Pharmaceutical composition containing cycloserine and lysine modified amino tetraphenylporphyrin and application thereof

Abstract

The invention discloses a pharmaceutical composition containing cycloserine or a stereoisomer of the cycloserine or a pharmaceutically acceptable salt of the cycloserine and lysine modified amino tetraphenyl porphyrin or a stereoisomer of the lysine modified amino tetraphenyl porphyrin or a pharmaceutically acceptable salt of the lysine modified amino tetraphenyl porphyrin. The preparation method comprises the following steps: mixing cycloserine or a stereoisomer of the cycloserine or a pharmaceutically acceptable salt of the cycloserine and lysine-modified amino tetraphenyl porphyrin or a stereoisomer of the lysine-modified amino tetraphenyl porphyrin or a pharmaceutically acceptable salt of the lysine-modified amino tetraphenyl porphyrin according to a mass ratio of 512: 1-1: 2000; or a pharmaceutically acceptable carrier is added to prepare a clinically acceptable dosage form. The invention further provides application of the pharmaceutical composition in preparation of bacterial infection resisting drugs and tissue repair promoting drugs. The pharmaceutical composition disclosed by the invention has a synergistic antibacterial effect and is good in biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin and its use, and more particularly to a pharmaceutical composition prepared by combining the antibiotic cycloserine and the photosensitizer lysine-modified aminotetraphenylporphyrin, as well as the use of the pharmaceutical composition in preparing drugs for treating bacterial infections and trauma. Background Art

[0002] Bacterial infections caused by opportunistic or invasive pathogens are major infectious diseases worldwide, causing numerous diseases, including pneumonia, periodontitis, tuberculosis, conjunctivitis, gastroenteritis, and sepsis, posing a serious threat to human health. These infections not only impact patient quality of life and survival, but also place a significant burden on public health systems.

[0003] According to the World Health Organization (WHO), bacterial resistance is becoming increasingly serious and has become a major challenge to global public health. With the widespread use of antibiotics, inappropriate use has also increased, and bacterial resistance is constantly increasing. In some countries and regions, the emergence and spread of multidrug-resistant (MDR) bacteria has become a serious problem. These MDR bacteria are resistant to multiple antibiotics, rendering previously effective treatments ineffective, leading to increased treatment costs, prolonged treatment cycles, and even incurable conditions.

[0004] Currently, antibiotics are the mainstay of treatment for bacterial infections. They work by destroying bacterial cell walls, inhibiting protein synthesis, and interfering with bacterial metabolism. However, the efficacy and safety of existing antibiotics are limited by the following issues:

[0005] ① Side effects: Antibiotic treatment may cause a series of adverse reactions, such as allergic reactions, gastrointestinal discomfort, liver and kidney function damage, etc.

[0006] ② Decreased efficacy: With the increase in bacterial resistance, the efficacy of some commonly used antibiotics has gradually decreased and has no therapeutic effect on certain bacterial strains. For multidrug-resistant bacterial infections, the failure rate of existing antibiotics has increased significantly, seriously threatening patients' lives.

[0007] ③ Double infection: Large-scale use of antibiotics can lead to intestinal flora imbalance, antibiotic-associated diarrhea, and aggravate the condition.

[0008] ④ Development of bacterial resistance: Long-term and excessive use of antibiotics has accelerated the development of bacterial resistance, leading to the continuous emergence of new resistant strains.

[0009] Against this backdrop, the search for new antibacterial drugs, particularly those with novel, unconventional mechanisms of action that overcome the limitations of existing antibiotics, has become a research hotspot in the medical field. Photodynamic antimicrobial chemotherapy (PACT), as a novel, unconventional mechanism and an antibacterial alternative to antibiotic therapy, has attracted widespread attention in recent years. The basic principle of PACT is the photochemical reaction between a photosensitizer (PS), oxygen, and light. Compared to traditional antibiotic therapy, PACT offers the following advantages:

[0010] ① Not prone to drug resistance: The bactericidal mechanism of PACT mainly relies on the reactive oxygen species (such as singlet oxygen and free radicals) produced by the photosensitizer under light to cause oxidative damage to microbial cells. This process is different from the bactericidal mechanism of antibiotics, so bacteria are not prone to drug resistance.

[0011] ② High selectivity: PACT utilizes the photodynamic effect to kill microorganisms only within the area illuminated by light of a specific wavelength, minimizing damage to surrounding normal tissue. Traditional antibiotics, on the other hand, may be widely distributed throughout the body, affecting unspecific microbial populations and sometimes causing side effects on normal tissues.

[0012] ③ Broad spectrum: PACT is effective against a wide range of Gram-positive and Gram-negative bacteria, including some drug-resistant bacteria. Some traditional antibiotics may not be as effective against certain types of bacteria.

[0013] ④ Local treatment with minimal toxicity and side effects: PACT is typically a local treatment, with the photosensitizer acting at a specific site, resulting in minimal systemic side effects. In contrast, traditional antibiotic treatment may require systemic administration, sometimes leading to systemic side effects.

[0014] ⑤ Promote wound healing: While killing bacteria, PACT also helps the wound healing process due to its local treatment characteristics, while traditional antibiotic treatment may sometimes be detrimental to wound healing.

[0015] Cycloserine (D-Cycloserine, DCS) is an anti-tuberculosis drug that inhibits cell wall synthesis and also has good antibacterial effects against other bacteria. Our recent research has discovered that the lysine-modified aminotetraphenylporphyrin compound 5,10,15,20-tetrakis{4-[(S)-2,6-diaminohexanamido]phenyl}porphyrin (LD4) has a unique chemical structure and exhibits significant photosensitizing antibacterial activity against a variety of bacteria, but its effectiveness against Mycobacterium tuberculosis is not ideal.

[0016] In order to solve this problem, the present invention provides a novel photodynamic synergistic antibacterial infection pharmaceutical composition, which is composed of a 5,10,15,20-tetrakis{4-[(S)-2,6-diaminohexanamido]phenyl}porphyrin compound and cycloserine. Through this unique drug combination, we can not only give full play to the broad-spectrum antibacterial properties of cycloserine, but also discover its role as a sensitizer of the compound 5,10,15,20-tetrakis{4-[(S)-2,6-diaminohexanamido]phenyl}porphyrin for Mycobacterium tuberculosis. This synergistic effect significantly improves the antibacterial activity and reduces the dosage of the drug, thereby effectively reducing the risk of drug resistance caused by cycloserine in bacteria. Therefore, the pharmaceutical composition provides new possibilities for the treatment of bacterial infections, especially for those Mycobacterium tuberculosis infections that are not sensitive to single photodynamic therapy, and its efficacy is significantly enhanced. Summary of the Invention

[0017] In view of the problem that the compound 5,10,15,20-tetrakis{4-[(S)-2,6-diaminohexanamido]phenyl}porphyrin (LD4) in the prior art is insufficiently effective in combating Mycobacterium tuberculosis infection, the present invention proposes an innovative antibacterial infection pharmaceutical composition and a method for preparing the same. The pharmaceutical composition of the present invention is characterized in that it comprises cycloserine (DCS) and 5,10,15,20-tetrakis{4-[(S)-2,6-diaminohexanamido]phenyl}porphyrin (LD4), and the combination of the two exhibits synergistic efficacy in the field of antibacterial infection. For the first time, it has been demonstrated that the combined use of DCS and LD4 can significantly enhance the efficacy against bacteria such as Mycobacterium tuberculosis. The pharmaceutical composition provided by the present invention opens up new perspectives and paths for the synergistic medication strategy of antibacterial infection drugs.

[0018] The object of the present invention is to overcome the deficiencies of the prior art and to provide a pharmaceutical composition for use in combating bacterial infections, comprising cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin.

[0019] A second object of the present invention is to provide a pharmaceutical composition for use in preparing antibacterial infection drugs containing cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin for antibacterial infection.

[0020] A third object of the present invention is to provide a pharmaceutical composition containing cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin for use in the preparation of a drug for promoting cell proliferation and tissue repair.

