1. INTRODUCTION:

Pharmaceutical moieties are subjected to changes in their properties due to several factors like, temperature, humidity, stability issues and so on. Changes in drug properties results in pharmacokinetic and pharmacodynamics variation. Where, pharmacokinetics deals with absorption, distribution, metabolism and excretion of the drug and pharmaco-dynamics with drug response to the body’s active site. It also includes variation in the stability, solubility, melting point, dissolution, micrometrics, hygroscopicity, etc¹.

According to the definition of solid-state chemistry, polymorphism is the capacity of a molecule for the development of multiple organizations to their crystalline lattice². Polymorphism helps to generate physical, chemical and physico-chemical properties of a molecule³. Various modification of crystals i.e. polymorphs exhibit subsequent changes in solubility, stability, optical properties and melting temperature and these characteristics are carried into great consideration in pharmaceutical industries as they typically alter drug bioavailability in greater extent^4,5. The Bioavailability problems can be resolved by polymorphic form such as solvate, hydrate and de-solvate form and there will be an issue related to both physical and chemical stability of the active pharmaceutical ingredients (API) throughout the formulation, packaging and storage during that formulation life cycle⁶.

Acetaminophen (paracetamol), also commonly known as Tylenol, is the most commonly taken analgesic worldwide and is recommended as first-line therapy in pain conditions by the World Health Organization (WHO)⁷. It is also used for its antipyretic effects, helping to reduce fever. This drug was initially approved by the U.S. FDA in 1951⁸. Pure paracetamol is in crystalline nature and exhibits three polymorphic forms which can be supercooled and an amorphous form^9,10,11. The stability range of these polymorphs and amorphous paracetamol are in the following order, form I > form II > form III > amorphous whose stability depends on standard temperature and pressure^12,13. Evidently, minor changes in the experimental conditions affects the crystallisation behaviour of paracetamol and leads to different crystallisation pathway resulting in different polymorphs^14,15. Though crystalline nature of drug offers advantages like high purity and physical or chemical stability, its crystal lattice energy acts as a major constrain for dissolution of the drug¹⁶. And the challenges of polymorphism in pharmaceutical industry also impels difficulty in manufacturing of crystalline drugs¹⁷.

On the other hand amorphous state exhibits a disordered structure compared to crystalline form and possess higher free energy and leading to high water solubility, rate of dissolution, oral absorption and bioavailability. Pure amorphous drugs are not used in formulation of pharmaceutical drugs due to their inherent physical and chemical instability¹⁸. The instability of amorphous nature is due several factors like, glass transition temperature, Preparation method, Humidity mechanical stress, and temperature¹⁹. The thermodynamic stability of amorphous state clearly depicted by glass transition temperature. Below this temperature it exists in amorphous form and above this temperature it exists as a supercooled amorphous liquid state. A higher-class transition temperature indicates delayed recrystallization process and thus enhancing the stability of amorphous form²⁰. The solubility advantages of the amorphous forms has encouraged the development of amorphous solid dispersions which can be retained effective strategies. Solid dispersions are manufactured by different techniques like fusion method, solvent method, and novel strategies like supercritical fluid method. Thus, the study is concentrated in formulation of amorphous paracetamol by fusion method using PEG 4000 as a polymer in order to increase the solubility and eventually enhanced bioavailability. The formulated amorphous paracetamol are characterised by thermal method in order to determine the solubility.

2. PREFORMULATION STUDIES:

2.1 Standard calibration curve:

Calibration curves of the drugs are plotted to know the linearity of the drug when observed at different concentrations by using UV- Visible spectrophotometer. To plot the calibration curves the drug has to be dissolved in a particular solvent and then it has to be diluted with the solvent to the required concentrations.

10mg of paracetamol was dissolved in 10ml of methanol which formed a concentration of about 1000mcg/ml. Then from this stock solution 0.5ml, 1ml, 1.5ml, 2ml, 2.5ml and 3ml of the solution was taken in different volumetric flasks of 10ml volume. These solutions were made up to 10ml with the solvent giving the concentration of 5mcg/ml, 10mcg/ml, 15mcg/ml, 20mcg/ml, 25mcg/ml and 30mcg/ml. These formulations were analyzed for absorbance at 249nm and the graph was plotted for the assessment of linearity.

2.2 Microscopic Examination:

Pure paracetamol and polyethylene glycol 4000 were taken in separate glass slides, a smear was made and was examined under the microscope.

Two batches of different ratios (1:0.5, 1:1, 1:2, 1:4) were prepared using the above method and amorphous form of paracetamol were obtained.

