Application of Indocyanine Green in the Treatment of Amanitin Poisoning

Abstract

The present invention belongs to the field of pharmaceutical technology, and specifically relates to the application of indocyanine green in the treatment of amatoxin poisoning. In order to further search for a specific antidote for amatoxin poisoning, through in vitro cell, organoid experiments and in vivo mouse experiments, the present invention discovers that indocyanine green can reduce the killing effect of amatoxin on cells and block the cytotoxic effect of amatoxin on liver organoids. At the same time, it has a protective effect on mice poisoned by amatoxin, and can significantly reduce the liver and kidney damage caused by amatoxin poisoning in mice, and improve the long-term survival rate after poisoning. The present invention not only broadens the application field of indocyanine green, but also provides a new solution for amatoxin poisoning, provides a strong theoretical basis and practical basis for the further research and development of amatoxin antidote drugs, and has important research value and development significance.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of indocyanine green in the treatment of amatoxin poisoning. Background Art

[0002] Mushroom poisoning is one of the main causes of death from food poisoning incidents worldwide. Among all mushroom species, the death toll from poisoning by mushrooms containing amatoxins exceeds 90% of the total number of mushroom poisoning cases, making it the main cause of mushroom poisoning. After being absorbed by the human body, amatoxins can cause severe acute liver damage and quickly endanger life.

[0003] Currently, there is no good solution for the treatment of amatoxin poisoning in clinical practice. Usually, symptomatic treatment is carried out by using conventional gastrointestinal poison clearance methods (such as gastric lavage, emesis), or some non-specific drugs are used for treatment, but the treatment effects are not good. Since amatoxin poisoning seriously threatens people's life, health and safety, and there is currently a lack of specific antidotes for the treatment of amatoxin poisoning, it is very necessary to further search for specific antidotes for amatoxin poisoning.

[0004] Indocyanine green is a fluorescent dye, which was approved by the FDA as a diagnostic drug for human use in 1956 and is currently widely used in clinical practice for ophthalmic angiography, liver function evaluation, etc. However, there is currently no study proving that indocyanine green can be used for the treatment of mushroom poisoning or other similar effects. Summary of the Invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention proposes a new application of indocyanine green (ICG), that is, ICG is a new antidote for amatoxin (AMA) poisoning, providing a new and effective solution for amatoxin detoxification.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides the application of indocyanine green in the preparation of drugs for the treatment of amatoxin poisoning.

[0008] Preferably, the treatment is to reduce the killing effect of amatoxin on cells.

[0009] Preferably, the treatment is to block the cytotoxic effect of amatoxin on liver organoids.

[0010] Preferably, the treatment has a protective effect on amatoxin poisoning, can significantly reduce the liver and kidney damage caused by amatoxin poisoning, and improve the long-term survival rate after poisoning.

[0011] The structure of indocyanine green is shown as follows:

[0012]

[0013] Indocyanine green (ICG) is a fluorescent dye that was approved by the FDA in 1956 for diagnostic use in humans and is currently widely used clinically for ophthalmic angiography, liver function assessment, etc. There is currently no report on the use of this compound for the treatment of amatoxin poisoning or other similar effects. Therefore, through cell experiments, the present invention confirmed that indocyanine green can reduce the cytotoxic effect of amatoxin on cells and can effectively block the cytotoxic effect of amatoxin on liver organoids; through mouse experiments, it was confirmed that ICG has a protective effect on mice poisoned with amatoxin, can significantly reduce the liver and kidney damage of amatoxin-poisoned mice, and improve the long-term survival rate of poisoned mice. The present invention provides a new strategy for the research and development of amatoxin antidotes and has important potential application value.

[0014] In addition, the present invention also found through research that within a certain concentration range of ICG, the antidote effect of ICG on amatoxin poisoning has a dose-dependence, and the earlier the drug is administered, the better the antidote effect. Therefore, ICG can also be used as a drug for preventing amatoxin poisoning.

[0015] The second aspect of the present invention provides an amatoxin poisoning antidote, and the antidote uses indocyanine green as the main active ingredient.

[0016] Preferably, the antidote further includes other drug components that can synergistically treat amatoxin poisoning with indocyanine green. In principle, after compounding indocyanine green with this drug component, the therapeutic effect on amatoxin poisoning is greater than that when indocyanine green is used alone.

