Pharmacokinetic and Pharmacodynamic studies of Omeprazole Co-crystals dry suspension

Madhuri Gaddam¹*, Nagaraju Ravouru²

¹Department of Pharmaceutics, Rao’s College of Pharmacy, Nellore, Andhra Pradesh, India.

²Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam,

Tirupati- 517502, Andhra Pradesh, India.

*Corresponding Author E-mail:

ABSTRACT:

A novel omeprazole co-crystals instant dry suspension dosage was formulated by applying co-crystallization which is a particle engineering technique to enhance the physicochemical properties. As omeprazole has major critical draw backs in their solubility and stability (acidic pH degradation, thermal sensitivity, photosensitivity and highly hygroscopic nature) bioavailability of omeprazole was drastically reduced and purpose of administration of drug was not fulfilled. There by this formulation was invented to check maid these problems. In this present study in-vivo evaluation were performed. Pharmacokinetic parameters and pharmacodynamic parameters were determined and statistically analysed. The prepared formulation of omeprazole produced reliable satisfaction in solving the drawback problems of pure omeprazole by enhancing bioavailability. Statistical analysis was done by one way ANOVA test with P value less than 0.0001, Dunnet multiple comparison test was performed between the groups with P value < 0.01 which was considered to be significant.

KEYWORDS: Omeprazole co-crystals, pharmacokinetic parameters, pharmacodynamic studies and ulcer index.

INTRODUCTION:

Gastric hyperacidity and gastric duodenal ulcer is a familiar global problem now-a- days. Stress, alcoholism, H.pylori infection, smoking, intake of drugs, usage of NSAIDS for various problems liberates the reactive free oxygen radical, most commonly hydroxyl radicals (-OH), generated causes gastric ulceration. Hyper secretion of gastric acid is a condition, which happened by uncontrolled secretion of hydrochloric acid from the parietal cells of gastric mucosa through the proton pumping H+ K+ ATPase channel. Not only secretion of gastric acid, but increases pepsin levels, inhibition of prostaglandin synthesis, cell proliferation growth, reduces gastric blood flow and gastric motility¹.

Omeprazole is a class of benzimidazole substituted antiulcer drug which is a potent anti-gastric acid secretary agent by binding to the active proton pumps (H+ K+ ATPase) in secretory membrane of parietal cells, so called as proton pump inhibitor. It is also used in various gastric acid related disorders like duodenal ulcers, reflux esophagitis, Zollinger-Ellison syndrome^2-6.

As omeprazole was unstable in acidic pH of stomach (acid-liable), uncoated or unprotected active drug upon exposure to gastric acid contents consequences to degradation and lose of antigastric secretory activity of about less than 50% by oral dosage administration. Thus, this formulation of omeprazole co-crystal instant dry suspension preparation will have a noticeable effect on bioavailability and antiulcer activity of omeprazole.

In this present study we have to access the pharmacokinetic parameters and pharmacodynamic effect of omeprazole co-crystal dry suspension formulation and histopathological study of gastric mucosal lining cells, which was compared to the pure omeprazole formulation.

MATERIALS AND METHODS:

Experimental Animals: 24 Healthy male albino rats of about 150-200 gm body weight were selected for experiments. The experimental protocol was approved by the Institutional Animal Ethical Committee of Sanzyme Labs Pvt Ltd. Hyderabad with the registered no. 1688/PO/Rc/8/13/2011/CPCSEA.