[0021] The purpose of the present invention is mainly achieved through the following technical solutions:

[0022] The present invention provides a pharmaceutical composition for resisting bacterial infection, comprising cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin. The pharmaceutical composition can be prepared by mixing cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt with lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin in a mass ratio of 512:1 to 1:2000, or adding a pharmaceutically acceptable carrier to prepare a clinical The pharmaceutically acceptable dosage forms include tablets, pills, capsules, granules, suspensions, aerosols, oral solutions, ointments, gels, patches, injections, infusions, freeze-dried powder injections, and sustained-release preparations. The pharmaceutically acceptable carriers include conventional diluents (such as water for injection, microcrystalline cellulose, etc.), fillers (such as mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, petrolatum, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO, etc.), binders (such as carbomer, gum arabic, starch, cellulose, gelatin, polyvinyl pyrrolidone, polyacrylamide, etc.), disintegrants (such as carboxymethyl at least one of starch sodium, cross-linked sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, etc.), lubricant (such as talc, magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silicon dioxide, micro-powdered silica, etc.), wetting agent (such as propylene glycol, glycerin, ethanol, etc. at least one), stabilizer (such as disodium edetate, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, ethanolamine, sodium bicarbonate, sodium acetate, niacinamide, vitamin C, etc. at least one), osmotic pressure regulator (such as sodium chloride, glucose, etc. at least one), pH regulator (such as triethanolamine, sodium hydroxide, sodium citrate, etc. at least one), preservative (such as chlorobutanol), , paraben, ethylparaben, benzalkonium bromide, etc.), the above-mentioned excipients can be commonly used in dosages, mixed with cycloserine or its stereoisomer or its pharmaceutically acceptable salt and lysine-modified aminotetraphenylporphyrin or its stereoisomer or its lysine-modified aminotetraphenylporphyrin in commonly used ratios, when the dosage of cycloserine or its stereoisomer or its pharmaceutically acceptable salt and lysine-modified aminotetraphenylporphyrin or its stereoisomer or its lysine-modified aminotetraphenylporphyrin is determined, the ratio between the pharmaceutical excipients can be appropriately adjusted as needed, the structure of the cycloserine is The structure of the lysine-modified aminotetraphenylporphyrin is

[0023] The present invention provides a pharmaceutical composition for resisting bacterial infection, comprising cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin. The mass ratio of the cycloserine or the stereoisomer of cycloserine or the pharmaceutically acceptable salt to the lysine-modified aminotetraphenylporphyrin or the stereoisomer of lysine-modified aminotetraphenylporphyrin or the pharmaceutically acceptable salt to methicillin-resistant Staphylococcus aureus is 512:1 to 1:8; the mass ratio of the two to Pseudomonas aeruginosa is 512:1 to 1:8; the mass ratio of the two to Escherichia coli is 512:1 to 1:8; and the mass ratio of the two to Mycobacterium tuberculosis is 1:2000 to 256:125.

[0024] The present invention provides an antibacterial infection pharmaceutical composition containing cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin in the preparation of an antibacterial infection drug.

[0025] The present invention provides a pharmaceutical composition containing cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin, and its use in preparing a drug for promoting cell proliferation and tissue repair.

[0026] Beneficial effects of the present invention:

[0027] (1) The present invention discovered for the first time that a pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin can significantly inhibit the growth of methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Mycobacterium tuberculosis, has a synergistic antibacterial effect, and the antibacterial effect is better than that of a single drug, providing a new solution for the treatment of bacterial infections.

[0028] (2) The present invention demonstrates through in vitro cell and hemolysis experiments that the pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin has high safety and good biocompatibility for normal tissue cells at effective drug concentrations, and has good application prospects in the treatment of bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is a heat map of the synergistic antibacterial effect of the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin of Examples 24-27 of the present invention.

[0030] Figure 2 This is the effect of the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin of Example 28 of the present invention on the growth of mouse brain hemangioendothelioma cells.

[0031] Figure 3 This is the effect of the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin of Example 28 of the present invention on the growth of mouse embryonic fibroblasts.

[0032] Figure 4 This is the effect of the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin of Example 28 of the present invention on the growth of mouse mononuclear macrophages.

[0033] Figure 5 This is the effect of the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin of Example 28 of the present invention on the growth of human hepatocytes.

[0034] Figure 6 These are the results of a hemolytic experiment on the pharmaceutical composition containing cycloserine- and lysine-modified aminotetraphenylporphyrin according to Example 29 of the present invention.

[0035] Figure 7 These are the results of a bacterial resistance experiment of the pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin according to Example 30 of the present invention.

[0036] Figure 8 This is the therapeutic effect (wound healing rate, wound colony count and blood cell count) of the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin of Example 31 of the present invention on bacterially infected wounds in mice.

[0037] Figure 9 This is a bacterial scanning electron microscope image showing the antibacterial effect of the pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin according to Example 32 of the present invention.

[0038] Figure 10 This is a transmission electron microscopy image of bacteria showing the antibacterial effect of cycloserine in Example 33 of the present invention. Specific implementation plan

[0039] The specific embodiments of the present invention are further described in detail below in conjunction with the examples and drawings. The purpose is only to better understand the content of the present invention but not to limit the scope of protection of the present invention. Reagents not provided with the source in the present invention are all commercially available reagents, and methods not described in detail are all conventional well-known experimental methods. The present invention is a pharmaceutical composition for antibacterial infection containing cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin, wherein the structure of the cycloserine is The structure of the lysine-modified aminotetraphenylporphyrin is

[0040] Example 1 Pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin

[0041] Cycloserine and lysine-modified aminotetraphenylporphyrin are mixed in mass ratios of 512:1, 256:1, 128:1, 64:1, 32:1, 16:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, and 1:8, respectively.

[0042] Example 2 Pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin

[0043] Cycloserine and lysine-modified aminotetraphenylporphyrin are mixed in mass ratios of 1:2000, 1:1000, 1:500, 1:250, 1:125, 2:125, 4:125, 8:125, 16:125, 32:125, 64:125, 128:125, and 256:125, respectively.

[0044] Example 3 Pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin

[0045] Mix cycloserine and lysine-modified aminotetraphenylporphyrin in a mass ratio of 8:1 and dissolve in PBS to obtain a pharmaceutical composition. Specifically, mix 31.25 mg of cycloserine and 3.91 mg of lysine-modified aminotetraphenylporphyrin and dissolve in 1 L of PBS to obtain a pharmaceutical composition with a concentration of 31.25 μg / mL cycloserine and 3.91 μg / mL lysine-modified aminotetraphenylporphyrin. Mix 62.50 mg of cycloserine and 7.81 mg of lysine-modified aminotetraphenylporphyrin and dissolve in 1 L of PBS to obtain a pharmaceutical composition with a concentration of 62.50 μg / mL cycloserine and 7.81 μg / mL lysine-modified aminotetraphenylporphyrin. 125 mg of cycloserine and 15.63 mg of lysine-modified aminotetraphenylporphyrin were mixed and dissolved in 1 L of PBS to obtain a pharmaceutical composition with a concentration of 125 μg / mL cycloserine and 15.63 μg / mL lysine-modified aminotetraphenylporphyrin.

[0046] Example 4 Tablets

[0047] 250.00 mg of cycloserine and 0.49 mg of lysine-modified aminotetraphenylporphyrin were mixed, and 500 mg of starch, 100 mg of magnesium stearate, 200 mg of carboxymethyl cellulose, 2.7 g of ethanol (70% by volume), 2.5 g of microcrystalline cellulose, and 3.2 g of corn starch were added. The mixture was thoroughly stirred to form wet granules, which were then dried at 60-70°C for 2-4 hours and compressed into tablets. The excipients used in the tableting process included magnesium sulfate, corn starch, and talc.

[0048] Example 5 Tablets

[0049] Take 250.00 mg of cycloserine and 0.98 mg of lysine-modified aminotetraphenylporphyrin, mix them, add 100 mg of hydroxypropyl cellulose, 100 mg of sodium carboxymethyl starch, and 240 mg of starch in equal and increasing amounts, mix them evenly, and then dry granulate them; add 30 mg of magnesium stearate, mix them evenly, and press them into tablets.

