INTRODUCTION:

Low aqueous solubility drugs are major problem for research scientist in the field of pharmaceutics.¹In the pharmaceutical market the more number of poorly water soluble drug substances has been constantly growing from past decades. Consequently, many perspective has been taken in consideration for improvement of apparent solubility and enhancement of dissolution rate.^2,3There are many perspective have been claimed for enhancement of dissolution profile and solubility such as formation of soluble salts of drugs, use of surfactant, reduction of crystals size, conversion of drugs into prodrugs,⁴ use of amorphous forms, cosolvation, impregnating liquid drugs or drug solution in porous powders,^5,6 micronization, formation of inclusion complexes, formation of amorphous drug, emulsification and formation of solid dispersion (SD).^7,8 The conceptualization of SD was introduced in 1960s by obi and sekiguchi as the solid dispersion; mixer of active ingredients and inert carrier as a solid state.⁹ The SD are one of the optimistic technique to enhance the solubility of low water soluble drug which stands for most of the drugs that are currently investigated. In an aqueous environment when SD introduced its dissolves the carrier and leave the active pharmaceutical ingredient as fine colloidal particles. This idea has number of advantages like flexibility in rate of drug, toxic constituents not use and process is simple.¹⁰

For the treatment of erectile dysfunction (ED), Phosphodiesterase – 5 inhibitor tadalafil has been used as a first line agent.¹¹ Tadalafil belongs to BCS class II. Possession of various credits, such as high selectivity, long duration of action and strong efficacy tadalafil may cause highly variable drug plasma levels and therapeutic failure.¹² So, it is necessary to develop formulation techniques for solubility and dissolution rate enhancement of drug and ultimately to improve the bioavailability.^13-15 Various technique to improving the dissolution rate of tadalafil were reported including the use of solid dispersions using HPMC, PVP, PVP-VP and poloxamer, Size reduction, complexation with cyclodextrins, inclusion in microporoussilicas, liquisolid technique, formulation of nanosuspension, and self-nanoemulsifying drug delivery systems.^16,17

Additionally SD preparation has advantages like simple formulation and nontoxicity. None of the efforts have been done to utilize skimmed milk to prepare SD as a carrier. The merits of the skimmed milk are cheap, easily available, biodegradable and nontoxic nature. Various polymers are used to enhance the solubility of drug but as the amount of polymer increase there is increase in the viscosity of the formulation these is the main drawback of polymer. Formulation of SD with the carrier skimmed milk is recommended to solve the issue of low solubility and it is a cost effective method.¹⁸

The aim of research work was to increase the dissolution profile and solubility profile of low water soluble drug by formulating SD of tadalafil with carrier skimmed milk. The skimmed milk is a colloidal suspension of lipoprotein particle, casein micelles and globular proteins.¹⁹The main casein fractions are κ-casein, α-s2, α-s1and β-casein. Amphiphilic β-casein acts as a detergent molecule which has a surfactant property. The whey protein of milk has the main fractions of α- lactalbumin, immunoglobulin, bovine serum albumin and β-lactoglobulin.²⁰These molecules were found to be surface active and has a superior solubility than caseins. Evaluation parameters were checked in terms of the solubility and dissolution enhancement of physical mixer (PM) and SD with the carrier skimmed milk (SKM). Solid state properties were monitored by x-ray powder diffraction (X-RPD) and differential scanning calorimetry (DSC) in PM and SD in comparison with the pure drug tadalafil.

MATERIALS AND METHODS:

Materials

Tadalafil was been procured as a gift sample from Bharat Parenteral Pvt. Ltd. Vadodara, Gujarat, India. All the other chemicals and solvents used were analytical grade.

SKM powderpreparation:²¹

Accurately measured SKM of 100ml of was spray dried at flowrate of 5ml/min and qualified spray drying parameter like inlet temperature 140 ̊C, outlet temperature 90 ̊C using spray drier (Labultima, India). The 100ml SKM yielded 9.2*g of free flowing powder. (* it is mean of three experiments) and passed through sieve # 65. Powder stored properly for further use.

PM preparation:

Physical mixer were prepared by mixing drug tadalafil and carrier using a mortar and pestle in ratios of 1:1, 1:3, 1:5, 1:7, 1:9 and 1:10. The obtained mixers were put in a desiccator over calcium chloride solution at a room temperature for 64 h and then it was passed through sieve # 65. The powder stored properly the further use.

SD preparation:^14,21

Drug tadalafil and SKM carrier SD were prepared in different ratios of 1:1, 1:3, 1:5, 1:7, 1:9 and 1:10 by using spray drying method. Drug tadalafil to carrier SKM specific ratio were taken. SKM and Tadalafil were dissolved in water and ethanol respectively. Solution was sonicating for 15 min. The prepared solution was spray dried at flow rate of 5 ml/min, inlet temperature 140 ̊C and outlet temperature 90 ̊C in labultima spray drier. The obtained powder was free flowing in nature and store for further use.