Pharmacokinetic studies^{7, 8}:

All animals were maintained under standard conditions. They were housed in appropriate cages with standard rat pellet diet with water and libitum. Animal room was maintained at 20-25ᴼC with 12:12 hrs light and dark cycles. The total number of rats was divided into 4 groups, each group of which contains 6 rats. The first group was given with Normal feed, water and serve as normal control. The second group was administered with pure omeprazole instant dry suspension equivalent to 72 mg/kg; the third group was administered with omeprazole co-crystals dry suspension equivalent to 72 mg/kg. Final group was administered with marketed formulation omeprazole sustained release dry suspension equivalent to 72 mg/kg body weight. The omeprazole (72 mg/kg) was given in the morning of the day of the experiment after 12 hrs fasting. Immediately after drug administration, few drops (0.3 ml) of rat’s blood were withdrawn from the tail after 15min, 30min, 45min, 1, 1:30, 2, 2:30, 4, 6, 8 and 10 hours and kept in heparinised eppendorf tubes. Each blood sample was centrifuged after adding 100μl of ethyl acetate at 6000 rpm for 10 minutes to obtain the plasma volume of (100-125μl), and then supernatant plasma was collected with a micropipette. After filtration 5μl of sample was injected to the HPLC system.

Mobile phase:

Mobile phase was prepared by mixing of methanol and 0.1% formic acid water (60:40v/v).

Preparation of omeprazole standard calibration curve:

The pure form of omeprazole (10mg) is dissolved in 10 ml of methanol/water (60:40, v/v). From this stock (1mg/ml) solution, serial dilutions were prepared as 1000ng/ml, 3000ng/ml, 5000ng/ml, 7000ng/ml, and 9000ng/ml. These dilutions were added to 200 µl of drug free rat plasma and vortexed for 15 minutes and 600 µl of ethyl acetate was added and vortexed for 15 min and then centrifuged at 6000 rpm for 10 min, then analysed by HPLC method at 301 nm.

Table no 1: Summary of conditions

Pump flow rate	0.5 ml/min
Autosampler injection volume	5 μl
Autosampler temp.	4°C
Mobile phase	Mixture of [60% methanol and 40% (0.1%) formic acid]
Column type	C18 column (150×4.8 mm, 5 μ)

Pharmacodynamic studies:

Ethanol Induced gastric ulceration in rats:

The main principle involved in alcohol induction ulceration, was due to decrease in stomach enzyme activity and the alcohol intake causes disruption of mucosal parietal cells, thus produces increase in mucosal permeability and provokes bleeding in to stomach. There by increase in secretion of gastric juices and decreases the mucosal resistance. This increased gastric secretion contains leakage of plasma proteins into stomach leads to reduction of mucosal resistance by the parietal cells in the mucosal layer of stomach, hence mucosal cells were exposed to excess of acid leads to damage of cells further to ulcers. Activeness of neutrophilis at the site of damage triggers high level of oxygen species concentration and mediators of inflammation causes oxidative damage on cells, ultimately leads to gastric mucosal damage and ulcer formation²

Procedure:

Male healthy albino rats weighing between (150-200 gm) were divided into 4 groups each containing six animals. These were fasted for 24 hrs with free access of water. Animals in group II, group III, group IV were administered by normal water (control), pure Omeprazole suspension and omeprazole cc suspension as test drug respectively at early in the morning times, after 1 hrs administration of samples 1 ml/200 gm of 99.8% ethanol was administered to each animal. After 1 hrs of ethanol administration, the animals was sacrificed and stomach was isolated, opened by cutting along the greater curvature and pasted on soft board. The gastric lesion formed due to the alcohol induction was observed and size of ulceration was scored, then ulcer index, percentage of inhibition and gastric juice pH were determined. Stomachs were immersed in 10% Formalin for 24hrs to study the histopathological changes in treated and ulcerated rats. ^{4,
9, 10}

Table no: 2 Protocol to carry out the Pharmacodynamic studies

S. no.	Group	Drug	Dosage(mg/Kg)	Purpose
1	I	Blank	---------	To serve as normal
2	II	Placebo	20	To serve as control
3	III	Pure Omeprazole suspension	Equivalent to 20	To serve as standard
4	IV	Omeprazole co-crystals suspension	Equivalent to 20	To serve as test

Mode of administration:

The drug suspension formulation was dispersed in 2 ml of water, and then it was administered through oral route to the animal.

