1. INTRODUCTION:

MWI could be a well-known methodology for heating and drying materials. Microwave magnetic force irradiations are situated between the infrared and radio frequencies within the vary of 0.3–300 GHz, that corresponds to wavelengths of 01mm to 01m. Microwaves, is directly reworked into heat within the fabric due to their ability to penetrate into any substance, this is often because of moment of molecules that absorbs microwave energy and converts it into heat. Therefore, it's doable to accomplish fast and uniform heating even in materials exhibiting low heat physical phenomenon.¹ This mechanism is employed wide for drying, compound cross linkages, drug–polymer interaction and modification of the structure of drug crystallites through its effects of heating and/or electromagnetic field on the dosage forms.

MWI offers many benefits such as: such as: rapid volumetric heating, no overheating at the surface, addressable heating, energy-saving and low operating cost. In addition the main advantage of not using organic solvents is the absence of any risk originating from residual solvents along with much shorter time.² The microwave heating is eco-friendly method or additionally referred to as green chemistry, this can be as a result of it doesn't produce any fuming gas or any risky by product along with risk of solvent entrapment along with API is also avoided.^3,4

2. MATERIAL AND METHODS:

2.1 Materials:

Analytical-quality chemicals were utilized as received.. RIT was received as bequest trial from Lupin Pharmaceuticals, Aurangabad. Polymers were purchased from Research lab, fine Chem Industries, Mumbai, India.

2.2. Methods:

2.2.1. Compatibility studies:

2.2.1.1. Fourier Transform Infrared Spectroscopy (FTIR) Analysis:

The FTIR studies of RIT, beta cyclodextrin (β-CD) and PM were carried out using (FTIR 4100 spectrophotometer, Jasco Corporation, Tokyo). RIT was subjected to Fourier transform infra-red spectroscopy studies to check the characteristic sharp peak of functional group. The potassium bromide (kbr) disk method was used for the preparation of sample. The sample of RIT was ground and mixed with potassium bromide in 1:9 ratio. The scanning range was 400-4000nm^-1 using potassium bromide as blank. ^5-8

2.2.1.2. Phase solubility Studies:

The phase-solubility technique permits the analysis of the affinity between β-CD and RIT in ethanol. Phase-solubility studies were performed according to the method reported by Higuchi and Connors.⁹

2.2.2. Experimental Design:

2.2.2.1. Preparation of Solid Dispersions

2.2.2.1.1. Solvent method:

RIT and water soluble carrier β-Cyclodextrin was accurately weighed in 1:1 ratio and transferred into Petri plate and dissolved in sufficient quantity of ethanol. The solution was stirred for 15 minutes. It was evaporated with constant stirring .the resulting SD was stored in a desiccator to harden for 4 days. The mass was passed through sieve No. 80 and 100.It was then stored in desiccator.¹⁰

2.2.2.1.2. Microwave method:

Sds of RIT with β-CD were prepared using 3² full factorial experimental designs with the intention of investigating the joint influence of formulation, and process variables using Design Expert® (Version 12). In this study design, 2 factors are assessed, each at three tiers, and investigational trials were carried out in the nine possible combinations. The independent variables were the time of exposure (X1) and power of radiation (X2) at atmospheric pressure as depicted in Table.1 and the % dissolution rate (Y1) was preferred as the dependent variable. SD of Drug and polymer were prepared by microwave induced fusion method.^11-13

Table. 1: 3² Factorial Design for Microwave Solid Dispersion

Factorial Batches	Coded Form		Actual Form
RIT: β-CD (1:1)	X1	X2	X1 (min)	X2 (Power)
F1	-1	-1	4	300
F2	-1	0	4	450
F3	-1	+1	4	600
F4	0	-1	6	300
F5	0	0	6	450
F6	0	+1	6	600
F7	+1	-1	8	300
F8	+1	0	8	450
F9	+1	+1	8	600

3. EVALUATION OF SD:

3.1. Drug Content Analysis:

The drugs-CD complex equivalent to 50mg the RIT for SD by solvent method and all batches of MWI technique were precisely weighed and transferred to a 10ml graduated flasks. 5ml ethanol was added individually and continuously shaken for 20min, The solutions were sonicated for 5 minutes and the final volumes were made up to the mark with ethanol for both the drug. The solutions were filtered by Whatman filter paper 0.45μm size. Then 1ml sample solutions were withdrawn and diluted by 0.1N HCl individually for preparing the desired concentration for spectrophotometric analysis at 240nm of RIT. Every study was tried triplicate. The percentage of drugs content were calculated by the equation -02.^14-16

% Drug content (DC) = M _act÷M_t x100-----Equation 02

3.2. FTIR Analysis:

The FTIR studies of pure RIT along with SD prepared by solvent method and MWI technique were carried out by using similar method describe earlier in compatibility study.