Saturation solubility studies:^22,23

Solubility study method was reported by Hecq et al. By using this method saturation solubility studies were performed. Equivalent amount of raw tadalafil, PM and SD formulation kept in glass stopper flask which contains 0.5% SLS solution in water. Agitate the sample by putting the sample on shaker and agitated at 37±0.5 ̊ C for 48 h till the equilibrium was achieved. Nylon filter paper of 0.45µm was used to filter the samples. According to the requirement the samples were diluted and final sample were assayed. UV- visible spectrophotometer used to determine the sample. (Electro lab, India)

Drug content determination:²²

Tadalafil 10mg equivalents for PM and SD were dissolved in 10ml of water and shake the sample for 10 min than add 30ml methanol, sonicate it for 10 min and finally make up the volume with 0.5 % SLS solution. The solution was further diluted up to until it fell under the standard curve absorbance range. The solution was filter using 0.45µm filter paper. UV spectrophotometer used to evaluate drug content at 284nm. Error of protein in skimmed milk was minimizing by evaluating the blank formulation in same manner like tadalafil drug formulation.

DSC analysis:²⁴

Perkin Elmer 4000 was used to recorded DSC thermograms to determine tadalafil drug, skimmed milk carrier and its formulation. PYRIS version – 11.1.0.0488, 2009, Perkin Elmer, Inc software was used for DSC analysis. Around 1mg of sample was heated between the temperature range of 30-350 ̊C and rate of heating was 10 ̊C/min. It was analyzed under a nitrogen atmosphere and on aluminum pan under nitrogen purging and 20ml/min flow rate.

FTIR analysis:¹⁴

FTIR spectra of moisture-free pure Tadalafil powder, Skimmed milk and formulations were obtained using a FTIR spectrophotometer using KBr as a reference.²² The scanning range was 400–4,000cm ^-1(Shimadzu 8400s).

X-RPD analysis:²⁵

X-RPD of tadalafil, carrier SKM, SD and PM was recorded with an X-ray diffractometer (Shimadzu, Maxima XRD - 7000, Japan) under influence of “Cu” radiation target having nickel filter, 30mA current.

In-vitro dissolution:^26,27

Equivalent amount of 10mg Tadalafil was taken for in-vitro dissolution study. Samples were added to 900ml of 0.5% SLS in water at 37±0.5 ̊C and speed of rotation was 50rpm using paddle type apparatus (USP type II). Withdraw the samples at specific time intervals of 5, 10, 15, 30 and 45 min. Sink condition was maintain by replacing with the fresh dissolution media. After the filtration of samples, diluted till require. UV spectroscopy was used to analyze the sample at 284nm. Percent cumulative drug release versus time profiles was plotted. The study was done in triplicate for each formulation.

RESULTS AND DISCUSSION:

Drug content and solubility profiles

The present study to prepare SD is to enhance the solubility and dissolution rate of low soluble tadalafil drug. Raw tadalafil, PM and SD drug content and solubility data is given in table 1. Drug content from the results shown that prepared PM and SD was in the range of ± 5% of theoretical amount, indicates that prepared method was reproducible and suitable. Saturation solubility of raw tadalafil was found to be 0.0026±0.15 mg/ml. The solubility was enhanced by 0.0719±0.06 mg/ml using SD technique and the solubility of the PM was enhanced by 0.0133±0.17mg/ml. Particle size reduction and amorphous state formation may be the responsible factor for the enhancement of solubility. Surface active agent is responsible for better solubility in case of PM due to presences of surface active agent and amino acid content of milk. The hydrophilic casein micelles entrap tadalafil to enhanced solubility.²⁸ The results reveled that SKM was capable to increase the solubility of drug tadalafil. As skimmed milk mostly consists of globular particles and lipoprotein which may constrain with spectroscopic studies, the relevant blank formulations were treated in the same manner as the test formulation.

Table No 1: Drug content and solubility of PM and SD

Sr. No.	Code	Ratio	Drug content %	Solubility mg/ml (0.5% SLS)
1	TSPM 1	1:1	99.45±0.13	0.0026±0.03
2	TSPM 2	1:3	98.67±0.06	0.0028±0.08
3	TSPM 3	1:5	96.54±0.34	0.0064±0.04
4	TSPM 4	1:7	96.40±0.16	0.0081±0.16
5	TSPM 5	1:9	97.43±0.08	0.0120±0.08
6	TSPM 6	1:10	98.19±0.14	0.0133±0.17
7	TSSD 1	1:1	96.54±0.46	0.0254±0.05
8	TSSD 2	1:3	98.64±0.79	0.0425±0.23
9	TSSD 3	1:5	96.82±0.54	0.0498±0.21
10	TSSD 4	1:7	98.98±0.84	0.0637±0.26
11	TSSD 5	1:9	97.49±0.49	0.0698±0.15
12	TSSD 6	1:10	99.24±0.74	0.0719±0.06

In-vitro dissolution:

