INTRODUCTION:

Oral drug absorption is a very important physiological process for the transport of active pharmaceutical ingredients into the bloodstream and allows the distribution, metabolism, and excretion out of the body ¹. Drug absorption can occur through two important stages. First, the drug must be released from its dosage form and dissolved in gastric or intestinal fluid. This stage is followed by the permeation of active substances through the gastrointestinal membrane, through an active transport process or concentration gradient.

Thus, solubility and permeability are two physical properties that are the main keys to drug absorption¹^,².

According to the Biopharmaceutics Classification System (BSC), pharmaceutical active ingredients are classified into, (1) High solubility - High permeability; (2) Low solubility - High permeability; (3) High solubility - Low permeability; and (4) Low solubility - low permeability³. Interestingly, the majority of pharmaceutical active ingredients are in class II BSC. Low-solubility drugs require the frequency of high doses to achieve therapeutic plasma concentrations after oral administration. Therefore, an increase in solubility is very important for developing formulations of drugs with low solubility⁴. Various formulation technology approaches can be used to improve oral bioavailability from active class II BSC pharmaceutical ingredients. The approaches referred to are micronizing, nanosizing, crystalline engineering, solid dispersion^1,5 cyclodextrin, solid lipid nanoparticles, and colloidal drug delivery systems such as microemulsions, self-emulsifying drug delivery systems, self-micro emulsifying drug delivery systems and liposomes^6,7. Other approaches to improve solubility are cocrystallization, inclusion complexes, and solubilizing agent techniques^8,9. This approach uses excipients and ingredients known to be safe (GRAS = Generally Recognized as Save)¹⁰.

The bioavailability of the majority of APIs in biological systems is determined by their dissolution profiles¹¹. Modification or engineering of crystalline forms can increase the molecular solubility of active pharmaceutical ingredients because it can affect solvation and lattice energy⁸. The rearrangement of molecular packaging in the crystal lattice can dramatically change the solid-state thermodynamics and therefore produce modifications to the relevant physicochemical properties, such as solubility and dissolution rates⁹. In many cases, molecular rearrangement is facilitated by combining the second molecule into the crystal lattice to form multi-component crystals^1,7,12.

The cocrystallization of pharmaceuticals has now been studied as an attractive alternative to increase in solubility of BCS class II drugs^13,14. Pharmaceutical cocrystal is defined as multicomponent crystals of two or more solid-state compounds in ambient conditions, in stoichiometric ratios¹⁵ and kinetic through hydrogen bonds where at least one neutral component and at least one component are pharmaceutical active ingredients^16-21. Research has shown that pharmaceutical cocrystals can change the physical and chemical properties including the solubility of drug molecules. The strategy of using pharmaceutical cocrystals has been successful in increasing water solubility for class II BCS medicine. For example, carbamazepine which has very low solubility in water and also has a low potential for salt formation has been successfully cocrystallized with various components forming the cocrystal^1,19.

Piroxicam is one of the most potent anti-inflammatory non-steroidal drugs in the treatment of musculoskeletal, bone, and joint injuries including ankylosing spondylitis, osteoarthritis, and rheumatoid arthritis. Based on the BCS, piroxicam is a member of the class II drug substance, which has poor solubility and good permeability. Due to its poor solubility in the body fluids, its absorption will be low and the onset of action will be slower^22,23. Piroxicam is a non-steroidal anti-inflammatory drug derived from oxicam with poor aqueous solubility (0.22 mg/ml), thus requiring certain techniques to increase its solubility. Several methods that have been reported include solid dispersion methods²⁴ and nanoparticles²⁵. Rapid onset and good bioavailability of piroxicam are expected to obtain good pharmacological effects. Therefore, a scientific effort must form piroxicam which has a supporting dissolution rate for formulations in oral dosage forms. The recent research that we have done is the formation of piroxicam cocrystals using a malic acid co-former, which aims to increase the dissolution rate of the active substance piroxicam, to improve its pharmacokinetic characteristics.

MATERIALS AND METHODS:

Materials:

Piroxicam (Nantong Jinghua Pharmaceutical CO.LTD) and malic acid (Amresco.LLC 0664) and methanol. All chemicals are analytical grade.

Methods of Crystallization Formation:

Preparation of cocrystal was conducted by the procedure described by^26,27 with slight modification. Piroxicam cocrystals were made with malic acid as conformer using solvent drop grinding method with various ratios (1:1, 1:2: and 2:1). Piroxicam and malic acid were added to 50mL of methanol pa then crushed for 10 minutes constantly. After the treatment, the powder was dried and stored at room temperature.

SEM study:

This analysis was carried out on the co-crystal using a scanning electron microscope (SEM). The Electron Microscope has an increase of 100,000 multiple times by using electrons instead of light on an optical microscope. Several samples were placed on an aluminium sample holder, then coated with gold-palladium (Au) particles with a thickness of ±10nm using a vacuum, the samples were then observed in various magnification of the SEM (BRUKER®).

