INTRODUCTION:

Preformulation studies are carried out soon after the completion of pre clinical / clinical trial and before undertaking actual formulation and development of dosage form. Preformulation is one of the phase of R & D where physical and chemical characterization of a drug molecule in undertaken¹.

Preformulation testing is designed to judge the influence of physicochemical properties of drug substances and excipients on formulation properties of dosage form, method of manufacture and pharmacokinetic–biopharmaceutical properties of the resulting product. Preformulation studies help us to determine compatibility of API (new drug entity) with common excipients². It guides formulator to choose correct form of a drug substance and to improve bioavailability³. It assists to gather complete knowledge of new drug entity. It also aids in determining the method of storage of formulation. Finally we can say preformulation studies will assists us in developing safe, effective and stable dosage form¹.

Glipizide is an oral hypoglycemic agent and belongs to second-generation sulfonylurea⁴ drug class that is used to control blood sugar levels in patients with type 2 diabetes mellitus. Glipizide appears to lower blood glucose acutely by stimulating the release of insulin from the pancreas, an effect dependent upon functioning beta cells in the pancreatic islets. Gastrointestinal absorption of glipizide is uniform, rapid, and essentially complete⁵. The absolute bioavailability of glipizide in patients with type 2 diabetes receiving a single oral dose was 100%. Glipizide belongs to BCS class II drug which has short elimination half life. Objective of the project was to conduct preformulation study of Glipizide in order to development of a stable, robust as well as the therapeutically effective and safe dosage form of Glipizide⁶.

In order to achieve this objective characterization of glipizide was done by finding out Bulk Density, Tapped Density, Compressibility Index and Hausner’s Ratio⁷. Solubility of drug in various solvents of varying pH was determined. Infrared spectrum was done to determine purity of drug⁸ and UV Spectra was developed which will help in further analytical studies. Loss on Drying (LOD) was carried out to calculate assay compensation. Finally drug-excipients compatibility studies were carried out to rule out any drug – excipient interactions⁹.

MATERIAL AND METHODOLOGY:

API AMSA Italy); Polyethylene Oxide-Sentry polyox WSR N80 and Polyethylene Oxide-polyox WSR 303 (Glipizide Dow Chemical company); Sodium Chloride (Merck); Hypromellose-METHOCEL TM E5 Premium LV, Opadry White YS–27063 and Opadry Blue OY–LS20921 (Colorcon); Magnesium stearate (Mallinkrodt), Iron Red oxide (Sicovit); Cellulose acetate-CA–398–10 (Eastman Chemical Company); Polyethylene glycol 400 (BASF); Polyethylene glycol 6000 and Polyethylene glycol 3350 (Clarient).

PREFORMULATION STUDIES:

Organoleptic properties:

Organoleptic properties of the drug sample were studied by visual inspection.

Characterization of Drug Glipizide:

The drug substance was characterized for following parameters.

Bulk Density: 25 g of Glipizide was accurately weighed, sifted through #18 and transferred into 100 ml graduated measuring cylinder. The volume of the mass without compacting was observed. Then unsettled apparent volume level was noted, V_o. The bulk density was calculated by following formula.¹⁰

B.D = (M) / (V_o) Where, M = Weight of the test sample V_o = Unsettled apparent volume

Tapped Density:

Accurately weighed 25 gm of candidate drug was screened through #18 ASTM and transferred into a 100 ml graduated measuring cylinder without compacting. Cylinder containing the sample was tapped mechanically by raising the cylinder and allowing it to drop under its own weight using a suitable mechanical tapped density tester that provides a fixed drop of 14 ± 2 mm at a nominal rate of 300 drops per minute. Cylinder was tapped 500 times initially and tapped volume, V_a was measured to the nearest graduated unit. Tapping was repeated an additional 500 times¹⁰ and tapped volume, V_b was measured to the nearest graduated unit. When the two volumes is less than 2%, V_b was declared as final tapped volume, V_f 1250 taps was incremented till the difference between succeeding measurements was observed more than 2%. Tapped density measured in g per ml. It was calculated by the following formula¹¹:

T.D = (M) / (V_f) Where, M= Weight of the test sample, V_f = Final tapped volume.

