Application of recombinant III-type humanized collagen in injection

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of recombinant III-type humanized collagen in an injection. The invention provides an injection, the injection comprises recombinant III-type humanized collagen, the amino acid sequence of the recombinant III-type humanized collagen comprises n repetitions of the sequence as shown in SEQ ID NO.1, n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repetitions are directly connected; moreover, when the injection is applied in one application period, the total application amount of the recombinant III-type humanized collagen is less than or equal to 3640 mg / kg. The recombinant III-type humanized collagen has high-dose injection safety, and can be applied in a single high-dose manner or continuously applied to realize high-dose application in a total amount level.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of recombinant humanized type III collagen in injections. Background Art

[0002] Collagen is the most abundant protein in the human body, accounting for 25%-30% of total protein. Type III collagen is widely distributed in blood vessels, new skin, and scar tissue, and has important biological functions. Synthetic biology and genetic engineering techniques enable the large-scale production of recombinant humanized collagen. Furthermore, by optimizing the amino acid sequence of collagen, its hydrophilicity, stability, and tissue compatibility can be improved. Recombinant humanized collagen has been widely used in the pharmaceutical field.

[0003] However, although recombinant humanized collagen is similar to the body's own natural collagen in structure and function, its amino acid sequence is not a full-length human amino acid sequence and may have undergone sequence modifications and artificially designed mutations. When applied to the human body, the human immune system may recognize it as a foreign substance, thereby generating an immune response, leading to local or systemic allergic reactions such as redness, swelling, pain, itching, and rash. In severe cases, breathing difficulties and anaphylactic shock may occur. At the same time, the production process of recombinant humanized collagen is complex, involving various raw materials and equipment. During the microbial fermentation and production process, there may also be the risk of contamination by microorganisms such as viruses and bacteria, as well as other impurities. These factors will lead to a decrease in the purity of the final recombinant humanized collagen product, making it extremely easy to cause infection and pathogenicity when applied to the human body. Therefore, there are still many unknown risks in the process of in vivo administration of recombinant humanized collagen, such as when injected in large doses.

[0004] Regarding large-dose injections, especially intravenous injections, large amounts of protein substances may cause vascular embolism. This can lead to local tissue ischemia and hypoxia, resulting in serious consequences such as pain and skin necrosis. If the embolism occurs in the blood vessels of vital organs such as the eyes or brain, it may also cause life-threatening complications such as blindness and cerebral infarction. Furthermore, the long-term safety of injectable substances requires research, and currently, long-term safety studies on large-dose injections of recombinant humanized collagen are relatively limited. Furthermore, in the long term, large-dose injections may have chronic effects on the structure and function of local tissues, such as tissue fibrosis and decreased elasticity. Especially when administered intravenously, they may have even more serious long-term harms to overall health, such as affecting the normal function of the immune system and increasing the risk of certain diseases.

[0005] Currently, there are some approved injectables for recombinant humanized collagen, such as:

[0006] Medical device registration number 20253130751 is a recombinant humanized type III collagen gel for injection. It consists of recombinant humanized type III collagen and 0.9% saline, with a collagen concentration of 16.00 mg / ml, supplied in a sterile, prefilled syringe. The product is indicated for injection into the midface, subcutaneously to the supraperiosteal layer, to correct midfacial volume loss and / or midfacial contour defects.

[0007] National Medical Device Registration No. 20233131245 is a recombinant type III humanized collagen solution for injection, 2mg / ml, specifications: 0.5ml / vial, 1.0ml / vial, 1.5ml / vial, 2.0ml / vial, 2.5ml / vial, 3.0ml / vial, 3.5ml / vial, 4.0ml / vial, 4.5ml / vial, 5.0ml / vial, 6.0ml / vial. The product is a colorless or off-white liquid, composed of recombinant type III humanized collagen (type A) and 0.9% normal saline, with a collagen concentration of 2mg / ml. The product is pre-filled in a sterile pre-filled syringe, the product is filtered and sterilized and produced by an aseptic process for single use. The product is used for facial dermal tissue filling to correct dynamic wrinkles on the forehead (including frown lines, forehead lines and crow's feet).

[0008] National Medical Device Registration No. 20213130488, this product is freeze-dried recombinant humanized type III collagen fibers. Available in 2 mg / vial, 4 mg / vial, 6 mg / vial, 8 mg / vial, 10 mg / vial, 12 mg / vial, 16 mg / vial, 20 mg / vial, and 24 mg / vial sizes. This product is a white, spongy solid composed of recombinant humanized type III collagen. It is packaged in single-use glass bottles and manufactured using a sterile process. It is used for facial dermal tissue augmentation to correct dynamic wrinkles on the forehead (including glabellar lines, forehead lines, and crow's feet).

[0009] Among the above-mentioned approved Class III medical devices related to recombinant humanized type III collagen, the collagen concentration is mostly 16.00 mg / ml or 2 mg / ml, which is a relatively low concentration. It is mostly used for facial muscle injection or dermal tissue filling, and the dosage is small. There are currently no related products and dosage references for systemic intravenous injection.

[0010] For other protein or high molecular weight injections, the existing ones include immunoglobulin injections, hyaluronic acid (sodium) injections, etc. Human hemoglobulin (also known as immunoglobulin) is a class of antibody-containing proteins extracted from healthy human plasma. It is an important class of immune active substances in human plasma. The main component is IgG antibodies (accounting for more than 80%), which contain antibodies against a variety of pathogens (such as viruses and bacteria). These antibodies bind to pathogens to prevent them from invading cells or activate the immune system to clear infections. It is mainly used to enhance immunity, treat immunodeficiency or infectious diseases. It works by neutralizing pathogens and regulating immune responses. Commonly used for the elderly, children or frail people, human hemoglobulin is injected by intravenous drip, which can significantly enhance immunity, supplement nutrition, promote wound healing, improve anemia symptoms and maintain plasma colloidal osmotic pressure. Sodium hyaluronate (also known as sodium hyaluronate) is a physiologically active substance widely present in the human body that promotes wound healing. It is a high-molecular-weight, linear mucopolysaccharide composed of disaccharide units composed of glucuronic acid and acetylhexosamine. It forms a thick, viscous, elastic solution in water with physiological pH and ionic strength. Sodium hyaluronate is a non-pharmaceutical, neutral medium. Sodium hyaluronate injection is a colorless, clear, viscous liquid. Due to its variable molecular form, it can be injected with a thinner needle.

[0011] At present, there are many approved human immunoglobulin injections, such as National Medicine Approval No. S20170002, National Medicine Approval No. S19993017, National Medicine Approval No. S20053060, National Medicine Approval No. S19993012, National Medicine Approval No. S20083099 and National Medicine Approval No. S19990002, with various specifications, including 300mg / bottle (10%, 3.0ml), 300mg / syringe (10%, 3.0ml), 10% (0.15g / bottle), 50g / L (1.25g), 2.5g / bottle (5%, 50ml), 10g / bottle (5%, 200ml), 2.5g / bottle, etc.

[0012] For sodium hyaluronate injection, the currently approved ones include, for example, National Medical Device Injection No. 20163131492, National Medical Device Injection No. 20203130096 and National Medical Device Injection No. 20243131359, with various specifications, including 20 mg / mL (0.25 mL, 0.5 mL, 0.6 mL, 0.75 mL, 0.9 mL, 1 mL, 1.1 mL, 1.25 mL, 1.4 mL, 1.5 mL), 24 mg / mL (0.5 mL / vial, 1.0 mL / vial, 2.0 mL / vial, 3.0 mL / vial), 12 mg / mL (0.5 mL / vial, 0.75 mL / vial, 1.0 mL / vial, 1.5 mL / vial, 2.0 mL / vial, 2.5 mL / vial, 5.0 mL / vial), etc.

[0013] In addition, US20250122270A1 discloses a method for intradermal administration of immunoglobulin G (IgG) preparations, wherein the IgG preparation has an IgG concentration of 15%-30% (w / v), and a maximum of 10 mL is administered to each skin site. CN102229705A discloses a collagen thermosensitive hydrogel preparation, wherein 0.5wt%-2.0wt% sodium hyaluronate solution, 1.0wt%-5.0wt% collagen solution and 20wt%-50wt% sodium β-glycerophosphate solution are mixed in a volume ratio of (1-5): (1-3): (1-2). CN103333349A discloses a hyaluronic acid-collagen composite hydrogel for injection, wherein the mass concentration of sodium hyaluronate is 6%-20%, and the mass concentration of collagen is 1%-6%.

[0014] From the above content, it can be seen that the concentration of commonly used hyaluronic acid injections with repair functions is about 10-20 mg / mL, and the mass fraction of its pharmaceutical salt in the preparation is below about 20wt%. It is often used for intradermal dermal injection filling in parts of the human neck, face, nose and lips, etc., and has not been used for large-dose intravenous injection. Summary of the Invention

[0015] Problems to be solved by the invention

[0016] Recombinant humanized collagen has a wide range of applications, but because it is not naturally present in the human body, it can still trigger an immune response when administered internally. Furthermore, the preparation process for recombinant humanized collagen is complex, posing the risk of infection from microorganisms and other sources. Therefore, currently, there are only a few recombinant humanized collagen injections available, and most are for topical administration with limited dosages. The development and research of recombinant humanized collagen products that can be administered systemically at high doses remains to be seen.

[0017] In this regard, the present inventors have conducted extensive research and found that the recombinant humanized type III collagen disclosed in ZL201811438582.6 is safe for large-dose injection and can be administered in a single large dose or continuously to achieve large-dose administration at the total amount level.

[0018] Based on this, the purpose of the present invention is to provide the use of the recombinant humanized type III collagen in injections, including corresponding injections and pharmaceutical uses.

[0019] Solutions for solving problems

[0020] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0021] [1]. An injection,

[0022] The injection comprises recombinant humanized type III collagen, wherein the amino acid sequence of the recombinant humanized type III collagen comprises n repeats of the sequence shown in SEQ ID NO. 1, where n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeats are directly connected; and when the injection is administered within one administration cycle, the total administration amount of the recombinant humanized type III collagen is less than or equal to 3640 mg / kg based on the body weight of the administered subject;

[0023] Preferably, when the subject is a human, the single administration amount of the recombinant humanized type III collagen is less than or equal to 590 mg / kg based on the subject's body weight;

[0024] Preferably, the amino acid sequence of the recombinant humanized type III collagen comprises the amino acid sequence shown in SEQ ID NO.2.

[0025] [2] The injection according to [1], wherein

[0026] The injection is an injection for single-dose administration;

[0027] Preferably, in the single-dose injection, when the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 90-110 mg / mL;

[0028] Preferably, when the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 2000 mg / kg based on the body weight of the subject;

[0029] More preferably, when the subject of the single-dose injection is a human, and the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 162 mg / kg based on the body weight of the subject.

[0030] [3] The injection according to [1], wherein

[0031] The injection is an injection for multiple doses;

[0032] Preferably, in the multi-dose injection, when the multi-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 6-29 mg / mL;

[0033] Preferably, when the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 130 mg / kg based on the body weight of the subject;

[0034] More preferably, when the subject of the multi-dose injection is a human, and the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 21 mg / kg based on the body weight of the subject;

[0035] Preferably, when the injection is administered in multiple doses, the administration frequency is once a day and the administration cycle is 1 to 5 weeks.

