BE1030308A1 - TEMPERATURE SENSITIVE IN-SITU GEL, TEMPERATURE SENSITIVE MELATONIN IN-SITU GEL AND METHOD FOR PRODUCING THE SAME - Google Patents

Abstract

The present invention provides a temperature-sensitive in-situ gel, a melatonin-temperature-sensitive in-situ gel and a method for production, wherein a temperature-sensitive melatonin in-situ gel consists of the components with the following weight percents: melatonin 0, 05% to 10% Poloxam 407 21% to 23% Poloxam 188 1.5% Hydroxypropylmethylcellulose 0.4% to 0.6% Polyethylene glycol 4000 0.3% to 0.5% The remainder is water for injections. The temperature-sensitive melatonin in situ gel according to the present invention is capable of achieving slow release of melatonin, effectively solving the problem of low usage rate and frequent administration of melatonin due to rapid metabolism.

Description

l BE2023/5564

TEMPERATURE SENSITIVE IN-SITU GEL, TEMPERATURE SENSITIVE

MELATONIN IN SITU GEL AND METHOD FOR PRODUCING SAME

TECHNICAL FIELD

The present invention relates to the field of veterinary medicine, temperature-sensitive in-situ

Gel, temperature-sensitive melatonin in situ gel and method for producing the same.

STATE OF THE ART

Melatonin (MLT), also known as cerebrolysin or pmealin, is a neuroendocrine hormone with a wide range of physiological and pharmacological effects, produced by the

Pineal gland is secreted in mammals and humans. In recent years the...

Application of melatonin in livestock and poultry farming is increasingly being studied, and it has been proven that melatonin plays an important role in regulating the immunity of

animals, improving the reproductive performance of animals, combating stress, as

Antioxidant and scavenging free radicals. In livestock farming, the ability of animals to scavenge free radicals decreases under external heat stress conditions. Among the enzymes that

In addition to peroxidase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) are also involved in heat stress. Melatonin has broad and potent antioxidant effects and is the most powerful endogenous free radical scavenger known with minimal side effects. Melatonin not only intercepts free radicals, but also has an anti-damage effect. During aerobic

Metabolism creates a variety of reactive substances during the oxidative decomposition of organic substances

Oxygen radicals (ROS) such as H2O2, -OH and O2-. Melatonin is the only known antioxidant that

ROS is eliminated by providing electrons and exerting its antioxidant properties

Functions rely entirely on the provision of electrons between biological macromolecules. By directly neutralizing ROO- and OH- and activating the activity of the

Glutathione peroxidase (GSH-Px) is able to remove melatonin oxidation products, oxidative

Inhibit stress and provide antioxidant protection.

Temperature-sensitive in situ gels are a new class of drug delivery forms that use polymeric materials to act at the site of administration in response to the

To complete a phase transition from a liquid state at a low temperature

Temperature to form a gel is reversible at gelling temperature. The preparation of the drug into temperature-sensitive in situ gel can improve the half-life and bioavailability of the drug in the organism, and the gel has better fluidity compared to the original solution and offers the advantages of easy administration and uniformity

Distribution of the drug so that the temperature-sensitive in situ gel drug is good

has development prospects.

2 BE2023/5564

In livestock production, melatonin is mainly administered through dosing, feeding or intravenous (subcutaneous) injection, which is quick but still presents many problems.

First, the absorption and utilization of melatonin is easily influenced by the environment

Digestive tract influenced, such as by the influence of rumen microorganisms, and its

Bioavailability is dose dependent; secondly, the half-life of melatonin is short and its

Bioavailability is influenced by dosage or feeding; thirdly, intravenous (subcutaneous) injection of melatonin is a stressful and painful injection and the dosage is not easy to control. Based on this, the present invention provides a temperature-sensitive in situ gel, a melatonin temperature-sensitive in situ gel and

Process for manufacturing available.

CONTENT OF THE PRESENT INVENTION

The present invention provides a temperature-sensitive in situ gel, a melatonin temperature-sensitive in situ gel and a method for producing which solve the problem of low usage rate and frequent administration of melatonin due to the rapid

Can solve metabolism.

In order to solve the above technical problems, the temperature-sensitive melatonin in situ gel according to the present invention is realized as follows:

A temperature-sensitive melatonin in situ gel containing the following substances

Weight percent:

Melatonin 0.05% to 10%

Poloxam 407 21% to 23%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.4% to 0.6%

Polyethylene glycol 4000 0.3% to 0.5%

The rest is water for injections.

Optionally, the melatonin temperature-sensitive In-srtu gel contains the following substances

Weight percent:

Melatonin 0.15%

Poloxam 407 22%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.5%

Polyethylene glycol 4000 0.4%

The rest is water for injections.

In order to solve the above technical problems, the method for producing a temperature-sensitive melatonin in situ gel according to the present invention is realized as follows: a method for producing a temperature-sensitive melatonin in situ gel, comprising:

Weigh Poloxam 407, add water for injections, dissolve and

3 BE2023/5564 then placed in a refrigerator at 4°C overnight; Removing the Poloxam 407

solution, bringing it back to room temperature and then adding Poloxam 188,

Polyethylene glycol 4000 and melatonin one at a time and stirring to dissolve; then adding hydroxypropyl methylcellulose, stirring to dissolve and then adding water for

Injection purposes for volume adjustment and placing in a refrigerator; The temperature-sensitive melatonin in situ gel solution is prepared in 24 hours through filtration and decontamination.

Optionally, filtration and sterilization can be done through a 0.22um filter membrane.

In order to solve the above-mentioned technical problems, the temperature-sensitive in-situ gel according to the present invention is realized as follows: a temperature-sensitive melatonin in-situ gel consisting of the following components

Weight percent:

Poloxam 407 21% to 23%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.4% to 0.6%

Polyethylene glycol 4000 0.3% to 0.5%

The rest is water for injections.

Optionally, the temperature-sensitive melatonin in situ gel consists of the following components by weight:

Poloxam 407 22%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.5%

Polyethylene glycol 4000 0.4%

The rest is water for injections.

In order to solve the above technical problems, the method for producing a temperature-sensitive in-situ gel according to the present invention is realized as follows: a method for producing a temperature-sensitive in-situ gel, comprising: weighing

Poloxam 407, adding water for injections, dissolving and then placing in a refrigerator at 4°C overnight; Take out the Poloxam 407 solution, put it back on

room temperature and then adding Poloxam 188 and polyethylene glycol 4000 one by one and stirring to dissolve; then adding hydroxypropyl methylcellulose,

Stir to dissolve and then add water for injections

Adjusting the volume and placing it in a refrigerator; The temperature-sensitive in-situ gel is produced in 24 hours through filtration and decontamination.

Optionally, filtration and sterilization can be carried out using a 0.22 µm filter membrane.

The temperature-sensitive melatonin in situ gel prepared by the present invention is at

Room temperature a homogeneous, translucent gel solution that appears after entering the body of the

Animal quickly becomes a gel, and the release time of melatonin in the

4 BE2023/5564 temperature-sensitive melatonin in situ gel can be up to 5 days, and it is tested and proven that the present invention effectively addresses the problems of low usage rate and frequent administration due to rapid metabolism in the prior art can solve.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of different concentrations of P407 provided by the present invention on the gelling temperature.

Figure 2 shows an effect of various concentrations of P188 provided by the present invention on gelling temperature.

Figure 3 shows the effect of different concentrations of HPMC (15KM) provided by the present invention on the gelling temperature.

Figure 4 shows an effect of different concentrations of chitosan provided by the present invention on the gelling temperature.

Figure 5 shows an effect of various concentrations of PEG400 provided by the present invention on gelling temperature.

Figure 6 shows an effect of various concentrations of PEG4000 provided by the present invention on the gelling temperature.

Figure 7 shows a graph of the standard curve of melatonin in a temperature-sensitive in situ gel provided by the present invention.

Figure 8 shows a line graph of the in vitro release of the temperature sensitive melatonin in situ gel provided by the present invention.

Figure 9 shows a standard curve plot of melatonin in plasma provided by the present invention.

Figure 10 shows a comparative diagram of the melatonin in situ gel provided by the present invention before and after the gelled state; A: Melatonin in situ gel before gelling; B:

Melatonin in situ gel in the gelled state.

