Acquired Immunodeficiency Syndrome (AIDS) has been one of the most devastating pandemic diseases over the last few decades caused by its etiologic agent Human Immunodeficiency Virus (HIV). Latest reports reveal that globally 40 million people are infected with HIV including 2.1 million from India in 2013 [
HIV protease inhibitors (PIs) currently are the key components of first-line therapy in both treatment-resistant and treatment-experienced patients. The introduction of novel second-generation PIs such as Darunavir Ethanolate (DRV) with activity against wild type HIV-1 virus and multidrug resistant strains requires at least four concomitant mutations in the viral genome for resistance development, thus providing clinicians with superior drugs to counter the development of resistance [
The current clinical antiretroviral therapies have suboptimal therapeutic effect attributed to poor bioavailability of anti-HIV drugs which is due to either their poor solubility, extreme first pass metabolism, extrusion into intestine lumen by efflux transporters, drug metabolization by enzymes, or poor permeability. Therefore, there is a need for a delivery system to overcome such solubility and bioavailability issues [
Second approach to increase the systemic availability of DRV is to hamper the drug efflux through P-gp. Coadministration of P-gp inhibitors (therapeutic agents) would result in increase in bioavailability but the toxicity associated with their high dose (required for P-gp inhibition) limits their usage. Ritonavir is the most widely used therapeutic agent for the inhibition of P-gp efflux pumps, thus contributing as a pharmacokinetic booster when given with antiretroviral therapies [
Simple carriers without surfactant properties have been used earlier in order to enhance bioavailability but the carriers with surfactant properties have not been investigated to a wide extent as they possess potential to achieve anticipated bioavailability. These carriers with surfactant like properties have given manifold improved bioavailability in comparison to simple carriers and hence can be preferred over them. Interestingly, utilizing the carriers which possess P-glycoprotein inhibitory activity could potentially enhance the bioavailability of substrate drugs and thus will further add to the therapeutic effect. Therefore, in the present work, Kolliphor as the carrier which exhibits surfactant and P-gp inhibitory activity has been used in order to enhance solubility resulting in enhancement of related bioavailability of an anti-HIV drug, Darunavir (DRV). However, there is no previous study reporting the preparation of solid dispersion of DRV. Some formulation approaches attempted in the past aiming to increase the bioavailability of DRV were complexation of DRV to
In the present study, SDs of DRV were prepared by employing design of experiment (DoE) to investigate different methods of preparation (like melt, solvent evaporation, and spray drying) and to screen and optimize carriers which are surface-active agents and P-gp inhibitors. The effect of food on absorption of drug was also studied both
Till date, third-generation carrier has not been used for the bioavailability enhancement of an anti-HIV agent. Furthermore, Darunavir is an anti-HIV agent which has not been extensively worked upon. Only few research papers are available whose rationale was entirely different from the rationale of the present work. Although the concept of using P-gp inhibitor has also been investigated by a large number of research groups, the use of third-generation carrier with P-gp inhibiting activity for the solubility and bioavailability enhancement of an anti-HIV agent has not been studied previously. Therefore, the present work is novel and remains critically unexplored so far.
Darunavir Ethanolate, Ritonavir, and Atazanavir Sulphate were received as a gift sample from Ranbaxy Research Laboratory (Gurgaon, India). Soluplus, Kolliphor TPGS (d-alpha-tocopheryl polyethylene glycol 1000 succinate), and Poloxamer 188 were obtained from BASF (Ludwigshafen, Germany). PVP K30 (Polyvinylpyrrolidone K30) was obtained from ISP (New Jersey, USA) and HPMC E5 (hydroxypropyl methylcellulose) from Dow Chemical Company (Michigan, USA). Diethyl ether and ethanol were obtained from Merck (Mumbai, India). SIF (Simulated Intestinal Fluid) powder was from Phares AG (Basel, Switzerland). Other chemicals and reagents used were from SD Fine Chemicals, Ltd. (Mumbai, India), and Qualigens Fine Chemical (Mumbai, India) and were of analytical grade. All drug solutions and buffer solutions were freshly prepared before use.