DSC Study:

Thermal tests were carried out on piroxicam, malic acid, and co-crystal using Differential Scanning Calorimetry (Perkin Elmer®). Sample 3-5mg was on the crimped sample pan which was coated by a 10µL pan crucible pressed. Then, the sample was heated at 26-400^oC at the heating speed of 20^oC per minute. Nitrogen is a gas purge that was used at a flow rate of 20ml/minute.

XRD study:

X-ray diffraction tests were carried out using X-ray Diffractometry (Rigaku®). The sample was placed on a sample holder (glass container) and noticed that the sample volume does not decrease during preparation. X-ray diffraction analysis was run at room temperature with the controlled system (Cu metal as a target, Kα filter, voltage at 40kV on 20-40mA, relative humidity 50%, and analysis carried out in the range 2θ 5-35^oC).

FTIR Study:

The analysis was carried out using the Fourier Transform Infra-Red (FTIR) (Thermo Fisher Scientific®). Preparation of piroxicam, malic acid, and co-crystal was done by forming a KBr plate with a sample ratio: KBr (1:10) at 4000-400 cm^-1_.

In vitro Dissolution Study:

The dissolution rate of Piroxicam cocrystals was determined using United State Pharmacopeia (USP) Dissolution Testing Apparatus 1(rotating basket method). The medium of dissolution test was prepared using 5.8ml hydrochloric acid 37% in 1000ml purified water. The dissolution test was performed using 900ml of the dissolution medium, at 37±0.5°C and 50rpm. A sample (5ml) of the solution was withdrawn from the dissolution apparatus every 15 minutes and the samples were replaced with a fresh dissolution medium. The absorbance of these solutions was measured at 334nm using a UV/Visible spectrophotometer. The percentage of drug release was plotted against time to determine the rate of dissolution profile.

RESULT:

SEM study:

The result of the SEM method on standard piroxicam, malic acid, and cocrystal are seen in Figure 1. Figure a is a crystalline form of Piroxicam, and figure b is a surface form of malic acid, figures c and d are a surface form of the cocrystal.

Figure 1: Characterization of cocrystal by using of SEM (Scanning Electron Microscope) (a) Piroxicam sized 10 µm; (b) Malic acid sized 10µm; (c) Cocrystal sized 10 µm; dan (d) Cocrystal sized 5µm

DSC Study:

The Thermogram of piroxicam shows sharp exothermic peaks at 211.95^oC with a peak onset of 199.60^oC and peak at 216.81°C. The thermogram of malic acid is on exothermic peaks of 155.26^oC with an onset peak of 133.47^oC and an end peak of 161.80^oC. The Thermogram of cocrystal ratio 2:1 showed an exothermic peak of 142.41^oC with an onset peak of 129.07^oC and the end peak of 149.41^oC (figure 2).

Figure 2: Characterization of cocrystal by using DSC (Differential Scanning Calorimetry) (a) Piroxicam; (b) Malic acid; (c) Cocrystal ratio 1:2

XRD Study:

Piroxicam-malic acid cocrystal diffraction shows some typical peaks as seen in Figure 3.

Figure 3: Piroxicam and Cocrystal 1:2 Diffractogram

FTIR spectroscopy:

FTIR spectrum as seen in Figure 4 shows that there is a peak at wave number 3321.4cm^-1 in the form of a single peak, where this peak indicates the presence of NH groups in the standard piroxicam as secondary amines. Next (1750 - 1625cm^-1) there is a peak at 1640.25 cm^-1 which represents the presence of carbonyl bonds (C = O) on the piroxicam standard. Furthermore, peaks appeared at 1528.68 cm^-1 indicating the bending NH group, and peaked at 1262.64 cm^-1 indicating the presence of pyridine groups. In figure 5 which is the result of FT-IR examination of malic acid, it shows that there is a wide peak in the area of 3500-2500 cm^-1 which is at 3446.89 cm^-1, this peak indicates the presence of OH groups in compounds, also found peaks in the area of 1755-1691 cm-1 which is at 1731.22 cm-1 which indicates the presence of carbonyl groups (C = O).

Figure 4: The spectrum of Cocrystal Piroxicam-Malic acid by FTIR

In vitro dissolution study:

The result of the dissolution test on Figure 5 shows that there are increasing of the dissolution for all co-crystal and the highest rate is indicated by ratio 1:2. The ratio 1:2 has a linear dissolution rate which exhibits the enhancement of dissolute sample concentration compared with piroxicam.

Tabel 1: The Result of Dissolution Rate Determination

Period (min)	Concentration of drug
Period (min)	Piroxicam	Cocrystal 1:1	Cocrystal 1:2	Cocrystal 2:1
0	0	0	0	0
15	4,17	6,19	5,49	6,28
30	5,09	7,95	8,35	6,59
45	6,90	9,69	10,33	8,37
60	7,69	10,62	11,72	8,86
75	8,75	11,31	12,30	9,67

Figure 5:The Dissolution Rate Curve

DISCUSSION:

As seen on figure 1, the surface image of cocrystal, there is a change in the structure surface of the two forming materials. As seen on the surface image of cocrystal, there is a change in the structure surface of the two forming materials. The co-crystallization reaction causes particle size reduction, as a result of the breakdown of the crystal lattice and the formation of an amorphous phase. This amorphous phase then forms a new crystalline phase³. The formation of a new crystalline phase indicates the occurrence of a physicochemical bond between piroxicam and malic acid in the crystalline phase.