3.3. Optimization of the MWI SD Batches of RIT with β-CD by Design-Expert (DoE) Software:

All the factorial batches of RIT SD by microwave technique were additionally optimized on the basis of evaluation parameter with the help of DoE (version 12). It is fascinating to develop a suitable pharmaceutical formulation while not wastage of staple in shortest potential time.^17-18

3.4. In vitro comparative drug release studies:

The comparative drug release rate of Pure RIT, SD by solvent method and optimized batch of microwave SD’s were find out using the USP type I basket dissolution apparatus using basket of 100 mesh size . The dissolution medium was 900ml of 0.1N HCl (pH 1.2) at 100rpm at a temperature of 37±0.5°C. Samples of 10 ml were calm at time interval of 5, 10, 15, 20, 30, and 60 min, and analyzed using UV visible spectrophotometer at 239nm.¹⁹

3.5. Powder X-ray diffraction (PXRD) studies:

PXRD patterns of pure RIT, SD by solvent method and optimized batch of microwave SD’s of were determined employing a diffractometer (Bruker, AXS D-8 Advance SPPU) equipped with a rotating target thermionic tube and a camera lens direction finder.^20-21

3.6. Differential Scanning Calorimetry (DSC):

The thermal behavior of pure RIT, SD by solvent method and optimized batch of microwave SD’s of were determined using differential scanning calorimetry. 1-2 mg of samples were weighed into aluminum pan and sealed with the lid having a pinhole within the center. Sealed pan-lid was then loaded on DSC instrument and heated from 0°C to 300°C at a heating rate of 10°C/min. Nitrogen was used as the purge gas for the instrument.^20-21

3.7. Scanning Electron Microscopy (SEM):

SEM is used to observe sample surfaces. The surface morphology of the pure RIT and PM powder optimized batch of microwave SD’s and were characterized by scanning electronic microscopy (FEI Nova NanoSEM 450 Bruker X Flash 6130) operating at of 10-kV accelerating voltage.^20-21

4. RESULTS AND DISSCUSSION:

4.1. Compatibility studies:

4.1.1. Phase solubility Studies:

The phase solubility diagram was got through by plotting the molar concentration of RIT versus the molar concentration of the β-CD used. Fig. 01 illustrate the phase solubility diagram of RIT with, β-CD.

Fig. 1: Phase solubility analysis of RIT with β-CD

The slope of the lines was found to be 0.282 for RIT. The slope for the line is less than unity, it was surmised that the enhance in the solubility was noted because of the formation of a 1:1 molar complex calculated from the slope of the phase solubility diagram were 747 M-1 for RIT, which pointed toward a appropriate and stable complex formation. Cyclodextrin-drug complexes with Ks values are reported to be in the range of 200 to 5000 M-1 confirm enhanced dissolution properties. Solubility constant (Kc) value for RIT is in the range of reported values, Therefore, a 1:1 ratio of β-CD was selected for RIT for other studies.

4.1.2. FTIR Analysis:

Compatability studies were performed to reveal and study any possible interaction between the RIT with excipient β-CD. FTIR of RIT (Table. 02, Fig 2) Showed strong characteristic peaks of RIT i.e. 1726, 1625, 1527 also remain present in the PM of RIT with β-CD in a 1:1 ratio indicate no interaction between drug and polymer. Fig.2 shows the characteristic peaks and functional groups which are present and responsible for the antiviral activity of RIT.