From literature search, six different carriers possessing P-gp inhibiting activity like Poloxamer 188, Kolliphor TPGS, Tween 80, Soluplus, Povidone (PVP K30), and Hypromellose (HPMC E5) were selected and were screened out by phase solubility study to determine the most appropriate and suitable carrier. In phase solubility study, an excess amount of DRV (100 mg) was added to 10 mL of distilled water, each containing different concentrations of carriers (i.e., 0.5%, 1%, 1.5%, and 2% w/v). The samples were prepared in triplicate and shaken in an oscillating water bath (Metrex Scientific Instrument, New Delhi, India) thermostatically controlled at
Soluplus and Kolliphor TPGS were selected based on the phase solubility study as a carrier. From the design of experiment (DoE), custom design was applied to screen (i) method of preparation of solid dispersion, (ii) polymer as a carrier, and (iii) drug to polymer ratio as input variables. The custom design studied the interaction of various categorical and continuous parameters and their effect on the dissolution rate of the drug and on cumulative percentage drug release. The polymer (Soluplus and Kolliphor TPGS) and the method of preparation (melt, solvent evaporation, and spray drying) were selected as the categorical factor and the drug : polymer ratio (1 : 0.5, 1 : 1.25, and 1 : 2) was selected as the continuous factor. The response factor (output variable) of the study was % cumulative drug release at 30 min and 60 min. A set of 18 experiments were generated by software (JMP software version 9) (Table
Saturation solubility and % drug content of eighteen batches of solid dispersion (as predicted by Simple Custom Design) using Soluplus and Kolliphor TPGS. Data presented as mean ± SD,
Exp. | Formulation code | Polymer | Polymer : drug | Method | Saturation solubility (mg/ml) mean ± SD | % drug content |
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1 | SD1 | P1 | 0.5 : 1 | SE |
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2 | SD2 | P1 | 2 : 1 | S.D |
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3 | SD3 | P2 | 0.5 : 1 | MM |
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4 | SD4 | P2 | 0.5 : 1 | SE |
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5 | SD5 | P1 | 0.5 : 1 | S.D |
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6 | SD6 | P2 | 1.25 : 1 | SE |
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7 |
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P2 | 2 : 1 | S.D |
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8 | SD8 | P1 | 2 : 1 | SE |
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9 | SD9 | P1 | 2 : 1 | MM |
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10 | SD10 | P2 | 2 : 1 | SE |
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11 | SD11 | P2 | 1.25 : 1 | MM |
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12 | SD12 | P1 | 1.25 : 1 | SE |
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13 | SD13 | P2 | 1.25 : 1 | S.D |
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14 | SD14 | P1 | 1.25 : 1 | MM |
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15 | SD15 | P2 | 0.5 : 1 | S.D |
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16 | SD16 | P2 | 2 : 1 | MM |
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17 | SD17 | P1 | 1.25 : 1 | S.D |
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18 | SD18 | P1 | 0.5 : 1 | MM |
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P1 = Soluplus, P2 = Kolliphor TPGS, MM = melt method, SE = solvent evaporation, and SD = spray drying.
Saturation solubility study was carried out to determine the increase in the solubility of pure drug in the prepared SDs. Excess amount of the prepared SDs was added to 10 ml of distilled water in glass vials. Samples were kept in triplicate on a water bath shaker at
SDs equivalent to 80 mg of DRV were added to 250 ml of methanol contained in a volumetric flask. Samples were sonicated for 15 min and filtered using a 0.45
Dissolution studies were performed using a USP II Paddle dissolution apparatus (Distek, USA). The prepared SDs were filled in 00 size capsules and placed in Japanese sinkers. The sinkers were then placed in dissolution vessels containing 900 ml of acetate buffer pH 4.5, at 75 rpm and temperature
The dissolution profile of the SD7 was also performed in FaSSIF (fasted state simulated intestinal fluid) and FeSSIF (fed state simulated intestinal fluid) media to determine the effect of food on the absorption of DRV in the intestinal tract. These two biorelevant media, FaSSIF and FeSSIF, were developed to simulate the condition of the intestine in the fasted and fed states [
Blank FaSSIF was prepared by dissolving 6.186 g NaCl, 4.47 g NaH2PO4, and 0.348 g NaOH in 900 ml of distilled water and the pH was adjusted to 6.5 by using 1 N NaOH or HCl using pH meter. The volume was made up to 1000 ml using distilled water.
