repose determines the inter-particle friction or cohesiveness of the particles. The value of θ was ≤25 for all three batches indicating non-cohesive particles and excellent flow.

Fourier Transform Infrared (FTIR) Spectroscopy

FTIR spectroscopy showed the functional peaks of TFL in the fingerprint region, the peak for secondary amine (N-H str) at (3,116.4 cm⁻¹), aliphatic-alkyl stretching (2,258.23 cm⁻¹), amide ketone (1,873.51 cm⁻¹), aromatic (C = C) at (1,752.01 cm⁻¹). Moreover, (1,586.16–1,503.24 cm⁻¹), (1166.72–1,136.83 cm⁻¹) for ketone (C-O-C str symmetry) and aromatic ring present in the structure of the pure drug. This data is in good agreement with the pure TFL FTIR reported spectrum. The FTIR spectra of kolliphor^®-188 showed peaks at 2,885 cm⁻¹ and 1,468 cm⁻¹ for O-H and C=O stretching, respectively. Kollidon 30 exhibited peaks at 2,957 cm⁻¹ (OH str) and 1,665 cm⁻¹ (CO str). KollidoneVA64 exhibited 2,954 cm⁻¹ (OH str) and 1673 cm⁻¹ (CO str), which are an agreement with reported spectra (Poudel & Kim, 2021; Aminu et al., 2021).

However, the FTIR spectrums of the binary SDs of TFL with Kolliphor^® P188 (TK188)/ Kollidon^® VA64 (TK64)/ Kollidon^® 30 (TK30) is the sum of spectra of drug and polymer dispersions in which the prominent functional bands of the drug with reduced intensities observed. All the spectrums of TFL and SDs presented in Fig. 2. The FTIR spectra of TK188, TKVA64 and TK30, SDs indicated presence of polymer peaks in each respective SDs. This indicated that there were no interaction of TFL with polymers in the SDs.

Differential scanning calorimetric analysis

All the three batches of SDs showed uniform distribution of TFL as a very fine microcrystalline structure within the hydrophilic polymer matrix of Kolliphor^® P188/ Kollidon^® VA64/ Kollidon^® 30, Fig. 3. The sharp endothermic peak of TFL was observed at 307.5 °C, corresponding to the melting of TFL as reported previously. The reduced intensity of the thermal peak could be due to the complete liquefaction of the TFL crystal and reflects a more amorphous nature.

X-ray powder diffraction (XRD) analysis

Prepared binary SDs represent the microcrystalline structure of TFL within the polymeric matrix, which was further confirmed with SEM. X-rays irradiated to the TFL generate a series of distinct peaks on 7.30°, 10.70°, 12.60°, 13.30°, 14.60°, 15.60°, 17.00°, 18.50°, 21.80°, 24.30° and 25.10° at diffraction angles at 2-theta axis indicating crystallinity nature of the pure drug. In the prepared SDs these peaks turn to broad and reduced in the intensity inferred as less crystalline (Fig. 4).

Scanning electron microscopy

The microstructure of TFL active pharmaceutical ingredient (API) and its binary SDs was studied by photomicrographs captured by scanning electron microscope, represented in Fig. 5. SEM photomicrographs TFL showed the rod-shaped crystalline drug form, whereas SDs represent the ratio of drug-polymer homogeneous smooth irregular and homogeneous aspects. TK188 SDs signify a transformation of the crystalline drug into a microcrystalline/amorphous state, SDs with reduced crystallinity reported to show improved dissolution rate and bioactivity of the poorly water-soluble drug.

In-vitro dissolution, release kinetic models and dissolution parameters

Dissolution profiles of pure drugs (TFL) and three batches have been depicted in Fig. 6. The SDs of TK188, TK66, and TK30 showed 97.17 ± 2.43%, 84.67 ± 2.99%, and 92.67 ± 3.23% dissolution at the end of 120 min, respectively. However, the dissolution from the crystalline TFL was 32.76 ± 2.65% due to its hydrophobicity. Improved dissolution profiles were observed for all the SD batches compared to pure TFL; the drug has changed its state from crystalline to amorphous, which was formerly confirmed by XRD and DSC studies.

The enhanced dissolution rate of SDs can be due to converting a crystalline drug into semi-crystalline to an amorphous or molecularly dispersed state, forming a higher effective surface area; more contact with the dissolution medium resulted in improved dissolution.