The dissolution profiles of pure drug, PM and SD are manifested in figure 1. Pure drug showed dissolution of only 28.56 % ± 0.54 within 45 min. The dissolution of PM formulation was in range of 35.56% to 83.54% where an SD formulation was in range of 49.45% to 98.65%. It was higher than the pure drug. The highest dissolution was shown in batch TSSD 6 and TSPM 6; 98.65% and 83.54% respectively. It is corroboration that increasing the hydrophilic carrier matrix, increase the dissolution of drug. % CDR of the drug increased with increasing the SKM. The rise in dissolution and solubility displayed by SD can be elucidated by the creation of fluffy and porous particle by spray drying technique. Dissolution rate of drug increased, due to higher surface area and ultimately surface free energy. The SD mixer showed greater dissolution rate as compare to PM. Presence of inhibits crystal growth of the drug assist higher dissolution. Moreover, existence of carrier suppresses aggregation of drug particle and improves the dissolution. Wetting property of carrier has higher influence on dissolution. Carrier also having surfactant property, which reduced the interfacial tension between dissolution medium and drug, gives better dissolution. Therefore TSPM 6 and TSSD 6 were chosen for additional studies.

Figure 1: In-vitro dissolution profiles of tadalafil PM and SD

Solid state characterization

Solid state properties of the system were scrutinized by the means of DSC and XRD.

DSC thermograms:

To determine solid state characterization, DSC studies have been a key tool to signalize solid state interaction of tadalafil and SKM. The thermograms are shown in figure 2, it were analyzed by investigating the peak temperature and endothermic transition profile. The DSC curve of tadalafil shows endothermic peak at 307.56 ̊ C commensurate to its melting point that specify the crystalline nature of drug indicated in figure 2 (A). The skimmed milk evinced characteristic endothermic peak at 58.85 ̊ C and 197.36 ̊ C elucidate the amorphous nature of skimmed milk indicated in figure 2 (D). The thermograms of TSPM 6 showed the peak at 295.66 ̊ C, 192.67 ̊ C, 60.16 ̊ C blend of skimmed milk and drug peaks indicated in figure 2 (B). On the contrary, drug peak is not present in TSSD 6 thermograms enlighten, the drug has interacted with the carrier that is skimmed milk has converted to amorphous nature indicated in figure 2 (C).²⁹

Figure 2: DSC thermograms of tadalafil (A), TSPM 6 (B), TSSD 6 (C) and skimmed milk (D)

Figure 3: XRD of tadalafil (A), TSPM (B), TSSD (C) and skimmed milk (D)

XRD diffractograms:

XRD pattern of tadalafil, TSPM 6, TSSD 6 and skimmed milk are given in figure 3. Tadalafil figure 3 (A) showed sharp and intense peak at diffraction angles of 7.33°, 12.62°, 14.47°, 14.56°, 18.49° and 21.14°, which reflect high crystalline nature of tadalafil. The skimmed milk diffractograms favor a typical amorphous material lacking of any characteristic peak figure 3 (D). The XRD diffractograms delineated by TSPM 6 divulge a decrease in the peaks number which indicates reduction in crystallinity figure 3 (B). The diffractograms of TSSD 6 indicate decrease in the intensity of the peaks, which represents the conversion of drug amorphous from figure 3 (C). These results are in conformity with those explained previously in DSC study. These indicate decrease crystalline nature of tadalafil because of hydrophobic core of SKM is casein micelles.

FTIR spectroscopy:

The FTIR spectra of samples are shown in figure 4. FTIR tadalafil spectra evince characteristic peaks of C-N stretching, C=C aromatic, C=O amide, C-H stretching aromatic, N-H stretching secondary amine, benzene, C-O single bond, C-H stretching aliphatic CH₃ at 1435.09, 1645.33, 1668.48, 3072.71, 3327.32, 742.62, 1043.52, 2893.32 wave number respectively. In spectra of TSSD 6 formulation, the SKM carrier characteristic peak were present at nearly same positions and peaks of tadalafil were also present but at lessen absorption rate indicate drug trapping in carrier. None of the spectra showed any peak other than drug and SKM indicating absences of chemical interaction.

CONCLUSION:

In the conclusion, the current study investigation suggested the probability of preparation of an SD of tadalafil with carrier skimmed milk by spray drying technology. Crystalline form of tadalafil converted to amorphous form. The DSC and XRD studies indicated formulation of SD which significantly improves the solubility of poorly soluble drug and dissolution profile of drug.

ACKNOWLEDGEMENTS:

The authors thank Bharat parenterals Pvt Ltd, Vadodara, for providing tadalafil drug as gift samples for this work. They also thank Parul institute of pharmacy and research, parul university, Vadodara, for providing required facilities to carry out this research work.

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interest.

ABBREVIATIONS:

SD: Solid dispersion

ED: Erectile dysfunction

PM: Physical mixture

SKM: Skimmed milk powder

DSC: Differential scanning calorimetry

X-RPD: X- ray powder diffraction

FTIR: Fourier transforms infra-red spectroscopy.

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Received on 27.11.2019 Modified on 21.01.2020

Research J. Pharm. and Tech. 2020; 13(12):6212-6217.

DOI: 10.5958/0974-360X.2020.01083.5