Figure 11 shows an investigation of the in-vivo and in-vitro gelling ability, which is carried out by the present

Invention is provided: A. aqueous melatonin solution; B. Melatonin gel solution; C. Melatonm-1n vivo gel.

DETAILED DESCRIPTION

The technical solution of the embodiment of the present invention is explained clearly and completely below, so that the aim, the technical solution and the advantages of the

Embodiment of the present invention will become clearer. Obviously, the exemplary embodiments described do not represent all exemplary embodiments, but only a part of them

Embodiments of the present invention. All other embodiments through

> BE2023/5564 obtained by one of ordinary skill in the art based on the embodiments in the present invention should be considered to be within the scope of the present invention.

The reagents and devices affected by the present invention:

Melatonin (MT) standard (HPLC > 98%) manufactured by Sigma, USA; Poloxam 407 (P407) manufactured by BASF, Germany; Poloxam 188 (P188), chitosan, polyethylene glycol 400 (PEG400), manufactured by Beijing Solarbio GmbH; Hydroxypropylmethylcellulose (HPMC, 15KM),

Polyethylene glycol 4000 (PEG4000) manufactured by Shanghai Maclean GmbH; methanol and

Chromatography grade acetonitrile manufactured by Thermo Fisher Scientific, Shanghai;

Sodium hydrogen phosphate and potassium dihydrogen phosphate manufactured by Tianjin Zhiyuan Chemical

Reagent Co;glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), catalase (CAT),

Malondialdehyde (MDA) and superoxide dismutase (SOD) were from Nanjing Jiancheng Institute of

Biological Engineering provided; the high-speed freezer centrifuge was developed by

Hunan Xiangxin Instrument GmbH.

Below, the present invention provides detailed embodiments illustrating a temperature-sensitive melatonin in-situ gel, a temperature-sensitive in-situ gel and a manufacturing method therefor.

Example 1

A temperature-sensitive melatonin in situ gel of the present exemplary embodiment, containing the substances in the following weight percents:

Poloxam 407 22%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.5%

Polyethylene glycol 4000 0.4%

The rest is water.

Weigh 22g of Poloxam 407, add water, dissolve and then place in a refrigerator at 4°C overnight; Taking out the cooled Poloxam 407 solution,

Return to room temperature and then add 1.5 g of Poloxam 188 and 0.4 g

Polyethylene glycol 4000 one at a time and stirring to dissolve; then add 0.5 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adding water for

Injection purposes to adjust the volume to 100 ml and place in a refrigerator; in 24 hours a temperature-sensitive in situ gel is obtained by filtration and sterilization via eme

Filter membrane made of 0.22um.

If it is necessary to contain therapeutic agents such as in the temperature-sensitive in-situ gel

Melatonm, to add, the addition of melatonin occurs when adding Poloxam 188 and

Polyethylene glycol 4000.

Embodiment 2

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A temperature-sensitive melatonin in situ Ge of the present exemplary embodiment, containing the substances in the following weight percents:

Poloxam 407 21%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.6%

Polyethylene glycol 4000 0.3%

The rest is water.

Weigh 21g of Poloxam 407, add water, dissolve and then place in a refrigerator at 4°C overnight; Taking out the cooled Poloxam 407 solution,

Bring back to room temperature and then add 1.5 g of Poloxam 188 and 0.3 g

Polyethylene glycol 4000 one at a time and stirring to dissolve; then add 0.5 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adding water for

Injection purposes to adjust the volume to 100 ml and place in a refrigerator; in 24 hours a temperature-sensitive in situ gel is obtained by filtration and sterilization via eme

Filter membrane made of 0.22um.

If it is necessary to contain therapeutic agents such as in the temperature-sensitive in-situ gel

Melatonin, the addition of melatonin occurs when adding Poloxam 188 and

Polyethylene glycol 4000.

Example 3

A temperature-sensitive melatonin in situ Ge of the present exemplary embodiment, containing the substances in the following weight percents:

Poloxam 407 23%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.4%

Polyethylene glycol 4000 0.5%

Weigh out 23g of Poloxam 407, add water, dissolve and then place in a refrigerator at 4°C overnight; Taking out the cooled Poloxam 407 solution,

Return to room temperature and then add 1.5 g of Poloxam 188 and 0.5 g

Polyethylene glycol 4000 one at a time and stirring to dissolve; then adding 0.4 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adding water for

Injection purposes to adjust the volume to 100 ml and place in a refrigerator; in 24 hours a temperature-sensitive in situ gel is obtained by filtration and sterilization via eme

Filter membrane made of 0.22um.

If it is necessary to contain therapeutic agents such as in the temperature-sensitive in-situ gel

Melatonm, to add, the addition of melatonin occurs when adding Poloxam 188 and

Polyethylene glycol 4000.

Example 4

7 BE2023/5564

The melatonin temperature-sensitive in situ gel provided by the exemplary embodiment contains the substances in the following weight percentages:

Melatonin 0.05%

Poloxam 407 21%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.6%

Polyethylene glycol 4000 0.3%

The rest is water.

In particular, the preparation of a temperature-sensitive melatonin in situ gel is as follows:

Weigh out 21g of Poloxam 407, add water, dissolve and then place in a refrigerator overnight; Take out the cooled Poloxam 407 solution, put it back on

room temperature and then adding 1.5 g of Poloxam 188, 0.3 g of polyethylene glycol 4000 and 0.05 g of melatonin one after the other and stirring to dissolve; then adding 0.6 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adjusting the volume to 100 ml and placing in a refrigerator; In 24 hours, filtration and sterilization are carried out to produce a temperature-sensitive melatonin-in-SRTU gel solution.

Example 5

The melatonin temperature-sensitive in situ gel provided by the exemplary embodiment contains the substances in the following weight percentages:

Melatonin 0.15%

Poloxam 407 22%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.5%

Polyethylene glycol 4000 0.4%

The rest is water.

In particular, the preparation of a temperature-sensitive melatonin in situ gel is as follows:

Weigh 22g of Poloxam 407, add water, dissolve and then place in a refrigerator at 4°C overnight; Take out the Poloxam 407 solution, put it back on

room temperature and then adding 1.5 g of poloxam 188, 0.4 g of polyethylene glycol 4000 and 0.15 g of melatonin one by one and stirring to dissolve; then add 0.5 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adjusting the volume to 100 ml and placing in a refrigerator; In 24 hours, filtration and sterilization are carried out to produce a temperature-sensitive melatonin-in-SRTU gel solution.

Example 6

A Melatonm temperature-sensitive in situ gel provided by the exemplary embodiment contains the substances in the following weight percents:

Melatonin 0.5%

Poloxam 407 23%

8 BE2023/5564

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.4%

Polyethylene glycol 4000 0.5%

The rest is water.

Specifically, the preparation of a temperature-sensitive melatonin-in-srtu gel is as follows:

Weigh out 23g of Poloxam 407, add water, dissolve and then place in a refrigerator overnight; Take out the cooled Poloxam 407 solution, put it back on

room temperature and then adding 1.5 g of Poloxam 188, 0.5 g of polyethylene glycol 4000 and 0.5 g of melatonin one after the other and stirring to dissolve; then adding 0.4 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adjusting the volume to 100 ml and placing in a refrigerator; In 24 hours, filtration and sterilization are carried out to produce a temperature-sensitive melatonin in situ gel solution.

Example 7

The melatonin temperature-sensitive in situ gel provided by the exemplary embodiment contains the substances in the following weight percentages:

Melatonm 1%

Poloxam 407 22%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.5%

Polyethylene glycol 4000 0.4%

The rest is water.

In particular, the preparation of a temperature-sensitive melatonin in situ gel is as follows:

Weigh out 22g of Poloxam 407, add water, dissolve and then place in a refrigerator overnight; Take out the cooled Poloxam 407 solution, return it to

room temperature and then adding 1.5 g of poloxam 188, 0.4 g of polyethylene glycol 4000 and 1 g of melatonin one by one and stirring to dissolve; then add 0.5 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adjusting the volume to 100 ml and placing in a refrigerator; In 24 hours, filtration and sterilization are carried out to produce a temperature-sensitive melatonin in situ gel solution.