[0017] Preferably, the antidote further includes pharmaceutically acceptable excipients. The excipients are diluents, binders, lubricants, disintegrants, solubilizers, stabilizers, etc. that can be used in the pharmaceutical field and some pharmaceutical matrices; they can also be functional pharmaceutical excipients available in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), poly(lactic-co-glycolic acid) copolymer (PLGA), hyaluronic acid, etc.

[0018] Preferably, the dosage forms of the antidote include injections, powder for injection, tablets, granules, capsules, dripping pills, sustained-release agents, oral liquid preparations.

[0019] The above dosage forms refer to the dosage forms commonly used clinically. The pharmaceutical preparations can be administered orally or parenterally (such as intravenously, subcutaneously, intraperitoneally or locally), that is, they can be flexibly administered by various methods such as oral administration, subcutaneous injection, intramuscular injection and intravenous injection. If some drugs are unstable under gastric conditions, they can be prepared into enteric-coated tablets.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The present invention discloses a new application of indocyanine green, specifically discloses the application of indocyanine green in the treatment of amanitin poisoning. Indocyanine green can reduce the cytotoxic effect of amanitin on cells, block the cytotoxic effect of amanitin on liver organoids, has a protective effect on amanitin poisoning, can significantly reduce the liver and kidney damage caused by amanitin poisoning in mice, and improve the long-term survival rate after poisoning. The present invention not only broadens the application field of indocyanine green, but also provides a new solution for amanitin poisoning, provides a strong theoretical basis and practical basis for the further research and development of amanitin antidotes, and has important research value and development significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Survival rate of α-amanitin poisoned cells after treatment with different concentrations of indocyanine green (left figure is the treatment group of HAP1 cells, right figure is the treatment group of HepG2 cells);

[0023] Figure 2 Survival rate of α-amanitin poisoned cells after treatment at different time points of indocyanine green administration (left figure is the treatment group of HAP1 cells, right figure is the treatment group of HepG2 cells);

[0024] Figure 3 Indocyanine green can reduce the toxicity of α-amanitin to mouse liver organoids (a is a representative image of mouse liver organoids after treatment with α-amanitin and / or indocyanine green for 3 days; b is the maximum diameter of the organoids, n = 8; c is the result of calcein / propidium iodide viability assay of the organoids; d is a representative HE image of the organoids);

[0025] Figure 4 Results of the effect of indocyanine green on blood biochemical indexes of α-amanitin poisoned mice (n = 6);

[0026] Figure 5 Results of the effect of indocyanine green on the 30-day survival curve of α-amanitin poisoned mice (n = 6). DETAILED DESCRIPTION OF THE INVENTION

[0027] The following further describes the specific embodiments of the present invention. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all available through conventional commercial channels unless otherwise specified.

[0029] Example 1 In Vitro Study on the Treatment of α-Amanitin (AMA) Cytotoxicity with Different Concentrations of Indocyanine Green (ICG)

[0030] (1) Research Method

[0031] Cell culture: In this example, human haploid cells HAP1 and human hepatoma cells HepG2 were used as research objects. The complete medium formula for HAP1 cells was: IMDM medium + 10% fetal bovine serum + 1% penicillin / streptomycin, and the complete medium formula for HepG2 cells was: DMEM medium + 10% fetal bovine serum + 1% penicillin / streptomycin.

[0032] HAP1 and HepG2 cells were seeded in 96-well plates at a density of 1.5×10 4 / well. After seeding for 12 h, the medium was discarded, and different concentrations of the antidote ICG were added for action. After adding the antidote for 12 h, AMA (3 μM for HAP1 cells and 5 μM for HepG2 cells) was added and cultured for 72 h. Then, the culture solution was discarded, and complete medium containing 10% CCK8 was added and cultured for 2 h. The relative cell viability at 450 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader to determine the cell survival rate.