[0050] Example 6 Pills

[0051] Mix 250.00 mg of cycloserine and 1.95 mg of lysine-modified aminotetraphenylporphyrin, add 1.8 g of polyethylene glycol, heat to 90°C to melt, so that the raw materials and excipients are fully mixed, drop the melt into dimethyl silicone oil receiving liquid, and cool naturally to form pellets.

[0052] Example 7 Capsules

[0053] Take 250.00 mg of cycloserine and 3.91 mg of lysine-modified aminotetraphenylporphyrin, mix them, add 7 mg of magnesium stearate, 70 mg of carboxymethyl cellulose, and 160 mg of microcrystalline cellulose, stir and mix thoroughly to form wet granules, dry the prepared wet granules directly at 60-70°C for 2-4 hours, and then fill them into empty capsule shells to obtain the product.

[0054] Example 8 Granules

[0055] Mix 250.00 mg of cycloserine and 7.81 mg of lysine-modified aminotetraphenylporphyrin, add 200 mg of magnesium stearate, 1.8 g of carboxymethyl cellulose, and 2 g of microcrystalline cellulose, stir thoroughly, sieve through a 12-14 mesh sieve, and granulate. Dry at 60-70°C for 2-4 hours to obtain the product.

[0056] Example 9 Suspension

[0057] 250.00 mg of cycloserine and 15.63 mg of lysine-modified aminotetraphenylporphyrin were mixed, and 42 g of pure water, 39 g of glycerol, 7 g of sorbitol and 10 g of propylene glycol were added to prepare a suspension uniformly dispersed in an aqueous carrier.

[0058] Example 10 Aerosol

[0059] Mix 250.00 mg of cycloserine and 31.25 mg of lysine-modified aminotetraphenylporphyrin, dissolve in propylene glycol, add 500 mg of vitamin C and 0.2 mL of chlorobutanol, mix to form a transparent solution, and charge into a pressure vessel together with compressed nitrogen according to specifications.

[0060] Example 11 Aerosol

[0061] Mix 125.00 mg of cycloserine and 0.49 mg of lysine-modified aminotetraphenylporphyrin, add 2 g of glycerol, 1 g of propylene glycol, 10 mg of Tween 80, 20 mg of menthol and 1 g of pure water, stir evenly, and put into a container with a nozzle to obtain the product.

[0062] Example 12 Oral Liquid

[0063] 125.00 mg of cycloserine and 0.98 mg of lysine-modified aminotetraphenylporphyrin were mixed, 74.5 g of pure water and 25 g of edible sugar were added, stirred to dissolve, and the mixture was divided and sterilized to obtain the product.

[0064] Example 13 Ointment (Oily Base)

[0065] Heat 5 g of vaseline and 4 g of liquid paraffin to melt, filter while hot to remove impurities, then heat to 150°C for about 1 hour to sterilize and remove moisture, and cool naturally; take 125.00 mg of cycloserine and 1.95 mg of lysine-modified aminotetraphenylporphyrin and place them in a mortar, add appropriate amount of liquid paraffin and grind into a paste, add vaseline in batches, and grind until evenly mixed.

[0066] Example 14 Ointment (o / w type emulsifier base)

[0067] Place 1g of stearic acid, 700mg of glyceryl monostearate, 700mg of white petrolatum, and 600mg of liquid paraffin in a beaker and heat to approximately 80°C on a water bath. Stir to melt. Place 5mg of Tween 80 and 6mL of distilled water in another small beaker and heat to approximately 80°C on a water bath. Stir thoroughly. At the same temperature, add the aqueous phase to the oil phase in a thin stream and stir continuously in the same direction on a water bath until a milky white semisolid forms. Continue stirring at room temperature until nearly condensed. Place 125.00mg of cycloserine and 3.91mg of lysine-modified aminotetraphenylporphyrin on an ointment plate and in a mortar. Add the prepared O / W emulsifier base in portions and grind until thoroughly combined.

[0068] Example 15 Gel

[0069] Sprinkle 1g of Carbomer 934 in portions into an appropriate amount of distilled water and allow it to slowly swell. Add 10g of glycerol and stir to form a transparent gel matrix. Dissolve menthol in ethanol and 1g of boric acid in an appropriate amount of water. Combine the above and stir thoroughly. While stirring, sprinkle in 125.00mg of cycloserine and 7.81mg of lysine-modified aminotetraphenylporphyrin. Adjust the pH to 4.5-5.5 with triethanolamine. Add water to 100mL, stir thoroughly, and aliquot.

[0070] Example 16 Patch

[0071] Weigh 4 g of carbomer, propylene glycol, Tween-80, and distilled water in a ratio of 10:20:3:12, heat to 60°C, and stir for 20 minutes. Add 125.00 mg of cycloserine and 15.63 mg of lysine-modified aminotetraphenylporphyrin, and mix well. Apply the mixture onto the lining cloth in batches until the desired thickness is reached, air-dry or oven-dry, and cut according to the preparation specifications.

[0072] Example 17 Sustained-release tablets

[0073] Take 125.00 mg of cycloserine and 31.25 mg of lysine-modified aminotetraphenylporphyrin, add 700 mg of hydroxypropyl methylcellulose, 2.5 g of carboxymethyl cellulose, 3.7 g of ethanol (70% by volume), 1 g of magnesium stearate, etc., stir and mix thoroughly to form wet granules, dry at 60-70°C for 2-4 hours, and press into tablets.

[0074] Example 18 sustained-release tablets

[0075] Take 62.50 mg of cycloserine and 0.49 mg of lysine-modified aminotetraphenylporphyrin, add 2.80 g of hydroxypropyl methylcellulose, 300 mg of stearic acid, 800 mg of lactose, and 100 mg of magnesium stearate, stir and mix thoroughly, dry granulate, and press into tablets to obtain the product.

[0076] Example 19 Injection

[0077] Dissolve 30 mg of vitamin C in 28 mL of water for injection, heat to 30°C-40°C, add 62.50 mg of cycloserine and 0.98 mg of lysine-modified aminotetraphenylporphyrin, add 180 mg of Tween-80, cool to room temperature, add 270 mg of sodium chloride, adjust the pH to 7.0-7.5 with 5% sodium citrate solution, and make up to 30 mL with water for injection; dispense 3-5 mL into ampoules according to the dosage form specifications, seal the bottles, and sterilize with circulating steam at 100°C for 30 minutes.

[0078] Example 20 Injection

[0079] Take 62.50 mg of cycloserine and 1.95 mg of lysine-modified aminotetraphenylporphyrin, 5 g of glucose and 94 g of water for injection, add a 1:1 volume ratio of hydrochloric acid aqueous solution dropwise, adjust the pH to 4.0, add 100 mg of activated carbon, boil for 30 minutes under stirring, filter to remove the activated carbon, and then filter and sterilize with a 0.22 μm filter membrane to obtain a clear sterile solution, encapsulate, and sterilize by autoclave at 115°C for 30 minutes.

[0080] Example 21 Freeze-dried powder injection

[0081] Take 62.50 mg of cycloserine and 3.91 mg of lysine-modified aminotetraphenylporphyrin, and 400 mg of mannitol, add them to 9.1 g of water for injection, stir to dissolve, add hydrochloric acid aqueous solution with a volume ratio of 1:1, adjust the pH to 6.2, then add 500 mg of activated carbon for injection, stir at room temperature for 20 minutes, filter to remove the activated carbon, and then filter with a 0.22 μm filter membrane for sterilization to obtain a clear sterile solution, pour into 5 mL tube bottles, 3 mL per bottle, and place in a vacuum freeze dryer for freeze drying to obtain the product.

[0082] Example 22 Infusion

[0083] Take 62.50 mg of cycloserine and 7.81 mg of lysine-modified aminotetraphenylporphyrin, add 400 mg of polyethylene glycol, 300 mg of lecithin, 600 mg of sodium acetate, 600 mg of sodium sulfite and 90 g of sodium chloride, dissolve in water for injection, filter, fill, sterilize and package to obtain the product.