Embodiment 8

A melatonin temperature-sensitive in situ gel provided by the exemplary embodiment contains the substances in the following weight percents:

Melatonin 3%

Poloxam 407 22%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.5%

Polyethylene glycol 4000 0.4%

The rest is water.

9 BE2023/5564

In particular, the preparation of a temperature-sensitive melatonin in situ gel is as follows:

Weigh out 22g of Poloxam 407, add water, dissolve and then place in a refrigerator overnight; Take out the cooled Poloxam 407 solution, put it back on

room temperature and then adding 1.5 g of Poloxam 188, 0.4 g of polyethylene glycol 4000 and 3 g of melatonin one after the other and stirring to dissolve; then add 0.5 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adjusting the volume to 100 ml and placing in a refrigerator; In 24 hours, filtration and sterilization are carried out to produce a temperature-sensitive melatonin in situ gel solution.

Embodiment 9

The Melatonm temperature-sensitive in-situ gel provided by the exemplary embodiment contains the substances in the following weight percentages:

Melatonin 5%

Poloxam 407 22%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.5%

Polyethylene glycol 4000 0.4%

The rest is water.

In particular, the preparation of a temperature-sensitive melatonin in situ gel is as follows:

Weigh out 22g of Poloxam 407, add water, dissolve and then place in a refrigerator overnight; Take out the cooled Poloxam 407 solution, return it to

room temperature and then adding 1.5 g of Poloxam 188, 0.4 g of polyethylene glycol 4000 and 5 g of melatonin one after the other and stirring to dissolve; then add 0.5 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adjusting the volume to 100 ml and placing in a refrigerator; In 24 hours, filtration and sterilization are carried out to produce a temperature-sensitive melatonin 1n-sttu gel solution.

Embodiment 10

A melatonin temperature-sensitive in situ gel provided by the exemplary embodiment contains the substances in the following weight percents:

Melatonin 10%

Poloxam 407 22%

Poloxam 188 1.5%

Hydroxypropylmethylcellulose 0.5%

Polyethylene glycol 4000 0.4%

The rest is water.

In particular, the preparation of a temperature-sensitive melatonin in situ gel is as follows:

Weigh out 22g of Poloxam 407, add water, dissolve and then place in a refrigerator overnight; Take out the cooled Poloxam 407 solution, put it back on

Room temperature and then adding 1.5 g of Poloxam 188, 0.4 g of polyethylene glycol 4000

10 BE2023/5564 and 10 g melatonin one after the other and stir to dissolve; then add 0.5 g

Hydroxypropylmethylcellulose, stirring to dissolve and then adjusting the volume to 100 ml and placing in a refrigerator; In 24 hours, filtration and sterilization are carried out to produce a temperature-sensitive melatonin-in-SRTU gel solution.

To further illustrate the test method and effect of the present invention, the following experimental examples are described in detail.

Experimental Example 1: Screening of the components of the temperature-sensitive in-situ gel according to the present invention

The types and amounts of excipients commonly used in slow release in situ gel formulations such as Poloxam 407, Poloxam 188,

Hydroxypropylmethylcellulose [HPMC(15KM)], chitosan, polyethylene glycol 4000 (PEG400) and

PEG4000, have been studied and the gel temperature is measured using the inverted test tube method to determine whether they can be used as starting materials for the production of temperature-sensitive in situ gel and what concentration is required.

Test tube inversion method: an appropriate amount of gel is added to a centrifuge tube, then it is placed in a shaking oven in a constant temperature water bath and the

Temperature increases at a rate of 0.5°C/min. For every 0.5°C increase in temperature the centrifuge tube is inverted 90 degrees and the flow of the gel is observed, if the gel does not flow the temperature is the gelling temperature. The temperature at this time is recorded. pH test of the gel: the pH value of the temperature-sensitive in-situ gel is determined with a

Acidity meter is measured, and it is optimal if the range of the individual excipients of the gel is 7.0 to 7.4. 1.1 Preliminary screening of P407 concentrations

Nine groups of P407 gels with concentrations of 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22% and 23% are configured with three replicates each and their gelling temperatures are determined.

The results are shown in Table 1 and Figure 1. The temperature is between 28-30°C and the temperature of the temperature-sensitive in situ gel is prone to phase change in vitro, therefore the overall gelling temperature of the gel excipients before dilution by body fluid is set at 28-30°C in this test . Previous studies have shown that the temperature of P407 before its concentration reaches gelation is best 5-10% lower than total

Gelling temperature is, and the optimal ratio is that the gelling temperature with added excipients is within the setting range, and in conjunction with the optimal range of pH values, the concentration range of P407 is set at 21-23%.

Table 1 Results of the different concentrations of P407 on the gelling temperature

Il BE2023/5564

P407 concentration 15 16 17 18 19 20 21 22 23%)

Sample 1 42.0 38.0 32.0 30.0 28.0 26.0 24.0 22.0 20.0

Gelling temperature (°C)

Sample 1 42.5 37.5 32.5 30.5 28.5 26.5 24.5 21.0 20.0

Gelling temperature (°C)

Sample 1 43.0 37.0 31.0 30.8 28.6 26.2 24.0 21.0 19.8

Gelling temperature (°C)

Mean 42.0+0.5 37.505 31.8#0.7 30.4+0.4 28.3+0.3 26.3+0.3 24 240.3 21.3+0.6 20.0+0.1 pH value 68 6.9 7.0 7.0 6.9 6.9 7.0 7.1 7.2 1.2 Influence of P188 on gelling temperature

The concentration of P407 is set at 21% and five groups of P407 gels are configured, with P188 added to each sample group at concentrations of 1%, 1.5%, 3%, 5% and 8%, with three in each group Repetitions are carried out and the

Gelling temperature is measured.

The results are shown in Table 2 and Figure 2. P188 is able to regulate the gelling temperature and gel strength of a temperature-sensitive gel and produce a temperature-sensitive in-situ gel. 21% P407 is used as a reference for the preferential selection of other excipients, combined with the optimal range of pH values, the concentration of P188 is set at 1.5%.

Table 2 Results of the different concentrations of P188 on the gelling temperature

P188- 1 1.5 3 5 8

Concentration (%)

Sample 1 25.0 28.0 30.0 33.0 36.0

Gelling temperature (°C)

Sample 1 248 29.0 31.0 34.0 35.0

Gelling temperature (°C)

Sample 1 25.0 29.5 31.0 33.5 35.5

Gelling temperature (°C)

Mean 24.9+0.1 28.8+0.8 30.7+0.5 33.5+0.5 35.5+0.5 pH value 6.9 7.1 7.5 7.5 7.6 1.3 Influence of HPMC (15KM) on the gelling temperature

The concentration of P407 is set at 21% and six groups of P407 gels are run

Addition of HPMC (15KM) configured in concentrations of 0.4%, 0.6%, 0.8%, 1.5%, 2% and 3%.

HPMC must be dissolved in advance by adding water for injection at 85°C, using three

12 BE2023/5564

Repeats are performed in each group and placed in the refrigerator at 4°C

Placed overnight to prepare it into a gel and to measure the gelling temperature.

The results are shown in Table 3 and Figure 3. As the dosage of HPMC (15KM) increases, the gelling temperature of the gel increases, and in conjunction with the optimal range of pH values and the overall gelling temperature, 0.4-0.6% HPMC (15KM) represents the optimal range.

Table 3 Results of the different concentrations of HPMC (15KM) on the

gelling temperature

TC HPMCBKM) 0406 OBS

Concentration(%)

Sample 1 30 310 20 390 000 MO

Gelling temperature (°C)

Sample 1 28.5 31.0 32.0 39.5 40.5 41.5

Gelling temperature (°C)

Sample 1 29.0 30.0 32.5 39.0 40.8 41.5

Gelling temperature (°C)

Mean 28.5+0.5 30.640.6 232.2+0.3 39.1+0.3 40.4+0.4 41.6+0.3 pH value 7.0 7.2 7.5 7.6 7.7 7.6 0 1.4 Influence of chitosan on the gelling temperature

The concentration of P407 is set at 21% and five groups of P407 gels are run

Addition of chitosan in concentrations of 0.1%, 0.15%, 0.2%, 0.3% and 0.5%, with three

Replicates configured in each group to prepare the gels and measure their gelling temperatures.

