Patent Application
20250000837
The present disclosure belongs to the field of biological medicine, and particularly relates to a treatment method for retinal degeneration and use of an inhibitor or microbicid for a microorganism in preparation of a drug for treating retinal degeneration.
Inherited retinal degeneration (IRD) is a group of hereditary diseases characterized by progressive loss of photoreceptor cells, including Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), early-onset rod-cone cytodystrophy, rod-rod dystrophy, congenital stationary night blindness and colour blindness and Stargardt disease (Broadgate, et al., 2017). It is the most common cause of visual loss of workers in industrialized countries and has an estimated incidence rate of 1:2000 (Kutluer, et al., 2020). Due to heterogeneity of heredity and clinics, treatment of the IRDs needs a highly personalized treatment strategy. Though neuroprotection, a gene therapy and a cell replacement therapy are proposed methods for treating the different stages of the IRD, until now, only one gene therapy (Luxturna) for correcting RPE65 gene mutation has been approved by FDA for treating the LCA (Botto, et al., 2021; Ikelle, et al., 2020; Kutluer, et al., 2020). Therefore, a treatment method for saving CRB1-related visual loss is an urgent need.
In a previous study PCT/CN2018/112022 (its content is incorporated by reference in its entirety) of the inventor, it is discovered that a microorganism, when administered in a live state, may activate a complement system and induce a drusen-like lesion in a macaque body. Besides, administering a vancomycin antibiotic to vitreous humor for killing this type of microorganism or inhibiting its growth may reduce a size of the drusen-like lesion in retinal tissue of a macaque compared with a control, and an agent for killing this type of microorganism, such as Bacillus megaterium, or inhibiting its growth may be used for treating age-related macular degeneration.
In previous studies PCT/CN2018/118929, PCT/CN2019/070572 and PCT/CN2019/117444 (their contents are incorporated by reference in their entirety) of the inventor, a compound/composition capable of being used for killing the microorganism or inhibiting its growth and a method for treating a microorganism infection and treating or preventing a disease or a disease symptom such as AMD related to this type of infection by using the compound/composition are reported.
Based on the previous studies, the inventor further discovers that an inhibitor or microbicid for a microorganism may be used as a drug for treating retinal degeneration.
The present disclosure proves that an inhibitor or microbicid for a microorganism may be used as a drug for treating retinal degeneration first by establishing an eye disease model or an eye disease model carrier, especially, a retinal degeneration model or model carrier.
Objectives of the present disclosure are implemented through the following technical solutions.
The present disclosure first provides a treatment method for retinal degeneration, including: administering an inhibitor or microbicid for a microorganism to eyes or a gastrointestinal tract of a patient.
Preferably, the retinal degeneration is progressive retinal degeneration; more preferably, the retinal degeneration is inherited retinal degeneration; more preferably, the inherited retinal degeneration includes Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), early-onset rod-cone cytodystrophy, rod-rod dystrophy, congenital stationary night blindness and colour blindness and Stargardt disease.
Preferably, the microorganism is one or a combination of two or more of bacteria, archaebacteria, protists, fungi or viruses. The microorganism is the bacteria, and the bacteria are selected from: one or two or more of Anearostipes, Bifidobacterium, Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillussp., Clostridium, Acinetobacter, Streptococcus, Mannheimia, Fibrobacter, Prevotella, Campylobacter, Actinomyces, Hymenobacter, Escherichia, Tissierella, Klebsiella, Porphyromonas, Azospira, Aquimarina, Achromobacter, Acidithiobacillus, Burkholderia, Marinobacter, Treponema, Actinosporangium, Vibrio, Ruminococcus, Methanobrevibacter, Shigella, Frankia, Streptomyces, Anaeroplasma and Coprococcus. More preferably, the bacteria are selected from: Anearostipeshadrus, Bifidobacterium pseudocatenulatum, Nitrosomonas sp.Is79A3, Oscillibactervalericigenes, Tatumella sp.TA1, Megamonasfuniformis, Thiobacillus denitrificans and Akkermansia muciniphila.
Preferably, the inhibitor or microbicid for the microorganism includes a compound, polypeptide, a polynucleotide, a natural plant or a natural plant extract.
More preferably, the inhibitor or microbicid for the microorganism is an antibiotic, for example, the antibiotic is one or two or more of a β-lactam antibiotic, an aminoglycoside antibiotic, a tetracycline antibiotic, a chloramphenicol antibiotic, a macrolide antibiotic, a glycopeptide antibiotic, a quinolone antibiotic, a nitroimidazole antibiotic, a rifamycin antibiotic, an echinocandin antibiotic, a polyene antibiotic, a pyrimidine antibiotic, an allylamine antibiotic, an azole antibiotic and other antibiotics.