The Thermogram of standard piroxicam shows sharp exothermic peaks at 211.95^oC with a peak onset of 199.60^oC and peak peaks at 216.81°C. The Thermogram of malic acid is on exothermic peaks of 155.26^oC with an onset peak of 133.47^oC and an end peak at 161.80^oC. The Thermogram of cocrystal ratio 2:1 showed an exothermic peak of 142.41^oC with an onset peak of 129.07^oC and the end peak at 149.41^oC. Based on these data, it is known that cocrystal ratio 1: 2 has a shift in the melting point to be lower than the standard piroxicam, which is equal to 142.41^oC. According to previous research, materials that have a polymorphism form can melt at temperatures of 162-175^oC²⁸. Cocrystal melting point is between the melting point of the active ingredient or conformer, or under active ingredients and co-formers. This is indicated by the correlation between melting point and solubility that a higher melting point is related to the decrease of cocrystal solubility¹⁵^,¹⁸. The decrease in the melting temperature of the crystalline is affected by efforts to increase the solubility of the material by crystallization²⁹. This data is in line with the research findings of a study by Muddukrishna et al. (2014), which demonstrated that the melting point of paclitaxel changed as the cocrystal phase formed³⁰ .

An analysis of the purity of the crystalline phase was carried out using the XRD instrument based on the observation of the characteristic peaks of the pure components³¹. There are peak positions and peak intensity is formed. The diffraction pattern between standard piroxicam and piroxicam cocrystal: Malic acid 1: 2 at a peak of 10, 2 20; 20, 277 (piroxicam) shows an intensity of 20.29(82) cps. In area 2θ; 19, 973, cocrystal shows a very dramatic increase in peak intensity up to 27, 743(304) cps. Except for peak 10, on cocrystal ratio 1: 2, there was an increase in peak intensity at 15(2θ; 23407), 18(2θ; 27,247), and found a peak shift in the form of a new peak at 20(2θ; 27, 497) and at 29(2θ; 37.36). Diffractograms can show the formation of cocrystal piroxicam and malic acid as indicated by peak intensity shift and also the discovery of new peaks. If cocrystal is formed through the interaction of two raw components, it will show a significant difference through X-Ray diffraction between its components⁴. X-Ray Diffraction (XRD) is a method for analysing polymorphism and determining whether there is a change like a crystal or whether a new phase of crystals is formed from the previously carried out crystallization process. Whereas the appearance of a new crystalline phase on the diffractogram indicates the formation of a cocrystal³².

In figure 4 there is an interaction between piroxicam and malic acid in the form of hydrogen bonds. This is indicated by the presence of NH peaks at 3392.15 cm^-1 and OH at 2607.99 cm^-1. The shift of the C = O absorption band to a frequency lower than 1731.22 to 1727.65 cm^-1 and from 1292.64 cm^-1 to 1291.93 cm^-1. This indicates the expected formation of hydrogen bonds, the C = O group from amide (on piroxicam) shifts causing the energy of the C and O bonds to decrease and resulting in a decrease in the intensity of the wavenumber. A shift towards smaller wavenumbers is possible if the C = O group in malic acid binds to NH groups on piroxicam supramolecular heterosynthon so that it requires greater vibrational energy that it causes a shift in wavenumber C = O. In addition, there was an increase in peak intensity of NH from 3391.24 cm^-1 to 3392.15 cm^-1 and a decrease in peak OH intensity from 3446.89 cm^-1 to 2607.99 cm^-1.

The difference in the dissolved percentage of cocrystal is due to differences in the stoichiometric ratio of piroxicam and malic acid. Cocrystal can be in the stoichiometric ratio of different components in the lattice. The difference in the ratio causes differences in the specific physical-chemical properties of cocrystal¹⁵ ³³.

CONCLUSION:

Preliminary characterization of solid-state grinding piroxicam and malic acid showed the formation of a new crystalline phase confirmed by the SEM. Decreasing the melting point as measured by DSC, illustrates the increase in solubility due to the formation of the crystalline phase. XRD data shows a diffractogram peak shift and a new peak appears which indicates the formation of a crystal lattice. Hydrogen bond formation has been confirmed by the FTIR instrument as evidenced by a shift in the peak of the bond. The dissolution test shows that the formation of the crystalline phase between Piroxicam and Malic Acid causes an increase in the Piroxicam dissolution rate up to 61.5%.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 01.08.2023 Modified on 16.12.2023

Research J. Pharm. and Tech 2024; 17(7):3061-3066.

DOI: 10.52711/0974-360X.2024.00479