Animal Acute Toxicity Study of Pharmaceutical Excipient Hydroxypropyl Methylcellulose

Abstract: Objective: To investigate the acute toxicity of hydroxypropyl methylcellulose to mice. METHODS: The mice were given hydroxypropyl methylcellulose three times in one day, with a total dose of 3.15 g/(kg bw), and the behaviors of the mice were observed every day for the next 14 days , body weight, respiratory rate, pupillary condition, food intake, and mortality. Then the surviving mice were sacrificed and autopsied to observe whether there were lesions in the visceral tissues and organs of the mice. Results: In the case of a total dosage of 3.15 g/(kg bw), the behavioral activities, respiratory rate, pupil status, and food intake of the mice during the 14-day observation period were not abnormal, and the body weight was similar to that of the control group. During the observation period, no mice died, and there were no obvious pathological changes in the morphology of major organs during autopsy. Conclusion Hydroxypropyl methylcellulose is a pharmaceutical excipient with low toxicity and high safety.

Key words:hydroxypropyl methylcellulose; acute toxicity; mice

1.Introduction

In recent years, the pharmaceutical excipient industry has developed rapidly, and new pharmaceutical excipients have developed more than 40 types including encapsulation materials, coating materials, surfactants, and sustained-release, controlled-release, and targeted drug delivery materials. This variety provides new ideas and directions for the research and development of new drugs. Due to the rich variety of pharmaceutical excipients and their complex and diverse sources, their application must undergo strict toxicological testing and safety evaluation. At present, there are many methods for studying the acute toxicity of drugs and excipients at home and abroad. The classic ones include the half-lethal dose experiment represented by the Horn method and the Cole method, as well as the approximate lethal dose experiment, acute toxicity classification method, dose accumulation experiment, Acute toxicity detection methods such as fixed dose method and limit test.

Hydroxypropyl Methyl Cellulose (Hydroxy Propyl Methyl Cellulose, HPMC), Mr about 86000, is a polymer pharmaceutical excipient, which is obtained by etherification of highly pure cotton cellulose under alkaline conditions. The appearance of HPMC is white powder, which has strong stability to heat, light and humidity. HPMC dissolves in cold water to form a viscous gel solution. The aqueous solution has high transparency and strong surface activity. The solubility of HPMC in water generally decreases with the increase of solution viscosity, but is not affected by the pH value. Because of its chemical inertness, HPMC will not affect the properties of the drug itself, and at the same time it has good anti-allergic properties, and is widely used as various pharmaceutical excipients in pharmaceutical preparations. HPMC can be used as a coating film-forming material, which can effectively improve the stability of drugs; as a slow-release or controlled-release matrix material for drugs, to block or control the release of drugs; as a binder or disintegrant to improve the solubility of tablets ; It can also be used as a suspending agent. The flocculation particles formed are fine and easy to redisperse without sticking to the wall. The usual dosage is 0.5% to 1.5%.

As a pharmaceutical excipient, hydroxypropyl methylcellulose is often used as an excipient or excipient in oral drugs. For its acute toxicity research, attention should be paid to its medicinal route. The acute oral toxicity test is a basic experiment to evaluate and detect the toxic effect of the test substance. It can obtain the health hazard data of the test substance exposed to the test substance in a short period of time, and can also be used as the basis for grading the acute toxicity of the test substance. Among them, the median lethal dose (Median Lethal Dose, LD50) is a key indicator of traditional acute toxicity tests. In traditional acute toxicity experiments, accurate LD50 values are often obtained by the Horn method or the Cole method. For the safety evaluation of pharmaceutical excipients with low toxicity, it is necessary to refer to other parameters such as the maximum dosage experiment and the fixed dose method.

The purpose of this study is to determine the acute toxicity of hydroxypropyl methylcellulose to mice, to study the safety performance of hydroxypropyl methylcellulose in drug use, and to determine the acute toxicity of hydroxypropyl methylcellulose to oral administration. Conduct investigations to provide a basis for experimental data in terms of clinical safety medication.

2. Materials and Instruments

2.1 Reagents

Domestic hydroxypropyl methylcellulose (SH-K4M) was donated by KIMA CHEMICAL CO.,LTD;

Imported hydroxypropyl methylcellulose (METHOCEL K4M CR grade) was donated by KIMA CHEMICAL CO.,LTD;

Potassium dihydrogen phosphate (Xilong Chemical Co., Ltd.); pancreatin (Beijing Suolaibao Technology Co., Ltd.).

2.2 Instruments

Mouse gavage sets, balances, forceps, mouse cages, dissecting utensils, and various other glassware.

2.3 Experimental animals

Healthy and clean Kunming mice were purchased from the Animal Experiment Center of Shandong University (certificate number: SCXK (Lu) 2014 0007), weighing about 20 g, and were adaptively fed for one week before the experiment, with a normal diet.

3. Experimental method

3.1 Solution preparation

Preparation of artificial intestinal juice: Weigh 6.8 g of potassium dihydrogen phosphate, add distilled water to 500 mL, and after it is completely dissolved, use 4% sodium hydroxide solution to adjust the pH to 6.8. Weigh 10 g of pancreatin, add an appropriate amount of distilled water to dissolve it, mix the two liquids, add water to make up to 1000 mL, and obtain artificial intestinal juice.

Preparation of hydroxypropyl methylcellulose: Weigh 5 g, 6 g, and 7 g of hydroxypropyl methylcellulose respectively, add them to 200 ml of distilled water, stir a small amount several times until dissolved, and when there are bubbles in the solution, Stand still until the bubbles disappear and stir several times to prepare 25 g•L-1, 30 g•L-1, 35 g•L-1 hydroxypropyl methylcellulose solutions.