Compressibility Index:

Particle–Particle interactions affect bulking properties and powder flow. Comparing bulk and tapped densities will give us a measure of the relative importance of these interactions in a given powder¹⁰. Compressibility Index was calculated by the following formula:

CI (%) = (Tapped density– Bulk density) Χ 100 / Tapped density

Hausner’s Ratio (H):

As the difference between the bulk and tapped density increases flow property decreases. These difference is reflected in Hausner Ratio¹². Hausner Ratio was calculated by the following formula¹²:

Hausner’s ratio = Tapped Density / Bulk density

Determination of pH Solubility:

Solubility is an essential and extensively studied preformulation parameter¹³. The solubility of drug Glipizide was checked in methylene chloride, acetone and ethanol 95%. Further solubility was determined as per BCS classification system¹⁴. The solubility was checked in 250 ml different medium and water¹⁵. The highest amount of dose was accurately weighed and transferred in individual volumetric flask containing different buffer solutions and sonicated for 30 minutes. The extracted drug samples were suitably diluted and analyzed with HPLC technique¹⁶.

Infrared spectrum:

The infrared spectrum of Glipizide was carried out using potassium bromide disk method. The samples were prepared on KBr-press and over wave number range of 4000 to 400cm^-1 it was scanned¹⁷.

UV Spectral Analysis¹⁸:

An accurately weighed amount (10mg) of Glipizide was transferred to 100 ml volumetric flask. The drug was dissolved in methanol and volume was made up to 100 ml with the same solvent (Methanol) to obtain a stock solution of 100mg/ml. From the standard stock solution, 1 ml was taken out in 10 ml volumetric flask and volume was made up to 10 ml with PBS pH 7.5. The resulting solution containing 10mg/ml was scanned over complete UV range (i.e. 200–400 nm)¹⁹ using Shimadzu UV–Visible spectrophotometer for determination of λ maxof the glipizide. This stock solution was diluted with PBS pH 7.5 to give concentrations in the range of 10mg/ml-40mg/ml. Absorbance of these samples at varying concentrations were determined and data was used to plot calibration curve.

Loss on Drying (LOD)

The loss on drying (LOD) of the glipizide was determined by the infrared moisture balance. Infrared Moisture Balance is a very reliable and sturdy instrument for an accurate determination of moisture contents of materials which do not undergo chemical change when exposed to infrared radiation²⁰. Observations can be taken quickly and the results obtained are accurate²⁰. One gram of glipizide was taken and placed evenly as a layer on the balance and heated at 105^oC for 5 min. Then, after 3 minutes LOD was directly noted from the value displayed by instrument. Percent loss of moisture on drying was calculated using following formula²¹

% LOD = Weight of moisture in sample Weight of sample before drying × 100

Assay Compensation (Calculations for assay on anhydrous basis):

Monograms in several pharmacopoeias and many raw material manufacturers defines content limit in assay test on anhydrous basis²². In routine practices however assay test is not carried out making substance anhydrous. These types of assay are termed as assay on as-is basis. The water present in a pharmaceutical use substance is not considered as an impurity and hence the result of water content test is accounted in the result of assay on as-is basis²³. In industry assay on anhydrous basis is more acceptable. In this results obtained from assay on as-is basis is put in the formula (assay on as-is basis × 100) / (100 - % water) and outcome of formula is termed as assay on anhydrous basis²³. Accordingly we need to determine the quantity of API to be dispensed based on assay on dried basis and moisture content by using following formula:

Qty of API =

(Dose x 100 x 100) / {Assay X (100– Loss on Drying)}

Drug: excipients compatibility study:

Drug excipient compatibility studies are critical for well–formulated final dosage forms where the drug reside in contact with one or more excipients during process scale–up from clinical trials through commercial to consumer²⁴. The study was designed with different ratio for drug and excipients as per their functionality. The weighed amount of API was mixed well with a proposed proportion of individual excipients (Table 5). Blend was filled and sealed in 5 ml glass vials. Vials were subjected to 40°C ± 2°C/75% ± 5% RH and 25°C 60% RH for 4 weeks conditions. The initial samples were analyzed immediately and used as control²⁵. The samples were observed for physical changes²⁶ like discoloration, liquefaction and analyzed for related substance by HPLC (Table No. 1).