For example, in some embodiments, the antibiotic may include one or more of the followings: β-lactam antibiotics, including penicillins (e.g., penicillin V), amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, pivampicillin, pivmecillinam, ticarcillin, cephalosporins such as cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, cefuzonam, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetime, ceftioxide, thienamycins, monobactams, β-lactamase inhibitors, methoxypenicillins, etc.; Aminoglycoside antibiotics: including streptomycin, gentamicin, kanamycin (e.g., kanamycin A), tobramycin, amikacin, neomycin (e.g., neomycin B, neomycin C and neomycin E), ribomycin, micronomicin, azithromycin, dibekacin, sisomicin, netilmicin, paramomycin, bramycin, etc.; Tetracycline antibiotics: including tetracycline, oxytetracycline, chlortetracycline and doxycycline; chloramphenicol antibiotics: including chloramphenicol, thiamphenicol, etc.; macrolide antibiotics: including erythromycin, leucomycin, odorless erythromycin, acetylspiramycin, medimycin, josamycin, azithromycin, clarithromycin, dirithromycin, oxithromycin, telithromycin, etc.; glycopeptide antibiotics: including vancomycin, norvancomycin, teicoplanin, etc.; quinolone antibiotics: including norfloxacin, ofloxacin, ciprofloxacin, pefloxacin, gatifloxacin, enoxacin, lomefloxacin, nalidixic acid, levofloxacin, moxifloxacin, besifloxacin; nitroimidazole antibiotics: including metronidazole, tinidazole, ornidazole, etc.; rifamycinoid antibiotics: including rifampicin; echinocandin antibiotics; polyene antibiotics; pyrimidines antibiotics; allylamine antibiotics; azole antibiotics; other antibiotics: fosfomycin, capreomycin, cycloserine, lincomycin, clindamycin, mitomycin, actinomycin D, bleomycin, doxorubicin, isoniazid, pyrazinamide, cyclosporine, polymyxin B combinations such as polymyxin B/trimethoprim, polymyxin B/bacitracin, polymyxin B/neomycin/gramicidin, etc.
In some embodiments, the antibiotic may be selected from Amikacin, Amoxicillin, Ampicillin, Arsphenamine, Azithromycin, Azlocillin, Aztreonam, Bacitracin, Capreomycin, Carbenicillin, Cefaclor, Cefadroxil, Cefalexin, Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime, Cefprozil, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefuroxime, Chloramphenicol, Cilastatin, Clarithromycin, Clavulanate, Clindamycin, Clofazimine, Cloxacillin, Colistin, Cycloserine, Dalfopristin, Dapsone, Daptomycin, Dicloxacillin, Dirithromycin, Doripenem, Doxycycline, Erythromycin, Ethambutol, Ethionamide, Flucloxacillin, Fosfomycin, Furazolidone, Fusidic acid, Gentamicin, Imipenem, Isoniazid, Kanamycin, Lincomycin, Linezolid, Loracarbef, Mafenide, Meropenem, Methicillin, Metronidazole, Mezlocillin, Minocycline, Mupirocin, Nafcillin, Neomycin, Netilmicin, Nitrofurantoin, Oxacillin, Oxytetracycline, Paromomycin, Penicillin G, Penicillin V, Piperacillin, Platensimycin, Polymyxin B, Pyrazinamide, Quinupristin, Rapamycin, Rifabutin, Rifampicin, Rifampin, Rifapentine, Rifaximin, Roxithromycin, Silver sulfadiazine, Spectinomycin, Streptomycin, Sulbactam, Sulfacetamide, Sulfadiazine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Tazobactam, Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracycline, Thiamphenicol, Ticarcillin, Tigecycline, Tinidazole, Tobramycin, Trimethoprim, Troleandomycin Vancomycin, enoxacin, lomefloxacin, nalidixic acid, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, ofloxacin, norfloxacin, Cefotetan, Cefonicid, Cephradine, Cephapirin, Cephalothin, Cefmetazole, Cefotaxime, Moxalactam, Cefepime, Ceftaroline fosamil, Ceftobiprole, Dalbavancin, Demeclocycline, Metacycline, Ertapenem, Fidaxomicin, geldanamycin, herbimycin, Posizolid, Radezolid, Torezolid, Oritavancin, Spiramycin, Sulfadimethoxine, Sulfonamidochrysoidine, Gemifloxacin Nadifloxacin Trovafloxacin Grepafloxacin Sparfloxacin Temafloxacin, Teixobactin, Malacidins, and combinations thereof.