[0036] [4] The injection according to any one of [1] to [3], wherein

[0037] The injection is an intravenous injection, a subcutaneous injection, an intramuscular injection or an intraperitoneal injection;

[0038] Preferably, the injection is an intravenous injection or a subcutaneous injection.

[0039] [5] The injection according to any one of [1] to [4], wherein

[0040] The injection has no cytotoxicity, organ toxicity, blood toxicity and immunotoxicity.

[0041] [6]. Use of recombinant humanized type III collagen in the preparation of injections,

[0042] The injection comprises recombinant humanized type III collagen, wherein the amino acid sequence of the recombinant humanized type III collagen comprises n repeats of the sequence shown in SEQ ID NO. 1, where n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeats are directly connected; and when the injection is administered within one administration cycle, the total administration amount of the recombinant humanized type III collagen is less than or equal to 3640 mg / kg based on the body weight of the administered subject;

[0043] Preferably, the amino acid sequence of the recombinant humanized type III collagen comprises the amino acid sequence shown in SEQ ID NO.2;

[0044] Preferably, the injection is used for any one of the following uses (a)-(e):

[0045] (a) preventing and / or treating ocular inflammation;

[0046] (b) preventing and / or treating gastric cancer;

[0047] (c) prevention and / or treatment of breast diseases;

[0048] (d) prevention and / or treatment of ovarian diseases;

[0049] (e) preventing and / or inhibiting and / or treating cervical cancer;

[0050] (f) prevention and / or treatment of cardiovascular disease, repair of vascular damage, or prevention and / or treatment of vascular damage;

[0051] (g) Inhibit esophageal squamous cell carcinoma.

[0052] [7] The use according to [6], wherein

[0053] The injection is an injection for single-dose administration;

[0054] Preferably, in the single-dose injection, when the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 90-110 mg / mL;

[0055] Preferably, when the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 2000 mg / kg based on the body weight of the subject;

[0056] More preferably, when the subject of the single-dose injection is a human, and the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 162 mg / kg based on the body weight of the subject.

[0057] [8] The use according to [6] or [7], wherein

[0058] The injection is an injection for multiple doses;

[0059] Preferably, in the multi-dose injection, when the multi-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 6-29 mg / mL;

[0060] Preferably, when the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 130 mg / kg based on the body weight of the subject;

[0061] More preferably, when the subject of the multi-dose injection is a human, and the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 21 mg / kg based on the body weight of the subject;

[0062] Preferably, when the injection is administered in multiple doses, the administration frequency is once a day and the administration cycle is 1 to 5 weeks.

[0063] [9]. The use according to any one of [6] to [8], wherein

[0064] The injection is an intravenous injection, a subcutaneous injection, an intramuscular injection or an intraperitoneal injection;

[0065] Preferably, the injection is an intravenous injection or a subcutaneous injection.

[0066]

[10] . The use according to any one of [6] to [9], wherein

[0067] The injection has no cytotoxicity, organ toxicity, blood toxicity and immunotoxicity.

[0068]

[11] . A medicine box,

[0069] The medicine kit comprises a syringe pre-filled with the injection according to any one of [1] to [5].

[0070]

[12] The medicine box according to

[11] , wherein

[0071] The kit satisfies the following conditions (i) or (ii):

[0072] (i) When the injection is a single-dose injection, the medicine kit contains only one syringe I prefilled with the single-dose injection, and the total amount of the recombinant humanized type III collagen contained in the medicine kit is less than or equal to 2000 mg / kg based on the body weight of the subject; preferably, when the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen in each syringe I is 90-100 mg / mL;

[0073] (ii) When the injection is a multi-dose injection, the medicine kit contains multiple syringes II of the same specifications pre-filled with the multi-dose injection, and the total amount of the recombinant humanized type III collagen contained in the medicine kit is less than or equal to 3640 mg / kg based on the body weight of the subject, and the amount of the recombinant humanized type III collagen contained in each syringe II is less than or equal to 130 mg / kg; preferably, when the multi-dose injection is a liquid, the concentration of the recombinant humanized type III collagen in each syringe II is 6-29 mg / mL;

[0074] Preferably, when the subject of administration of the drug kit is a human, the drug kit satisfies the following conditions (iii) or (iv):

[0075] (iii) When the injection is a single-dose injection, the medicine kit contains only one syringe I prefilled with the single-dose injection, and the total amount of the recombinant type III humanized collagen contained in the medicine kit is less than or equal to 162 mg / kg based on the body weight of the subject; preferably, when the single-dose injection is a liquid, the concentration of the recombinant type III humanized collagen in each syringe I is less than or equal to 30 mg / mL; preferably, when the single-dose injection is a lyophilized preparation, the content of the recombinant type III humanized collagen in each syringe I is 9720 mg based on the body weight of the subject being 60 kg / person;

[0076] (iv) When the injection is a multi-dose injection, the medicine kit contains a plurality of syringes II of the same specifications pre-filled with the multi-dose injection, and the total amount of the recombinant type III humanized collagen contained in the medicine kit is less than or equal to 590 mg / kg, and the amount of the recombinant type III humanized collagen contained in each syringe II is less than or equal to 21 mg / kg, based on the body weight of the subject. Preferably, when the multi-dose injection is a liquid, the concentration of the recombinant type III humanized collagen in each syringe II is 6-29 mg / mL. Preferably, when the multi-dose injection is a lyophilized preparation, based on the body weight of the subject being 60 kg / person, the content of the recombinant type III humanized collagen in each syringe II is 1260 mg.

[0077] More preferably, when the medicine kit meets condition (ii) or (iv), the medicine kit contains 7-35 syringes II; even more preferably, when the medicine kit meets condition (ii) or (iv), the medicine kit contains 7, 14, 21 or 28 syringes II.

[0078]

[13] The kit according to

[11] or

[12] , wherein:

[0079] The kit is for a single administration cycle.

[0080] Effects of the Invention

[0081] Through the implementation of the above technical solution, the present invention has achieved at least the following beneficial effects:

[0082] The recombinant humanized type III collagen used in the present invention has excellent hydrophilicity and stability, and its amino acid composition is 100% identical to the corresponding part of the natural collagen amino acid sequence. In addition, its production and preparation process is carried out in a strictly controlled bioreactor, and the cell lines and raw materials used are strictly screened and tested. The collagen product is highly pure when leaving the factory, and is non-toxic and non-immunogenic, and can be used for the production and use of injections.

[0083] The injection containing recombinant humanized type III collagen provided by the present invention can be used for single-dose administration or multiple-dose administration, and in both administration methods, the recombinant humanized type III collagen is injection safe and does not produce cytotoxicity, organ toxicity, blood toxicity and immunotoxicity during injection. The experimental data showed that in these two administration methods, recombinant type III humanized collagen had a significant effect on the behavior, appearance, pathological anatomy, organ examination, weight gain, food intake, coagulation indicators (including plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT)), hematological indicators (including white blood cell count (WBC), red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT), mean platelet volume (MPV), hemoglobin (HB), neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO), basophils (BA), R There were no adverse effects on serum biochemical parameters (including albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), calcium (Ca), creatine kinase (CK), creatinine (CR), glucose (GLU), lactate dehydrogenase (LDH), total bilirubin (TBIL), serum total cholesterol (TCHO), triglycerides (TG), serum total protein (TP), blood uric acid (UA), blood urea nitrogen (UREA)), urine parameters (including urobilinogen (UBG), bilirubin (BIL), ketone bodies (KET), occult blood (BLD), protein (PRO), nitrite (NIT), white blood cells (LEU), glucose (GLU), urine specific gravity (SG), pH value, color, turbidity), serum electrolyte levels (including chloride ion, sodium ion, potassium ion), organ weights and organ coefficients. At the same time, experimental data showed that when administered as a single dose, the maximum tolerated dose of recombinant humanized type III collagen injected into the tail vein of ICR mice was greater than 2000 mg / kg; when administered in multiple doses, administered once a day for 4 weeks, the single dose with essentially no toxic reactions in SD rats was 130 mg / kg. By verifying the safety of recombinant humanized type III collagen when injected in large doses (including safety when administered as a single large dose and safety when administered in large doses at the total amount through continuous administration), a theoretical basis is provided for the development and utilization of injectables, and a feasibility and safety basis is laid for the delivery of drugs to tissues or organs throughout the body.

[0084] The recombinant humanized type III collagen used in the present invention can improve skin tissue structure, promote skin and vascular damage repair, or prevent and / or treat eye inflammation, gastric cancer, breast disease, ovarian disease, cervical cancer, or esophageal squamous cell carcinoma. The injection containing recombinant humanized type III collagen provided by the present invention can safely and efficiently deliver recombinant humanized type III collagen in large doses, thereby promoting terminal tissue repair after vascular and venous delivery. This solves the problem of protein decomposition or loss during delivery of low-dose recombinant humanized type III collagen, which can prevent it from reaching the designated terminal tissue site. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 : Body weight changes of ICR mice after single-dose toxicity test of recombinant humanized type III collagen injected into tail vein.

[0086] Figure 2 : Changes in body weight of female SD rats after repeated toxicity test of recombinant humanized type III collagen injected into tail vein for 4 weeks.

[0087] Figure 3 : Changes in body weight of male SD rats after repeated toxicity test of recombinant humanized type III collagen injected into tail vein for 4 weeks.

[0088] Figure 4 : Changes in feed consumption in female SD rats after repeated toxicity test of recombinant humanized type III collagen injected into tail vein for 4 weeks.

[0089] Figure 5 : Changes in feed consumption in male SD rats after repeated toxicity test of recombinant humanized type III collagen injected into tail vein for 4 weeks. DETAILED DESCRIPTION

[0090] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the invention. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0091] In the present invention, the terms "comprising," "having," "including," or "containing" may be inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprising," "having," "including," or "containing" may also be closed-ended, excluding additional, unrecited elements or method steps.

[0092] In the present invention, the use of "may" includes both the meanings of performing a certain process and not performing a certain process.

[0093] In the present invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0094] In the present invention, numerical ranges expressed using “numerical value A to numerical value B,” “numerical value A - numerical value B,” or “numerical value A or more / or less” refer to ranges including the endpoints A and B.

[0095] Throughout the present invention, the term "about" is used to define numerical ranges and parameters of the present invention. Numerical values ​​are approximate, and the relevant numerical values ​​in the specific embodiments are presented as accurately as possible. Unless otherwise expressly stated, all ranges, quantities, values, and percentages used herein are to be understood as modified by the word "about." As used herein, "about" generally refers to the actual value being within ±5%, ±3%, ±1%, or ±0.5% of a particular value or range. Furthermore, all numerical values ​​and numerical ranges presented herein should be understood to include the inevitable systematic errors that occur in industrial production.

[0096] In the present invention, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0097] In the present invention, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used represents weight or mass percentage.

[0098] In the present invention, "single-dose administration" refers to an administration method in which an effective amount or a target dose of a drug, therapeutic agent, diagnostic agent, medical device, cosmetic, or composition is administered once within one administration cycle.