3.2 Pre-experiment

Preliminary experiments were conducted to observe the acute toxicity of hydroxypropyl methylcellulose to determine whether an accurate semi-lethal dose (LD50) value could be obtained. Divide the mice into 3 groups, observe the death of the mice by administering different concentrations of hydroxypropyl methylcellulose solutions, and determine the experimental method of the formal experiment.

Take 12 mice, half male and half male. Each mouse was marked, weighed, and fasted for 18 hours before the experiment. The mice were divided into 3 groups with 4 mice in each group and marked as group 1, group 2 and group 3. Groups 1, 2 and 3 were distributed by intragastric administration of hydroxypropyl methylcellulose solution at doses of 1000 mg/(kg·bw), 1200 mg/(kg·bw) and 1400 mg/(kg·bw), and observed The death and pathological symptoms of the mice within one week of intragastric administration of the test drug.

3.3 Maximum dosage experiment

According to the results of the pre-experiment, the acute toxicity of hydroxypropyl methylcellulose is small, and the accurate semi-lethal dose cannot be measured by intragastric administration. Therefore, the maximum dosage experiment was used to divide the mice into different control groups and After the experimental group, the maximum dose of hydroxypropyl methylcellulose solution was given by intragastric administration.

Take 40 mice, half male and half male, and check that all female mice are not pregnant. The mice were fasted for 18 hours, and each mouse was marked and weighed. Randomly grouped according to gender and body weight, divided the mice into 4 groups with 10 mice in each group, and marked them as distilled water control group (gavage with equal volume of distilled water), artificial intestinal juice control group (gavage with equal volume of artificial intestinal juice), Domestic hydroxypropyl methylcellulose experimental group (gastric administration dose 1050 mg / (kg bw)) and imported hydroxypropyl methylcellulose experimental group (gastric administration dose 1000 mg/(kg bw)), according to the above differences The grouping doses were intragastrically administered 3 times within 24 hours, and the interval between each time was not less than 6 hours. The final cumulative intragastric dosage of each mouse was 3150 mg / (kg bw), which was 3.15 g / (kg bw).

From the first gavage administration, observe and record the behavior, respiratory rate, food intake and activity of mice in each group, observe continuously for two weeks and record the behavior of mice at the beginning, third day, seventh day and tenth day Body weight for four days.

During the experimental observation period, the mice were fed normally, and their behaviors such as eating and drinking were not restricted.

After the end of the observation period, the mice that did not die were weighed and sacrificed for autopsy, and whether there were any pathological changes in the morphology of the major organs of the mice was observed with the naked eye.

4. Results and Discussion

4.1 Pre-experimental results

None of the mice in the 3 groups died or showed symptoms of poisoning, so the maximum dosage test was used in the formal experiment.

4.2 Experimental results of the maximum dosage

During the observation period, none of the mice in the four groups died, so the LD50 could not be measured.

The results of this experiment showed that the maximum tolerated dose of hydroxypropyl methylcellulose in mice was 3.15 g/(kg bw).

1) Behavioral activities

Comparing the 4 groups of mice, observing the conditions of the mice in each group, it was found that the living conditions of the mice in the administration group were good, and there were no abnormalities in behavioral activities, shiny fur, no allergies and dullness to external stimuli, and no loose fur , cyanosis, hyperemia and rash and other adverse phenomena. The mice in each group did not experience irritability or inhibition.

2) Weight

During the experiment, the weight of the mice in each group increased naturally, and there was no growth slowdown, stagnation or weight loss. Compared with the control group, the body weight of the mice in the HPMC experimental group had no significant difference.

3) Respiratory rate and pupil

Comparing the 4 groups of mice, observing the conditions of the mice in each group, the breathing and respiratory rate of the mice in both the control group and the experimental group were normal, and no obvious abnormal breathing was found in the mice during the observation period. Observe the pupils of the mice, the pupils on both sides are equal in size, still sensitive to light, and there are no adverse phenomena such as eyelid drooping or eyeball hyperemia.

4) Food intake

During the experiment period, the food intake and water intake of the mice in each group were observed, and it was found that there was no significant difference between the experimental group and the control group.

5) Organizational form

After the experiment, the mice were executed for autopsy, whether domestic or imported, the tissue morphology of the main organs (heart, liver, spleen, lung, kidney) of the mice in the two groups of hydroxypropyl methylcellulose experimental groups were observed by naked eyes , No obvious pathological changes were found. After comparing with the control group, no obvious difference was found.

4.3 Discussion

The safety performance of pharmaceutical excipients is an important part of the development of pharmaceutical excipients. In order to more comprehensively ensure the safe application of drugs, the safety testing methods of pharmaceutical excipients are also advancing with the times. Because the traditional acute toxicity test methods such as Horn's method and Kou's method need to measure the accurate LD50 value, resulting in a large number of experimental animals, an alternative method was developed later, such as the maximum dosage test method , up-down method (UDP), fixed dose method, acute toxicity classification method, etc., can minimize the death of experimental animals and are more suitable for application. The results of this study showed that hydroxypropyl methylcellulose did not cause death in mice within two weeks under the condition of oral daily dose of 3.15 g/(kg·bw), and did not produce obvious acute toxicity, including There were no visible pathological changes in physiological activities and organ tissue morphology. Therefore, mice were orally administered hydroxypropyl methylcellulose

The LD50 of the element is greater than 3.15 g/ (kg·bw). As a pharmaceutical excipient, hydroxypropyl methylcellulose is basically non-toxic and safe. In the process of drug preclinical safety evaluation, it will be more comprehensive and reasonable to conduct toxicity evaluation based on the characteristics of pharmaceutical excipients. In-depth research on the properties, safety and new formulation technology of hydroxypropyl methylcellulose will help the application of pharmaceutical excipients hydroxypropyl methylcellulose in new dosage forms and new drug delivery systems.