Preferably, the inhibitor or microbicid for the microorganism is a compound of a formula I (e.g., a formula I-1, a formula I-2, a formula I-3, a formula I-4, and a formula I-5), a formula II (e.g., a formula II-1, a formula II-2, a formula II-3, a formula II-4, a formula II-5, a formula II-6, a formula II-7, a formula II-8, a formula II-9, and a formula II-10), a formula III (e.g., a formula III-1, a formula III-2, and a formula III-3), a formula IV-1 or IV-2 (e.g., a formula IV-3, a formula IV-4, a formula IV-5, and a formula IV-6), a glycoside (e.g., a formula V), or a pharmaceutically acceptable salt or ester thereof, where an aglycone of the glycoside is a phenolic compound, a flavonoid, coumarin, benzoic acid, or a sterol, where the formula I, the formula II, the formula III, the formula IV-1, the formula IV-2, the formula V, and subformulae thereof are defined as follows.
In some embodiments, the compounds of the present disclosure may be represented by having the formula I, or a pharmaceutically acceptable salt or ester thereof:
For the avoidance of doubt, in the formula I, a cyclic structure Cy1 is connected with another cyclic structure Cy2, which may be the same or different, through two linkers, L and L′, which form an additional ring structure between Cy1 and Cy2. It should be understood that both Cy1 and Cy2 are separately a ring structure, which is independent of L and L′.
In the formula I, Cy1 and Cy2 are each independently an optionally substituted cycloalkyl ring (e.g., C3-7 cycloalkyl ring), an optionally substituted heterocyclic ring, such as an optionally substituted 4-7 membered heterocyclic ring (e.g., having one or two ring heteroatoms independently selected from N, O, and S), an optionally substituted aryl ring (e.g., C6-10 aryl ring (e.g., phenyl)), or an optionally substituted heteroaryl ring, such as an optionally substituted 5-10 membered heteroaryl ring (e.g., 5- or 6-membered heteroaryl ring with one or two ring heteroatoms independently selected from N, O, and S);
L and L′ are each independently null or a linker (e.g., described herein); as used herein, the term “linker” is not restricted to any particular types of linking groups. For example, in some embodiments, the linker may also form a ring structure with one of the moieties that it is attached to, for example, L and Cy1 may form a ring structure independent of Cy2;
L2 is null, optionally substituted C1-6 alkylene, optionally substituted C1-6 heteroalkylene, optionally substituted C2-6 alkenylene, optionally substituted C2-6 alkynylene, optionally substituted C3-6 cycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, or optionally substituted 4-7 membered heterocyclylene;
W is —OR1; —COR2; —COOR1a; —OCOOR1a; —NR3R4; —CONR3aR4a; —OCONR3bR4b; —SO2NR3CR4c; —OSO2NR3dR4d; —SR5; —SO2R5a; —OCOR2a; —OSO2R5a or
where:
without showing optional substituents, may be any of the following:
where L2-W may be attached to either the left or the right ring, where L and L′ may be any of those described herein and suitable substituents for the rings are described herein.
In some embodiments, both Cy1 and Cy2 in the formula I may be an aryl or heteroaryl ring. For example, in some embodiments, the compound of the formula I may have a formula I-1:
In some embodiments, Ar1 and Ar2 in the formula I-1 are each independently an optionally substituted C6-10 aryl ring, or an optionally substituted 5-10 membered heteroaryl ring. In some embodiments, Ar1 and Ar2 in the formula I-1 are each independently an optionally substituted phenyl ring or a 5 or 6 membered heteroaryl ring. For example, in some embodiments, Ar1 and Ar2 in the formula I-1 are each independently an optionally substituted phenyl ring, an optionally substituted thienyl ring, an optionally substituted furanyl ring, an optionally substituted pyridyl ring, or an optionally substituted pyrimidinyl ring.
The formula I-1 typically has a polycyclic core structure. For example, in some embodiments, Ar1 and Ar2 are such that the core structure of the formula I-1,
without showing optional substituents, may be any of the following:
where L2-W may be attached to either the left or the right ring, where L and L′ are defined herein and suitable substituents for the rings are described herein.
In some embodiments, the compound of the formula I may have a formula I-2:
where:
where R1, R1a, R2, R2a, R2b, R3, R4, R3a, R3b, R3c, R3d, R3e, R4a, R4b, R4c, R4d, R4e, R5, R5a, and R5b are defined herein, see e.g., the formula I.
It should be noted that each instance of the structural units-L″-W′ and -L2-W is independently selected and may be the same or different.
In some embodiments, Cy1 in the formula I-2 is an optionally substituted phenyl ring, an optionally substituted thienyl ring, an optionally substituted furanyl ring, an optionally substituted pyridyl ring, or an optionally substituted pyrimidinyl ring. In some embodiments, Cy1 in the formula I-2 is an optionally substituted C3-6 cycloalkyl ring or an optionally substituted 4-7 heterocyclic ring with 1 or 2 ring heteroatoms independently selected from N, O, and S.
In some embodiments, Cy1 is such that the core structure of the formula I-2 may be any of the following:
where -L2-W is attached to the right phenyl ring, L and L′ are defined herein and suitable substituents for the rings are described herein.