[0033] (2) Experimental Results

[0034] In this example, human haploid cells HAP1 and human hepatoma cells HepG2 were used as research objects to investigate the toxicity of different concentrations of ICG to cells and its reversing effect on the cytotoxicity caused by AMA. Figure 1 (Left) showed that administration of 0 - 30 μM ICG alone had no obvious cytotoxicity to HAP1 (Control), while after incubating with 0 - 30 μM ICG for 12 h in advance and then adding 3 μM AMA, the killing effect of AMA on cells decreased with the increase in the concentration of ICG. Meanwhile, the phenomenon in HepG2 cells was the same as that in HAP1 cells. Administration of 0 - 200 μM ICG alone had no obvious cytotoxicity to HAP1 (Control). After incubating with 0 - 200 μM ICG for 12 h in advance and then adding 5 μM AMA, the killing effect of AMA on cells decreased with the increase in the concentration of ICG ( Figure 1 (Right)). The above results prove that ICG has good safety, low toxicity, and a new use as an antidote for AMA poisoning.

[0035] Example 2 In Vitro Study on the Treatment of α-Amanitin Cytotoxicity with Indocyanine Green at Different Time Points

[0036] (1) Research method

[0037] HAP1 and HepG2 cells were seeded into 96-well plates at a density of 1.5×10 4 / well. After 24 hours of seeding, the culture medium was discarded, 3 μM AMA was added, and ICG (10 μM for HAP1 cells and 100 μM for HepG2 cells) was added at -12 h, 0 h, 4 h, 8 h, and 12 h respectively. At 72 h, the culture solution was discarded, and complete medium containing 10% CCK8 was added and cultured for 2 h. The relative cell viability at 450 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader to determine the cell survival rate.

[0038] (2) Experimental results

[0039] In this example, HAP1 cells and human hepatoma cells HepG2 were used as research objects to investigate the reversal effect of adding ICG at different time points on the cytotoxicity caused by AMA. Figure 2 (Left) shows that the killing effect of AMA on HAP1 cells increased with the delay of ICG incubation time from -12 h to 12 h. Meanwhile, the phenomenon in HepG2 cells was the same as that in HAP1 cells, except that the killing effect of AMA on HepG2 cells advanced by 12 h and delayed by 12 h after adding ICG, and also increased with the delay of ICG incubation time ( Figure 2 (Right)). The above results prove that ICG has a new use as an antidote for AMA poisoning, and in poisoned cells, the earlier the drug is used, the better the antidote effect.

[0040] Example 3 Study on the therapeutic effect of indocyanine green on α-amanitin-poisoned organoids

[0041] (1) Research method

[0042] The liver of CD-1 mice (25 - 35 g) purchased from the Experimental Animal Center of Sun Yat-sen University was cut into cubes of 1 - 2 mm3 and washed twice in PBS. Then the tissue fragments were placed in 10 mL of digestion buffer (1 mg / mL collagenase I, 0.1 mg / mL hyaluronidase, 0.1 mg / mL Dnase I) and incubated at 37 °C for 1.5 hours. After digestion, the tissue suspension was filtered through a 40-μm cell strainer, and the suspension was centrifuged and resuspended in PBS to 10 mL after centrifugation. The resulting single-cell suspension was seeded in complete organoid medium (DMEM / F12 medium containing 5 μg / mL insulin, 250 ng / mL amphotericin B, 10 μg / mL gentamicin, 0.125 ng / mL EGF, 25 ng / mL hydrocortisone, 10 μM Y-27632). After 14 days, α-amanitin and / or indocyanine green were added (for HAP1 cells, AMA was 3 μM and ICG was 10 μM; for HepG2 cells, AMA was 5 μM and ICG was 100 μM). Three days later, the size of the organoids in different groups was observed, and calcein / propidium iodide staining and hematoxylin-eosin staining (HE) were performed.

[0043] (2) Experimental results

[0044] It was found that indocyanine green could effectively block the cytotoxic effect of α-amanitin on liver organoids. The organoids treated with indocyanine green had a more compact morphology and larger volume than those without indocyanine green addition ( Figure 3 a, 3b). At the same time, through the calcein / propidium iodide staining test, it was found that indocyanine green treatment significantly reduced the cell death rate induced by α-amanitin ( Figure 3 c). By observing the treatment effect of indocyanine green through HE staining, it could also be seen that indocyanine green blocked the toxic effect of α-amanitin on organoids ( Figure 3 d).