[0099] In the present invention, "multiple-dose administration" refers to a method of administration in which a drug, therapeutic agent, diagnostic agent, medical device, cosmetic or composition is repeatedly administered at a certain dose and at a certain interval within one administration cycle, so that the blood drug concentration reaches and remains within the therapeutic window or the total amount of administration reaches an effective amount or a target dose, also known as repeated administration.

[0100] In the present invention, "administration cycle" refers to a course of medication for the purpose of improvement, prevention or treatment. The administration cycle may vary with the different improvement, prevention or treatment purposes, the different half-life of the drug, the different drug administration targets, etc.

[0101] As used herein, the term "effective amount" encompasses an amount sufficient to ameliorate or prevent a physiological symptom or condition. The effective amount for a particular subject may vary depending on factors such as the symptom to be ameliorated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0102] In the present invention, the terms "administer", "administer" and "treat" when applied to animals, humans, experimental subjects, cells, tissues or organs, etc., refer to the contact of exogenous drugs, therapeutic agents, diagnostic agents, medical devices, cosmetics or compositions with animals, humans, experimental subjects, cells, tissues or organs.

[0103] In the present invention, "individual," "object," "patient," or "subject" includes mammals, including but not limited to humans, monkeys, gorillas, cats, dogs, cows, sheep, pigs, horses, camels, rabbits, mice, rats, or guinea pigs.

[0104] In the present invention, the term "identity" refers to the percentage of identical amino acids between two or more polypeptides. Sequence identity between two or more polypeptides can be determined by aligning the amino acid sequences of the polypeptides and scoring the number of positions containing identical amino acid residues in the aligned polypeptides and comparing this score to the number of positions containing different amino acid residues in the aligned polypeptides. Sequence identity can be calculated by dividing the number of positions containing identical amino acid residues by the total number of amino acid residues in the polypeptides.

[0105] In the present invention, amino acid addition may refer to the addition of 1, 2 or 3 or more amino acids at the C-terminus, N-terminus or any position between the C-terminus and the N-terminus of the amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0106] In the present invention, amino acid substitution may refer to the replacement of an amino acid at a certain position in an amino acid sequence by another amino acid, as long as the altered sequence retains the activity of the original amino acid sequence in whole or in part. Amino acid substitution may be a conservative amino acid substitution, which means that compared with the original amino acid sequence, several amino acids are replaced by amino acids with similar or similar properties to form a peptide (conservative variant peptide). Exemplary, these conservative variant peptides can be produced based on the following amino acid substitutions: substitution of Ala by Val, Leu or Ile, substitution of Arg by Lys, Gln, Asn or His, substitution of Asn by Gln, His, Lys or Arg, substitution of Asn by Glu or Asn, substitution of Cys by Ser or Ala, substitution of Gln by Asn or Glu, substitution of Glu by Asp or Gln, substitution of Gly by Ala, substitution of His by Asn, Lys, Gln or Arg, substitution of Leu, Met, Ala, Val, Phe or norleucine for Cys. The amino acid substitutions may also be non-conservative amino acid substitutions.

[0107] In the present invention, amino acid deletion may refer to the deletion of 1, 2 or 3 or more amino acids from an amino acid sequence, as long as the altered sequence fully or partially retains the activity of the original amino acid sequence.

[0108] In the present application, "hybridization" means the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions, without non-specific binding occurring between the polynucleotide and non-complementary objects under these conditions. The terms "moderate stringency conditions", "moderate-high stringency conditions", "high stringency conditions" or "very high stringency conditions" as used in the present application describe the conditions of nucleic acid hybridization and washing. For example, the specific hybridization conditions are as follows: (1) low stringency hybridization conditions in 6x sodium chloride / sodium citrate (SSC) at about 45°C, then at least 50°C, washing in 0.2x SSC, 0.1% SDS twice (for low stringency conditions, the washing temperature can be raised to 55°C); (2) moderate stringency hybridization conditions in 6x SSC at about 45°C, then at 60°C, washing in 0.2x SSC, 0.1% SDS once or more; (3) high stringency hybridization conditions in 6x SSC at about 45°C, then at 65°C, washing in 0.2x SSC, 0.1% SDS once or more and preferably; (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, then at 65°C, washing in 0.2x SSC, 1% SDS once or more.

[0109] Unless otherwise defined, other technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0110] I. Recombinant humanized collagen type III

[0111] The recombinant humanized collagen type III described in the present application is recorded in the patent with the application number 201811438582.6 and the invention name "Polypeptide, its production method and use", the content of which is incorporated into the present application by reference.

[0112] In some embodiments, the amino acid sequence of the recombinant humanized collagen type III comprises n repeats of the sequence shown in SEQ ID NO. 1, n being an integer greater than or equal to 1, wherein when n is an integer greater than or equal to 2, each repeat sequence is directly connected.

[0113] SEQ ID NO. 1: GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0114] In some embodiments, the amino acid sequence of the recombinant humanized collagen type III comprises:

[0115] a) the amino acid sequence shown in SEQ ID NO. 2;

[0116] b) an amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO. 2, which retains the cell adhesion effect of the amino acid sequence shown in SEQ ID NO. 2;

[0117] c) an amino acid sequence in which one or more amino acid residues are added, substituted or deleted in the amino acid sequence shown in SEQ ID NO. 2, and which retains the cell adhesion effect of the amino acid sequence shown in SEQ ID NO. 2; or

[0118] d) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2 under stringent conditions, and the amino acid sequence retains the cell adhesion effect of the amino acid sequence shown in SEQ ID NO. 2, wherein the stringent conditions are moderately stringent conditions, medium-high stringent conditions, high stringent conditions, or very high stringent conditions.

[0119] SEQ ID NO.2: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGP AGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGER GAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0120] In some embodiments, the amino acid sequence of the recombinant humanized type III collagen comprises the sequence shown in SEQ ID NO.2.

[0121] In some embodiments, the amino acid sequence of the recombinant humanized type III collagen is the sequence shown in SEQ ID NO.2.

[0122] The recombinant humanized type V collagen of the present invention can be prepared by conventional methods in the art. For example, it can be prepared by the following steps: (1) construction of genetically engineered Escherichia coli; (2) fermentation and cultivation of the genetically engineered Escherichia coli; (3) induced expression of the recombinant humanized type V collagen; and (4) purification and optional enzymatic digestion of the recombinant humanized type V collagen.

[0123] II. Injections containing recombinant humanized type III collagen

[0124] The present invention provides an injection comprising the recombinant humanized type III collagen described in Part I above.

[0125] In some embodiments, the recombinant humanized type III collagen used in the injection is recombinant humanized type III collagen freeze-dried fiber, which is produced according to the preparation process disclosed in CN114632022A, the content of which is incorporated into the present invention by reference.

[0126] In some specific embodiments, the purity of the recombinant humanized type III collagen freeze-dried fibers is less than or equal to 100%; preferably greater than or equal to 70%, 80% or 90%; more preferably 100%.

[0127] In some embodiments, the injection further comprises a pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier includes any one or more of an osmotic pressure regulator, a pH regulator, a solubilizer, a cosolvent, an antioxidant, an antibacterial agent, an emulsifier, and a suspending agent.

[0128] In some embodiments, the injection is a lyophilized injection.

[0129] In some embodiments, the injection is an injection solution.

[0130] In some embodiments, the injection comprises the recombinant humanized type III collagen and sodium chloride injection.

[0131] In some embodiments, the injection is an intravenous injection, a subcutaneous injection, an intramuscular injection, or an intraperitoneal injection; preferably, the injection is an intravenous injection or a subcutaneous injection; more preferably, the injection is an intravenous injection.

[0132] In some embodiments, the injection is an injection for mammals; preferably, the injection is an injection for humans.

[0133] In some embodiments, the injection is free of cytotoxicity, organ toxicity, hemotoxicity, and immunotoxicity.

[0134] In some embodiments, the method for preparing the injection comprises the step of dissolving the recombinant humanized type III collagen freeze-dried fibers using sodium chloride injection.

[0135] In some embodiments, when the injection is administered within one administration cycle, the total administration amount of the recombinant humanized type III collagen is less than or equal to 3640 mg / kg based on the body weight of the subject; preferably, the upper limit of the total administration amount is for rats.

[0136] In some embodiments, the present invention converts the dosage for rats and humans based on body surface area, and the human equivalent dose (mg / kg) = rat dose (mg / kg) × (rat Km coefficient / human Km coefficient), wherein the rat Km coefficient is 6 and the human Km coefficient is 37.

[0137] In some embodiments, when the subject of the injection is a human, when the injection is administered within one administration cycle, the total administration amount of the recombinant humanized type III collagen is less than or equal to 590 mg / kg based on the body weight of the subject.

[0138] The present invention does not impose any particular limitation on the total administration amount of recombinant humanized type III collagen within one administration cycle, and those skilled in the art may adjust the dosage according to the administration goal (eg, treatment endpoint, administration subject, etc.).

[0139] In some embodiments, the injection is a single-dose administration injection.

[0140] In some specific embodiments, in the single-dose injection, when the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 90-110 mg / mL, for example, it can be 90 mg / mL, 91 mg / mL, 92 mg / mL, 93 mg / mL, 94 mg / mL, 95 mg / mL, 96 mg / mL, 97 mg / mL, 98 mg / mL, 99 mg / mL, 100 mg / mL, 101 mg / mL, 102 mg / mL, 103 mg / mL, 104 mg / mL, 105 mg / mL, 106 mg / mL, 107 mg / mL, 108 mg / mL, 109 mg / mL or 110 mg / mL, etc.; preferably 95-105 mg / mL.

[0141] In some specific embodiments, in the single-dose injection, when the subject of the single-dose injection is a human and the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is less than or equal to 30 mg / mL, for example, it can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL or 30 mg / mL, etc.

[0142] In some specific embodiments, when the injection is administered in a single dose, the single administration amount of the recombinant type III humanized collagen is less than or equal to 2000 mg / kg based on the body weight of the subject; preferably, the upper limit of the single administration amount is for mice.

[0143] In some embodiments, the present invention converts the dosage for mice and humans based on body surface area, and the human equivalent dose (mg / kg) = mouse dose (mg / kg) × (Km coefficient for mice / Km coefficient for humans), where the Km coefficient for mice is 3 and the Km coefficient for humans is 37.

[0144] In some specific embodiments, when the subject of the single-dose injection is a human, and the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 162 mg / kg based on the body weight of the subject.

[0145] In some specific embodiments, when the injection is administered in a single dose, the present invention does not particularly limit the specific single administration amount of the recombinant humanized type III collagen, which can be selected according to the specific administration object and usage scenario.

[0146] In some specific embodiments, when the injection is administered in a single dose, the total amount of the recombinant humanized type III collagen administered is less than or equal to 2000 mg / kg based on the body weight of the subject; preferably, the upper limit of the total amount is for mice.

[0147] In some specific embodiments, when the subject of the single-dose injection is a human, and the injection is administered in a single dose, the total administration amount of the recombinant humanized type III collagen is less than or equal to 162 mg / kg based on the body weight of the subject.

[0148] In some embodiments, the injection is a multi-dose injection.