Blank FaSSIF medium (500 ml) was taken and 2.240 g SIF powder was added to it and this solution was magnetically stirred until the powder completely dissolved to obtain a clear micellar solution. The volume was made up to 1000 ml using the buffer. The solution was allowed to stand for 2 h; it becomes slightly opalescent and thereafter used. A volume of 500 ml is recommended for dissolution for simulating fasted state condition
Blank FeSSIF was prepared by dissolving 8.65 g glacial acetic acid, 11.874 g NaCl, and 4.04 g NaOH in 900 ml of distilled water and the pH was adjusted to 5.0 by using 1 N NaOH or HCl using pH meter. The volume was made up to 1000 ml using distilled water.
Blank FeSSIF medium (500 ml) was taken and 11.20 g SIF powder was added to it and this solution was magnetically stirred until the powder completely dissolved to obtain a clear micellar solution. The volume was made up to 1000 ml using the buffer and thereafter used. For simulating fed state condition 1 L dissolution fluid is recommended.
The experiment was conducted on three formulations including pure drug DRV (15 ml of 1 mg/ml solution), the conventional treatment, that is, DRV + Ritonavir (RTV) (15 ml of 1 mg/ml DRV + 1.7 ml of 1 mg/ml RTV calculated according to the dose of 8 mg/kg) and SD7 (15 ml equivalent to 1 mg/ml of DRV). Permeability coefficients (
The pharmacokinetic studies were performed to compare the plasma concentration profiles of the optimized SD with the pure drug and conventional therapy (DRV + RTV) to check the bioavailability difference between the two. The animal species used for
A rapid, simple, specific, and accurate high performance liquid chromatography (HPLC) with UV detector method was employed for quantification of DRV in SD formulation in blood plasma as reported by Takahashi et al., 2007 [
The albino Wistar rats were divided into two groups: one group contains the rats which were fasted overnight (fasted state), while the other group contains the rats which were fasted overnight and then fed for 15 min prior to dosing (fed state).
The dose was calculated according to the formula:
The fasted and fed groups were further subdivided into four groups with three animals in each group, one for control, the second for pure drug solution of DRV, third for the conventional treatment DRV + RTV, and fourth for the optimized preparation SD7. All the formulations were administered at the required dose (70 mg/kg of DRV, 8 mg/kg of Ritonavir) and were given orally using oral feeding sonde. The blood samples collected at appropriate time intervals were then analysed by HPLC and the pharmacokinetic parameters were estimated.
On the basis of the phase solubility study of DRV in different polymers, Soluplus and Kolliphor TPGS were selected as the two carriers for the formulation of SD based on the highest increase in the solubility of the drug in their respective aqueous solutions. The solubility of the drug in pure water was 0.15 mg/ml and in Soluplus and Kolliphor TPGS solution at drug : polymer ratio of 1 : 2 was 0.823 and 0.922 mg/ml, respectively. Thus, there was 5-6 times increase in the solubility of drug in water using these carriers
The response variables generated for the runs of the custom design were analyzed to get the optimized formulation. The output variables (response variables) of the study were % cumulative drug release at 30 and 60 min. Regression plot for different output variables was obtained and the
The solubility profile of the prepared SDs was found to be directly proportional to the polymer concentration showing an increase with the increase of polymer concentration from 0.5 parts to 2 parts of the drug (Table
The results revealed that the drug content was found to be more in the SDs prepared using Kolliphor TPGS than with Soluplus. The highest % drug content was found to be
The goal of the dissolution study was to illustrate the improvement in dissolution rate of various SDs over pure drug. Impact of polymer and method of SD preparation on drug release was studied through design of experiment. The output variables (response variables) of the design were % cumulative drug release at 30 and 60 min. The influence of individual parameters and their interaction terms on the response variables is known as Parameters Estimates.
Parameters for which
The prediction profiles were generated through the software during the custom design analysis as shown in Figure
Prediction profiles generated by JMP software (version 9).
In Figures
In Figures
Drug release was found to be the highest 98.8%, for the dispersion prepared by spray drying method using Kolliphor TPGS as the polymer in the drug : polymer ratio of 1 : 2 (SD7) which was almost 3 times of the drug release obtained with the drug alone i.e. 30.9%. However, the maximum drug release obtained by the dispersions using Soluplus as the polymer (SD2) by the same preparation method (spray drying) after 90 min was comparatively less i.e. 56.3% (Table
Comparative dissolution profile of nine batches of SD prepared using polymer P1 (Soluplus) and P2 (Kolliphor TPGS) in acetate buffer pH 4.5. Data presented as mean ± SD,
Formulation code | Polymer | Cumulative % drug release (±SD) |
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After 30 min | After 60 min | After 90 min | ||
SD1 | P1 |
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SD2 | P1 |
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SD3 | P2 |
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SD4 | P2 |
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SD5 | P1 |
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SD6 | P2 |
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SD7 | P2 |
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SD8 | P1 |
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SD9 | P1 |
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SD10 | P2 |
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SD11 | P2 |
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SD12 | P1 |
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SD13 | P2 |
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SD14 | P1 |
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SD15 | P2 |
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SD16 | P2 |
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SD17 | P1 |
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SD18 | P1 |
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P1 = Soluplus; P2 = Kolliphor TPGS.