[0045] Example 4 Study on the short-term therapeutic effect of indocyanine green on α-amanitin-poisoned mice

[0046] (1) Research methods

[0047] Twenty-four CD-1 mice (25 - 35 g) purchased from the Experimental Animal Center of Sun Yat-sen University were randomly divided into 4 groups, namely (i) control group (0.9% NaCl, i.p. at 0, 4, 8, and 12 h); (ii) ICG group (0.9% NaCl, i.p. at 0 h; 5 mg / kg ICG, i.v. at 4, 8, and 12 h); (iii) AMA group (0.33 mg / kg AMA, i.p. at 0 h; 0.9% NaCl, i.v. at 4, 8, and 12 h); (iv) AMA + ICG group (0.33 mg / kg AMA, i.p. at 0 h; 5 mg / kg ICG, i.p. at 4, 8, and 12 h). Among them, i.p. is intraperitoneal injection and i.v. is tail vein injection. All mice were anesthetized and sacrificed at 24 h, and plasma supernatants were collected for biochemical detection.

[0048] (2) Experimental results

[0049] The liver and kidneys are the main target organs of AMA. To evaluate the protective effect of ICG on the AMA-exposed liver and kidneys, liver and kidney injuries were analyzed by measuring plasma biomarkers. The results showed that after AMA administration, the liver biomarkers aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were significantly increased ( Figure 4 a, 4b, 4d), and the AST / ALT ratio was significantly decreased ( Figure 4 c), indicating that the liver was severely damaged by AMA toxicity. It was shown that ICG treatment significantly reduced AST, ALT, and ALP, indicating that ICG could block AMA-induced liver injury ( Figure 4 a - d). At the same time, similar results were also observed in the kidneys. In AMA-treated mice, ICG treatment significantly reduced the levels of the kidney biomarkers blood urea nitrogen (BUN) and creatinine (Cre) ( Figure 4 e, 4f). The above results demonstrated that ICG had a protective effect on AMA-poisoned mice and could significantly reduce liver and kidney injuries in AMA-poisoned mice.

[0050] Example 5 Study on the long-term therapeutic effect of indocyanine green on α-amanitin-poisoned mice

[0051] (1) Research methods

[0052] Twenty-four mice purchased from the Experimental Animal Center of Sun Yat-sen University were randomly divided into four groups: (i) control group (0.9% NaCl, i.p. at 0, 4, 8, and 12 h); (ii) ICG group (0.9% NaCl, i.p. at 0 h; 5 mg / kg ICG, i.v. at 4, 8, and 12 h); (iii) AMA group (0.33 mg / kg AMA, i.p. at 0 h; 0.9% NaCl, i.v. at 4, 8, and 12 h); (iv) AMA + ICG group (0.33 mg / kg AMA, i.p. at 0 h; 5 mg / kg ICG, i.p. at 4, 8, and 12 h). Among them, i.p. is intraperitoneal injection and i.v. is tail vein injection. The survival of all mice was observed within 30 days, and the remaining mice were anesthetized and sacrificed at 30 days.

[0053] (2) Experimental results

[0054] In this example, long-term (30-day) survival analysis was performed to evaluate whether ICG can also protect mice from the toxicity of AMA. The experimental results are as Figure 4 shown, demonstrating that ICG treatment can significantly improve the survival rate of mice poisoned with AMA.

[0055] In summary, indocyanine green (ICG) is a new antidote for α-amanitin (AMA) poisoning, which is expected to provide a strong theoretical and practical basis for the further research and development of amanitin antidote drugs, and has important research and development value and significance.

[0056] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations of these embodiments still fall within the protection scope of the present invention.

Claims

1. Use of indocyanine green in the preparation of a medicament for treating amatoxin poisoning.

2. The application according to claim 1, characterized in that, The treatment is to reduce the cytotoxic effect of amatoxin on cells.

3. The application according to claim 1, characterized in that, The treatment is to block the cytotoxic effect of amatoxin on liver organoids.

4. The application according to claim 1, characterized in that The treatment has a protective effect on amatoxin poisoning, can significantly reduce the liver and kidney damage caused by amatoxin poisoning, and improve the long-term survival rate after poisoning.

5. The application according to claim 1, wherein The medicament also includes pharmaceutically acceptable excipients.

6. The application according to claim 1, characterized in that, The dosage forms of the medicament include injection, powder for injection, tablets, granules, capsules, dropping pills, sustained-release agents, oral liquid preparations.

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