Table No. 1: Drug–Excipients time–point testing schedule

Conditions	Time Zero	1 week	2 weeks	4 weeks
25°C and 60%RH	––	––	––	––
40°C and 75%RH	X Z	X	X	XZ

X= Visual Observation; Z= RS by HPLC method

RESULTS AND DISCUSSION:

Organoleptic properties:

Drug under consideration was found to be white crystalline powder.

Characterization and Identification of Glipizide Physical characterization of Glipizide:

Physical parameters such as bulk density, tapped density, Carr’s index and Hausner’s ratio of glipizide were determined as per method described in previous section. From data obtained it can be interpreted that Glipizide has poor flow properties²⁷ and one has to go for granulation technique in order to increase flow properties during tablet manufacturing

Results obtained were tabulated in Table No. 2

Table No.2: Physical properties of candidate drug

Parameters	Observed Value
Bulk density (g/mL)	0.222
Tapped density (g/mL)	0.425
Carr’s index (%)	47.78
Hausner’s ratio	1.915

Solubility profile:

The drug was insoluble in water and ethanol 95%. Solubility (mg/250ml) of glipizide in different pH buffer solutions is as shown in table 4. Data indicates that solubility of glipizide increases with increase in pH (Table 3).

Table No. 3: Solubility of glipizide at different pH buffer solutions

Medium	Solubility (mg/250ml)
0.1N HCL	0.30
Phosphate buffer pH 7.4	10.00
Phosphate buffer pH 7.5	10.30
Phosphate buffer pH 6.8	9.50
Acetate buffer pH 4.5	0.50

Infrared spectrum:

Fourier transform infrared spectroscopy (FTIR) spectrum of drug under consideration (Figure 1) showed characteristic bands at 1689 cm^-1 which is due to C = O stretch of amides, band at 3325 cm^-1 may be attributed to N-H stretching of sulphonamido group, a band present at 1650cm^-1 is due to C=O of urea, 2942.8cm^-1 and 2854.2cm^-1 bands are due CH stretching of CH². Presence of characteristics shows that the drug sample is of pure glipizide. The wave number and assignment of the most important bands are shown Table no 4.

Table No. 4 : Symmetric and asymmetric vibration

IR Values (cm^-1)	Functional Groups
3325.0	NH str. of sulphonamido gr.
3250.6	NH str. of amido gr.
2942.8	CH str. of CH2
2854.2	CH str. of CH2
1688.9	C=O of amide
1333.43,1160	O=S=O
1650	C=O urea

Figure No. 1: IR spectrum of glipizide

UV spectral analysis:

UV spectrum of glipizide was recorded on UV-VIS spectrophotometer at 200-400 nm. It was found that the drug obeyed Beer’s law and showed good correlation. It showed absorption maxima at 276 nm in methanol (Figure no. 2) which is similar to reported value thus suggesting purity of the drug under consideration. The developed method was validated with respect to linearity, accuracy and precision in accordance with the requirements of ICH guidelines. The linearity was observed between 10-40μg/ml was having line equation y = 0.0144x +0.0078 with correlation coefficient of 0.998 (Figure no. 3) which suggests that it obeys the Beer-Lamberts law²⁸. This analytical method is having potential for routine laboratory estimation of glipizide after converting into formulation because of its simplicity, rapidity, precision and accuracy.

Figure No. 2: UV spectrum of glipizide

Figure No. 3: UV Calibration Curve.

Determination of LOD:

LOD of drug sample was found to be less than 0.5% w/w.