[0084] Example 23 Infusion

[0085] Take 62.50 mg of cycloserine and 15.63 mg of lysine-modified aminotetraphenylporphyrin, add 400 mg of polyethylene glycol, 400 mg of lecithin, 400 mg of sodium acetate, 800 mg of sodium sulfite and 500 g of glucose, dissolve in water for injection, filter, fill, sterilize and package to obtain the product.

[0086] Example 24 In vitro antibacterial activity (MRSA) of a pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4)

[0087] The in vitro antibacterial evaluation of a pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin against methicillin-resistant Staphylococcus aureus comprises the following steps:

[0088] In this experiment, the methicillin-resistant Staphylococcus aureus (MRSA) standard strain ATCC 43300 was selected as the research object, which was provided by Beijing 304 Hospital.

[0089] Preparation of bacterial suspension: Using aseptic operation, restore MRSA bacteria frozen in glycerol to 37℃, take 20μL and add 10mL sterile LB liquid medium, place in a 37℃ shaker at 180rpm and culture overnight. Inoculate the bacterial suspension onto three LB solid agar media by plate streak method, place in a 37℃ bacterial incubator and incubate for 24h, pick a single colony with an inoculation loop, inoculate it into 10mL LB liquid medium, place in a 37℃ shaker at 180rpm and culture overnight until the bacteria are in the logarithmic growth phase, and dilute the bacterial suspension to 10 with LB medium. 6 CFU / mL is reserved.

[0090] Drug solution preparation: First, dissolve LD4 in sterile phosphate-buffered saline (PBS) to a 10 mg / mL stock solution. Then, using a two-fold dilution method, prepare the drug solution in PBS to concentrations of 62.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, and 0.49 μg / mL. Dissolve DCS in sterile PBS to a 10 mg / mL stock solution. Then, using a two-fold dilution method, dilute the solution in LB liquid medium to concentrations of 500.00, 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, and 3.91 μg / mL for later use.

[0091] Determination of MIC and MBC: The experiment was carried out in a 96-well plate, with a negative control group, a positive control group, and an experimental group. Three independent replicates were performed for each concentration. 200 μL of LB medium was added to the wells of the negative control group, 100 μL of LB medium and 100 μL of bacterial suspension were added to the wells of the positive control group, and 100 μL of bacterial suspension and 100 μL of drug solution of corresponding concentration were added to the wells of the experimental group, so that the final concentrations of the LD4 drug solution were 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, and 0.49 μg / mL, respectively. After mixing evenly, the mixture was placed in a 37°C bacterial incubator in the dark for incubation for 30 min, and then taken out and irradiated under a 650 nm laser for 10 min with a light dose of 6 J / cm 2 After irradiation, the plates were incubated in a 37°C incubator for 24 hours. The growth of the strain in each well was observed. The first well to transition from turbid to clear was the minimum inhibitory concentration (MIC). Twenty microliters of the bacterial solution from each well with a concentration greater than or equal to the MIC was inoculated onto LB solid medium. The plates were incubated in the dark for 24 hours. The growth of the bacteria in each dish was observed. The concentration at which 5 or fewer colonies were observed was the minimum bactericidal concentration (MBC). The MIC and MBC determination methods for cycloserine were the same as above, with the final concentrations of DCS solution being 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, and 3.91 μg / mL, respectively, but laser irradiation was not required.

[0092] Combined drug MIC determination: The chessboard method was used for determination. The final concentrations of LD4 were set to 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, and 0 μg / mL, and the final concentrations of DCS were set to 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, 3.91, and 0 μg / mL. A total of 64 wells were set up in an 8×8 well format. Different concentrations of LD4 were used as the rows, and different concentrations of DCS were used as the rows. 100 μL of bacterial solution, 50 μL of DCS solution, and 50 μL of LD4 solution were added to each well. After mixing, the wells were placed in a 37°C bacterial incubator in the dark for 30 minutes. The wells were then taken out and irradiated with a 650 nm laser for 10 minutes at a light dose of 6 J / cm 2 After illumination, the cells were placed in a 37°C incubator for 24 h, the results were taken out for observation, and a heat map was drawn.

[0093] The fractional inhibitory concentration index (FICI) was calculated using the following formula:

[0094]

[0095] Among them, MIC协同A is the MIC value of drug A in combination with other drugs, MIC 单药A is the MIC value of drug A when used alone, MIC 协同B is the MIC value of drug B in combination therapy, MIC 单药B is the MIC value of drug B when used alone, FICI ≤ 0.5 indicates synergistic effect, 0.5 < FICI ≤ 1 indicates additive effect, 1 < FICI ≤ 2 indicates irrelevant effect, and 2 < FICI ≤ 4 indicates antagonistic effect.

[0096] The MIC (μg / mL) and FICI values of LD4 and DCS against MRSA are shown in Table 1 , and the bacterial growth is shown in Figure 1 , where red represents wells with bacterial growth and blue represents wells without bacterial growth.

[0097] Table 1 MIC (μg / mL) and FICI values of LD4 and DCS against MRSA. Interaction: Synergistic (Syn.) and additive (Add.).

[0098]

[0099] Example 25 In vitro antibacterial activity of a pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) (P. aeru)

[0100] The in vitro antibacterial evaluation of a pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin against Pseudomonas aeruginosa comprises the following steps:

[0101] In this experiment, the standard strain of Pseudomonas aeruginosa (P.aeru) ATCC27853 was selected as the research object, which was provided by Beijing 304 Hospital.

[0102] Preparation of bacterial suspension: Aseptically restore P. aeru bacteria frozen in glycerol to 37°C, take 20 μL and add it to 10 mL of sterile LB liquid medium, and culture it on a shaker at 37°C at 180 rpm overnight. Inoculate the bacterial suspension onto three LB solid agar plates using the plate streak method, incubate them in a 37°C bacterial incubator for 24 hours, pick a single colony with an inoculation loop, inoculate it into 10 mL of LB liquid medium, and culture it on a shaker at 37°C at 180 rpm overnight until the bacteria are in the logarithmic growth phase, and dilute the bacterial suspension to 10 with LB medium. 6 CFU / mL is reserved.

[0103] Drug solution preparation: First, dissolve LD4 in sterile phosphate-buffered saline (PBS) to a 10 mg / mL stock solution. Then, using a two-fold dilution method, prepare the drug solution in PBS to concentrations of 62.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, and 0.49 μg / mL. Dissolve DCS in sterile PBS to a 10 mg / mL stock solution. Then, using a two-fold dilution method, dilute the solution in LB liquid medium to concentrations of 500.00, 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, and 3.91 μg / mL for later use.

[0104] Determination of MIC and MBC: The experiment was carried out in a 96-well plate, with a negative control group, a positive control group, and an experimental group. Three independent replicates were performed for each concentration. 200 μL of LB medium was added to the wells of the negative control group, 100 μL of LB medium and 100 μL of bacterial suspension were added to the wells of the positive control group, and 100 μL of bacterial suspension and 100 μL of drug solution of corresponding concentration were added to the wells of the experimental group, so that the final concentrations of the LD4 drug solution were 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, and 0.49 μg / mL, respectively. After mixing evenly, the mixture was placed in a 37°C bacterial incubator in the dark for incubation for 30 min, and then taken out and irradiated under a 650 nm laser for 10 min with a light dose of 6 J / cm 2 After irradiation, the plates were incubated in a 37°C incubator for 24 hours. The growth of the strain in each well was observed. The first well to transition from turbid to clear was the minimum inhibitory concentration (MIC). Twenty microliters of the bacterial solution from each well with a concentration greater than or equal to the MIC was inoculated onto LB solid medium. The plates were incubated in the dark for 24 hours. The growth of the bacteria in each dish was observed. The concentration at which 5 or fewer colonies were observed was the minimum bactericidal concentration (MBC). The MIC and MBC determination methods for cycloserine were the same as above, with the final concentrations of DCS solution being 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, and 3.91 μg / mL, respectively, but laser irradiation was not required.