The results are shown in Table 4 and Figure 4. With the gradual increase in the

The gel temperature of the 21% P407 solution tends to decrease with the amount of chitosan, and all pH values are too low for in vivo injection, so chitosan is not suitable for addition to the gel.

Table 4 Results of the different concentrations of chitosan on the gelling temperature

Concentration(%)

Sample 30 30 30 000 80

gelling temperature

CC)

Sample 1 34.0 27.0 22.0 20.2 18.5

gelling temperature

CC)

Sample 1 34.3 28.2 21.5 20.6 19.0

Gelling temperature (°C)

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Mean 33.7Æ0.6 27.7+0.6 22.1+0.7 20.3+0.3 18.5+0.5 pH value 4.5 40 3.6 3.5 3.0 1.5 Influence of PEG400 on the gelling temperature

The concentration of P407 is set at 21% and five groups of P407 gels are run

Addition of PEG400 at concentrations of 0.5%, 0.6%, 0.7%, 0.8% and 1% with three replicates configured in each group to prepare the gels and measure their gelling temperatures.

The results are shown in Table 5 and Figure 5. The amount of PEG400 gradually increased and the gel temperature of the 21% P407 solution did not change in any case. In conjunction with the optimal range of pH values, it is considered that PEG400 is not suitable for addition to the gel.

Table 5 Results of the different concentrations of PEG400 on the gelling temperature

TT PEG00 0506 OT O8 LO

concentration(%)

Sample 260 260260260260

Gelling temperature (°C)

Sample 1 26.1 26.0 26.0 26.0 26.0

Gelling temperature (°C)

Sample 1 26.0 26.1 26.1 26.1 26.1

Gelling temperature (°C)

Mean 26.0+0.1 26.0+0.1 26.0+0.1 26.0+0.1 26.0+0.1 pH value 7.5 7.5 7.6 7.5 7.5 1.6 Influence of PEG4000 on the gelling temperature

The concentration of P407 is set at 21% and five groups of P407 gels are run

Addition of PEG4000 configured in concentrations of 0.3%, 0.5%, 0.7%, 0.9% and 1%, where in

Three repetitions are performed for each group and the gelling temperature is measured. the results are shown in Table 6 and Figure 6. The gelling temperature of the 21% P407

Solution gradually increases with increasing dosage of PEG4000, combined with the optimal range of pH values and overall gelling temperature, 0.3-0.5% PEG4000 represents the optimal range.

Table 6 Results of the different concentrations of PEG4000 on the gelling temperature

U PEG4000-0308009 LO

Concentration (%) ‘Proel 280800 800 360 380

Gelling temperature (°C)

Sample 1 27.5 31.2 32.0 36.5 38.5

Gelling temperature (°C)

Sample 1 28.0 30.5 32.5 36.8 38.0

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Gelling temperature (°C)

Mean 27.8+40.3 30.6+0.6 32.1+0.4 36.4+0.4 38.2+0.3 pH value 7.1 7.3 7.5 7.6 7.6

So far, in the present invention, the substances for producing a temperature-sensitive in-situ gel and the concentration range of each substance in the temperature-sensitive in-situ gel are examined: the concentration range of P407 is 21% to 23%, the concentration of P188 is 1 .5%, the concentration range of HPMC (15KM) is 0.4% to 0.6% and the concentration range of PEG4000 is 0.3% to 0.5%.

Experimental example 2: Production and concentration optimization of the temperature-sensitive

Melatonin in situ gels according to the present invention 2.1 Establishment of an HPLC method for determining melatonin in a temperature-sensitive in situ gel 2.1.1 Selection of the wavelength of melatonin: 10 mg of melatonin standard are weighed and in

methanol to prepare a 0.1 mg/ml solution of the standard, which was treated with a UV

Spectrophotometer 1m wavelength range of 230-450 nm is scanned, with the maximum

Absorption wavelength of the melatonin standard is measured at 230 nm. 2.1.2 Chromatographic conditions for high-performance liquid chromatography (HPLC):

Column: Agela Odssil C18 column (250x4.6mm, 5um); column temperature 25°C; flow rate 1 mL/min; mobile phase methanol: water = 60:40 (v/v); Injection volume 10 uL. 2.1.3 Creation of the standard curve: The melatonin standards are precisely measured and added

Melatonin standard solution of 100 ug/mL, 75 ug/mL, 50 ug/mL, 25 ug/mL, 10 ug/mL, 7.5 ug/mL, 5 ug/mL, 1 ug/mL and 0.5 ug /mL, with the injection volume being 10 uL each. With the peak area Y as the ordinate and the measured concentration of the melatonin standard (ug/mL) as

The abscissa shows the standard curve in Figure 7: Y=57.988x-9.9134, R2=0.9997, with good

Linearity 1m range from 0.5-100 ug/mL for melatonin standard. 2.1.4 Specificity test: 10 mg melatonin standard is accurately weighed and given an appropriate

Amount of methanol is diluted to obtain a standard solution with a concentration of 1 mg/mL, a temperature-sensitive gel and a temperature-sensitive in situ gel with melatonin are prepared and dissolved in the mobile phase, the sample is passed through a 0.22-um filter membrane and the volume of 1 mL of the filtrate is adjusted to 10 mL. 10 uL of each

Melatonin standards, the temperature-sensitive in-situ gel and the temperature-sensitive in-situ gel with melatonin are precisely aspirated and chromatographically tested according to the above-mentioned

Conditions determined. The chromatogram is recorded and the results show good

Specificity of the method. 2.1.5 Precision test: Three standard solutions of melatonin in low, medium and high

Concentration (2 µg/mL, 25 µg/mL and 75 µg/mL) are prepared; the peak values are measured in 0, 2, 4, 6 and 8 hours respectively to calculate the precision within a day, and then in 1,

15 BE2023/5564 2, 3, 4 and 5 days to calculate the precision between days. The results are shown in Tables 7 and 8. The RSDs of the peak areas of the intraday precision test and the

Peak areas of the interday precision test are below 2%, indicating good precision of the method.

Table 7 Intraday precision results (n=5)

Concentration I (og/m) TN ae ee a OO (201009 1082 1021 1008 105 08 25 1260 1258 1236 1233.6 1255.6 0.99 75 4408 4403 4106 4314 4320 0.85

Table 8 Interday precision test (n=5) “Concentration a peak area RS DO (ng/ml) ld à UC SA (287 1005 1019 1007 1016 080 25 1261 1272 1275.6 1273.9 1276.5 0.99 75 4300 4315 4307 4 316 4418 0.88 0 2.1.6 Stability test: 10 µl of the Melatonm standard solution is precisely aspirated and into the

Liquid chromatographs injected 0, 2, 4, 6 and 12 hours after configuration, and the

Peak area is measured and RSD is calculated, the results are shown in Table 9, the RSD is less than 2%, indicating that the melatonin standard solution is stable within 12 hours.

Table 9 Results of the stability test of melatonin “Concentration peak area RSD) (ng/ml) Oh 2h 4h 6h 12h

TS 3759 36933673209 2.1.7 Sample recovery rate test: 5 parts each of temperature-sensitive melatonin in situ gel injection with known melatonin content (2 mg, 4 mg, 6 mg) are measured (see Table 10), 1 ml of the standard solution is added and the recovery rate of the sample is calculated according to the above chromatographic conditions. The results show that the mean

Recovery rate of the known sample determinations is 96.86% and the RSDs are all within 2%, indicating that the sample recovery rate test of this method meets the requirements of HPLC determination with high accuracy, and the results are in

Table 10 shown below.

16 BE2023/5564

Table 10 Sample recovery rate test results

TE Medium

Added Measured Recovery /

Amount (mg) Amount (mg) rate (%) Recovery rate RSD(%) (%)

RS

1.94 97 2 1.92 96 96.44 0.97 1.93 96.6 1.95 97.5 3.8 95 3.81 95.2 4 3.85 96 96 0.96 3.9 97.5 3.85 96.3 5.8 96.6 5.85 97.5 6 59 99.2 98.14 0.87 5.95 99.2 5.89 98.2 2.2 Box-Behnken reaction surface design method for optimizing the optimal concentration of temperature-sensitive in-situ gels 2.2.1 Experimental plan

For the production of a temperature-sensitive melatonin in situ gel, a 3-factor-3

Level response surface method is used, and the conditions for the preparation of a temperature-sensitive melatonin in situ gel are optimized based on the response values of the composite scores. a: P407 (21-23%) b: PEG4000 (0.3-0.5%) and c: HPMC15 (KM) (0.4-0.6%), the experimental plan is shown in Table 11.