Table. 02: Characteristic Peak for RIT Infrared Absorption Bands

Sr. No	Experimental Frequency (cm^-1)	Characteristic Peak
RIT
01	3458	O-H stretching
02	2962	N-H stretching
03	1746	Ester linkage
04	1625	C=C stretching conjugated alkenes
05	1570	C=C stretching alkenes
06	1223	C-N stretching
β-CD
01	1447	Overtones or combination bands of –C-C stretching
02	1199	C-O-C stretching

Fig. 2: IR spectra of (A) Pure RIT (B) β CD (C) PM (D)SD (E) SD-MWI

4.2. Drug Content:

Table 03 shows the average drug content of RIT in PM was 90.32%, and in SD was found to be 94.71% . where as in SD prepared by MWI technique it is 87.23 PM and SD showed the presence of more than 85 % of the drug content and less than ±3 standard deviations from the results.

Table. 03: Drug Content for PM, SD by Solvent and MWI method.

Sr. No	Composition	% Drug Content N=3
01	RIT:β-CD (1:1) PM	90.32± 2.5
02	RIT:β-CD (1:1) SD	94.71 ± 2.5
03	Optimized Batch MWI	87.23 ± 0.58

4.3. Optimization of the MWI SD Batches of RIT with β-CD by DoE:

The statistical assessment of dependent variables were carried out by analysis of variance (ANOVA) using DoE version 12. The ANOVA outcome (P value) of the variables on percentage drug dissolved of solid dispersion by MWI technique are shown in Table 4. The comprehensive outline for results of regression analysis of RIT-SD by MWI is revealed in Table 4. The coefficients for the equations representing the quantitative effect of the independent variables on percentage drug dissolved in 0.1 N HCl for each factors are shown in Table 5. The equations for factor can be generated by putting values of coefficients in Equation 03.

Y = A⁰ + A1X1+ B2X2 + A1B2 X1 X2 + A1²X1²+ B2² X2² ------------------- Equation 03

Table. 04 . Analysis of Variance Results (P value) Effect of the Variables on Percentage Drug release of Solid Dispersions by MWI technique

Source	Coefficient Estimate	F-value	p-value	Suggestion
Intercept	43.77	20.93	0.0154	Significant
A-Power	-3.58	11.84	0.0412	Significant
B-Time	-4.82	21.50	0.0189	Significant
AB	-3.33	6.84	0.0793
A²	-12.21	45.92	0.0066	Significant
B²	7.76	18.55	0.0230	Significant

Since the P-values less than 0.0500 indicates the input value of factors selected for RIT to prepare SD by MWI i.e. Time = 4 min and Power 450 watts as this variable code batch F2 give the higher percentage of drug release that is significant. The small changes in the value of factor effect the study i.e. dissolution rate get affect.

Fig. 3: Predicted v/s Actual Value of Dissolution Rate Of SD of RIT

Fig.3 showing the prediction for % dissolution rate at the dimensions of X1 and X2. The expected worth then compared with the predicted worth of response. These established the closeness in the relationship between the actual and predicted value of the dependent variable. In Fig. 3 prediction response is clear that the factor X1 i.e. Time of exposure increases the dissolution rate response decreases, while another factor X2 i.e. Power exposure as the power is increasing dissolution rate is also increases up to intermediate after that as power value is increases the dissolution rate response is decreases. Thus the actual value ( X1= 4 min, X2 = 450 watt) plotted it shows the resemblance with predicted value.

Fig. 4: Response Surface Plot (3D Surface) of RIT-SD by MWI

From Response surface plot analysis in fig 4 its concluded that the actual value show the similar response as predict for the model study and the model for experimental design selected is significant. Hence optimized batch (F2 in Table 01 value i.e. X1= 4 min, X2 = 450 watt) is selected with reference of statically model is significant by the design expert software in the preparation of SD of RIT with β-CD in 1:1 ratio by MWI technique.

4.4. In vitro comparative drug release studies:

The comparative study of dissolution profile of RIT , PM along with SDs prepared by solvent method and optimized MWI batch shown in fig. 5 from the figure it indicates that pure RIT has a very low dissolution profile of less than 10 % in one hour whereas in the case of PM, the release profile for RIT was about 22% . The SD prepared by Solvent method showing the 47 % release of RIT in 60 min, While SD prepared by MWI technique give 57.87 % release of RIT in 60 min.

