USE OF NOCARDIA RUBRA CELL WALL SKELETON IN TREATMENT OF THERMAL INJURY

Abstract

Provided is the use of a Nocardia rubra cell wall skeleton in the preparation of a drug for treating thermal injury.

Claims

1. Use of Nocardia Rubra cell wall skeleton in the preparation of a medicament, wherein the medicament is for the treatment of thermal injury.

2. The use according to claim 1, wherein: the thermal injury is selected from the group consisting of burn, scald and chemical burn; preferably, the thermal injury involves tissue(s) selected from the group consisting of: epidermis, dermis, mucosa and subcutaneous tissue; preferably, the thermal injury is selected from the group consisting of: first degree, second degree, third degree, and more preferably deep second degree.

3. The use according to claim 1, wherein the medicament is formulated into a dosage form selected from the group consisting of ointment, cream, emulsion, suspension, paste, gel, lotion, tincture, oil, liniment, powder, tablet, suppository, film, patch and dressing.

4. The use according to claim 1, wherein the unit dose of the medicament comprises 1 μg to 1000 μg of Nocardia rubra cell wall skeleton; preferably 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg.

5. The use according to claim 1, wherein the Nocardia rubra cell wall skeleton is a commercial Nocardia rubra cell wall skeleton.

6. The use according to claim 1, wherein the Nocardia rubra cell wall skeleton can be obtained by the following method comprising or consisting of the following steps: 1) providing a Nocardia rubra; 2) disrupting the Nocardia rubra to obtain a disrupted product; 3.1) optionally, removing lipids from the disrupted product; 3.2) optionally, removing nucleic acids from the disrupted product; 3.3) optionally, removing proteins from the disrupted product; 3.4) obtaining a product derived from the Nocardia rubra cell wall; 4) optionally, performing aliquoting; 5) optionally, lyophilizing the product derived from the Nocardia rubra cell wall; wherein, steps 3.1), 3.2) and 3.3) are interchangeable in order or performed in parallel, step 4) and step 5) are interchangeable in order; the average particle size of the disruption is 10 nm to 1000 nm, preferably 10 nm to 800 nm, more preferably 10 nm to 500 nm; preferably, the aliquoting refers to aliquoting into containers; preferably, the container is selected from the group consisting of: vial, tube, package, bag, plate, ampoule, injection device, aluminum-plastic packaging, dressing, capsule and film.

7. A method for the treatment of thermal injury, including the following steps: making a subject in contact with a therapeutically effective amount of Nocardia Rubra cell wall skeleton; the Nocardia rubra cell wall skeleton is formulated into a form selected from the group consisting of: ointment, cream, emulsion, suspension, paste, gel, lotion, tincture, oil, liniment, powder, tablet, suppository, film, patch and dressing; the contact is performed by administering twice a day, or once a day, or once every two days, or once every three days, or once a week; the contact lasts for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks or longer; the thermal injury is selected from the group consisting of burn, scald and chemical burn; preferably, the thermal injury involves tissue(s) selected from the group consisting of: epidermis, dermis, mucosa and subcutaneous tissue; preferably, the thermal injury is selected from the group consisting of: first degree, second degree, third degree; and most preferably, deep second degree.

8. The method according to claim 7, wherein the Nocardia rubra cell wall skeleton is a commercial Nocardia rubra cell wall skeleton.

9. The method according to claim 7, wherein the Nocardia rubra cell wall skeleton is obtained by the following method comprising or consisting of the following steps: 1) providing Nocardia rubra; 2) disrupting the Nocardia rubra to obtain a disrupted product; 3.1) optionally, removing lipids from the disrupted product; 3.2) optionally, removing nucleic acids from the disrupted product; 3.3) optionally, removing proteins from the disrupted product; 3.4) obtaining a product derived from the Nocardia rubra cell wall; 4) optionally, performing aliquoting; 5) optionally, lyophilizing the product derived from the Nocardia rubra cell wall; wherein, steps 3.1), 3.2) and 3.3) are interchangeable in order or performed in parallel, step 4) and step 5) are interchangeable in order; the average particle size of the disruption is 10 nm to 1000 nm, preferably 10 nm to 800 nm, more preferably 10 nm to 500 nm; preferably, the aliquoting refers to aliquoting into containers; preferably, the container is selected from the group consisting of: vial, tube, package, bag, plate, ampoule, injection device, aluminum-plastic packaging, dressing, capsule and film.