And these products were subjected for the evaluation studies like solubility, DSC, stability studies and X-ray powder diffraction test. Results were collected and compared with the standard

2.3 Solubility studies:

Solubility of the paracetamol were determined in Millipore Water by through adding overload quantity of drug to 10ml volumetric flask. This was set and kept aside for 72 hours at room temperature to get the poise. The supernatants were taken and the next concentration of paracetamol was determined by appropriate dilutions and the absorbance were obtained using UV-Visible spectroscopy at 249nm.

2.4 Compatibility studies:

Compatibility of the drug and the excipient were studied using Perkin Elmer spectrum 2(FTIR) and DSC.

2.4.1 Differential scanning calorimeter:

Predictable and high-speed DSC experiments were carried out using DSC by TA$ instruments. Paracetamol, polyethylene glycol and the physical mixture (1:1) were evaluated for their thermal behavior. samples were placed in crimp reference aluminium pan and heat up to 20-400C at the heating rate of 10C/min and passing the nitrogen gas 30ml/mi. Blank pans were sealed s similar to that of the samples. DSC was obtained by mechanical thermal analyzer system. Calibration of temperature were done through calibration of the reference standard. Thermograms were obtained from the DSC and it’s used for comparing and finding compatibility of both standards and the physical mixture.

2.4.2 FTIR measurements:

The FTIR was used to identify the compatibility of paracetamol and polyethylene glycol 4000 and their physical mixture. The IR spectra of the standards and physical mixture were collected using FTIR spectrometer.

3. METHODOLOGY:

3.1 Method A:

Polyethylene glycol 4000 was taken in beaker. The beaker was kept in a boiling water bath of temperature around 50°C. PEG starts melting and to that paracetamol was added and mixed well. Solid dispersion was expected but it gave a sticky mass of paracetamol and PEG 4000.

Table 1: Composition of SD formulation using method A

Batch			Paracetamol	PEG 4000
Method A	Method B	Method C	Paracetamol	PEG 4000
F1	F5	F9	1	0.5
F2	F6	F10	1	1
F3	F7	F11	1	2
F4	F8	F12	1	4

3.2 Method B:

PEG 4000 was taken in a beaker. To that paracetamol was added and mixed well. The mixture was triturated until a fine powder was obtained. Uniform mixture of solid dispersion was expected but it was not obtained.

3.3 Method C –PEGlyation:

Paracetamol was taken in a china dish and the china dish was exposed to Bunsen burner. Once the paracetamol started to melt, PEG was added. The ingredients were mixed well until uniform distribution was attained.

3.4 Solubility studies:

Solubility of the prepared paracetamol-PEG mixture using method C were determined in Millipore Water through adding overload quantity of drug to 10ml volumetric flask. This was set and kept aside for 72 hours at room temperature to get the poise. The supernatants were taken and the next concentration of paracetamol was determined by appropriate dilutions and the absorbance were obtained using UV-Visible spectroscopy at 249nm.

3.5 Differential scanning calorimeter:

Predictable and high-speed DSC experiments were carried out using DSC by TA instruments. The prepared mixtures of paracetamol-PEG with different ratios using method C were evaluated for their thermal behavior. samples were placed in crimp reference aluminium pan and heat up to 20-400C at the heating rate of 10C/min and passing the nitrogen gas 30ml/mi. Blank pans were sealed s similar to that of the samples. DSC was obtained by mechanical thermal analyzer system. Calibration of temperature were done through calibration of the reference standard. Thermograms were obtained from the DSC and it’s used for comparing and finding compatibility of both standards and the physical mixture.

4. RESULTS AND DISCUSSION:

4.1 Preformulation studies:

4.1.1 Calibration curve:

Paracetamol was dissolved in water at required dilutions and the absorbance observed was tabulated in the table. The observed absorbance in y axis and the concentration in the x axis Calibration curve was plotted and the linearity was found to be R2=0.9995.

Figure 1: Calibration curve of Paracetamol and microscopic examination of pure paracetamol

Table 2: Linearity curve of paracetamol pure drug

Standard calibration curve
Concentration (mcg/ml)	Absorbance
0	0
5	0.316
10	0.531
15	0.761
20	0.897
25	1.181
30	1.513

4.1.2 Microscopic examination:

The image Fig 1 here shows the microscopic structure of pure paracetamol which exhibits in needle shaped crystals.

4.1.3 Solubility Study:

The aqueous solubility of standard paracetamol was found to be 4.15mg/ml.

4.2 Compatibility studies:

4.2.1 Differential Scanning Calorimeter:

The studies were performed as per the procedures given in experimental session. The thermogram of paracetamol alone and the physical mixture of paracetamol and polyethylene glycol was analyzed considering the melting point (thermal reaction) of the individual components. The thermogram obtained from analysis shows that no interaction between paracetamol and PEG 4000 .since there were no peaks shifting of paracetamol and PEG 4000 in analyzing the mixtures with pure paracetamol and PEG4000 the melting point lies in the expected range and the melting point of PEG4000 were also in the proper range shows there was no interaction.