[0105] Combination drug MIC determination: The checkerboard assay was used. Final LD4 concentrations were set at 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, and 0 μg / mL, and final DCS concentrations were set at 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, 3.91, and 0 μg / mL. An 8×8 well plate was set up, with 64 wells, each containing different LD4 concentrations. 100 μL of bacterial solution, 50 μL of DCS solution, and 50 μL of LD4 solution were added to each well. After mixing, the wells were incubated in a 37°C incubator in the dark for 30 min. The wells were then exposed to a 650 nm laser for 10 min at a dose of 6 J / cm². After exposure, the wells were incubated in a 37°C incubator for 24 h. The results were observed and plotted as a heat map.

[0106] The MIC (μg / mL) and FICI values of LD4 and DCS against P. aeru are shown in Table 2 , and the bacterial growth is shown in Figure 1 , where red represents wells with bacterial growth and blue represents wells without bacterial growth.

[0107] Table 2 MIC (μg / mL) and FICI values of LD4 and DCS against P. aeruginosa. Interaction: Synergistic (Syn.) and additive (Add.).

[0108]

[0109] Example 26 In vitro antibacterial activity (E. coli) of a pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin (LD4)

[0110] The in vitro antibacterial evaluation of a pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin against Escherichia coli comprises the following steps:

[0111] In this experiment, the standard strain of Escherichia coli (E. coli) ATCC25922 was selected as the research object, which was provided by Beijing 304 Hospital.

[0112] Preparation of bacterial suspension: Using aseptic operation, E. coli bacteria frozen in glycerol were restored to 37℃, 20μL was added to 10mL sterile LB liquid medium, and cultured at 37℃ on a shaker at 180rpm overnight. The bacterial suspension was inoculated onto three LB solid agar media by plate streak method, and incubated in a 37℃ bacterial incubator for 24h. A single colony was picked with an inoculation loop and inoculated into 10mL LB liquid medium, and cultured at 37℃ on a shaker at 180rpm overnight until the bacteria were in the logarithmic growth phase. The bacterial suspension was diluted to 10 with LB medium.6 CFU / mL is reserved.

[0113] Drug solution preparation: First, dissolve LD4 in sterile phosphate-buffered saline (PBS) to a 10 mg / mL stock solution. Then, using a two-fold dilution method, prepare the drug solution in PBS to concentrations of 62.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, and 0.49 μg / mL. Dissolve DCS in sterile PBS to a 10 mg / mL stock solution. Then, using a two-fold dilution method, dilute the solution in LB liquid medium to concentrations of 500.00, 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, and 3.91 μg / mL for later use.

[0114] Determination of MIC and MBC: The experiment was carried out in a 96-well plate, with a negative control group, a positive control group, and an experimental group. Three independent replicates were performed for each concentration. 200 μL of LB medium was added to the wells of the negative control group, 100 μL of LB medium and 100 μL of bacterial suspension were added to the wells of the positive control group, and 100 μL of bacterial suspension and 100 μL of drug solution of corresponding concentration were added to the wells of the experimental group, so that the final concentrations of the LD4 drug solution were 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, and 0.49 μg / mL, respectively. After mixing evenly, the mixture was placed in a 37°C bacterial incubator in the dark for incubation for 30 min, and then taken out and irradiated under a 650 nm laser for 10 min with a light dose of 6 J / cm 2 After irradiation, the plates were incubated in a 37°C incubator for 24 hours. The growth of the strain in each well was observed. The first well to transition from turbid to clear was the minimum inhibitory concentration (MIC). Twenty microliters of the bacterial solution from each well with a concentration greater than or equal to the MIC was inoculated onto LB solid medium. The plates were incubated in the dark for 24 hours. The growth of the bacteria in each dish was observed. The concentration at which 5 or fewer colonies were observed was the minimum bactericidal concentration (MBC). The MIC and MBC determination methods for cycloserine were the same as above, with the final concentrations of DCS solution being 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, and 3.91 μg / mL, respectively, but laser irradiation was not required.

[0115] Combined drug MIC determination: The chessboard method was used for determination. The final concentrations of LD4 were set to 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, and 0 μg / mL, and the final concentrations of DCS were set to 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, 3.91, and 0 μg / mL. A total of 64 wells were set up in an 8×8 well format. Different concentrations of LD4 were used as the rows, and different concentrations of DCS were used as the rows. 100 μL of bacterial solution, 50 μL of DCS solution, and 50 μL of LD4 solution were added to each well. After mixing, the wells were placed in a 37°C bacterial incubator in the dark for 30 minutes. The wells were then taken out and irradiated with a 650 nm laser for 10 minutes at a light dose of 6 J / cm 2 After illumination, the cells were placed in a 37°C incubator for 24 h, the results were taken out for observation, and a heat map was drawn.

[0116] The MIC (μg / mL) and FICI values of LD4 and DCS against E. coli are shown in Table 3 , and the bacterial growth is shown in Figure 1 , where red represents wells with bacterial growth and blue represents wells without bacterial growth.

[0117] Table 3 MIC (μg / mL) and FICI values of LD4 and DCS against E. coli. Interaction: Synergistic (Syn.) and additive (Add.).

[0118]

[0119] Example 27 In vitro antibacterial activity of a pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) (Mtb.)

[0120] The in vitro antibacterial evaluation of a pharmaceutical composition containing cycloserine- and lysine-modified aminotetraphenylporphyrin against Mycobacterium tuberculosis comprises the following steps:

[0121] The Mycobacterium tuberculosis (Mtb.) standard strain H37Rv was selected as the research object in this experiment and was provided by Tianjin Haihe Hospital.

[0122] Preparation of bacterial suspension: Using aseptic operation, pick the colonies on the modified Roche medium from the Mtb. standard strain frozen in a -80℃ refrigerator, disperse them in 2mL of normal saline, place them in an ultrasonic disperser for ultrasonication, disperse the bacterial mass, set the program to ultrasonicate for 5s, stop for 5s, ultrasonicate for 1min, measure its McFarland concentration, and adjust it to 1MCF. Pipette 50μL of bacterial solution, inoculate it on the modified Roche medium, place it in a 37℃ bacterial incubator and incubate it for 21 days until cheese-like colonies grow. Use an inoculation loop to pick the colonies, place them in 2mL of normal saline, disperse the bacterial mass with an ultrasonic disperser, set the program to ultrasonicate for 5s, stop for 5s, ultrasonicate for 1min, measure the McFarland concentration, and adjust it to 1MCF, i.e. 3×10 8 CFU / mL, and then diluted to 10 with 7H9 liquid medium. 5 CFU / mL is reserved.

[0123] Drug solution preparation: First, dissolve LD4 in sterile phosphate-buffered saline (PBS) to a 10 mg / mL stock solution. Then, using a two-fold dilution method, prepare the drug solution in PBS to concentrations of 1000.00, 500.00, 250.00, 125.00, 62.50, 31.25, 15.63, and 7.81 μg / mL. Dissolve DCS in sterile PBS to a 10 mg / mL stock solution. Then, using a two-fold dilution method, dilute the solution in 7H9 liquid medium to concentrations of 32.00, 16.00, 8.00, 4.00, 2.00, 1.00, 0.50, and 0.25 μg / mL for later use.

[0124] Determination of MIC and MBC: The experiment was carried out in a 96-well plate, and a negative control group, a positive control group and an experimental group were set up respectively. The experiment was repeated three times for each concentration. 200 μL of 7H9 medium was added to the wells of the negative control group, 100 μL of 7H9 medium and 100 μL of bacterial suspension were added to the wells of the positive control group, and 100 μL of bacterial suspension and 100 μL of drug solution of corresponding concentration were added to the wells of the experimental group, so that the final concentrations of LD4 drug solution were 500.00, 250.00, 125.00, 62.50, 31.25, 15.63 and 7.81 μg / mL, respectively. After mixing evenly, the mixture was placed in a 37°C bacterial incubator in the dark for incubation for 30 min, and then taken out and irradiated under 650 nm laser for 10 min with a light dose of 6 J / cm 2After irradiation, the plates were placed in a 37°C incubator for 21 days. The growth of the strain in each well was observed. The first well to transition from turbid to clear was the minimum inhibitory concentration (MIC). Twenty microliters of the bacterial solution from each well with a concentration greater than or equal to the MIC was inoculated onto modified Roche solid medium. The plates were incubated in the dark for 24 hours. The bacterial growth in each dish was observed. The concentration at which 5 or fewer colonies were observed was the minimum bactericidal concentration (MBC). The MIC and MBC determination methods for cycloserine were the same as above, with the final concentrations of DCS solutions being 16.00, 8.00, 4.00, 2.00, 1.00, 0.50, and 0.25 μg / mL, respectively, but laser irradiation was not required.