Fig. 5: Dissolution profile of RIT , PM and SDs prepared by Solvent and MWI method

4.5. PXRD studies:

Fig. 6 shows X-ray diffraction pattern for pure RIT showed numerous strong distinctive sharp peaks of 1360, 1513 at ~ 21° indicating the high crystalline nature of pure ritonavir. The typical peak intensities in the case of the PM were lower i.e. 1190 ~ 21° than those of pure drug at indicating partial crystallinity in this system. This indicates that, PM of RIT at amorphous as well as crystalline level with the carrier β-CD. The X-ray diffraction pattern of SD showed no strong distinctive peaks like 1513, 1360 and shows the weak intensity peaks 669, and 619 at ~ 18°, 19°, representing that the crystalline nature of the RIT transformed in amorphous structure. The X-ray diffraction pattern of SD prepared by MWI shows the weak intensity peaks 561, and 508 at ~ 17°, 19° also strong distinctive sharp peaks of 1360, 1513 at ~ 21° appear as low intensity of 396 at ~ 21° and indicating that crystalline nature of the drug, totally converted in amorphous form using MWI technique.

Fig. 6 : XRD profile of (A) RIT (B) β-CD (C) PM (D) SD (E) SD-MWI

4.6. DSC:

In Fig. 7 the DSC thermograms of RIT showed a sharp endothermic peak at 125.33°C which is corresponding to their melting point. The thermogram of the PM of RIT slightly shifts with a weak endothermic peak at 124.67°C. The SD prepared by solvent method show shifting of the endothermic peak at 122.46°C and SD prepared by MWI method show shifting of the endothermic peak at 119.56°C. indicates a strong interaction between RIT with β-CD hence confirms complex formation and indicates loss of crystallinity and thus support also with XRD and dissolution studies.

Fig. 7: DSC Thermograms of (A) RIT (B) β-CD (C) PM (D) SD (E) SD-MWI

4.7. SEM:

In Fig. 8, SEM shows photomicrographs initial shape of RIT particles has appeared as non-porous needle-shaped crystals at the resolution of 1µm (fig 08-A) which was observed more clearly as a non-porous, needle-like shape at 500nm resolution (fig 08-A1). After β-CD was added to in 1:1 ratio as a PM, the surface of the rod-shaped particles seemed the smooth exhibited size of between 428 and 536nm particle size (fig 08 B and B1) indicates smoothness in needle structure on same resolutions. The morphological analysis of SD of RIT with 1:1 ratio with β-CD shows cluster of smooth porous edge rod indicates loss of needle structure indicates loss of crystallinity (fig 08 C and C1). The morphological analysis of SD of RIT with 1:1 ratio with β-CD prepared by MWI method shows a group of smooth spongy edges and total loss of needle structure indicates loss of crystallinity (fig 08 D and D1), thus supporting with XRD studies.

Fig. 8 : SEM Analysis of RIT (A, A1), PM (B, B1), SD (C, C1) and SD by MWI (D, D1)

5. CONCLUSION:

The solubility and dissolution release of RIT can be improved through preparation of SD by β-CD. On the comparison among the two techniques, MWI was found to improve dissolution rate to an extent better than in case of solvent method. FTIR and DSC studies depicted that there were no interactions between the RIT and the carrier used. XRD studies specify feasible destruction in the crystal lattice structure of the drug in to amorphous state. SEM studies showed that pure RIT is in crystalline state and SD prepared is irregular form and in amorphous state indicates drug dispersed in carrier. Remarkably SD prepared by MWI technique avoids the use of organic solvent hence the risk of solvent entrapment along with API is avoided with shorter time of exposure of API to microwave. In addition, the technique is environmentally friendly or further called green chemistry because it does not produce fumigating gas or any hazard by product.

6. ACKNOWLEDGMENT:

The authors would like to thank, Lupin Pharmaceutical Aurangabad, for providing gift sample of Ritonavir.

7. AUTHORS CONTRIBUTION:

All the author contributed equally

8. CONFLICT OF INTERESTS:

The authors state that there is no conflict of interest in publishing this paper.

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Received on 02.03.2022 Modified on 08.06.2022

Research J. Pharm. and Tech 2023; 16(6):2643-2648.

DOI: 10.52711/0974-360X.2023.00434