The number of ulcers per stomach were noted and severity of the ulcers scored by

0= Normal stomach

0.5 = red colouration

1 = spot ulcers

1.5 = Haemorrhagic streaks

2 = Ulcer > 3 mm <5 mm

3= Ulcers > 5 mm

Ulcer Index = U_N + U_S + U_PX10^-1

Where U.I = Ulcer Index, U_{N =}Average number of ulcer per animal, U_S= Average number of severity score, U_P= Percentage of animals with ulcers.¹¹

Percentage protection⁶ = [U.I _control – U.I _treated] /U.I _control×100

Histopathological studies:

Gastric juices from the stomach was washed and tissue samples were preserved in 10% formalin for 24 hrs, then the tissues were embedded in paraffin take a cross section of 3-5 µm was stained with haematoxylin and eosin dye. Finally, the histochemically stained sections were evaluated under optical microscopy².

RESULTS AND DISCUSSION:

Table no: 3 Comparative pharmacokinetic parameters of pure, micronized complex omeprazole formulations with marketed formulation.

S. no	P.K parameter	Pure omeprazole formulation	Micronized complex Omeprazole formulation	Marketed formulation
1.	C_max(µg/ml)	732±18.67	829.75±16.09*	776±50.84
2.	T_max(hrs)	1±0.04	1±0.03	1±0.02
3.	AUC(µg/ml.hrs)	1185.260±77.27	2193.587±186.5*	4171.604±334.6
4.	T_1/2(hrs)	3.07±0.124	3.14±0.1	4.825±0.26
5.	Cl(ml/min)	8.515±0.57	4.74±0.11*	2.75±0.246
6.	V_d(ml)	37.772±1.77	20.8±2.184	18.437±1.55

*Indicates significant change

Figure no: 1 Comparative plasma concentration of pure omeprazole and omeprazole co-crystal formulations with marketed formulation

Table no: 4 Antiulcer activity of pure and omeprazole co-crystal formulations.

S.no.	Group	Ulcer index	% Protection	Gastric pH
1	Normal	00.00±0	0	2.1±0.5
2	Control (placebo)	30.74±1.86	------	1.5±0.63
3	Standard (omeprazole-pure)	8.655±0.713	71.84	3.5±0.47
4	Test (omeprazole co-crystal formulation)	6.116±0.747*	80.104*	4.0±0.53*

*Indicates significant

DISCUSSION:

In-vivo pharmacokinetic studies were performed plasma drug concentrations of omeprazole formulations was compared. Omeprazole co-crystal instant dry suspension formulation had increased the bioavailability level when compared to pure omeprazole, due to the co-crystallization technique which had changed the crystal habitant of pure omeprazole to micron in size with irregular sphere in shape. In addition to this there is an increased acid stability at gastric pH conditions, thus it produces some stability in stomach thereby sustained its activity when compared to that of pure one. Pharmacodynamic studies for anti-ulcer activity were performed to omeprazole formulations. The omeprazole co-crystal formulation produced the better anti-ulcer activity when compared to pure omeprazole formulation which was found statistically extremely significant. This may be due to enhanced acidic stability, dissolution and solubility parameters of the omeprazole co-crystals, leading to better absorption by the stomach. The histopathological studies states, there is a severe damage to the gastric sub mucosa and epithelial cells of control group animals when compared to the normal intact group of animals. The group of animals treated with standard and test drugs had a protective effect on the gastric mucosal cells, which was shown in figures by reduction of gastric sub mucosal edema and damage to epithelial cell of stomach.

CONCLUSION:

The prepared novel formulation of omeprazole had produced the significant enhancement in bioavailability; this was evaluated by performing pharmacokinetic and pharmacodynamic studies. The results were analyzed statistically.

NO CONFLICTS OF INTEREST:

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Received on 24.06.2020 Modified on 25.08.2020

Research J. Pharm. and Tech. 2022; 15(1):494-498.

DOI: 10.52711/0974-360X.2022.00080