[0149] In some specific embodiments, in the multiple-dose injection, when the multiple-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 6-29 mg / mL, for example, it can be 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL or 29 mg / mL, etc.; preferably, it is 6.5-26 mg / mL.

[0150] In some specific embodiments, when the injection is administered in multiple doses, the single administration amount of the recombinant type III humanized collagen is less than or equal to 130 mg / kg based on the body weight of the subject; preferably, the upper limit of the single administration amount is for rats.

[0151] In some specific embodiments, when the subject of the multi-dose injection is a human, and the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 21 mg / kg based on the body weight of the subject.

[0152] In some specific embodiments, when the injection is administered in multiple doses, the amount of the recombinant humanized type III collagen administered each time may be the same or different; preferably, the amount administered each time is the same.

[0153] In some specific embodiments, when the injection is administered in multiple doses, the present invention does not particularly limit the specific single administration amount of the recombinant humanized type III collagen, which can be selected according to the specific administration object and administration scenario.

[0154] In some specific embodiments, when the injection is administered in multiple doses, the total administration amount of the recombinant humanized type III collagen is less than or equal to 3640 mg / kg based on the body weight of the subject; preferably, the upper limit of the total administration amount is for rats.

[0155] In some specific embodiments, when the subject of the multiple-dose injection is a human, and the injection is administered in multiple doses, the total administration amount of the recombinant humanized type III collagen is less than or equal to 590 mg / kg based on the body weight of the subject.

[0156] In some specific embodiments, when the injection is administered in multiple doses, the frequency of administration is once a day, and the administration cycle is 1-5 weeks, for example, 1 week, 2 weeks, 3 weeks, 4 weeks or 5 weeks; preferably 1-4 weeks.

[0157] In some specific embodiments, when the injection is administered in multiple doses, the present invention does not specifically limit the frequency and cycle of administration, which can be selected according to the specific administration object and administration scenario.

[0158] III. Use of recombinant humanized type III collagen in the preparation of injections

[0159] The present invention provides the use of the recombinant humanized type III collagen described in Part I above in the preparation of an injection.

[0160] In some embodiments, the recombinant humanized type III collagen used to prepare the injection is recombinant humanized type III collagen freeze-dried fiber, which is produced according to the preparation process disclosed in CN114632022A, the content of which is incorporated into the present invention by reference.

[0161] In some specific embodiments, the purity of the recombinant humanized type III collagen freeze-dried fibers is less than or equal to 100%; preferably greater than or equal to 70%, 80% or 90%; more preferably 100%.

[0162] In some embodiments, the injection further comprises a pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier includes any one or more of an osmotic pressure regulator, a pH regulator, a solubilizer, a cosolvent, an antioxidant, an antibacterial agent, an emulsifier, and a suspending agent.

[0163] In some embodiments, the injection is an injection solution.

[0164] In some embodiments, the injection comprises the recombinant humanized type III collagen and sodium chloride injection.

[0165] In some embodiments, the injection is an intravenous injection, a subcutaneous injection, an intramuscular injection, or an intraperitoneal injection; preferably, the injection is an intravenous injection or a subcutaneous injection; more preferably, the injection is an intravenous injection.

[0166] In some embodiments, the injection is free of cytotoxicity, organ toxicity, hemotoxicity, and immunotoxicity.

[0167] In some embodiments, when the injection is administered within one administration cycle, the total administration amount of the recombinant humanized type III collagen is less than or equal to 3640 mg / kg based on the body weight of the subject; preferably, the upper limit of the total administration amount is for rats.

[0168] In some embodiments, when the subject of the injection is a human, when the injection is administered within one administration cycle, the total administration amount of the recombinant humanized type III collagen is less than or equal to 590 mg / kg based on the body weight of the subject.

[0169] The present invention does not impose any particular lower limit on the total administration amount of recombinant humanized type III collagen within one administration cycle, and those skilled in the art can adjust it according to the administration goal (eg, treatment endpoint, administration subject, etc.).

[0170] In some embodiments, the injection is a single-dose administration injection.

[0171] In some specific embodiments, in the single-dose injection, when the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 90-110 mg / mL, for example, it can be 90 mg / mL, 91 mg / mL, 92 mg / mL, 93 mg / mL, 94 mg / mL, 95 mg / mL, 96 mg / mL, 97 mg / mL, 98 mg / mL, 99 mg / mL, 100 mg / mL, 101 mg / mL, 102 mg / mL, 103 mg / mL, 104 mg / mL, 105 mg / mL, 106 mg / mL, 107 mg / mL, 108 mg / mL, 109 mg / mL or 110 mg / mL, etc.; preferably 95-105 mg / mL.

[0172] In some specific embodiments, in the single-dose injection, when the subject of the single-dose injection is a human and the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is less than or equal to 30 mg / mL, for example, it can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL or 30 mg / mL, etc.

[0173] In some specific embodiments, when the injection is administered in a single dose, the single administration amount of the recombinant type III humanized collagen is less than or equal to 2000 mg / kg based on the body weight of the subject; preferably, the upper limit of the single administration amount is for mice.

[0174] In some specific embodiments, when the subject of the single-dose injection is a human, and the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 162 mg / kg based on the body weight of the subject.

[0175] In some specific embodiments, when the injection is administered in a single dose, the present invention does not impose any particular limitation on the lower limit of the single administration amount of the recombinant humanized type III collagen, which can be selected according to the specific administration object and usage scenario.

[0176] In some specific embodiments, when the injection is administered in a single dose, the total amount of the recombinant humanized type III collagen administered is less than or equal to 2000 mg / kg based on the body weight of the subject; preferably, the upper limit of the total amount is for mice.

[0177] In some specific embodiments, when the subject of the single-dose injection is a human, and the injection is administered in a single dose, the total administration amount of the recombinant humanized type III collagen is less than or equal to 162 mg / kg based on the body weight of the subject.

[0178] In some embodiments, the injection is a multi-dose injection.

[0179] In some specific embodiments, in the multiple-dose injection, when the multiple-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 6-29 mg / mL, for example, it can be 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL or 29 mg / mL, etc.; preferably, it is 6.5-26 mg / mL.

[0180] In some specific embodiments, when the injection is administered in multiple doses, the single administration amount of the recombinant type III humanized collagen is less than or equal to 130 mg / kg based on the body weight of the subject; preferably, the upper limit of the single administration amount is for rats.

[0181] In some specific embodiments, when the subject of the multi-dose injection is a human, and the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 21 mg / kg based on the body weight of the subject.

[0182] In some specific embodiments, when the injection is administered in multiple doses, the amount of the recombinant humanized type III collagen administered each time may be the same or different; preferably, the amount administered each time is the same.

[0183] In some specific embodiments, when the injection is administered in multiple doses, the present invention does not impose any particular limitation on the lower limit of the single administration amount of the recombinant humanized type III collagen, which can be selected according to the specific administration object and usage scenario.

[0184] In some specific embodiments, when the injection is administered in multiple doses, the total administration amount of the recombinant humanized type III collagen is less than or equal to 3640 mg / kg based on the body weight of the subject; preferably, the upper limit of the total administration amount is for rats.

[0185] In some specific embodiments, when the subject of the multiple-dose injection is a human, and the injection is administered in multiple doses, the total administration amount of the recombinant humanized type III collagen is less than or equal to 590 mg / kg based on the body weight of the subject.

[0186] In some specific embodiments, when the injection is administered in multiple doses, the frequency of administration is once a day, and the administration cycle is 1-5 weeks, for example, 1 week, 2 weeks, 3 weeks, 4 weeks or 5 weeks; preferably 1-4 weeks.

[0187] In some specific embodiments, when the injection is administered in multiple doses, the present invention does not specifically limit the frequency and cycle of administration, which can be selected according to the specific administration object and administration scenario.

[0188] IV. Kits containing injections

[0189] The present invention provides a medicine kit comprising the injection comprising recombinant humanized type III collagen as described in Part II above; more specifically, the medicine kit provided by the present invention comprises a syringe pre-filled with the injection comprising recombinant humanized type III collagen as described in Part II above.

[0190] In some embodiments, when the injection is an injection for single-dose administration, the kit satisfies the following condition (i):

[0191] (i) The medicine kit contains only one syringe I prefilled with the single-dose injection, and the total amount of the recombinant humanized type III collagen contained in the medicine kit is less than or equal to 2000 mg / kg based on the body weight of the subject; preferably, when the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen in each syringe I is 90-100 mg / mL.

[0192] In some specific embodiments, when the injection is a single-dose injection, and when the subject of the drug kit is a human, the drug kit satisfies the following condition (iii):

[0193] (iii) The medicine kit contains only one syringe I pre-filled with the single-dose injection, and the total amount of the recombinant type III humanized collagen contained in the medicine kit is less than or equal to 162 mg / kg based on the body weight of the subject; preferably, when the single-dose injection is a liquid, the concentration of the recombinant type III humanized collagen in each syringe I is less than or equal to 30 mg / mL; preferably, when the single-dose injection is a lyophilized preparation, based on the body weight of the subject being 60 kg / person, the content of the recombinant type III humanized collagen in each syringe I is 9720 mg.

[0194] In some embodiments, when the injection is an injection for multiple doses, the kit satisfies the following condition (ii):

[0195] (ii) The medicine kit contains multiple syringes II of the same specifications pre-filled with the multi-dose injection, and based on the body weight of the subject, the total amount of the recombinant type III humanized collagen contained in the medicine kit is less than or equal to 3640 mg / kg, and the amount of the recombinant type III humanized collagen contained in each syringe II is less than or equal to 130 mg / kg; preferably, when the multi-dose injection is a liquid, the concentration of the recombinant type III humanized collagen in each syringe II is 6-29 mg / mL.

[0196] In some specific embodiments, when the injection is a multi-dose injection and when the subject of the drug kit is a human, the drug kit satisfies the following condition (iv):

[0197] (iv) The medicine kit contains a plurality of syringes II of the same specifications pre-filled with the multi-dose injection, and based on the body weight of the subject, the total amount of the recombinant type III humanized collagen contained in the medicine kit is less than or equal to 590 mg / kg, and the amount of the recombinant type III humanized collagen contained in each syringe II is less than or equal to 21 mg / kg; preferably, when the multi-dose injection is a liquid, the concentration of the recombinant type III humanized collagen in each syringe II is 6-29 mg / mL; preferably, when the multi-dose injection is a lyophilized preparation, based on the body weight of the subject of administration of 60 kg / person, the content of the recombinant type III humanized collagen in each syringe II is 1260 mg.

[0198] The present invention does not impose any particular lower limit on the total amount of the recombinant humanized type III collagen contained in the kit, and those skilled in the art can adjust it according to the target of the kit (eg, treatment endpoint, administration subject, etc.).

[0199] In some embodiments, the kit is for use within a single administration cycle.

[0200] In some specific embodiments, when the kit satisfies condition (i) or (iii), the kit is a single-dose kit.

[0201] In some specific embodiments, when the medicine kit meets condition (ii) or (iv), the medicine kit contains 7-35 syringes II, for example, 7, 14, 21 or 28 syringes, and the medicine kit is a multiple-administration medicine kit, and one syringe II can be used each time, and the administration is once a day.

[0202] V. Uses of injections

[0203] The injection of the present invention contains or is prepared using the recombinant humanized type III collagen described in Part I above, and thus has the function of the recombinant humanized type III collagen.