[0125] Combined drug MIC determination: The chessboard method was used for determination. The final concentrations of LD4 were set to 500.00, 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, and 0 μg / mL, and the final concentrations of DCS were set to 16.00, 8.00, 4.00, 2.00, 1.00, 0.50, 0.25, and 0 μg / mL, respectively. A total of 64 wells were set up in an 8×8 well format, with different concentrations of LD4 as the row and different concentrations of DCS as the column. 100 μL of bacterial solution, 50 μL of DCS solution, and 50 μL of LD4 solution were added to each well. After mixing evenly, the well was placed in a 37°C bacterial incubator in the dark for 30 minutes. The well was then taken out and irradiated with a 650 nm laser for 10 minutes at a light dose of 6 J / cm 2 After illumination, the cells were placed in a 37°C incubator and incubated for 21 days. The results were taken out for observation and plotted into a heat map.

[0126] The MIC (μg / mL) and FICI values of LD4 and DCS against Mtb. are shown in Table 4 , and the bacterial growth is shown in Figure 1 , where red represents wells with bacterial growth and blue represents wells without bacterial growth.

[0127] Table 4 MIC (μg / mL) and FICI values of LD4 and DCS against Mtb. Interaction: Synergistic (Syn.) and additive (Add.).

[0128]

[0129] Example 28 In vitro cytotoxicity of a pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4)

[0130] The in vitro biocompatibility evaluation of a pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) comprises the following steps:

[0131] The mouse brain angioendothelioma cells bEnd.3, mouse embryonic fibroblasts NIH / 3T3, mouse mononuclear macrophages RAW264.7, and human hepatocytes L-02 used in this experiment were purchased from the National Biomedical Experimental Cell Resource Bank.

[0132] Cell recovery and plating: Cryopreserved cells were recovered using conventional methods and cultured in culture medium until the logarithmic growth phase. bEnd.3, NIH / 3T3 and L-02 cells were digested with trypsin to remove adherent cells. RAW264.7 cells were scraped off with a cell scraper and diluted into a cell suspension with culture medium containing 10% fetal bovine serum. Cells were counted and plated at a rate of 10 cells per well. 5 The cells were inoculated and placed in a cell culture incubator for 24 h to allow the cells to adhere.

[0133] Preparation of drug solution and cell administration: The experiment was divided into a control group and an experimental group. The control group was a PBS group, and the experimental groups were an LD4 group, a DCS group, and a group containing a drug composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4). The dosing concentrations of the LD4 group were 1.95, 3.91, 5.86, 7.81, and 15.63 μg / mL; the dosing concentrations of the DCS group were 15.63, 31.25, 62.50, 93.75, and 125 μg / mL; the dosing concentrations of the pharmaceutical composition group containing cycloserine and lysine-modified aminotetraphenylporphyrin (LD4) were 31.25 μg / mL cycloserine and 3.91 μg / mL lysine-modified aminotetraphenylporphyrin prepared in Example 3, a pharmaceutical composition of 62.5 μg / mL cycloserine and 7.81 μg / mL lysine-modified aminotetraphenylporphyrin, and a pharmaceutical composition of 125 μg / mL cycloserine and 15.63 μg / mL lysine-modified aminotetraphenylporphyrin. The culture medium was aspirated and 150 μL of the corresponding drug solution was added to each well according to the experimental groups. Each experimental group was further divided into a light reaction group and a dark reaction group. The light reaction group was first incubated in the dark for 30 min, and then the cells were exposed to 6 J / cm 2 Illuminate at an optical density of 100 nm for 10 min with a spot diameter of 7 cm. The dark reaction group is not illuminated and should be protected from light during the experiment. Six replicate wells are set for each concentration, and cells are incubated for 24 h.

[0134] MTT assay: After incubation, aspirate the supernatant, add 90 μL of complete medium and 10 μL of MTT solution (5 mg / mL) to each well, and incubate at 37°C for 4 h. Aspirate the supernatant, add 110 μL of Formazan dissolution solution to each well, and shake on a shaker for 10 min to fully dissolve the crystals. Measure the OD value at 490 nm on a microplate reader. Cells incubated without compound served as a blank control. Calculate cell viability (%) = OD value of the treatment group / OD value of the blank group × 100%.

[0135] The effects of each experimental group on the survival rate of normal cells are shown in Figure 2 . The results showed that at the set dosage concentration, the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) had no significant toxicity to the four cell types, indicating that the pharmaceutical composition has good safety. In addition, under the condition of no light, the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) can significantly promote the proliferation of NIH / 3T3 and RAW264.7 cells after 24 hours of action, and the cell survival rates are increased by 43.48±1.15% and 36.79±17.14%, respectively, indicating that the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) is not only non-cytotoxic, but also can promote the proliferation of normal cells.

[0136] Example 29 In vitro hemolytic activity of a pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4)

[0137] The in vitro hemolysis rate test of a pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) comprises the following steps:

[0138] Rabbit erythrocytes were purchased from Shanghai Yuanye Biotechnology Co., Ltd. and mixed before use. 10 mL of the solution was centrifuged to remove the supernatant and resuspended in 10 mL of normal saline. The solution was washed several times until no red color appeared in the supernatant and resuspended in sterile normal saline to prepare a 2% erythrocyte suspension for later use.

[0139] Preparation of drug solution and grouping: The experiment was divided into a control group and an experimental group. The control group included a negative control (0.9% normal saline NS) and a positive control (ultrapure water). The experimental groups included the LD4 group, the DCS group, and the group containing the drug composition modified with cycloserine and lysine aminotetraphenylporphyrin (LD4). The dosing concentrations of the LD4 group were 1.95, 3.91, 5.86, 7.81, and 15.63 μg / mL, the dosing concentrations of the DCS group were 15.63, 31.25, 62.5, 93.75, and 125 μg / mL, and the dosing concentrations of the drug composition group containing cycloserine and lysine-modified aminotetraphenylporphyrin (LD4) were 31.25 μg / mL cycloserine and 3.91 μg / mL lysine-modified aminotetraphenylporphyrin prepared in Example 3, 62.50 μg / mL cycloserine and 7.81 μg / mL lysine-modified aminotetraphenylporphyrin, and 125 μg / mL cycloserine and 15.63 μg / mL lysine-modified aminotetraphenylporphyrin. Each experimental group was also divided into a light reaction group and a dark reaction group. The light reaction group was first incubated in the dark for 30 minutes, and then irradiated with 6 J / cm 2 The light density was set to 0.01, the irradiation time was 10 min, the spot diameter was 7 cm, the dark reaction group did not need to be illuminated, and light should be avoided during the experiment.

[0140] Hemolysis rate determination: Centrifuge the treated red blood cell suspension, take the supernatant and place it in a 96-well plate. Add 100 μL to each well, set up four replicates for each group, and measure the absorbance at 545 nm using a microplate reader. Since LD4 has a certain absorption at 545 nm, it is necessary to first measure the absorbance of LD4 drug solutions of different concentrations and deduct it to obtain an accurate result. The formula for calculating the hemolysis rate is as follows:

[0141]

[0142] Hemolysis result determination: complete hemolysis: the solution is clear red, and no cells remain at the bottom of the centrifuge tube; partial hemolysis: the solution is clear red or brown, and a small amount of red blood cells remain at the bottom of the centrifuge tube; no hemolysis: all red blood cells sink, and the upper liquid is colorless and clear; coagulation: although there is no hemolysis, red blood cells agglutinate and cannot be dispersed after shaking, partial hemolysis or coagulation occurs, and it is not suitable for intravenous injection.