Figure 2. DSC thermogram of PEG 4000, Paracetamol and physical mixture

Thermogram of both the pure standards were obtained and the melting point of paracetamol was found to be 170C, the melting point of PEG 4000 was found to be 63.99C.

The products of different ratio 1:0.5, 1:1, 1:2, 1:4 were taken and evaluated under the DSC Q 200, Thermogram instrument. The thermograms obtained are listed in Fig 2. Where it showed the conversion of crystal paracetamol to amorphous form. The conversions were confirmed using the obtained thermograms below where it shows blend peaks rather than sharp peaks as shown in fig pure paracetamol.

4.2.2 FT-IR spectroscopy:

The interactions between paracetamol and polyethylene glycol 4000 in the solid state were probed by FTIR studies. The changes in the bandwidth and frequency are apparent of interacting groups in the spectrum. If the interactions of the paracetamol and PEG 4000 existed, the functional groups in the IR spectra should have shown the band shifts and augmentation as compared to the spectra of the pure paracetamol and PEG4000.

Figure 3. IR spectra of Paracetamol, PEG 4000 and physical mixture

Table 3: IR interpretation data

Peak	Paracetamol	PEG 4000	Physical mixture	Interference
O-H	3160.05	2883	3159.40	No
C=C	1649.6	1101
N-H	1435	-	1439
C-H	1171	-	-
C-O	-	1147	1147
CH2	-	1341	1341
C-C	-	-	1103

The peaks obtained for the standards and the physical mixture are analogues to each other. Hence Paracetamol and PEG4000 are compatible to each other. The drug paracetamol and physical mixture the spectra from IR studies at a wavelength from 4000 cm^-1 to 400 cm^-1. After the interpretation, it was observed that no functional group loss in between the spectra of drug and the physical mixture i.e. no major shifting occurred and it confirms that paracetamol is compatible with excipient.

4.3 Evaluation of amorphous paracetamol:

Three different ratios 1:1, 1:2, 1:4 were prepared by method A which did not gave a proper amorphous formation. In method B the excipients did not uniformly distributed. Hence the preparation method was changed. In method C the excipients were uniformly distributed. and gave a proper amorphous formation.

Table 4: Observation for Method A

Batch	Ratios prepared	Product observed
F1	1:0.5	Powder without uniform mixture
F2	1:1	Sticky mass
F3	1:2	Sticky wet mass
F4	1:4	Too hard to triturate into powder
F5	1:0.5	Powder without uniform mixture
F6	1:1	Powder without uniform mixture
F7	1:2	PEG remained separate
F8	1:4	Sticky wet mass
F9	1:0.5	Obtained the expected uniform amorphous mixture
F10	1:1
F11	1:2
F12	1:4

4.4 Solubility study of the products:

The solubility of samples was examined through UV visible spectroscopy to determine the concentration and the absorbance were obtained; concentration was calculated using the slope. Hence there is a gradual increase in the solubility as the ratio increases.

Table 5: Observation for Method C

S. No	Sample	Absorbance	Conc. of paracetamol (Mg/ml)
1	1:0.5	0.701	1.4122
2	1:1	0.784	1.5873
3	1:2	0.831	1.6864
4	1:4	1.021	2.0873

Figure 4. solubility study of amorphous paracetamol

4.5 Differential Scanning Colorimetry:

Paracetamol and PEG 4000 exhibits characteristic sharp endothermic peak at 170˚C and 53.14˚C respectively, indicating the decomposition of the substances and as well as the crystalline nature of the drug and PEG 4000. The PEGylated paracetamol exhibits broad and short endothermic peak, indicating the amorphous nature of the drug which is due to formation of solid dispersion. The shifting of melting points of PEGylated paracetamol of ratio 1:4 and 1:2 (51.14˚C) and (51.00˚C) indicates, increase in solubility of the drug.

5. CONCLUSION:

Amorphous paracetamol was formulated by fusion method using PEG 4000 as a polymer and the results revealed that it is technically feasible to prepare amorphous paracetamol with PEG4000 in a minimum ratio of 1:4. The product obtained by PEGylation under controlled parameters and environmental factors is suspected that paracetamol shows 1.47 folds increase in its aqueous solubility. Therefore it is ultimately expected that the increase in the solubility index of paracetamol-PEG complex would result in higher dissolution and bioavailability as compared with marketed available solid formulations.

6. ACKNOWLEDGMENTS:

The authors would like to thank Department of Science and Technology-Fund for Improvement of science and technology infrastructure in Universities and Higher Educational Institutions (DST-FIST), New Delhi for their infrastructure support to our department.

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Received on 25.11.2019 Modified on 31.03.2020

Research J. Pharm. and Tech 2021; 14(3):1487-1492.

DOI: 10.5958/0974-360X.2021.00264.X