Description

DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1A to FIG. 1D: Healing of deep second degree scald wounds in mice: wound on Day 21 in the broken wound treatment group (FIG. 1A); wound on Day 21 in the positive group (FIG. 1B); wound on Day 21 in the unbroken wound treatment group (FIG. 1C); wound on Day 21 in is the model group (FIG. 1D).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0063] Nocardia Rubra Cell Wall

[0064] In the present disclosure, the “Nocardia Rubra cell wall” can be interpreted as either a complete cell wall or an incomplete cell wall (for example, disrupted or partially degraded). Under the teaching of the present disclosure, the skilled person will understand that the components exhibiting the desired activity are derived from Nocardia Rubra cell wall (for example, the cell wall itself or components thereof). Therefore, complete cell wall, disrupted cell wall, incomplete degradation product of cell wall, cell wall components, cell wall extracts and other various forms are allowed to be used in clinical applications, which are all encompassed in the scope of the present disclosure.

[0065] Cell Wall Skeleton

[0066] A component that constitutes the main structure of the cell wall; however, it cannot be interpreted as merely representing the cross-linked network-like entity in the cell wall, and the skilled person understands that other cell wall components adsorbed by, bound to and carried by the cross-linked network-like entity are not excluded.

[0067] Unit Dose

[0068] The medicament or the medical device of the present disclosure can be prepared in the form of a unit dose (formulation unit).

[0069] “Optionally” means that the event following this term can happen, but not necessarily happen; it depends on the situation. For example, “optionally, performing aliquoting” means that the product is allowed to be aliquoted, but is not necessary to be aliquoted; whether the product is aliquoted or not does not affect the realization of the technical effects.

[0070] “A”, “an”, “single” and “the”, if not explicitly stated, also involve plural forms.

[0071] The present disclosure is further described below with reference to the examples. However, these examples do not limit the scope of the present disclosure. When the particular conditions are not specified, operation should be performed in accordance with the normal conditions and the conditions recommended by the raw material supplier. The reagents without giving specific sources are conventional reagents purchased on the market.

[0072] The skilled person especially understands that although a particular commercial cell wall product is used in the following particular examples, the realization of the technical effects is not limited to the particular commercial cell wall product, and any species classified as the Nocardia Rubra species is applicable.

EXAMPLES

Example 1. Commercial Nocardia Rubra Cell Wall Skeleton

[0073] The Nocardia rubra cell wall skeleton (trade name: NAKEJIA) was purchased from Liaoning Greatest Bio-pharmaceutical Co., Ltd., with the medicine permission No. 520030009 (2 ml/vial; lyophilized powder), which comprised 60 μg of active ingredients and 15 mg of dextran 40.

Example 2. Preparation of Nocardia Rubra Cell Wall Skeleton

[0074] 1. The bacteria were cultured by well-known methods and collected. The cells were disrupted (for example, but not limited to by sonication). Any appropriate well-known method in the art was also allowed to be applied in the disruption of the bacteria, for example CN101250490A or CN101323865A. The disruption state was checked under a microscope. There should be no more than 5 intact bacteria in each visual field. The disruption was considered as qualified when several (10 to 30) visual fields checked met this standard.

[0075] 2. Removal of nucleic acids: the supernatant after disruption was centrifuged. DNase and RNase were added to the obtained precipitate, and nucleic acids were removed according to the operation recommended by the supplier of the enzymes.

[0076] 3. Removal of proteins: commonly used protease (such as trypsin) was added to the precipitate, and proteins were removed according to the operation recommended by the supplier of the enzyme.

[0077] 4. Removal of lipids: an organic reagent (for example, but not limited to one or a combination of acetone, ether and ethanol) was added to the precipitate, and lipids were removed according to conventional operations in the art.

[0078] 5. Removal of cell membranes: Triton X-100 was added to the precipitate, and the precipitate was collected by centrifugation according to conventional operations in the art, and rinsed with PBS.

[0079] It should be understood that among the above steps for removing impurities, those skilled in the art can adjust the order of the steps to make them compatible with each other. After removing the non-cell wall components, the precipitate was re-dissolved in water for injection, and then kept for later use. Optionally, it could be sterilized at 115° C. for 20-30 minutes as the stock solution of the cell wall skeleton (mainly comprising the cell wall skeleton and components thereof).

Example 3. Preparation of the Pharmaceutical Composition or Medical Device

[0080] 1. The product obtained in Example 2 (active ingredient 60 μg to 120 μg, for example 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg) or the commercial product of Example 1 was coated on dressings (for example sterile gauzes) to prepare a medical device for external use.

[0081] 2. Or, the product obtained in Example 2 (active ingredient 60 μg) was formulated into a lyophilized powder and applied directly on the surface of the lesion.