The optimized formulation, that is, SD7 prepared using Kolliphor TPGS as the polymer in the drug : polymer ratio of 1 : 2 by spray drying method, was characterized by the following methods. DSC thermograms showed the melting endothermic peak for pure DRV at 103.3°C [Figure
DSC of (a) DRV (b) Kolliphor TPGS and (c) SD7 = SD of DRV with Kolliphor TPGS.
The FTIR spectra of SD7 showed disappearance of peaks at 3448 and 1710 cm−1 which are characteristic of DRV. No sharp peak of DRV appeared in this region. However, there is a shift in the peak of polymer in the spectra and peaks at 2868 and 1093 cm−1 characteristic of Kolliphor TPGS are retained in the SD (
Polymorphic transformation was observed with help of X-ray diffraction and results were found to be in good agreement with those of DSC. The crystalline nature of the drug was further confirmed by the appearance of sharp multiple peaks obtained in the XRD spectrum [Figure
XRD of (a) DRV (b) Kolliphor TPGS and (c) SD7 = SD of DRV with Kolliphor TPGS.
The SEM images indicated the morphology of the drug to be in crystalline rod shape [Figure
SEM images of (a) DRV and (b) SD7 = SD of DRV Kolliphor TPGS.
Comparison between the cumulative % drug release in FaSSIF and FeSSIF media. Data presented as mean ± SD,
Formulation code | Cumulative % drug release (±SD) |
Cumulative % drug release (±S.D.) |
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After 30 min | After 60 min | After 90 min | After 30 min | After 60 min | After 90 min | |
Drug |
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SD7 |
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SD7 = (DRV : Kolliphor TPGS = 1 : 2 w/w, spray drying).
Apparent permeability coefficients
Formulation code | Equation of graph between drug conc. inside sac ( |
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Apparent permeability coefficient |
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Drug |
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Drug + Ritonavir |
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SD7 |
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SD7 = (DRV : Kolliphor TPGS = 1 : 2 w/w, spray drying).
The plasma conc. profile of DRV in albino Wistar rats following oral administration of SD7 formulation was compared with the plasma profile obtained following administration of pure drug and drug with RTV in fasting and fed state [Figure
Results for various pharmacokinetic parameters by different formulations in Wistar rats. Data presented as mean ± SD,
Groups | Formulations | Pharmacokinetic parameters | ||||
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Group A (fasted) | DRV |
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DRV + RTV |
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SD7 |
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Group B (fed) | DAR |
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1 |
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DRV + RTV |
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1 |
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SD7 |
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1 |
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(
Plasma concentration profile of DRV following oral administration of different formulations in (a) fasted state and (b) fed state. Data presented as mean ± SD,
Therefore,
On the basis of phase solubility studies, Soluplus and Kolliphor TPGS reported the maximum solubility of DRV. However, the solubility of DRV obtained with the carrier Kolliphor TPGS was observed to be slightly higher as compared to that with Soluplus. These results were in accordance with the study reported by Ramesh et al. wherein Kolliphor TPGS showed an enhanced solubility profile as compared to Soluplus in increasing the solubility of Etravirine [
The custom design is applied to the 18 formulations of DRV prepared with Soluplus and Kolliphor TPGS as the carrier to obtain the optimized formulation. This design helped in determining model’s sensitivity to changes in the factor settings and to identify whether the model is fit for the experiment. The
Kolliphor TPGS was selected as the carrier for preparing solid dispersion of DRV attributed to high saturation solubility, drug content, and % cumulative drug release. Further,
The increase in saturation solubility with increase in polymer concentration might be attributed to the physical properties of solubilizing and emulsifying ability of Kolliphor TPGS. It can also be due to possible complexation of the poorly soluble drug with water soluble carrier. Shin and Kim, 2003, reported a marked enhancement in the solubility from 18.25 to 345.75