[0204] In some embodiments, the injection is used to prevent and / or treat cardiovascular disease, repair vascular damage, or prevent and / or treat vascular damage in a subject. For information about the applicability of recombinant humanized type III collagen to cardiovascular disease and vascular damage, reference may be made to CN118892536A, the contents of which are incorporated into the present invention by reference.

[0205] In some specific embodiments, the injection has an anti-endothelial cell damage effect.

[0206] In some specific embodiments, the endothelial cell damage is ultraviolet photoaging-induced damaged skin or diabetic infection wounds.

[0207] In some specific embodiments, the cardiovascular disease is a cardiovascular disease associated with endothelial cell damage; preferably, the endothelial cell damage is endothelial cell damage induced by angiotensin II or ROS, and / or the vascular damage is vascular damage induced by angiotensin II or ROS.

[0208] In some specific embodiments, the cardiovascular disease is selected from: arteriosclerosis, atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation and thrombotic disease; preferably, the hypertension is spontaneous hypertension.

[0209] In some specific embodiments, the vascular injury is vascular endothelial injury; preferably, the vascular endothelial injury is diabetic vascular endothelial injury.

[0210] In some embodiments, the injection is used to prevent and / or treat ocular inflammation. For information on the application of recombinant humanized type III collagen in ocular inflammation, please refer to CN120037359A, the content of which is incorporated into the present invention by reference.

[0211] In some specific embodiments, the ocular inflammation comprises any one or more of blepharitis, keratitis, conjunctivitis, scleritis, hordeolum, dacryocystitis, optic neuritis, and uveitis.

[0212] In some specific embodiments, the ocular inflammation is inflammation resulting from contact lens wear.

[0213] In some specific embodiments, the ocular inflammation is ocular surface inflammation, which includes any one or more of blepharitis, keratitis and conjunctivitis; preferably, the ocular surface inflammation is blepharitis, keratitis or conjunctivitis; more preferably, the blepharitis has symptoms including eyelid defect, the keratitis has symptoms including corneal ulcer, and the conjunctivitis has symptoms including conjunctival scarring.

[0214] In some specific embodiments, the prevention and / or treatment of ocular inflammation includes any one or more of the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more of eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more tissues in the eyelid, cornea and / or conjunctiva, and enhancing the stability of the tear film.

[0215] In some embodiments, the injection is used to prevent and / or treat gastric cancer. For information on the applicability of recombinant humanized type III collagen to gastric cancer, please refer to CN120114574A, the contents of which are incorporated herein by reference.

[0216] In some specific embodiments, the gastric cancer is selected from nodular gastric cancer, polypoid gastric cancer, liquid gastric cancer, gastric adenocarcinoma, and gastric adenocelluloma.

[0217] In some specific embodiments, the prevention and / or treatment of gastric cancer includes any one or more of the following manifestations: inhibiting gastric cancer cell proliferation, inhibiting gastric cancer cell DNA replication activity, and inhibiting gastric cancer cell migration.

[0218] In some embodiments, the injection is used to prevent and / or treat breast diseases. For information on the applicability of recombinant humanized type III collagen to breast diseases, reference may be made to CN116407620A, the contents of which are incorporated herein by reference.

[0219] In some specific embodiments, the breast disease is breast cancer.

[0220] In some specific embodiments, the breast cancer includes luminal A breast cancer, luminal B breast cancer, HER-2 overexpressing breast cancer, basal cell-like breast cancer, triple-negative breast cancer and normal breast-like breast cancer; preferably, the breast cancer is luminal A breast cancer, HER-2 overexpressing breast cancer, basal cell-like breast cancer or triple-negative breast cancer.

[0221] In some specific embodiments, the prevention and / or treatment of breast cancer includes any one or more of the following manifestations: inhibiting the proliferation ability of breast cancer cells, inhibiting the migration ability of breast cancer cells and / or promoting the dormancy of breast cancer cells; preferably, the inhibition of the proliferation ability of breast cancer cells includes inhibiting the DNA replication activity of breast cancer cells.

[0222] In some embodiments, the injection is used to prevent and / or treat ovarian diseases. For information on the applicability of recombinant humanized type III collagen to ovarian diseases, reference may be made to CN116617371A, the contents of which are incorporated herein by reference.

[0223] In some specific embodiments, the ovarian disease is ovarian cancer.

[0224] In some specific embodiments, the ovarian cancer includes ovarian epithelial tumors, ovarian germ cell tumors, and ovarian sex cord-stromal tumors.

[0225] In some specific embodiments, the prevention and / or treatment of ovarian cancer includes any one or more of the following manifestations: inhibiting the proliferation ability of ovarian cancer cells, inhibiting the migration ability of ovarian cancer cells, inhibiting the invasion ability of ovarian cancer cells and / or inhibiting the tumor-forming ability of ovarian cancer cells; preferably, the inhibition of the proliferation ability of ovarian cancer cells includes inhibiting the DNA replication activity of ovarian cancer cells; preferably, the inhibition of the tumor-forming ability of ovarian cancer cells includes inhibiting the ability of ovarian cancer cells to form tumors; preferably, the inhibition of the ability of ovarian cancer cells to form tumors includes inhibiting the number of cell clones formed by ovarian cancer cells and / or inhibiting the growth rate of ovarian cancer cells.

[0226] In some embodiments, the injection is used to prevent and / or inhibit and / or treat cervical cancer. For information on the applicability of recombinant humanized type III collagen to cervical cancer, please refer to CN119564836A, the contents of which are incorporated herein by reference.

[0227] In some specific embodiments, the cervical cancer includes any one or more of squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, clear cell carcinoma, small cell carcinoma and sarcoma; preferably, the cervical cancer includes squamous cell carcinoma.

[0228] In some specific embodiments, the cervical cancer is associated with persistent infection with high-risk human papillomavirus; preferably, the high-risk human papillomavirus includes any one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and HPV68; more preferably, the high-risk human papillomavirus includes any one or more of HPV18, HPV39 and HPV68.

[0229] In some specific embodiments, the prevention and / or inhibition and / or treatment of cervical cancer includes any one or more of the following manifestations: inhibiting the viability of cervical cancer cells, inhibiting the proliferation ability of cervical cancer cells, and inhibiting the migration ability of cervical cancer cells.

[0230] In some embodiments, the injection is used to inhibit esophageal squamous cell carcinoma. For information on the applicability of recombinant humanized type III collagen to inhibit esophageal squamous cell carcinoma, reference may be made to CN120131920A, the contents of which are incorporated herein by reference.

[0231] In some specific embodiments, the inhibition of esophageal squamous cell carcinoma includes inhibiting the growth of esophageal squamous cell carcinoma cells and / or inducing cell morphology changes; preferably, the esophageal squamous cell carcinoma cells include esophageal squamous cell carcinoma cells KYSE410 and esophageal squamous cell carcinoma cells KYSE510.

[0232] Example

[0233] The embodiments of the present invention will be described in detail below with reference to the examples. However, it will be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used were commercially available conventional products.

[0234] Example 1: Preparation and expression of recombinant humanized type III collagen

[0235] Recombinant type III humanized collagen freeze-dried fibers were produced according to the preparation process in CN114632022A. The specific process includes:

[0236] Outer packaging washing, drying, and sterilization process: After receiving the vials, they enter the unscrambling room, where they are unpacked and neatly stacked on an unscrambling tray. They are then transported via a conveyor belt to a bottle washer for cleaning. They then enter an oven set at 320°C and a mesh belt speed of 20-50Hz for drying until ready for use. Pretreatment for freeze-dried stoppers and aluminum-plastic caps: Sterilize at 121°C for 30 minutes.

[0237] The vial filling process begins with adjusting the fill volume. Once adjusted, close the fill volume control and begin normal filling operations. During the filling process, 10 samples are drawn every 30 minutes and the fill volume of each vial is measured on an electronic balance. The fill volume range for each vial complies with production instructions. At the start of filling, the fully automatic vacuum freeze dryer is activated, with the automatic freeze-drying cycle feed mode enabled. Once activated, filling is performed at a rate of 80-120 vials / minute.

[0238] Freeze dryer operation process: The freeze dryer undergoes CIP cleaning and SIP sterilization treatment, sets the freeze drying parameters, starts the freeze drying cycle, opens the small door to start the feeding program and waits for the filling to be completed. After the filled finished product enters the freeze drying box, the freeze drying program is automatically started and the freeze drying cycle is executed.

[0239] The pH value of the recombinant human type III collagen stock solution was adjusted to 7 with sodium dihydrogen phosphate and disodium hydrogen phosphate, and filled into 10 mL vials according to the above process, 4 mL per vial, and freeze-dried using the freeze-drying program. The freeze-drying program is shown in Table 1 below:

[0240] Table 1 Freeze-drying procedure

[0241]

[0242] After the freeze-drying is completed, nitrogen is flushed in and the box is plugged. When the ambient temperature and humidity meet the requirements, the freeze-drying box door is opened and the product is transported to the capping track for aseptic capping.

[0243] Example 2: Purity Detection of Recombinant Humanized Type III Collagen (Kjeldahl Nitrogen Method)

[0244] 1. The recombinant humanized type III collagen freeze-dried fiber sample prepared in Example 1 was prepared into a dispersion with a concentration of about 10 mg / ml as a test solution.

[0245] 2. Sample pretreatment:

[0246] After adding approximately 2 g of the test solution to each digestion tube (weight loss method), 10.0 ml of concentrated sulfuric acid and two Kjeldahl catalyst tablets were added. The samples were digested using an automated digester (model: SH520) at a temperature profile of 150°C for 5 minutes, 350°C for 5 minutes, and 400°C for 60 minutes. The solution was clear green after digestion.

[0247] 3. Detection:

[0248] The samples were transferred to a fully automatic Kjeldahl nitrogen analyzer (model: VAP500) for titration.

[0249] 4. Calculation:

[0250] Protein content (%) = average nitrogen content × 4.87 × 10; 4.87 is the protein conversion coefficient of recombinant humanized type III collagen.

[0251] Protein content (mg / ml) = protein content (%) × 10; 1 ml = 1 g.

[0252] 5. The results are shown in Table 2 below:

[0253] Table 2 Purity test results of recombinant humanized type III collagen

[0254] Specimen type Weight (g) <![CDATA[V2(ml)]]> <![CDATA[V1-V2(ml)]]> Nitrogen content N (%) Blank sample 0.266 Blank sample 0.268 Blank sample 0.263 Sample 1.9434 0.266 5.156 0.197 Sample 1.9497 0.266 5.179 0.197 Sample 1.9795 0.266 5.243 0.196

[0255] Nitrogen content calculation formula:

[0256]

[0257] The meaning of each symbol in the above formula is:

[0258] N: nitrogen content in the sample;

[0259] V1: The volume of sulfuric acid or hydrochloric acid standard titrant consumed by the sample;

[0260] V2: Volume of sulfuric acid or hydrochloric acid standard titrant consumed by reagent blank;

[0261] c: concentration of sulfuric acid standard titration solution;

[0262] m: mass of the sample;

[0263] 0.0140: The mass of nitrogen equivalent in 1.0 mL of hydrochloric acid (c(HCl) = 1.000 mol / L) or sulfuric acid (c(1 / 2H2SO4) = 1.000 mol / L) or standard titration solution

[0264] The protein content was calculated using the formula, yielding a recombinant humanized type III collagen concentration of 95.78 mg / ml. The product standard concentration is 100 mg / ml, with an error within ±5%, indicating the product is qualified. The error ranges for other dosage concentrations were similar, indicating the product was qualified.