Table 11 Response surface factor levels

Factomivem

factor

SO P407(A)% PEG4000B)% HPMC(15KM)(C)% — -1 21 0.3 0.4 0 22 0.4 0.5 1 23 0.5 0.6 2.2.2 Response surface method analysis to optimize the recipe

The data obtained are analyzed by multiple regression fitting using the software Design-

Expert.8.0.6 analyzes to find the theoretical optimal conditions and the predicted values for

17 BE2023/5564 to obtain and validate each factor. The gelling time coefficient is set to 0.4

Release weighting coefficient set to 0.4 and gelling temperature weighting coefficient to 0.2 according to the primary and secondary relationships. Total score = (difference of gelling time/shortest gelling time) x 100 x 0.4 + (difference of average

Release/uniform release) x 100 x 0.4 (difference in gelling temperature/fastest

Gelling temperature) x 100 x 0.2. The experimental design and the results are shown in Table 12.

Table 12 Response surface test analysis results

No. A B C Temperature Gelling time Day! Day2 Day4 Total Score _ (°C) (S) Release Release Release (%) (%) (%)

SO + 1 2 0 0 O0 325 75 18 47 82 96 3 1 - O0 335 85 20 51 91 85 1 4 - 0 1 321 68 22 54 92 82 1 5 1 0 - 33.0 72 20 52 90 88 1 6 1 1 0 318 76 18 48 93 88 7 0 - - 320 81 19 54 91 84 1 1 8 1 1 1 322 78 18 55 90 82 9 0 0 O0 331 75 18 46 87 93 0 - 1 313 75 19 54 9 3 81 1 11 - 0 - 323 75 18 55 92 83 1 1 12 0 0 0 327 76 18 46 86 97 13 - 1 0 327 93 20 51 91 84 1 14 - - 0 32.1 68 20 54 93 80 1 1 0 0 0 3 25 75 18 46 87 96 16 1 0 1 320 70 20 50 92 81 17 0 0 0 327 77 19 45 85 96 10 2.2.3 Analysis of variance

It can be seen from Table 13 that the significance level of the multiple regression model

P=0.0008<0.01, the developed regression model is highly significant and also shows that the

18 BE2023/5564

Regression equation is highly significant. The fitting error P=0.1482>0.05, the difference is not significant, indicating that the error is extremely small and the test optimization is high

level of trust. A regression adjustment is carried out for the experimental data using the software Design-

Expert 8.0.6 performed to determine the composite score: Y = 95.6 + 1.625A + 0.5B- 1.125C - 0.25AB - 1.5AC + 1.25BC - 4.8A2 - 6.55B2 - 7 ,3C2, R2 = 0.9515, which means that the change in response value of 95.15% depends on the selected factors, the equation is well fitted and well consistent with the actual situation. The binary multinomial model chosen for this test is statistically significant (p<0.01); the out-of-fit term p=0.1482>0.05, which means that there are no significant out-of-fit factors in the test.

Table 13 Analysis of variance results for the total score regression model for temperature-sensitive melatonin in situ gel “Source of variance Sum of degrees of freedom Mean F-P value Significance

Squares Variance Value (SS)

Model 60752 9 6750 1527 00008 +

A 21.13 1 21.13 4.78 0.0651

B 2.00 1 2.00 0.45 0.5228

C 10.13 1 10.13 2.29 0.1740

AB 0.25 1 0.25 0.057 0.8189

AC 9.00 1 9.00 204 0.1967

BC 6.25 1 6.25 1.41 0.2732

A? 97.01 1 97.01 21.94 0.0022 **

B? 180.64 1 180.64 4086 0.0004 **

CC? 224.38 1 224.38 50.75 0.0002 **

Residual 30.95 7 4.42

Misfit term 21.75 3 7.25 3.15 0.1482

Pure error 9.20 4 2.30

Total difference 638.47 16

Note: * means a significant difference (P < 0.05), ** means a highly significant difference (P < 0.01). 2.2.4 Experimental model fitting

Combined with the results in Table 13, it can be seen that the differences in the secondary terms of the model A2 (P = 0.0022 < 0.01), B2 (P = 0.0004 < 0.01), C2 (P = 0.0002 < 0.01) are all highly significant; the primary terms A (P = 0.0651 > 0.05), B (P = 0.5228 > 0.05), CP = 0.1740 > 0.05) and the interaction terms AB (P = 0.8189>). 0.05), AC (P = 0.1967 > 0.05) and BC (P = 0.2732 > 0.05) are not significantly different. This suggests that the main effect of A,

B and C on the overall result is significant and the main effect relationship of each factor is:

C>B>A. The analysis shows that P407 (A), PEG4000 (B) and HPMC (15KM) (C) each account for 22%

P407, 0.4% for PEG4000 and 0.5% for HPMC (15KM).

19 BE2023/5564 2.2.5 Preparation of a temperature-sensitive melatonin in situ gel: the ideal gel should be at

Be liquid at room temperature and quickly turn into a gel when injected into the animal, this temperature index serving as a screening criterion for the optimal prescribing conditions. Three batches of temperature-sensitive melatonin in situ gels are prepared according to the optimized recipes: MT 0.15%, P407 22%, P188 1.5%, PEG4000 0.4%, HPMC (15KM) 0.5% and water for injection purposes 75.45%. 2.2.6 Measurement of the in vitro dissolution and release of the temperature-sensitive melatonin in situ

Gels: the gel temperature and the gelling time are the same as those described in experimental example 1

Test tube inversion method determined. The in vitro dissolution of the gel is carried out using a film-free

Dissolution model examined the in vitro release behavior of the temperature-sensitive

Gels to be examined, i.e. the temperature-sensitive melatonin in situ gel is prepared according to the optimized recipe, 3 g of temperature-sensitive melatonin in situ gel are in em

Test tube is added and placed in a constant temperature shaker at 37°C for 5 minutes, waiting for gelling, then 2 mL of PBS buffer solution is added and 0.5 mL of the

Sample collected at 1, 12, 24, 36, 48, 72, 96, 104 and 120 hours in a 10 mL volumetric flask and the volume is adjusted with the mobile phase. 10 µl is placed in a high performance

Liquid chromatograph is injected, the concentration of the sample solution is determined and the

Release calculated according to the HPLC method for melatonin in gel in experimental example 2.1. It is found that the in vitro release of the preparation is proportional to the increase in the formulation

Slow release time increases, showing a more moderate release rate, and the preparation of the formulation is essentially fully released on the fifth day; the results are in

Table 14 and Figure 8 shown below.

Table 14 Results of the validation of the formulation for the temperature-sensitive melatonin in situ

gel

Number of 123 Mean Wen RSD(%)

Measurements

Gel temperature (°C) 328 324325325402

Gelling time (S) 76.0 75.7 75.5 75.7+0.3 -

Release on day 18.3 18.5 18.6 18,402 1 1 (%)

Release on day 46.0 47.0 46.5 46.5+0.5 1 2 (%)

Release on day 85.0 86.0 84.0 85.0+1.0 1.1 4 (%)

Release on day 100 99.9 100 99.9+0.6 0.6 5 (%)

The composition of the temperature-sensitive in situ gel provided by the present invention and the ratio range are determined by the first experimental example, with a

20 BE2023/5564

Concentration range of 21% to 23% for P407, 1.5% for P188, 0.3% to 0.5% for PEG4000 and 0.4% to 0.6% for HPMC (15KM); the optimal concentration of the temperature-sensitive 1n-situ gel with 0.15% melatonin provided by the present invention is determined by the second

Experimental example determined: MT 0.15%, P407 22%, P188 1.5%, HPMC (15KM) 0.5%, PEG4000 0.4% and water for injections 75.45%, and the temperature-sensitive Melatonm-m-s1tu -Gel is produced. The concentration range of the above substance components and the optimal ones

Ratios are capable of achieving the purpose of the present invention.