[0082] 3. Or, a method for the preparation of lotions known in the art can also be applied, for example:

[0083] Water and ethanol are mostly used as the dispersion medium in lotion; it is prepared from active ingredients, electrolytes, isotonicity regulators, etc. in the dispersion medium. During storage of emulsion type of lotions, the oil phase and the aqueous phase may separate, but they can be re-dispersed after shaking.

[0084] Test Example. The Therapeutic Effect on Thermal Injury

[0085] 1. Materials

[0086] 1.1 Drugs and Main Reagents [0087] Test drug: Nocardia rubra cell wall skeleton for external use (Liaoning Greatest Bio-pharmaceutical Co., Ltd., medicine permission No. 520030009, specification 60 μg/vial, batch number 201809004); [0088] Control drug: Ching Wan Hung ointment (Tianjin Darentang Jingwanhong Pharmaceutical Co., Ltd., medicine permission No. Z20023137, specification 30 g/tube, batch number 215054);

[0089] Veet Hair Removal Cream (Reckitt Benckiser (China) Co., Ltd.);

[0090] Ether (Sinopharm Chemical Reagent Co., Ltd.).

[0091] 1.2 Experimental animals

[0092] 20 clean-grade Kunming mice, weighing 26 g to 35 g, were provided by Liaoning Changsheng Biotechnology Co., Ltd., with certificate number 211002300051326.

[0093] 2. Experimental Methods

[0094] 2.1 Experimental Design

[0095] 20 Kunming mice were randomly divided into 4 groups, with 5 in each group:

[0096] 1) Model group;

[0097] 2) Unbroken wound treatment group (Nocardia rubra group);

[0098] 3) Broken wound treatment group (Nocardia rubra group);

[0099] 4) Positive control group (control drug).

[0100] 2.2 Preparation of Second Degree Scald Mouse Model

[0101] The mice were adapted to the environment and kept for one week. After shaving the skin on the back of mice with a shaver, a depilatory cream was used to remove the hair (in an area of about 3 cm×4 cm) and let stand for 180 s. The depilated area was washed with warm water at 30° C. to 40° C. and wiped dry with degreasing cotton.

[0102] After depilation, the mice were kept normally for 24 h and observed, confirming that there were no abnormal conditions such as redness, inflammation and broken skin at the depilated site. After 24 h, the mice were anesthetized with ether, placed on an operating table, the skin in the experimental area was disinfected with 75% ethanol, and the scald model was constructed.

[0103] The mice were placed on the device for scalds with a 1.2 cm hole in the middle, making the depilated skin on the back be at the place of the hole. The 1.2 cm hole with the back exposed was kept in water at a constant temperature of 75° C. for 10 s, thus making a round-shaped scald wound with a diameter of about 1.2 cm.

[0104] The injured mice were kept in separate cages, the wounds were treated with saline, feed and distilled water were given, and the bedding was ensured to be dry and clean, with good ventilation. According to the criteria of three degrees with four levels, the burn/scald was identified as deep second grade.

[0105] 2.3 Grouping and Administration

[0106] Administration started 24 h after the scald: [0107] Model group: physiological saline was applied to the scald wound on the skin of each mouse every day; [0108] Unbroken wound treatment group: the wound was first treated with physiological saline, and then 1 vial of Nocardia rubra cell wall skeleton was externally applied to every animal every day (0.25 ml of physiological saline was injected into the vial to completely dissolve the lyophilized powder, which was used to completely impregnate 3 layers of 1.2 cm×1.2 cm gauze for application on the wound, and then fixed with medical desensitization tape); [0109] Broken wound treatment group: the wound was first treated with physiological saline and made broken, and then 1 vial of Nocardia rubra cell wall skeleton was externally applied to every animal every day (0.25 ml of physiological saline was injected into the vial to completely dissolve the lyophilized powder, which was used to completely impregnate 3 layers of 1.2 cm×1.2 cm gauze for application on the wound, and then fixed with medical desensitization tape); [0110] Positive control group: the wound was first treated with physiological saline, and then Ching Wan Hung ointment was externally applied to every animal once every day.

[0111] The wound healing was observed on Day 1, 3, 7, 11 and 15 of the administration.

[0112] 3. Experimental Observation and Index Detection

[0113] 3.1 Determination of Wound Healing Time

[0114] For each group, the healing time was determined. The determination criteria include: [0115] Healed: the scabs completely fell off at the scald site, and the surface of the repaired tissue was fresh and relatively flat; [0116] Basically healed: the scabs intermittently fell off at the scald site, the is surface of the newly grown tissue was not very flat, with a small amount of exudate in a small area but no obvious infection focus; [0117] Infected: obvious redness and swelling appeared around the scabs, with pus or ulcer under the scab.