The relatively increased dissolution rate of Kolliphor^® P188 (TK188) compared to other polymers Kollidon^® VA64 and Kollidon^® 30 (TK64 and TK30) most likely due to its enhanced hydrophilic and surfactant properties compared to the other agents used. The most significant improvement in dissolution rate was observed in TK188 SD, which demonstrated a 2.96 fold increase in the dissolution percentage compared to pure TFL. The highest dissolution percentage of TK188 could be due to Kolliphor^® P188 wetting property, micelle formation at critical micelle concentration with hydrophilic surfaces and lipophilic core dramatically improved the solubility of hydrophobic TFL drug (Aldawsari et al., 2021). Model dependent kinetic equations were fitted with the dissolution data, and the regression parameters were calculated to analyze the type of drug release from binary SDs. The regression coefficient value (R²) of Korsmeyer–Peppas model was found to predominate, except for TK188, for which the R2 was 0.922, indicating first-order release in which the rate dissolution is a function of the amount of the TFL remaining in the SDs. The possible mechanism of drug release proposed based on the exponent “n” values which were 0.328–0.369, within the range within 0–0.5, suggesting a Fickian type of drug release from the SDs. Improved drug release of TFL from the TK188 SDs was also found to correlate with the findings of release kinetic analysis. Additionally, as per the model-independent parameters, MDT was 17.48, 27.99, 23.57 min, whereas DE was 84.53%, 76.01%, 79.61% for TK188, TK60, and TK30, respectively. Whereas for pure TFL, it was 39.46 min (MDT) and 66.66% (DE). The shortest MDT was obtained for TK188 reduces the MDT by 2.25 fold. Therefore, Kolliphor^® P188 enhances the dissolution efficiency (DE) by 1.26-fold for TK188 SDs compared to TFL alone (Table 3).

In-vivo aphrodisiac activity: sexual behavior parameters- measurement

Results of sexual behavior parameters replicated in Fig. 7, all these parameters reflect significant enhancement in the sexual behavior of male rats administered with the TK188 SDs. Mount frequency (ML) and mount latency (MF) of the test group (TK188) was found to be (48.5 ± 1.57 s), (14.5 ± 0.85), as compared to the STD (56.3 ± 2.15 s), (11.4 ± 0.52), and negative control (127.2 ± 7.16 s), (4.7 ± 0.35) groups respectively. Intromission latency for the negative control, STD, and test TK188 was found (226.8 ± 9.72 s), (91.85 ± 4.75 s), and (80.60 ± 3.10 s), respectively, the time of IL was found to be significantly less for the TK188 SDs. Whereas, intromission frequency (IF) more for the tested SDs it was (12.3 ± 0.52) for TK188, and for standard it was (7.9 ± 0.27), (2.5 ± 0.17) for the negative control. The significant increases in MF and IF with corresponding decreases in ML and IL are the signs of aroused male rats, reflecting the motivation and vigor. Increased duration of EL-1 (479.4 ± 11.50 s) and EL-2 (457.6 ±10.61 s), and decreased PEI (336.7 ± 11.78 s) were observed for TK188 as compared to the STD (EL-1 (435.8 ± 12.22 s) and EL-2 (422.5 ± 11.20 s) and decreased PEI (385.8 ± 11.81 s) and negative control. The CE and IC efficiency were improved showed CE (84.83 ± 4.35%) and IC (45.90 ±1.28%) in animals exposed with TK188 in comparison to the pure TFL (STD) that showed CE (69.30 ± 3.72%) and IC (40.93 ± 1.27%), whereas in negative control CE (53.19 ± 2.15%) and IC (34.72 ± 1.47%). The results indicated the significant effect of the TK188 SDs (P < 0.005). Improvement in the sexual behavior of the treated (TK188) in male rats expressed potential aphrodisiac action. The improved sexual desire could be due to the stimulation of TFL to increase testosterone hormone, as reported by Itoga et al. (2020). Testosterone is a steroidal hormone secreted from the testes, which assists in sexual function and desires.

Stability study

The dissolution profiles of TK188 SDs was plotted (Fig. 8), and the data fitted into the equation, the calculated f2 was 51.58, indicating the similarity between the cumulative percentage drug releases for the SDs (TK188) exposed for different temperature. Drug content was also estimated; only 0.9% amount of TFL was less as compared to the product content before the stability study. The observed difference in TMF content was insignificant for the TK188 SDs tested. Stability tests are carried out so that recommended storage conditions and shelf life can be included on the label to ensure that the medicine is safe and effective throughout its shelf life. The results represented that the prepared SDs (TK188) can be stable in all the climatic conditions. The data could be supportive of designating the product’s shelf-life and ensuring its effectiveness during the expiration duration. Moreover, stability study data is one of the regulatory requirements for marketing approval.