Glipizide potency calculation (LOD and assay compensation):

Actual quantity of active taken in any formulation was determined on the basis of its Loss on drying and the purity (% assay) as given in the equation below,

For 2.5 mg, actual quantity calculated as follows:

2.5 x 100 x 100

Drug Potency = ---------------------------------- = 2.501mg

100 x (100-0.06)

Drug-Excipients Compatibility Study:

Drug-excipient compatibility study was performed as per the method described in experimental. Visual observation of the samples was performed for discoloration, liquefaction and odor. The study showed no lumps / aggregate and discoloration at the end of 30 days, so it could be concluded that drug has no interaction with excipients.

Table No. 5: Result of Drug-Excipient Compatibility

Excipient	Ratio (D:E)	Initial	40^oC/75% RH
Excipient	Ratio (D:E)	Initial	15 Days	30 Days
Drug substance	--	White or almost white, crystalline flowable powder.	NC	NC
Drug + PEO WSR N80	1: 10	White or almost white free flowing powder	NC	NC
Drug + PEO WSR 303	1:10	White or almost white free flowing powder	NC	NC
Drug + NaCl	1:0.5	White or almost white free flowing powder	NC	NC
Drug + Ferric oxide	1:0.03	Red powder flowable, some part adhere to base of vial	NC	NC
Drug +Mg. Stearate	1:0.05	White or almost white poorly flowing powder	NC	NC
Drug + HPMC 5cps	1: 0.5	White or almost white free flowing powder	NC	NC
Drug + HPC SSL	1: 0.5	White or almost white free flowing powder	NC	NC
Drug + PEG 400	1: 0.01	White or almost white powder adhere to vial	NC	NC
Drug + PEG 6000	1: 0.02	White or almost white free flowing powder	NC	NC
Drug + PEG 3350	1: 0.02	White or almost white free flowing powder	NC	NC
Drug + Cellulose Acetate	1:0.5	White or almost white free flowing powder	NC	NC
Drug + Opadry white	1: 05	White or almost white free flowing powder	NC	NC

#NC- No change. *OA-Agglomeration.

CONCLUSION:

Preformulation study is most important phase in developing safe, effective and stable dosage form. Data generated through these studies has great impact on subsequent development of final dosage form. In the present work, the preformulation study of Glipizide was done. The organoleptic properties and LOD of drug sample, which was found to be less than 0.5% w/w, comply with Indian Pharmacopeia²⁵. Physical characteristics studies found that Glipizide has poor flow and compressibility properties and hence direct compression tablet compression technique should be ruled out. Solubility of drug was found to increase with increase in pH. The purity of drug was confirmed by infrared spectrum which showed characteristics peaks and by uv spectroscopy which exhibited maxima at 276 nm. The standard curve obtained was linear with correlation coefficient (R2 =0.998) and equation y = 0.0144x +0.0078. The potential excipients PEO N80 WSR 200000, PEO WSR 303, Nacl, Ferric oxide, Magnesium stearate, HPMC 5cps, HPC SSL, PEG 400, PEG 3350, PEG 6000, Cellulose Acetate 398–10, Opadry White which we are intending to use in formulating Osmotic drug delivery of were found to be compatible with drug Glipizide when subjected to different conditions. This study shows a satisfactory result for all characterization and on the basis of this study we concluded that the drug was suitable for choice of formulation.

CONFLICT OF INTEREST:

No conflicts of interest are mentioned by the researchers. The composition and writing of the document are the sole responsibility of the writer.

ACKNOWLEDGEMENT:

Authors are thankful to all who providing us with the required gift samples required for our studies. We also thank Management of School of Pharmacy, Dr. Vishwanath Karad, MIT World Peace University, MIT Campus, Kothrud, Pune, 411038 for providing necessary help in carrying out the study in their laboratories.

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Received on 05.11.2020 Modified on 27.03.2021

Research J. Pharm. and Tech 2022; 15(1):29-34.

DOI: 10.52711/0974-360X.2022.00006