[0143] To investigate the biocompatibility of the pharmaceutical composition, we determined the hemolysis rate of the pharmaceutical composition containing cycloserine (DCS) and lysine-modified aminotetraphenylporphyrin (LD4). Figure 6 The results showed that the deionized water group presented a clear, transparent red solution, indicating complete hemolysis with a hemolysis rate of 100%. The NS group presented a clear, colorless solution, indicating no hemolysis. The LD4 group did not experience hemolysis of rabbit blood cells under either illumination or non-illumination conditions. Because it is a porphyrin compound and appears light yellow in physiological saline, the solution color was observed to be darker than that of the NS group. At different concentrations, the hemolysis rate of the LD4 group did not exceed 5%, indicating that the hemolysis rate of LD4 alone was low and had good biosafety. The hemolysis rate of the DCS group was less than 3% within the concentration range of 15.63-125 μg / mL. The solution in the centrifuge tube was clear and colorless, indicating no hemolysis. There was no significant difference between the Laser group and the Dark group, indicating that illumination did not affect the hemolysis of DCS and that it had good safety. The hemolysis rate of the pharmaceutical composition group containing cycloserine (DCS) and lysine-modified aminotetraphenylporphyrin (LD4) was no different from that of the LD4 group and the DCS group. The hemolysis rates at different concentrations were all less than 5%, showing the same phenomenon as that of the NS group, indicating that no hemolysis occurred; this shows that the pharmaceutical composition containing cycloserine (DCS) and lysine-modified aminotetraphenylporphyrin (LD4) has good safety, no hemolysis occurs under light conditions, and has good biocompatibility.

[0144] Example 30 Drug resistance of a pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4)

[0145] The detection of drug resistance of a pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin (LD4) to MRSA, P.aeru, and E.coli comprises the following steps:

[0146] The drug concentration was set to 0.25MIC, 0.5MIC, 1MIC, 2MIC, 4MIC. Take 10μL of the concentration of 10 6 CFU / mL bacterial solution was added to 490 μL culture medium, and then 500 μL of drug solution of different concentrations was added, mixed evenly, and incubated in the dark for 30 min. 2 The cells were irradiated with an energy density of 100 nm for 10 min and incubated in a 37°C bacterial incubator for 24 h. The bacterial growth in each tube was observed and the MIC value of the drug for this generation of bacteria was measured. The bacterial solution with the second highest concentration that allowed bacterial growth was selected and mixed evenly according to the above method. The above steps of determining the MIC were repeated for 50 generations.

[0147] The results of the drug resistance test of the drug composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) on three bacteria are shown in Figure 7 The results showed that the use of LD4 alone would not cause bacterial resistance, while the MIC values of DCS alone against MRSA, P. aeru, and E. coli increased from 62.5 μg / mL to 4000 μg / mL, indicating that bacterial resistance increased with each generation; the drug composition containing cycloserine and lysine-modified aminotetraphenylporphyrin (LD4) did not produce resistance against MRSA, P. aeru, and E. coli, indicating that the drug composition can effectively inhibit the development of bacterial resistance, prevent the spread of super-resistant bacteria, and ensure the effectiveness of antibacterial treatment.

[0148] Example 31 Therapeutic Effect of a Pharmaceutical Composition Containing Cycloserine and Lysine Modified Aminotetraphenylporphyrin (LD4) on Bacterial Infection Wounds

[0149] An in vivo experiment on treating traumatic infection in mice using the pharmaceutical composition containing cycloserine and lysine-modified aminotetraphenylporphyrin (LD4) prepared in Example 3 comprises the following steps:

[0150] Balb / c male mice aged 6-8 weeks and weighing 20-22 g were selected. A bacterial infection wound model was constructed using the full-thickness skin defect method. On the day of the experiment, the mice were fasted and anesthetized with an intraperitoneal injection of 400 mg / kg of 4% chloral hydrate in saline. After entering the anesthesia state, the mice were placed in a prone position on a fixed board (disinfected with 75% ethanol in advance), and the back was shaved to an area of 4 cm × 2 cm. A 15 mm trephine was then used to press a circular mark perpendicular to the skin on the back of the mouse. Sterile surgical curved scissors were used to cut along the indentation, removing the full-thickness skin and part of the fascia to make a circular full-thickness skin damage wound, and then the skin was cut open with sterile curved scissors. The wound was covered with sterile gauze of similar size and 50 μL of the prepared mixed bacterial suspension of MRSA, P. aeru and E. coli was added dropwise (one colony was picked from the solid culture medium of the three strains and added to 40 mL of culture medium respectively, and cultured overnight in a shaker at 37°C and 180 rpm. An appropriate amount of bacterial suspension was taken and centrifuged at 9000 rpm for 1 min. The precipitate was collected and washed with PBS three times and then resuspended in PBS to an OD of 0. 600 =0.5, and finally the three bacterial solutions were mixed in a certain proportion to make the MRSA concentration 10 9 CFU / mL, P. aeru concentration was 5×10 8 CFU / mL, E. coli concentration is 5×10 8 CFU / mL). Secure the wound with gauze, house the mice in separate cages, provide them with ample food and water, and observe their condition. Observe the wound for infection 24 hours after modeling. Successful model establishment is considered complete if the wound is stained with yellowish-white pus, elevated skin temperature, and lethargy.

[0151] Experimental grouping and administration: The mice were randomly divided into 5 groups, including a model control group (NS, administered with normal saline), a DCS high-dose group (DH, administered with a cycloserine aqueous solution at a concentration of 125 μg / mL), a LD4 high-dose group (LH, administered with a LD4 aqueous solution at a concentration of 15.63 μg / mL), a low-dose group containing a pharmaceutical composition of cycloserine and lysine-modified aminotetraphenylporphyrin (LD4) (LD-L, administered with a pharmaceutical composition of 62.50 μg / mL cycloserine and 7.81 μg / mL lysine-modified aminotetraphenylporphyrin prepared in Example 3), and a high-dose group containing a pharmaceutical composition of cycloserine and lysine-modified aminotetraphenylporphyrin (LD4) (LD-H, administered with a pharmaceutical composition of 125 μg / mL cycloserine and 15.63 μg / mL lysine-modified aminotetraphenylporphyrin prepared in Example 3). 50 μL of normal saline was dripped onto each wound of mice in the NS group, and drug solution of corresponding concentration was dripped onto each wound of mice in the other groups. The wounds were incubated in the dark for 30 min, and a 650 nm laser was used at 60 J / cm 2The mice were exposed to light for 10 minutes at a dose of 500 mg / kg and kept away from light after treatment. After 24 hours, the mice were exposed to light again. The NS group was also given normal saline every other day without light treatment.

[0152] Data collection: ① Wound images were collected every two days, and the wound area was calculated using Image J. The wound healing rate was calculated using the following formula:

[0153]

[0154] ② Wound bacterial count;

[0155] ③ Blood cell count: Use a blood cell counter to count the blood cells of the experimental and control group mice, observe the differences in blood cells of the mice, and evaluate the treatment effect.

[0156] The antibacterial effect of the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) in treating bacterial infection wounds is shown in FIG. Figure 8 The results showed that the skin wound recovery effect of mice in all treatment groups was better than that in the control group, especially the high-dose group containing the pharmaceutical composition modified with cycloserine and lysine tetraphenylporphyrin (LD4), with a wound healing rate of 97%, a decrease in wound colony count by about 2.5 log units, and a basic recovery of blood cell count to the level of the normal group, indicating that the mice basically recovered after treatment, the inflammatory response in the body was mild, and the treatment effect was good. The wound healing rates of the high-dose DCS group and the high-dose LD4 group were poor, suggesting that the pharmaceutical composition containing cycloserine and lysine modified tetraphenylporphyrin (LD4) has a synergistic effect in promoting tissue repair.

[0157] Example 32 Study on the Anti-Infection Mechanism of a Pharmaceutical Composition Containing Cycloserine and Lysine Modified Aminotetraphenylporphyrin (LD4)

[0158] Scanning electron microscope (SEM) was used to capture images of bacteria after the action of cycloserine, LD4, and a drug composition containing cycloserine and lysine-modified aminotetraphenylporphyrin (LD4).