Experimental example 3 Pharmacokinetic test of melatonin in situ gel for veterinary medicine

In this comparative example, the

Differences in the relevant pharmacokinetic parameters between the veterinary temperature-sensitive melatonin-in-srtu gel of the present invention and the melatonin

Stock solution compared after subcutaneous administration to investigate the effectiveness of melatonin in the animal organism. 3.1 Establishment of the HPLC method for melatonin in plasma 3.1.1 Sample pretreatment: 100 µl of each sample is placed in a centrifuge tube, 1 mL

Acetonitrile is added, vortexed and mixed for 120 s, then centrifuged at 12000 rpm for 10 min. 100 µL methanol is added to the supernatant and centrifuged at 12000 rpm for 2 min. The supernatant is blown dry under nitrogen, redissolved with 500 μl of methanol and filtered through a 0.22 μm microporous membrane. 10 µl is precisely aspirated for HPLC analysis. 3.1.2 Chromatographic conditions: Agela Odssil C18 column (250 mmx 4.6 mm, 5 μm), methanol

Water=45:55, column temperature 28°C, flow rate 1 mL/min, excitation wavelength 286 nm,

Emission wavelength 352 nm, injection volume 10 uL. 3.1.3 Preparation of standard curve: Take a 2 mL centrifuge tube, add 200 µl of blank plasma and add melatonin standard solution in concentrations of 5, 10, 15, 20, 50, 75, 50, 100, 150, 200, 500 and 1000 ng/mL to quantify the peak area of the melatonin gel, and use the concentration X (ng/mL) to generate a standard curve for the peak area Y of the

to draw medicine. The results in Table 15 show good linearity in the range of 5-1000 ng/mL, and the standard curve for melatonin in plasma is shown in Figure 9.

Table 15 Results of linearity of melatonin in plasma

Concentration 5 10 15 20 58075 100 150 200 500 1000 (ng/m))

Peak area 3308 6166 9206 1219 30883 47506 60358 89032 120011 288001 56000

21 BE2023/5564 3.1.4 Specificity test: Melatonin standards, blank plasma and administered plasma (temperature sensitive melatonin in situ gel) are collected for HPLC analysis to determine whether there is interference between them. 10 µl each of the standard solution, des

Blank plasma and the plasma solution after drug administration are precisely collected and determined according to the chromatographic conditions in 3.1.2. The results show that the method has good specificity. 3.1.5 Accuracy and precision: The melatonin standards are dissolved in blank plasma and in graded concentrations of 75, 150 and 500 ng/ml with 5 replicates each

Concentration is configured and the intraday precision is calculated. 5 repetitions are performed in the same way on 7 consecutive days and the interday precision is calculated.

From Tables 16 and 17 it can be seen that the RSDs of the peak areas of the intraday

Precision tests and the peak areas of the interday precision test for melatonm in plasma are within 2%, indicating that the method has good precision.

Table 16 Intraday precision of MT in broiler plasma (n=5)

Concentration RDS (ng/mL) Peak area 0075 47199 46856 47076 4618 4675 11° 150 8906.2 8701 8903.7 8806.2 8906.3 1 500 28301.7 28101.4 28001.9 27902.3 28901.6 1.4

Table 17 Precision of interday MT in broiler plasma (n=5) “Concentration peak area RSD) (ng/mL) 75 4719.06 4719.08 4519.02 47193 4719.22 4718.82 4720.42 16 150 8844.9 8764.78 8544.88 8 734.98 8857.68 8843.9 8864.68 13 500 28679.98 28722.56 281631 2870174 2870226 29103.4 29103.4 1.1 3.1.6 Measurement of the lower limit of the specified amount: melatonin-containing plasma with a

Mass concentration of 1 ng/mL is assayed five times, HPLC analysis is performed under the conditions described above, and data is recorded. The signals from the above solutions are compared using the signal-to-noise ratio method. From Table 18, it can be seen that the RSD of the specified amount of samples is within 2%, indicating that the test for this method meets the requirements for HPLC determination.

Table 18 Results of measuring the lower limit of the specified amount of melatonin in

plasma

22 BE2023/5564

Concentration RD (ng/mL) Peak area

C1 681683 685 682 688 03 3.1.7 Recovery rate test:

The melatonin standards are dissolved in blank plasma and configured in graded concentrations of 75, 150 and 500 ng/mL with 5 replicates of each concentration. The ratio between the measured concentration and the added concentration is the relative one

Recovery rate. The results show (Table 19) that the average recovery rate of the

Sample is 96.86% and RSD is less than 2%, indicating that the method is good

Has stability.

Table 19 Sample recovery rate in broiler plasma “Concentration RSD) (ng/mL) Peak area 78 45585 46602 47191 47568 4719 16° 150 8905.6 87034 8802.3 8901.6 8900.7 1 500 28901 28905.6 28903.8 27901.9 28902.6 1.5 3.1.8 Stability test: The melatonin standards are in Broiler plasma dissolved and in graded

Concentrations of 75, 150 and 500 ng/mL were configured with 5 replicates of each concentration. The first three portions are placed at room temperature for Oh, 5h and 10h and then operated to examine the stability of the samples; the fourth portion is at -20°C for 5

Stored for days and then thawed in a water bath to determine concentration; the fifth

Portion is stored at -20°C for 10 days and then thawed to study stability. The

Results in Table 20 show that the melatonin plasma samples under different

Storage conditions at low, medium and high concentrations are not significant

show changes. Samples are stored at -20°C for 10 days, with an RSD of 2% or less, meeting the long-term stability requirement; Samples are stored at room temperature for 10 hours, with an RSD of 2% or less, which meets the requirement

Short-term stability met.

Table 20 Stability of MT in broiler plasma

Concentrate 0 RSD room temperature RSD room temperature RSD( -20°C(5d) RSD -20°C(10d) RSD ion ch) (%) atur(5h) (%) atur(10h)%) (%) (%) ( ug/mL) 75 47.44+0.49 1 47.17+0.91 1.9 47.19+0.89 1.8 47.18+0.54 11 47.18+0.89 1.9 150 89.81+1.29 1.4 88.43+1.34 1.5 87.05+1 .25 1.4 88.43+1.34 1.5 88.63+0.90 1 500 29.15+4.64 1.5 282.42+4.77 1.6 287.01+3.99 13 287.01+4.46 1.6 287.01+4.47 1.5

23 BE2023/5564 3.2 Pharmacokinetic study 3.2.1 Grouping of pharmacokinetic test animals: 20 broilers are selected based on similar age and weight, and randomly divided into two test groups A and B with 10 animals in each group. Group A is original melatonin solution (dissolved in 50% anhydrous ethanol), group B is the group of temperature-sensitive melatonin in situ.

Gel according to the present invention, both group A and B contains 3 mg of melatonm and uses subcutaneous injection. Blood will be taken before administration at 8am 0.08h, 0.16h, 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 4h, 6h , 8 h, 9 h, 24 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h and 120 h after the

administration, and a total of 1 ml of blood is collected at each time point from an underwing vein and anticoagulated with sodium heparin, centrifuged at 4000 r/min for 15 minutes, and the upper plasma layer is extracted and made available for HPLC analysis. 3.2.2 Pharmacokinetic analysis

The pharmacokinetic data is processed using the non-atrial model method using the WinNonlm software (5.2). The content of melatonin 1m broiler plasma of groups A and B is measured, and the mass concentration time of the drug in broiler chickens of groups A and B is plotted according to the change in blood concentration over time. Out of

Table 21, it can be seen that the drug increases faster in group À, while the

Drug in group B initially increases slowly after entering the system and then slowly decreases as it reaches the maximum mass concentration.