[0118] 3.2 Wound Healing Rate

[0119] The wound healing of mice was observed and recorded on Day 3, 7, 11 and 15 of the administration. Complete epithelialization and no exudation of the wound were regarded as complete healing of the wound. At the same time, the wound was observed for redness, swelling, infection, etc. The wound healing rate at each time point was calculated: the diameter of the wound was obtained according to the scale in the photo and by graphic processing softwares, and the wound area was calculated.

Wound healing rate %=(original wound area−unhealed wound area)/original wound area×100%.

[0120] 3.3 Statistical Methods

[0121] SPSS 17.0 statistical software was used for analysis, and one-way

[0122] ANOVA was used for comparison between groups. The data obtained was represented as X±s. P<0.05 was considered statistically significant.

[0123] 4. Experimental Results

[0124] 4.1 General Observations

[0125] All mice survived until the wounds healed, and there were no obvious differences in eating, drinking, mental state, activity, etc., and no obvious signs of infection.

[0126] 4.2 Wound Healing

[0127] 4.2.1 Wound Healing Time

[0128] Complete healing of the wounds and a relatively flat tissue surface of mice were considered as the standard for healing. Compared with the model group, the wound healing time of the broken wound treatment group and the positive group was shortened, and the difference was statistically significant P<0.05. Compared with the positive group, the healing time of the broken wound treatment group was slightly earlier than that of the positive group, and the difference was statistically significant P<0.05.

TABLE-US-00001 TABLE 1 Wound healing time of deep second degree scald in mice (x ± s) Group n Wound healing time/d Model group 5 22.4 ± 0.3  Unbroken wound treatment group 5 21.6 ± 0.3  Broken wound treatment group 5 16.2 ± 0.2* Positive group 5 17.6 ± 0.3* Note: compared with model group, *P < 0.05.

[0129] 4.2.2 Wound Healing Rate

[0130] Administration started 24 h after the model was established. The wound area before administration was set as the reference area. The wound area on Day 3, 7, 11 and 15 was recorded, and the healing rate was calculated.

[0131] Compared with the model group, the wound healing rate of the broken wound treatment group started to be significantly higher than that of the model group at Day 7 of administration, and the healing rate of the positive group and the unbroken wound treatment group also started to be higher than that of the model group at Day 11 of administration, with statistical significance P<0.05. Compared with the positive group, the wound healing rate of the broken wound treatment group started to be higher than that of the positive group at Day 7 of administration, and with statistical significance P<0.05.

TABLE-US-00002 TABLE 2 Wound healing rate of deep second degree scald in mice (x ± s) Healing rate (%) Group n 3 d 7 d 11 d 15 d Model group 5 13.14 ± 1.89 32.36 ± 6.98  58.47 ± 8.19  82.10 ± 6.47  Unbroken wound treatment group 5 11.97 ± 6.17 32.20 ± 13.92 66.33 ± 4.33* 89.03 ± 1.55* Broken wound treatment group 5 14.02 ± 4.57 38.80 ± 2.93* 76.44 ± 4.06* 97.83 ± 0.17* Positive group 5 12.50 ± 3.69 32.11 ± 6.30   67.66 ± 14.75* 91.89 ± 1.96* Note: compared with model group, *P < 0.05.

[0132] 4.2.3 Wound Healing State (FIG. 1A to FIG. 1D)

[0133] When the broken wound treatment group healed, the wound had no scar hyperplasia and was smooth, while other groups showed different degrees of scar hyperplasia; meanwhile, the wound site of the broken wound treatment group started to grow hair.

[0134] Not limited to particular theories, the main components of the Nocardia rubra cell wall skeleton are generally believed to include: muramic acid, arabinogalactan, mucopeptide, etc., which have a regulatory effect on the body's immune system and can enhance the activity of T cells, macrophages and natural killer cells, promote the production of cytokines, is and have a positive effect on the early stage of wound healing and reduction of inflammation.

[0135] In the broken wound treatment group of the present application, there was basically no scar after wound healing, while other groups all had different degrees of scars. Meanwhile, the wounds of the broken wound treatment group had grown a lot of hair on Day 21, demonstrating that the composition of the present application had an extremely strong repairing effect on hair follicles and other appendages of the skin while repairing the wounds.

[0136] In summary, during the healing process of deep second degree scalds in mice, the Nocardia rubra cell wall skeleton can effectively improve the healing rate, shorten the healing time, reduce the formation of scars after wound healing, while having a significant repairing effect on hair follicles and other appendages of the skin.