[0159] Preparation of strains: MRSA, P. aeru, and E. coli bacteria were separated from the culture medium using a refrigerated centrifuge (6000 rpm, 5 min), washed three times with PBS (pH = 7.4), and then diluted with PBS to the working concentration, i.e., OD 600 =0.2, add into 2mL centrifuge tubes, each tube has a volume of 500μL, centrifuge and set aside.

[0160] Solution preparation, grouping and illumination: The experiment was divided into two groups: dark reaction and light reaction. Each group was divided into PBS group, LD4 group, DCS group, and group containing the pharmaceutical composition of cycloserine and lysine-modified aminotetraphenylporphyrin (LD4). The LD4 group was administered at a concentration of 15.63 μg / mL, and the DCS group was administered at a concentration of 125 μg / mL. The pharmaceutical composition of cycloserine and lysine-modified aminotetraphenylporphyrin (LD4) was administered with the pharmaceutical composition of 125 μg / mL cycloserine and 15.63 μg / mL lysine-modified aminotetraphenylporphyrin (LD4) prepared in Example 3. 500 μL of the drug solution was added to each tube. The light reaction group was first incubated in the dark for 30 minutes, and then irradiated with 6 J / cm 2 The bacteria were irradiated with laser of high energy density for 10 min, and the dark reaction group was treated in the same way but without irradiation.

[0161] Sample processing: After the drug treatment, the samples were centrifuged in a refrigerated centrifuge (4°C, 6000 rpm, 5 min), the supernatant discarded, and washed three times with PBS. The samples were fixed with 2.5% glutaraldehyde for 4 h, washed three times with PBS, and dehydrated with a prepared gradient of ethanol (30%, 50%, 70%, 85%, 90%, and 95% ethanol once for 15 min each time, and 100% ethanol twice for 20 min each time). The ethanol was replaced once with a mixture of anhydrous ethanol and isoamyl acetate (v / v = 1:1) (30 min / time). The samples were treated with pure isoamyl acetate for 1 h. After each step, they were centrifuged at 8000 rpm for 5 min. The samples were frozen at -20°C, -40°C, and -80°C for 12 h, dried in a freeze dryer for 12 h, and sent for testing.

[0162] The SEM results of bacteria in the drug composition group containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) are shown in Figure 9 .

[0163] The results showed that compared with the control group, the LD4 group and the group containing the drug composition of cycloserine and lysine modified aminotetraphenylporphyrin (LD4) destroyed the bacterial morphology, causing the bacteria to shrink, sink and be damaged. Compared with the LD4 group and the DCS group, the group containing the drug composition of cycloserine and lysine modified aminotetraphenylporphyrin (LD4) destroyed the bacterial cell structure after irradiation, causing the cell wall to collapse and be more severely damaged, more bacterial contents to leak, and leading to bacterial death.

[0164] Example 33 Study on the antibacterial mechanism of a pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4)

[0165] Transmission electron microscope (TEM) photos of bacteria were taken to explore the antibacterial mechanism of the drug composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4).

[0166] Strain preparation: Use a sterile inoculating loop to pick standard Mycobacterium tuberculosis H37Rv from modified Roche medium, add it to an ultrasonic tube containing 4 mL of sterile saline, and place it in an ultrasonic disperser to disperse the bacterial pellet. Set the program to sonicate for 5 seconds, stop for 5 seconds, and sonicate for 1 minute. Measure the McFarland concentration and adjust it to 5 MCF. Add 1 mL of bacterial solution to each 1.5 mL centrifuge tube, centrifuge and remove the supernatant. Collect the bacterial pellet and set aside.

[0167] Drug Preparation, Administration, and Grouping: DCS was prepared in sterile PBS to 4 μg / mL and 8 μg / mL solutions for later use. Three groups were divided into the experiment: PBS (0 μg / mL), DCS-1 (4 μg / mL), and DCS-2 (8 μg / mL). One mL of DCS solution was added to the centrifuge tube containing the collected bacterial pellet. The PBS group was treated with 1 mL of PBS for 24 hours.

[0168] Sample processing: Place the centrifuge tube in a refrigerated centrifuge and centrifuge (6000 rpm, 15 min) to remove the supernatant, wash 3 times with PBS, centrifuge to remove the supernatant, fix the bacterial pellet with 5% glutaraldehyde at 4°C for 72 h, and send the sample for TEM examination.

[0169] TEM images of DCS acting on bacteria are shown in Figure 10 As can be seen from the figure, the bacterial structure of the PBS group is intact, the cell wall and cell membrane are tightly connected, and the cell contents are intact. However, after the action of the drug in the DCS-1 and DCS-2 groups, the bacterial cell wall and cell membrane will be peeled off, the cell wall will be damaged, and the cell membrane will become thinner. This may be related to the change in bacterial permeability. It is speculated that the mechanism of enhancing the antibacterial activity of the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) may be to increase the permeability of the bacterial cell membrane, thereby causing the bacteria to absorb more LD4. Therefore, under the same concentration conditions, the antibacterial effect of the pharmaceutical composition containing cycloserine and lysine modified aminotetraphenylporphyrin (LD4) is far better than that of the DCS group and LD4 group, and is significantly different from the control group.

[0170] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. In addition, it should be understood that although this specification is described in accordance with the implementation mode, it does not contain only one technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the attached claims.

Claims

1. A pharmaceutical composition for use in combating bacterial infection, comprising cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin, wherein the pharmaceutical composition comprises: Cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin are mixed in a mass ratio of 512:1 to 1:2000, or a pharmaceutically acceptable carrier is added to prepare a clinically acceptable dosage form, including tablets, pills, capsules, granules, suspensions, aerosols, oral liquids, ointments, gels, patches, injections, infusions, lyophilized powder injections and sustained-release preparations.

2. The pharmaceutical composition according to claim 1, wherein The chemical structure of cycloserine is The chemical structure of the lysine-modified aminotetraphenylporphyrin is 3. The pharmaceutical composition according to claim 1, wherein The pharmaceutically acceptable carrier includes a conventional diluent (such as water for injection, microcrystalline cellulose, etc.), a filler (such as mannitol, sucrose, lactose, polyethylene glycol, Tween 80, sorbitol, menthol, liquid paraffin, petrolatum, stearic acid, glyceryl monostearate, lanolin, mineral oil, DMSO, etc.), a binder (such as carbomer, gum arabic, starch, cellulose, gelatin, polyvinyl pyrrolidone, polyacrylamide, etc.), a disintegrant (such as sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, etc.), a lubricant (such as talc, magnesium stearate, calcium stearate, solid polyethylene glycol, lecithin, silicon dioxide, micropowdered silica, etc.), a wetting agent (such as propylene glycol, glycerol, ethanol, etc.), a stabilizer (such as disodium edetate, sodium thiosulfate, sodium metabisulfite, sodium sulfite, sodium bisulfite, acetone). The pharmaceutical excipients may include at least one of an amine, sodium bicarbonate, sodium acetate, nicotinamide, and vitamin C), an osmotic pressure regulator (such as at least one of sodium chloride and glucose), a pH regulator (such as at least one of triethanolamine, sodium hydroxide, and sodium citrate), and a preservative (such as at least one of chlorobutanol, paraben, ethylparaben, and benzalkonium bromide). The above excipients may be mixed with cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a lysine-modified aminotetraphenylporphyrin in commonly used dosages and ratios. Once the dosages of cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a lysine-modified aminotetraphenylporphyrin are determined, the ratios between the pharmaceutical excipients may be appropriately adjusted as needed.

4. Use of a pharmaceutical composition for antibacterial infection comprising cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin according to claim 1, 2 or 3 in the preparation of an antibacterial infection drug.

5. Use of a pharmaceutical composition comprising cycloserine or a stereoisomer of cycloserine or a pharmaceutically acceptable salt of cycloserine and lysine-modified aminotetraphenylporphyrin or a stereoisomer of lysine-modified aminotetraphenylporphyrin or a pharmaceutically acceptable salt of lysine-modified aminotetraphenylporphyrin according to claim 1, 2 or 3 in the preparation of a drug for promoting cell proliferation and tissue repair.

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