Table 21 Melatonin drug concentrations in broiler chickens after subcutaneous administration of

Melatonin stock solution and gel (n=10) 'Blood collection time A group(ug/mL) B group(ug/mL) (h) time 08 10234025 OO 0.16 295.36+1.05 0.64+0.02 0.25 431.33+0.18 1.24+0.025 0.5 890.2 2 +0.2 4 84+0.03 0.75 631.47+0.1 8.14+0.03 1 311.79+0.55 15.45+0.37 1.5 190.19+0.4 18.34+0.08 2 120.53+0.55 25.69+0.22 4 20. 62+0.27 45.59+0.24 6 0.77+0.04 55.47+0.25 8 65.34 +0.3 9 133.55+0.22 24 162.5+1.03 48 324.6+1.66 60 381.38+0.51 72 561.66+0.82

24 BE2023/5564

ON

96 232.76+1.59 108 223.37+1.64 120 138.11+1.05

The one-way ANOVA method in SPSS software is also applied to analyze the significance of the data of both groups A (melatonin stock solution) and B (melatonin in situ gel group). The test results are expressed as X+SD. The results are shown in Table 22. Compared with Group A, the half-life of Group B is greater than that of Group A and the Ke is less than that of Group A, indicating that Group A has a rapid

Excretion of the active ingredient and a short duration of action. Group B, on the other hand, has a delayed effect on the release of the active ingredient in vivo. The active ingredient peak concentration represents the maximum value of the blood active ingredient concentration after administration and reflects the

Absorption rate of the drug again, Cmax of group B (561.66+0.82) ug/ml is significantly lower (P<0.05) than that of group A (890.22+0.2) pg/ml, which suggests that the

Absorption rate of melatonin in broiler chickens is slowed down after gel preparation of melatonin. However, the AUC (0-0) (39068.75+1514) h*ug/m L of group B is extremely significantly higher than the AUC (0-0) (890.7+1.02) h*ug/m L of group À (P<0.01) and the Tmax is extremely significantly higher than that of melatonin stock solution (P<0.01), indicating that group B of

is well absorbed in broiler chickens.

Table 22 Pharmacokinetic parameters of melatonin stock solution and gel in the blood of

broiler chickens

Parameter Unit AGroup 1 BGmppe

Tm OSE

Cmax ug/m L 890.22+0.2* 561.66+0.82* ke 1/h 1.18+0.008 0.03+0.001

Tin h 0.58 22.49

AUC (0-00) h*ug/m L 890.7+1.02** 39068.75+1514**

AUMC (0-00) h*ug/m L 987.44+2.91 3017482.9+10305.74

MRI (0-0) h*ug/m L 1.108+0.002** 77.3242.59%*

Vz/F mL/kg 2.84+0.02 2.49+0.09

CL/F mL/h/kg 3.367+0.003 0.07+0.002

Note: * means a significant difference (P < 0.05), ** means a highly significant difference (P < 0.01).

25 BE2023/5564

Experimental Example 4 Test of the antioxidant effect of temperature-sensitive melatonin in situ

Gel 4.1 Grouping of test animals for the antioxidant test: 21 healthy broiler chickens with similar

Weights at 40 days of age will be selected for the test and randomly divided into 3

Divided into groups of 7 chickens each. The experimental chickens are kept and cared for by a dedicated person, fed three times a day at 6:00 a.m., 12:00 p.m. and 6:00 p.m. and have free access

Water. The heat stress conditions are simulated [in the experimental stable the average temperature is 31°C during the day and 24°C at night, the relative humidity is controlled at 55% -75%]. The first experimental group receives melatonin stock solution, the second group receives temperature-sensitive

Melatonin in situ gel melatonin and the third group a blank control (saline solution) over a test period of five days. On the first, third and fifth days, serum is collected and 2 ml of blood is taken from a vein under the wing to check the antioxidants

Determine parameters using an enzyme immunoassay (ELISA). 4.2 Antioxidant properties results on day 1

From Table 23 it can be seen, shows the GSH-Px of the stock solution group and the on the first day

Gel group shows an increasing trend and the MDA content shows a decreasing trend, both groups are highly significantly higher than the control group (P < 0.01), the SOD, T-AOC and CAT activities of the

Stock solution group and gel group are significantly higher than the control group (P<0.05); the

MDA, GSH-Px, SOD and CAT activities were not significantly different between the

Stock solution and the gel group (P>0.05).

Table 23 Determination of antioxidant activity on day 1 'group/index GSH-Px(u/mL) MDA(nmol/mL) SOD(wmL) T-AOC(u/mL) CAT(WmL) 'control gmp = 891254047585 3.0840.03 % 88.1940.50° 12.73+0.15° 10435036 pe

Stock solution 923.83£0.5544 1.85+0.06ÊP 90.70+0.502 13.56+0.362 11.0640.112 sgroup

Gel group 919.63+0.11% 2.09+0.0286 89.52+0.874 13.29+0.192 12.11+0.098

Note: Data in the same column with different lowercase letters indicates significant

differences (P<0.05), different capital letters indicate highly significant differences (P<0.01), and the same letters indicate no significant differences (P>0.05). 4.3 Antioxidant properties results on day 3

It can be seen from Table 24 that the SOD and CAT of the gel group increased on the third day

tend to have and their activities are extremely significantly higher than those of the stock solution group and the

control group (P<0.01); the MDA content of the gel group decreases and is extremely significantly higher than that of the stock solution group and the control group (P<0.01), and the T-AOC and GSH-Px

Activities of the gel group are significantly higher than those of the control group and the

26 BE2023/5564

stock solution group (P<0.05); the differences between the activities of CAT, GSH-Px, MDA,

T-AOC and SOD in the stock solution group and control group are not significant (P>0.05).

Table 24 Determination of antioxidant activity on day 3 “Group/Ind GSH-Px (u/mL) MDA (nmol/mL) SOD (u/mL) T-AOC(wmL) CAT (WmL) ex “Control gra 894.5240.36° 3.234002% 87342026 11.8440.23° 10854022" ppe

Root solution 899.94+0.45b 3.16+0.0242 87.93£0.3044 12.02+0.04" 11.06#0.11Fb

NEsgroup

Gel group 909.13+0.522 1.62+0.04P6 93.70+0.91Pb 13.36+0.282 17.59+0.2644 _ m

Note: Data in the same column with different lowercase letters indicates significant

differences (P<0.05), different capital letters indicate highly significant differences (P<0.01), and the same letters indicate no significant differences (P>0.05). 4.4 Antioxidant properties results on day 5

From Table 25, it can be seen that GSH-Px, T-AOC, CAT and SOD in the gel group show an increasing trend on the fifth day, and their activities are significantly higher than those of the stock solution group and the control group (P<0.05); the MDA content of the gel group decreases and is significantly higher than that of the stock solution group and the control group (P<0.05); the activities of GSH-Px,

MDA, SOD, T-AOC and CAT in the stock solution group and control group have no significant differences (P>0.05).

Table 25 Determination of antioxidant activity on day 5 'Group/inde GSH-Px (u/mL) MDA (nmol/mL) SOD (u/mL) T-AOC (u/mL) CAT (WmL) x “ Control Gu 895.0040. 25° 3.2240.03° 87000265 11.7840.32° 10.8440.23° ppe

Master solution 898.94+0.45b 3.15+0.03P 87.94+0.32P 12.01+0.06» 11,060.11"

BSETUPPC

Gel group 910.23+0.132 2.08+0.042 90.44+0.912 13.05+0.324 12.57+0.152

Note: Data in the same column with different lowercase letters indicates significant

differences (P<0.05), different capital letters indicate highly significant differences (P<0.01), and the same letters indicate no significant differences (P>0.05).

Experimental example 5 Quality testing of a temperature-sensitive melatonin in situ gel

The quality assessment and stability study of the temperature-sensitive melatonin in situ gel is carried out by examining the indicators of the temperature-sensitive melatonin in situ gel,

27 BE2023/5564 including appearance and shape, gelling temperature, gelling time and stability, carried out to ensure the

Control the quality of the gel. 5.1 Morphological study: Comparison of the appearance properties of temperature-sensitive melatonin gels at room temperature and 37°C. The gelling time is determined by adding 5 ml of gel to a brown Celine bottle, the Celme bottle is placed at an angle of 60°, placed in a constant temperature water bath of 37°C and removed after a certain time to observe the morphology . Figure 10 shows that the product is a homogeneous, semi-transparent gel solution with a uniform color and good

Flowability at room temperature and a semi-solid gel in the state (37 ° C). 5.2 Determination of pH: 2 g of the gel are taken from each batch in a beaker and processed according to the method specified in the Chinese Veterinary Pharmacopoeia (2015 edition).