The high percent drug content for the SDs containing Kolliphor TPGS indicated uniform distribution of the drug in this hydrophilic carrier without any drug degradation and/or precipitation. Also, low values of standard deviation indicated the reproducibility of the method. Some variations were observed for the dispersions prepared using Soluplus, thus indicating improper distribution of the drug within the carrier. Similar results were obtained by Barea and coworkers who reported the drug content of thalidomide to be
On the basis of DoE study, it was seen in Figure
The optimized formulation, that is, SD7, on characterization by DSC showed disappearance of sharp melting peak of drug indicating a reduced degree of crystallinity and that the drug is either solubilized due to the presence of used excipients or present in an amorphous form. Fule and Amin observed similar results with solid dispersion of Lafutidine (LAFT) prepared by hot melt processing approach. The disappearance of peak of LAFT confirmed its conversion to its amorphous form [
Absence of peak characteristic of DRV and shift in the peaks of Kolliphor in FTIR spectra indicated presence of some interaction between drug and polymer. Bond formation between the drug and polymer might have occurred between drug and polymer. Similarly, Li et al. elucidated the reason behind disappearance of peaks of Curcumin in FTIR study to be as a result of interaction due to phenolic, carbonyl, and H-bond between Curcumin and Eudragit® E PO [
XRD revealed there might be transformation of crystalline DRV into amorphous form during the spray drying process as there was disappearance of peaks characteristic to DRV. The reason for this could be that spray drying is an energy intensive process where solution passes from the state of relative unsaturation to supersaturation in a fraction of seconds. Further, rapid evaporation of solvent from the supersaturated atomized droplets of the solution seemingly interferes with the crystal building process leading to amorphization of the drug. These results were in accordance with the results of Shamma and Basha who found out that the diffractogram of the solid dispersion of Carvedilol (CAR) showed complete absence of the distinctive peaks of CAR. The typical diffuse pattern obtained with the solid dispersion indicated the disruption of crystalline nature of CAR and changing into an entirely amorphous state [
SEM microphotographs revealed the presence of drug particles dispersed in the polymer matrix which was confirmed by the smooth texture exhibited in SD. There is no evidence of drug crystals, which confirms the previous findings based on XRD patterns. Similar results were obtained with the SEM results of solid dispersion of CoQ10 which confirmed the existence of CoQ10 in an amorphous form or very fine crystalline form [
Increased release of DRV in FeSSIF confirms the higher concentration gradient of drug at the absorption site which will ultimately lead to an increased absorption. Furthermore, FeSSIF media facilitate this absorption by enhancing the solubilization of drug. So it was concluded that the formulation will be better absorbed when given with food. The study is in concordant with the study elucidated by Sinha and coworkers who demonstrated a better % drug release of solid dispersion of Ritonavir with Gelucire in FeSSIF media than FaSSIF indicating the effect of food on drug absorption [
The reason for the considerably lower value of
The study suggested that improved bioavailability in case of the SD of DRV in both fasted and fed states can be attributed primarily to the solubilizing activity and P-gp inhibiting action of Kolliphor TPGS incorporated in the dispersion. The carrier molecules fluidize the plasma membrane by inserting themselves between tails of the lipid bilayer, interacting with the bilayer’s polar heads and thus modifying the hydrogen/ionic bond forces which may add onto their inhibitory action [
The
Substantial solubility enhancement as well as P-gp inhibition was achieved for the drug DRV by formulating it into solid dispersion using Kolliphor TPGS as a carrier possessing P-gp inhibiting activity. The industrially scalable method, that is, spray drying, was optimized for the preparation of SD using DoE. Findings of DSC, XRD, FTIR, NMR, and SEM confirmed that DRV was present in an amorphous state in the spray dried SD at a drug : polymer ratio of 1 : 2 (w/w). The saturated solubility of DRV was markedly enhanced. The dissolution performed in biorelevant media exhibited maximum dissolution with FeSSIF media than in FaSSIF which confirmed food-related absorption of drugs. The P-gp inhibiting activity of the Kolliphor TPGS polymer was confirmed by both
The authors declare that there are no conflicts of interest regarding the publication of this article.
The authors are grateful to All India Council of Technical Education (AICTE), New Delhi, India, for providing financial assistance for the present study.