[0265] Example 3: Toxicity test of single high-dose administration of recombinant humanized type III collagen

[0266] Recombinant humanized type III collagen was injected into the tail vein of ICR mice to observe the possible poisoning symptoms, degree, nature, recovery and death of ICR mice, so as to understand the information related to acute poisoning and provide a reference for the preclinical safety evaluation dosage design and clinical safe use of the drug.

[0267] (A) Experimental procedures

[0268] 1. The animals used in this study and all related treatments met animal welfare requirements and underwent ethical review by a professional organization before the experiment was conducted.

[0269] SPF-grade ICR mice aged 21-34 days (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., license number: SCXK-(Beijing) 2021-0011, animal quality certificate number: 110011241108436386, 110011241108436253) were selected, half male and half female, 40 mice, and divided equally into two groups of 20 mice in each group. The animal quarantine observation period is 5 days. During the quarantine period, the animals' drinking water, food intake, and health status were observed, as well as the presence of signs of disease and death. During the quarantine period, the mice had smooth fur, normal behavior and appearance, no abnormal secretions, etc., and no abnormal animals were found.

[0270] The maximum tolerated dose method was used, and a sodium chloride injection control group (Shijiazhuang Siyao Co., Ltd., batch number 2403121601) and a recombinant type III humanized collagen administration group were set up. ICR mice were randomly divided into groups according to body weight, with 20 mice in each group, half male and half female. The dose of recombinant type III humanized collagen was 2000 mg / kg, and the drug concentration was 100 mg / ml (diluted with sodium chloride injection); the control group was given an equal volume of sodium chloride injection. The specific dosage design is shown in Table 3 below. Weigh before administration, calculate the dosage according to body weight, draw the drug solution with a disposable syringe, and perform tail vein injection. A total of 1 application, the observation period was 14 days, and the experiment was terminated after 14 days.

[0271] Table 3. Dose design for single-dose toxicity study of recombinant humanized type III collagen injected into the tail vein of ICR mice

[0272]

[0273] 2. Feeding conditions: Animals are kept in a barrier environment by qualified personnel. 12 hours of lighting and 12 hours of darkness. The temperature and humidity of the animal room are automatically monitored and recorded by the environmental monitoring platform. During the experiment, the lowest temperature in the feeding room was 19.6°C and the highest was 21.5°C; the lowest humidity was 27.6% and the highest was 70.4%. Animals are fed in standard feeding cages, 5 per cage, and cages are changed twice a week to keep the cages clean and dry. Clean and wipe the cages and countertops after daily operations, and wipe and disinfect them regularly with disinfectant. The disinfectants are dilute glutaraldehyde solution (produced by Shanxi Zhaoyi Biological Co., Ltd., batch number: 240402) and Sanisol (produced by Shandong Lierkang Medical Technology Co., Ltd., batch number: 20240312A).

[0274] 3. Feed: SPF maintenance feed for rats and mice produced by Beijing Ke'ao Xieli Feed Co., Ltd. (Production License No.: Jingshizheng (2014)06054; Feed Batch Nos.: 24073213, 24083213). Animals were fed ad libitum. Test results for moisture, crude protein, crude fat, crude fiber, crude ash, calcium, and total phosphorus in the feed all met the standards of GB14924.3-2010 and GB / T18823-2010. Test results for arsenic, lead, cadmium, mercury, BHC, DDT, and aflatoxin B1 all met the standards of GB14924.2-2001.

[0275] 4. Bedding: Clean-grade wood shavings produced by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd. are sterilized by cobalt-60 irradiation at the Irradiation Center of the China Institute of Radiation Protection (ICRP). Irradiation dose: 10 kGy. Sensory and moisture content testing of the bedding meets DB32 / T2129-2012 standards.

[0276] 5. Drinking water: High-pressure sterilized tap water is supplied in drinking water bottles. The tap water is inspected by the Taiyuan Monitoring Station of the National Urban Water Supply Quality Monitoring Network (Test Report No.: W20240617). The main control indicators include: color, turbidity, pH, total hardness, iron, manganese, copper, zinc, fluoride, cyanide, arsenic, selenium, mercury, cadmium, chromium, lead, aluminum, total colony count, total coliform group, heat-resistant coliform group, Escherichia coli group, etc., which meet the requirements of the "National Drinking Water Quality Standard" (GB5749-2022); the drinking water bottle should be replaced daily and the drinking water bottle should be cleaned and sterilized under high pressure before use.

[0277] (B) Experimental results and statistical analysis

[0278] Body weight was measured before administration and on days 1, 3, 8, and 14 after administration. The average values ​​of body weight for each gender in each group were calculated using SPSS 25.0 statistical software. The data were analyzed by one-way ANOVA if P≥0.05, and Dunnett-t test was performed if there were differences between the groups (P<0.05). If the normality or homogeneity of variance test was not met (P<0.05), the nonparametric Kruskal-Wallis test was performed.

[0279] The feed consumption of animals was measured before administration and on the 3rd and 12th day after administration. The calculation formula of feed consumption is:

[0280]

[0281] After administration, the animals were closely observed for toxic reactions and survival, including symptoms of poisoning, time of onset, duration, recovery period, etc. Pathological examination: At the end of the experiment, all animals were sacrificed by CO2 asphyxiation, and gross anatomical examinations were performed, and histopathological observations of tissues and organs were performed.

[0282] The statistical results of the weight changes of animals in the toxicity test of ICR mice injected with recombinant humanized type III collagen in the tail vein are shown in Table 4 and Figure 1 shown.

[0283] Table 4 Changes in body weight of animals in the toxicity test of a single dose of recombinant humanized type III collagen injected into the tail vein of ICR mice

[0284]

[0285] The statistical results of the changes in feed consumption of animals in the toxicity test of a single dose of recombinant humanized type III collagen injected into the tail vein of ICR mice are shown in Table 5 below.

[0286] Table 5 Changes in feed consumption of animals in the toxicity test of single administration of recombinant humanized type III collagen injected into the tail vein of ICR mice

[0287]

[0288] The results of the toxicity test of a single dose of recombinant humanized type III collagen injected into the tail vein of ICR mice are shown in Tables 6 and 7 below.

[0289] Table 6 Results of single-dose toxicity test of recombinant humanized type III collagen injected into the tail vein of ICR mice

[0290]

[0291] Table 7 Results of single-dose toxicity test of recombinant humanized type III collagen injected into the tail vein of ICR mice

[0292]

[0293] (C) Experimental conclusion analysis

[0294] From the time of administration until the end of the observation period, all animals showed no abnormalities, with normal behavior and activity, no toxic reactions, no abnormal physical signs, and normal fecal color and shape. There was no statistically significant difference in body weight between the treated and control groups on the day of administration and 1, 3, 8, and 14 days after administration (P>0.05). The trends in feed consumption in each treated group on days 3 and 12 after administration were essentially the same as those in the control group. The results showed that tail vein injection of 2000 mg / kg of recombinant humanized type III collagen had no effect on animal body weight or food intake, demonstrating that the maximum tolerated dose of recombinant humanized type III collagen injected into the tail vein of ICR mice is greater than 2000 mg / kg.

[0295] Example 4: Toxicity test of recombinant humanized type III collagen after repeated administration for 4 weeks

[0296] The toxicity test was conducted by injecting recombinant humanized type III collagen into the tail vein of SD rats for 4 weeks repeatedly to observe the nature and extent of possible toxic reactions in SD rats, the development and recovery of toxic reactions; determine the toxic dose-effect relationship; determine the target organs of toxic effects and the reversibility of their damage, and determine the non-toxic dose, providing reference data for dosage design for clinical safety of drugs and clinical toxic and side effect monitoring.

[0297] (A) Experimental conditions

[0298] 1. The animals used in this study and all related treatments met animal welfare requirements and underwent ethical review by a professional organization before the experiment was conducted.

[0299] Six-week-old SPF-grade SD rats (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021-0011, laboratory animal quality certificate number: 110011251100136182, 110011251100136071) were selected, half male and half female, and divided into four groups. The animal quarantine observation period was 6 days. During the quarantine period, the animals' drinking water, food intake, and health status were observed, as well as the presence of signs of illness and death. During the quarantine period, the rats had smooth fur, normal behavior and appearance, no abnormal secretions, etc., and no abnormal animals were found.

[0300] The experiment set up a control group (administered with the same volume of sodium chloride injection) and three experimental groups (administered with recombinant humanized type III collagen, diluted with sodium chloride injection). The doses of the high, medium and low dose groups were 130 mg / kg, 65 mg / kg and 32.5 mg / kg, respectively. The male and female animals were divided into groups according to the weight of the animals using a segmented balanced random grouping method. The control group had 15 males and 20 females in each administration group, of which 15 males and 15 females were the main experimental group, and the remaining 5 were spare. The doses of each group and their grouping are shown in Table 8. Weigh before administration, calculate the dosage according to the body weight, draw the liquid with a disposable syringe, and perform the tail vein injection operation. Administer once a day for a total of 4 weeks, and recover for 4 weeks after stopping the drug.

[0301] Table 8 Toxicity test dosage design of SD rats with tail vein injection of recombinant humanized type III collagen for 4 weeks

[0302]

[0303] 2. Housing Conditions: Animals were housed in a barrier environment by qualified personnel. The temperature and humidity of the animal room were automatically monitored and recorded by an environmental monitoring platform, with 30-minute intervals between temperature and humidity monitoring points. During the experiment, the minimum temperature was 17.9°C and the maximum was 24.6°C; the minimum humidity was 12.5% ​​and the maximum was 56.9%. Animals were housed in standard cages, with 5 animals per cage, and cages were changed twice weekly. During experimental operations, the room was ventilated ≥ 15 times / hour (not less than 10 times / hour during non-operating hours). Cages and countertops were cleaned and wiped after daily operations. Disinfectants were wiped and disinfected with disinfectant every Friday, rotating the types of disinfectants used. The disinfectants used were Sanisol (produced by Shandong Lierkang Medical Technology Co., Ltd., batch numbers: 20241012A, 20240502A) and dilute glutaraldehyde solution (produced by Shanxi Zhaoyi Biological Co., Ltd., batch number: 240801).

[0304] 3. Feed: SPF maintenance feed for rats and mice was produced by Keao Xieli (Tianjin) Feed Co., Ltd. (Production License Number: Jingshizheng (2020) 01005, SCXK (Beijing) 2020-0004, Experimental Feed Batch Number: 24123213). Animals ate ad libitum. Test results for moisture, crude protein, crude fat, crude fiber, crude ash, calcium, and total phosphorus in the feed all met the standards specified in GB14924.3-2010 and GB / T14924.1-2001. Test results for arsenic, lead, cadmium, mercury, hexachlorocyclohexane, DDT, and aflatoxin B1 all met the standards specified in GB14924.2-2001 and GB14924.1-2001.