Determination of the pH value. The results show that the pH values are in the range of 7.0-8.0, which corresponds to the norm. 5.3 Centrifugation test: 5 mL of the gel is placed in a 10 mL centrifuge tube and 20

Centrifuged at 4000 rpm for minutes to observe the condition of the gel. The

Melatonin content is determined by high-performance liquid chromatography. As shown in Table 26, no significant changes in drug content and appearance are observed after centrifugation, indicating that the prepared gel has good physical properties

Has stability.

Table 26 Centrifugation test results

Influencing factors Time Shape Delamination Homogeneity Imbalt%

TN

Centrifugation 20min transparent - homogeneous 99.69 transparent - homogeneous 99.68 5.4 High and low temperature tests: The in situ gel is stored in a brown Celine bottle at 40°C for 10

Stored away from light for days, samples are taken for examination on days 5 and 10 and the results are compared with day 0. It is stored at 4°C for 10 days protected from light

Samples are taken for testing on days 5 and 10 and the results are compared with day 0. It can be seen from Table 27 that the temperature-sensitive melatonin gel is stored at 45°C, and although the appearance does not change substantially, the content varies significantly, indicating that it cannot be stored at high temperatures. The temperature-sensitive melatonin gel shows essentially no change in appearance and content when stored at low temperatures, indicating that a

Long-term storage at 4°C is possible.

Table 27 High and low temperature test results

28 BE2023/5564

Factors influencing time fom delamination homogeneity 7

Contents %

TO transparent 99.70 homogeneous 40°C 5 transparent - homogeneous 99.01 10 transparent - homogeneous 98.50 0 transparent - 99.70 homogeneous 4°C 5 transparent - homogeneous 99.65 10 transparent - homogeneous 99.63 5.5 Acceleration test: from the influencing factor test it can be seen that melatonin is sensitive to light, therefore, care should be taken to avoid light during the accelerated test.

Five batches are made of in situ gels and filled into airtight Celine bottles. The temperature-sensitive gels are stored at 20+2°C and protected from light for 3 months. Samples will be taken after months 1, 2 and 3 to determine their appearance and their

examine content. From Table 28, it can be seen that the appearance of the temperature sensitive gel did not change significantly when accelerated for 3 months

conditions at 20+2°C, but the contents decrease in all cases, indicating that the gel is not suitable for storage at room temperature.

Table 28 Results of the acceleration test “Influencing factors Time Shape Delamination Homogeneity 7

Contents %

TE transparent 990 homogeneous 22°C 2 transparent - homogeneous 99.46 3 transparent - homogeneous 98.43 5.6 Long-term stability test: 10 g of temperature-sensitive melatonin in situ gel from 3 batches are removed and stored in brown Celine bottles at 4 + 2°C for several months. At the end of the 0th, 3rd, 6th, 9th and 12th months, samples are taken to determine the gelling temperature and the active ingredient content

Comparison to the gel samples from day 0. From Table 29, it can be seen that there are no significant changes in the appearance, homogeneity and content of the temperature-sensitive gel at 4+2°C, indicating that the temperature-sensitive melatonin

Gel has good stability under these storage conditions.

Table 29 Results of the long-term stability tests on temperature-sensitive melatonin in situ gel “Influencing factors Time Fom Delamination Homogeneity 0

Contents %

29 BE2023/5564 0 transparent 1 homogeneous 99.70 1 transparent - homogeneous 99.60 2 transparent - homogeneous 99.57 3°C 3 transparent - homogeneous 99.55 6 transparent - homogeneous 99.41 9 transparent - homogeneous 99.32 12 transparent - homogeneous 99.10 5.7 Determination of gelling ability in vitro and in vivo: 5.7.1 The in vitro gelling ability is determined by weighing an amount of methyl red dye into a specified amount of the temperature-sensitive gel and an equal amount

Methyl red dye is dissolved in a quantitative anhydrous ethanol solution. 5 ml of each of these two liquids are injected into a certain volume of PBS solution of 37°C and pH 7.4 and observe the gel and stock solution state. 5.7.2 In vivo gelling properties: 1 ml of the gel stored at 4°C is placed in a 10 ml syringe and injected under the skin of the broiler. 6 minutes later, the broiler is anesthetized and the marked area is opened to observe whether the gel has gelled. 5.7.3 Results of the in-vivo and in-vitro gelling ability determination: The gel solution and the aqueous

Solution gelled in vitro, as shown in Figure 11, after being extracted with a syringe and injected through the needle into a PBS buffer solution of 37 ° C and pH 7.4, which is linear and located in a line at the bottom of the bottle collects, indicating that the preparation also gels immediately in the animal and can collect and fixate at the administration site. Furthermore, this confirms

Evaluation indicator the temperature-sensitive properties of the preparation. The gelling state can be observed under the skin of the broiler organism. 5.8 Purity determination: three batches of the temperature-sensitive melatonin in situ gel are prepared and the amount of active ingredient contained in the gels is determined. The test results show that the amount obtained is 99.1%, 98.9% and 99.3% of the marked amount. This shows that the temperature-sensitive melatonin in situ gel prepared with the manufacturing method provided by the present invention is of high purity.

The above content is only preferred embodiment of the present invention. It should be noted that several improvements and modifications can be made by one of ordinary skill in the relevant technical field without departing from the technical principles of the present invention. The improvements and modifications should be considered as of

Scope of protection of the present invention can be considered. The scope of protection of this

However, the invention is not limited thereto, but any equivalent substitution or improvement etc. according to the technical solution of the present invention and its inventive concept, which corresponds to the spirit and principles of the present invention, falls within the scope of the present invention.

Claims (8)

30 BE2023/5564 CLAIMS 1. Temperature-sensitive melatonin 1n-sttu gel, characterized in that it consists of the components with the following weight percentages: Melatonin 0.05% to 10% Poloxam 407 21% to 23% Poloxam 188 1.5% Hydroxypropylmethylcellulose 0.4% up to 0.6% polyethylene glycol 4000 0.3% to 0.5% with the remainder being water for injections. 2. Temperature-sensitive melatonin in situ gel according to claim 1, characterized in that it consists of the components with the following weight percentages: melatonin 0.15% poloxam 407 22% poloxam 188 1.5% hydroxypropylmethylcellulose 0.5% polyethylene glycol 4000 0 .4% with the remainder being water for injection purposes. 3. A method for producing a temperature-sensitive melatonin in situ gel according to claim 1 or 2, characterized by weighing poloxam 407, adding water for injection purposes, dissolving it and then placing it in a refrigerator at 4 ° C overnight; Take out the Poloxam 407 solution, bring it back to room temperature and then add Poloxam 188, polyethylene glycol 4000 and melatonin one by one and stir to dissolve; then adding hydroxypropyl methylcellulose, stirring to dissolve and then adding water for injections to adjust the volume and placing in a refrigerator; In 24 hours, the temperature-sensitive melatonin in situ gel solution is prepared through filtration and decontamination. 4. A method for producing a temperature-sensitive melatonin in situ gel according to claim 3, characterized in that the filtration and sterilization can take place through a 0.22 µm filter membrane. 5. Temperature-sensitive in-sıtu gel, characterized in that it consists of the components with the following weight percentages: Poloxam 407 21% to 23% 31 BE2023/5564 Poloxam 188 1.5% Hydroxypropylmethylcellulose 0.4% to 0.6% Polyethylene glycol 4000 0.3% to 0.5% with the remainder being water for injections. 6. Temperature-sensitive in-sttu gel according to claim 5, characterized in that it consists of the components with the following percentages by weight: Poloxam 407 22% Poloxam 188 1.5% Hydroxypropylmethylcellulose 0.5% Polyethylene glycol 4000 0.4%, the rest being water is for injection purposes. 7. A method for producing a temperature-sensitive in-situ gel according to claim 5 or 6, characterized by weighing Poloxam 407, adding water for injection purposes, dissolving it and then placing it in a refrigerator at 4 ° C overnight; Take out the Poloxam 407 solution, bring it back to room temperature and then add Poloxam 188, polyethylene glycol 4000 and melatonin one by one and stir to dissolve; then adding hydroxypropyl methylcellulose, stirring to dissolve and then adding water for injections to adjust the volume and placing in a refrigerator; The temperature-sensitive in-situ gel is produced in 24 hours through filtration and decontamination. 8. A method for producing a temperature-sensitive in-situ gel according to claim 7, characterized in that the filtration and sterilization can take place through a 0.22 µm filter membrane.