[0305] 4. Bedding: Clean-grade wood shavings produced by Beijing Keao Xieli Feed Co., Ltd. are sterilized by cobalt-60 irradiation at the Irradiation Center of the China Institute of Radiation Protection (CIRP), with an irradiation dose of 10 kGy. Tests of sensory indicators, dust content, and water absorption of the bedding meet the company standards Q / TJKAF003-2023 and GB / T18823-2010.

[0306] 5. Drinking water: High-pressure sterilized tap water is supplied in drinking water bottles. The tap water is inspected by the Taiyuan Monitoring Station of the National Urban Water Supply Quality Monitoring Network (Test Report No.: W20241365). The main control indicators are: color, turbidity, pH, total hardness, iron, manganese, copper, zinc, fluoride, cyanide, arsenic, selenium, mercury, cadmium, chromium, lead, aluminum, total colony count, total coliform group, heat-resistant coliform group, Escherichia coli group, etc., which meet the requirements of the "National Drinking Water Quality Standard" (GB5749-2022); the drinking water bottle should be replaced daily and the drinking water bottle should be cleaned and sterilized under high pressure before use.

[0307] (B) Experimental results and conclusions

[0308] (B-1) General physiological index observation and conclusion

[0309] The mean values ​​of body weight, hematological indicators, serum biochemical indicators, coagulation indicators, organ weight, organ coefficient and other quantitative data were calculated using SPSS25.0 statistical software for each gender. The data were analyzed using the nonparametric Kruskal-Wallis test with P < 0.05. The results of urine and food consumption were analyzed using SPSS 25.0 statistical software with the nonparametric Kruskal-Wallis test. Significance was determined at P < 0.05.

[0310] After administration, the animals were closely observed for toxic reactions and survival / death, including symptoms of poisoning, onset time, duration, recovery period, etc. During the test, the animals' behavior and activities were normal, there were no toxic reactions, no deaths, no abnormal physical signs, and the color and shape of feces were normal.

[0311] Body weight was measured on days 1, 8, 15, 22, 29, 36, 43, and 50. Figure 2 and Figure 3The weight changes of female and male SD rats in the toxicity test after repeated injection of recombinant type III humanized collagen into the tail vein for 4 weeks were recorded. It can be seen that there is no statistical difference in the weight of animals in each administration group compared with the control group; the results show that the drug has no effect on the weight of animals.

[0312] Measure animal feed consumption once during the quarantine period and once a week during the administration and recovery periods. Measurement method: 1 day before, place 200±1.0g of feed in each cage, and weigh the remaining feed in each cage on the second day. Calculation formula for feed consumption:

[0313]

[0314] Table 11, Table 12, Figure 4 and Figure 5 The data from a 4-week repeated toxicity study of recombinant humanized type III collagen injected into the tail vein of SD rats showed changes in feed consumption in both male and female rats. The results showed no statistically significant difference in feed consumption between the treatment groups and the control group during the study. The results indicate that the drug had no effect on the animals' feed consumption.

[0315] Table 9 Changes in body weight of female rats in SD rat tail vein injection of recombinant humanized type III collagen for 4 weeks after repeated administration toxicity test

[0316]

[0317] Table 10 Changes in body weight of male rats in toxicity test of 4-week repeated administration of recombinant humanized type III collagen in tail vein of SD rats

[0318]

[0319] Table 11 Changes in feed consumption in female rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0320]

[0321] Table 12 Changes in feed consumption in male rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0322]

[0323] (B-2) Hematological and urine test results and conclusions

[0324] Instruments included: Konelab PRIME 30 fully automatic biochemistry analyzer (Thermo Fisher, USA), AC9900 electrolyte analyzer (Jiangsu Audicon Medical Technology Co., Ltd.), BH-5190Vet five-differential fully automatic hematology analyzer (Guilin Youlite Medical Electronics Co., Ltd.), XL3200c fully automatic coagulation tester (Beijing Zhongchi Weiye Technology Development Co., Ltd.), H-100 urine analyzer (Changchun Dirui Medical Technology Co., Ltd.), Histocentre 3 paraffin embedding machine (Shandon, UK), Finesse 325 paraffin sectioner (Shandon, UK), Excelsior fully automatic tissue dehydrator (Shandon, UK), BX53 fluorescence microscopy system (Olympus, Japan), YZ11 ophthalmoscope (Suzhou Liuliu Vision Technology Co., Ltd.). All other reagents and detection methods were performed according to conventional procedures in the field.

[0325] Blood index determination: once at the end of administration and once at the end of recovery period. Ten animals of each sex were taken from each group at the end of administration. All remaining animals were taken at the end of recovery period (4 weeks after drug withdrawal). (30 mg / kg), anesthesia was given by subcutaneous injection in the neck, and blood was collected through the abdominal aorta using a negative pressure tube. The blood collected using a sodium citrate anticoagulant tube was separated into plasma and then the coagulation parameters (including plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT)) were measured. A small amount of blood collected using an EDTAK2 anticoagulant tube was taken and stained with brilliant tar blue to measure the reticulocyte count (Ret), and the rest was used to measure hematological parameters (including white blood cell count (WBC), red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), Platelets (PLT), mean platelet volume (MPV), hemoglobin (HB), neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO), basophils (BA)). Blood collected using separation gel coagulation tubes was coagulated and then separated into serum for the determination of serum biochemical indicators (including albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), calcium (Ca), creatine kinase (CK), creatinine (CR), glucose (GLU), lactate dehydrogenase (LDH), total bilirubin (TBIL), serum total cholesterol (TCHO), triglycerides (TG), serum total protein (TP), blood uric acid (UA), and blood urea nitrogen (UREA)).

[0326] Urine analysis: 10 animals of each sex were collected from each group at the end of administration and at the end of the recovery period (4 weeks after drug withdrawal). Urine was collected from animals using metabolic cages, and the urine volume was recorded. The color and turbidity were visually inspected. Urine urobilinogen (UBG), bilirubin (BIL), ketone bodies (KET), occult blood (BLD), protein (PRO), nitrite (NIT), white blood cells (LEU), glucose (GLU), urine specific gravity (SG), and pH were measured using a test strip method (produced by Dirui Medical Technology Co., Ltd., batch number: 20240915) and an H-100 urine analyzer.

[0327] Experimental conclusion:

[0328] The results of hematological index testing are shown in Tables 13 and 14. There were no statistically significant differences in any hematological index between the female mice in each administration group and the control group at each examination stage. The results indicate that the drug had no effect on the hematological indexes of female mice. At the end of administration, hematological index testing of male mice showed an increase in EO% in the 130 mg / kg and 65 mg / kg groups compared to the control group (P < 0.05, P < 0.01), and an increase in BA% in the 130 mg / kg group compared to the control group (P < 0.05). These parameters remained normal at the end of the recovery period, and were determined to be transient changes without toxicological significance.

[0329] The results of serum biochemical indices are shown in Tables 15 and 16. At the end of administration, ALT levels in female mice in the 65 mg / kg group were higher than in the control group (P < 0.05), and TBIL levels were lower than in the control group (P < 0.01). These changes were transient and occurred in both sexes within a single dose group. There were no decreases in the associated indices AST, ALP, or TP. Furthermore, no abnormalities were observed at the end of the recovery period. Therefore, the changes in TBIL and ALT were considered to be toxicologically insignificant. There were no statistically significant differences in serum biochemical indices between the animals in each administration group and the control group at all other examination stages.

[0330] The results of the serum electrolyte index changes are shown in Tables 17 and 18. There were no statistically significant differences in the serum electrolyte indexes of the animals in each treatment group compared with the control group at each examination stage. The results showed that the drug had no effect on the serum electrolyte indexes of the animals.

[0331] The results of the coagulation index change test are shown in Tables 19 and 20. There was no statistical difference in the coagulation indexes of the animals in each treatment group compared with the control group at each examination stage. The results showed that the drug had no effect on the animal coagulation indexes.

[0332] The results of urine parameter changes are shown in Tables 21 and 22. At the end of administration, BLD in female mice in the 32.5 mg / kg group was higher than in the control group (P < 0.05), and LEU in male mice in the 130 mg / kg group was higher than in the control group (P < 0.05). These changes in parameters remained normal at the end of the recovery period and were considered transient. There were no statistically significant differences in urine parameters between the remaining treatment groups and the control group. At the end of the recovery period, there were no statistically significant differences in urine parameters between the treatment groups and the control group.

[0333] Table 13 Changes in hematological parameters in female rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0334]

[0335] Table 14 Changes in hematological parameters in male rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0336]

[0337] Table 15 Changes in serum biochemical status of female rats in toxicity test of 4-week repeated administration of recombinant humanized type III collagen in tail vein of SD rats

[0338]

[0339] Table 16 Changes in serum biochemical status of male rats after 4-week repeated administration of recombinant humanized type III collagen in tail vein injection in SD rats

[0340]

[0341] Table 17 Changes in serum electrolytes in female rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0342]

[0343] Table 18 Changes in serum electrolytes in male rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0344]

[0345] Table 19 Changes in coagulation parameters in female rats after 4-week repeated administration of recombinant humanized type III collagen in the tail vein of SD rats

[0346]

[0347] Table 20 Changes in coagulation parameters in male rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0348]

[0349] Table 21 Changes in urine parameters in female rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0350]

[0351] Table 22 Changes in urine parameters in male rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0352]

[0353] (B-3) Histopathological observation results and conclusions

[0354] At the end of the administration and recovery periods, the animals in each group were euthanized by subcutaneous injection in the neck. The animals were anesthetized, exsanguinated via the abdominal aorta, and then sacrificed. The tissues and organs were visually inspected for abnormalities. The body surface, subcutaneous tissues, abdominal cavity, and thoracic cavity, as well as the location, color, size, and texture of major tissues and organs were examined. Organs were removed, weighed, and the organ coefficient was calculated.

[0355] Organs include: brain, heart, liver, spleen, lungs, kidneys, adrenal glands, thymus, testicles, epididymis, uterus, and ovaries.

[0356] Organ coefficient calculation formula:

[0357]

[0358] The various tissues and organs were fixed and preserved, and the fixed tissues were cut into blocks, dehydrated with alcohol step by step, embedded in paraffin, sectioned, stained with hematoxylin-eosin (HE), and examined under a light microscope for pathological judgment.

[0359] Experimental conclusion:

[0360] The results of organ weight testing are shown in Tables 23 and 24. At the end of administration, the liver weight of female rats in the 65 mg / kg group was increased compared to the control group (P < 0.01), and the epididymal weight of male rats in the 65 mg / kg group was increased compared to the control group (P < 0.05). These changes were not dose-dependent and were determined to be non-toxicologically significant. There were no statistically significant differences in the organ weights of the animals in the remaining administration groups compared to the control group. At the end of the recovery period, there were no statistically significant differences in the organ weights of the animals in the administration groups compared to the control group. These results indicate that the drug has no effect on the organ weights of the animals.

[0361] The results of organ coefficient testing are shown in Tables 25 and 26. At the end of the administration, the liver coefficient of female mice in the 65 mg / kg group was higher than that of the control group (P < 0.01). This change had no dose-effect relationship and was determined to be of no toxicological significance. There were no statistically significant differences in the organ coefficients of the animals in the other administration groups compared with those in the control group. At the end of the recovery period, the spleen coefficient of female mice in the 130 mg / kg group was higher than that of the control group (P < 0.05), the ovary coefficient of female mice in the 32.5 mg / kg group was higher than that of the control group (P < 0.05), and the testis coefficient of male mice in the 32.5 mg / kg group was lower than that of the control group (P < 0.05). The above-mentioned changes were single-sex and single-dose group changes, and there was no dose-effect relationship, so it was determined to be of no toxicological significance. There were no statistically significant differences in the organ coefficients of the animals in the other administration groups compared with those in the control group.

[0362] Gross anatomical and histological examinations revealed mild bilateral thyroid enlargement in one female rat in the medium-dose group at the end of the administration period. No gross pathological changes were observed on the body surface, subcutaneous tissue, thoracic cavity, abdominal cavity, or tissues / organs in the remaining animals. At the end of the recovery period, anatomical examinations revealed mild bilateral thyroid enlargement in one rat in both the control and administration groups. No gross pathological changes were observed on the body surface, subcutaneous tissue, thoracic cavity, abdominal cavity, or tissues / organs in the remaining animals.

[0363] Histopathological examination revealed localized vascular damage at the administration site in both the control and high-dose groups at the end of the four-week treatment period. No significant abnormalities were observed at the administration site at the end of the recovery period. This damage was attributed to the repeated intravenous administration procedure and was unrelated to the drug.

[0364] In summary, the sporadic, irregular lesions found during gross anatomical examination and in the control and high-dose groups at the end of administration and recovery were not considered to be related to the test substance. Under the conditions of this study, compared with the control group, SD rats injected with recombinant humanized type III collagen via the tail vein for four weeks showed no toxic pathological changes related to the test substance.

[0365] Table 23 Changes in organ weights of female rats in a toxicity study of 4-week repeated administration of recombinant humanized type III collagen in the tail vein of SD rats

[0366]

[0367] Table 24 Changes in organ weights of male rats in a toxicity study of 4-week repeated administration of recombinant humanized type III collagen in the tail vein of SD rats

[0368]

[0369] Table 25 Changes in organ coefficients in female rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into the tail vein of SD rats

[0370]

[0371] Table 26 Changes in organ coefficients in male rats after 4-week repeated toxicity test of recombinant humanized type III collagen injected into tail vein of SD rats

[0372]

[0373] During the trial, the animals showed normal behavior and physical signs, and their feces were normal in color and shape. Examinations at each stage showed that the drug had no effect on the animals' ophthalmology, body weight, food intake, blood, serum electrolytes, and coagulation indicators.

[0374] At the end of the administration, the TBIL level of female mice in the 65 mg / kg group was lower than that in the control group (P < 0.01), and the ALT level was higher than that in the control group (P < 0.05). The BLD level of female mice in the 32.5 mg / kg group was higher than that in the control group (P < 0.05), the liver weight and coefficient of female mice in the 65 mg / kg group were higher than those in the control group (P < 0.01), and the epididymal weight of male mice in the 65 mg / kg group was higher than that in the control group (P < 0.05). At the end of the recovery period, the ovarian coefficient of female mice in the 32.5 mg / kg group was higher than that in the control group (P < 0.05), and the testicular coefficient of male mice in the 32.5 mg / kg group was lower than that in the control group (P < 0.05). There was no dose-response relationship between the changes in these indicators, and they were judged to be of no toxicological significance.

[0375] At the end of administration, the LEU of male rats in the 130 mg / kg group was higher than that in the control group (P < 0.05). At the end of the recovery period, the spleen coefficient of female rats in the 130 mg / kg group was higher than that in the control group (P < 0.05). These changes were all single-dose and single-sex changes. No histopathological changes in related organs were observed, and these changes were all mild. Therefore, they were determined to be of no toxicological significance. Histopathological examination results showed that SD rats did not develop toxic pathological changes related to the test substance after 4 weeks of tail vein injection of recombinant humanized type III collagen.

[0376] In summary, the animals showed normal behavior and physical appearance after administration, with normal fecal color and shape. The drug had no effect on ophthalmology, body weight, food intake, serum electrolytes, coagulation, serum biochemistry, urine, organ weights, or organ coefficients. Under these experimental conditions, the toxicity test of recombinant humanized type III collagen in SD rats was repeated for four weeks after tail vein injection. The minimum no-toxic dose (ADR) was 130 mg / kg.

Claims

1. An injection, The injection comprises recombinant humanized type III collagen, wherein the amino acid sequence of the recombinant humanized type III collagen comprises n repeats of the sequence shown in SEQ ID NO. 1, where n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeats are directly connected; and when the injection is administered within one administration cycle, the total administration amount of the recombinant humanized type III collagen is less than or equal to 3640 mg / kg based on the body weight of the subject; Preferably, the amino acid sequence of the recombinant humanized type III collagen comprises the amino acid sequence shown in SEQ ID NO.2; Preferably, the injection is free of cytotoxicity, organ toxicity, blood toxicity and immunotoxicity.

2. The injection according to claim 1, characterized in that The injection is an injection for single-dose administration; Preferably, in the single-dose injection, when the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 90-110 mg / mL; Preferably, when the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 2000 mg / kg based on the body weight of the subject; More preferably, when the subject of the single-dose injection is a human, and the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 162 mg / kg based on the body weight of the subject.

3. The injection according to claim 1, characterized in that The injection is an injection for multiple doses; Preferably, in the multi-dose injection, when the multi-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 6-29 mg / mL; Preferably, when the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 130 mg / kg based on the body weight of the subject; More preferably, when the subject of the multi-dose injection is a human, and the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 21 mg / kg based on the body weight of the subject; Preferably, when the injection is administered in multiple doses, the administration frequency is once a day and the administration cycle is 1 to 5 weeks.

4. The injection according to any one of claims 1 to 3, characterized in that The injection is an intravenous injection, a subcutaneous injection, an intramuscular injection or an intraperitoneal injection; Preferably, the injection is an intravenous injection or a subcutaneous injection.

5. Use of recombinant humanized type III collagen in the preparation of injections, The injection comprises recombinant humanized type III collagen, wherein the amino acid sequence of the recombinant humanized type III collagen comprises n repeats of the sequence shown in SEQ ID NO. 1, where n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeats are directly connected; and when the injection is administered within one administration cycle, the total administration amount of the recombinant humanized type III collagen is less than or equal to 3640 mg / kg based on the body weight of the subject; Preferably, the amino acid sequence of the recombinant humanized type III collagen comprises the amino acid sequence shown in SEQ ID NO.2; Preferably, the injection is used for any one of the following uses (a)-(e): (a) preventing and / or treating ocular inflammation; (b) preventing and / or treating gastric cancer; (c) prevention and / or treatment of breast diseases; (d) prevention and / or treatment of ovarian diseases; (e) preventing and / or inhibiting and / or treating cervical cancer; (f) prevention and / or treatment of cardiovascular disease, repair of vascular damage, or prevention and / or treatment of vascular damage; (g) inhibit esophageal squamous cell carcinoma; Preferably, the injection is free of cytotoxicity, organ toxicity, blood toxicity and immunotoxicity.

6. The use according to claim 5, characterized in that The injection is an injection for single-dose administration; Preferably, in the single-dose injection, when the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 90-110 mg / mL; Preferably, when the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 2000 mg / kg based on the body weight of the subject; More preferably, when the subject of the single-dose injection is a human, and the injection is administered in a single dose, the single administration amount of the recombinant humanized type III collagen is less than or equal to 162 mg / kg based on the body weight of the subject.

7. The use according to claim 5, characterized in that The injection is an injection for multiple doses; Preferably, in the multi-dose injection, when the multi-dose injection is a liquid, the concentration of the recombinant humanized type III collagen is 6-29 mg / mL; Preferably, when the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 130 mg / kg based on the body weight of the subject; More preferably, when the subject of the multi-dose injection is a human, and the injection is administered in multiple doses, the single administration amount of the recombinant humanized type III collagen is less than or equal to 21 mg / kg based on the body weight of the subject; Preferably, when the injection is administered in multiple doses, the administration frequency is once a day and the administration cycle is 1 to 5 weeks.

8. The use according to any one of claims 5 to 7, characterized in that The injection is an intravenous injection, a subcutaneous injection, an intramuscular injection or an intraperitoneal injection; Preferably, the injection is an intravenous injection or a subcutaneous injection.

9. A medicine box, The medicine kit comprises a syringe pre-filled with the injection according to any one of claims 1 to 4.

10. The medicine box according to claim 9, characterized in that The kit satisfies the following conditions (i) or (ii): (i) When the injection is a single-dose injection, the medicine kit contains only one syringe I prefilled with the single-dose injection, and the total amount of the recombinant humanized type III collagen contained in the medicine kit is less than or equal to 2000 mg / kg based on the body weight of the subject; preferably, when the single-dose injection is a liquid, the concentration of the recombinant humanized type III collagen in each syringe I is 90-100 mg / mL; (ii) When the injection is a multi-dose injection, the medicine kit contains multiple syringes II of the same specifications pre-filled with the multi-dose injection, and the total amount of the recombinant humanized type III collagen contained in the medicine kit is less than or equal to 3640 mg / kg based on the body weight of the subject, and the amount of the recombinant humanized type III collagen contained in each syringe II is less than or equal to 130 mg / kg; preferably, when the multi-dose injection is a liquid, the concentration of the recombinant humanized type III collagen in each syringe II is 6-29 mg / mL; Preferably, when the subject of administration of the drug kit is a human, the drug kit satisfies the following conditions (iii) or (iv): (iii) When the injection is a single-dose injection, the medicine kit contains only one syringe I prefilled with the single-dose injection, and the total amount of the recombinant type III humanized collagen contained in the medicine kit is less than or equal to 162 mg / kg based on the body weight of the subject; preferably, when the single-dose injection is a liquid, the concentration of the recombinant type III humanized collagen in each syringe I is less than or equal to 30 mg / mL; preferably, when the single-dose injection is a lyophilized preparation, the content of the recombinant type III humanized collagen in each syringe I is 9720 mg based on the body weight of the subject being 60 kg / person; (iv) When the injection is a multi-dose injection, the medicine kit contains a plurality of syringes II of the same specifications pre-filled with the multi-dose injection, and the total amount of the recombinant type III humanized collagen contained in the medicine kit is less than or equal to 590 mg / kg, and the amount of the recombinant type III humanized collagen contained in each syringe II is less than or equal to 21 mg / kg, based on the body weight of the subject. Preferably, when the multi-dose injection is a liquid, the concentration of the recombinant type III humanized collagen in each syringe II is 6-29 mg / mL. Preferably, when the multi-dose injection is a lyophilized preparation, based on the body weight of the subject being 60 kg / person, the content of the recombinant type III humanized collagen in each syringe II is 1260 mg. More preferably, when the medicine kit meets condition (ii) or (iv), the medicine kit contains 7-35 syringes II; even more preferably, when the medicine kit meets condition (ii) or (iv), the medicine kit contains 7, 14, 21 or 28 syringes II.

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