In more preferred embodiments, both Cy1 and Cy2 in the formula I are phenyl rings. For example, in some embodiments, the compound of the formula I-2 may have a formula I-3:
When the linker L or L′ forms a double bond with one of the ring carbons, it cannot be CR101R102 with both R101 and R102 present, as the valence of the carbon will exceed 4. In such cases, it should be understood that one of R101 and R102 is absent and L or L′ is CR101 or CR102 as defined herein. When L or L′ forms a double bond with one of the ring carbons, it may be NR100 with R100 typically being a lone pair. Other similar situations in the present disclosure should be understood similarly.
In some embodiments, L and L′ in the formula I are each independently null, —O—, —C(O)—, —S—, —NR100—, —S(O)—, —SO2—, or —CR101R102—. In some embodiments, the compound of the formula I has a formula according to any one of I-4 to I-5:
where:
In some embodiments, the compound has a formula I-4, where X3 and X4 are each independently —O—, —C(O)—, —S—, —NR100a—, or —SO2—. In some embodiments, the compound has a formula I-5, where X5 is —O—, —C(O)—, —S—, —NR100a—, or —SO2—. In some embodiments, R100a is hydrogen or optionally substituted C1-4 alkyl.
Various W groups are suitable for compounds of the formula I (e.g., any of the sub-formulae described herein, such as the formulae I-1 to I-5). In preferred embodiments, the W group at each occurrence is independently —OH, —NH2, —SO2NH2, —SO2NH(C1-4 alkyl), —SO2NH(C1-4 alkanoyl), —COOH,
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2, —OC(O)NH(C1-4 alkyl)-, —O—(CO)—(C1-4 alkyl), or —O—(C1-4 alkyl), where each of the C1-4 alkyl is independently optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine. In some embodiments, W in the formula I is —OH, —NH2, —SO2NH2, —SO2NH(Acetyl), —COOH,
As described herein, L″-W may in some embodiments be selected as a substituent for Cy1 or Cy2, such as for Ar1 or Ar2. When applicable, L″ in the formula I, including any of the sub-formulae described herein, such as the formulae I-1 to I-5, at each occurrence may be independently null, i.e., the W group is directly attached to Cy1 or Cy2, such as for Ar1 or Ar2, as applicable, or a C1-4 alkylene, C2-4 alkenylene, C2-4 alkynylene or C1-4 heteroalkylene. For example, the W group may be attached to Cy1 or Cy2, such as for Ar1 or Ar2, as applicable, through a methylene or vinyl group. When applicable, W′ in the formula I, including any of the sub-formulae described herein, such as the formulae I-1 to I-5, at each occurrence may be independently —OH, —NH2, —SO2NH2, —SO2NH(C1-4 alkyl). —SO2NH(C1-4 alkanoyl), —COOH,
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2, —OC(O)NH(C1-4 alkyl)-, —O—(CO)—(C1-4 alkyl), or —O—(C1-4 alkyl), where each of the C1-4 alkyl is independently optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine. In some embodiments, each instance of W′ in the formula I, when applicable, may be —OH, —NH2, —SO2NH2, —SO2NH(Acetyl), —COOH,
Various groups may be suitable for R10 and R11 in any of the applicable formula I (e.g., any of the sub-formulae described herein, such as the formulae I-2 to I-5, as applicable). In some embodiments, each of R10 and R11 at each occurrence may be independently F; C1; —OH; —NH2; —SO2NH2; —SO2NH(C1-4 alkyl):—SO2NH(C1-4 alkanoyl); —COOH;
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2; —OC(O)NH(C1-4 alkyl)-; —O—(CO)—(C1-4 alkyl); C1-4 alkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkenyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkynyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C3-6 cycloalkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; C3-6 cycloalkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; or C1-4 alkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine. In some embodiments, each of R10 and R11 at each occurrence may be independently —OH; —NH2; —SO2NH2; —SO2NH(C1-4 alkyl):—SO2NH(C1-4 alkanoyl); —COOH;
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2; —OC(O)NH(C1-4 alkyl)-; —O—(CO)—(C1-4 alkyl); C1-4 alkyl; or C1-4 alkoxy. In some embodiments, one or more instances of R10 and/or one or more instances of R11 may be independently selected from L″-W′ as described herein.
Typically, m, as applicable, is 0, 1, or 2; preferably, 1.
Typically, n, as applicable, is 0, 1, 2, or 3; preferably, 1 or 2.
In some embodiments, the compounds herein may be characterized by having a formula II, or a pharmaceutically acceptable salt or ester thereof:
Cy10-L10-Cy11-L11-W10 Formula II
where:
where:
R1 and R1a are each independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocyclyl;
In some embodiments, in the formula II, at least one of Cy10 and Cy11 is an optionally substituted C6-10 aryl ring, or an optionally substituted 5-10 membered heteroaryl ring. In some embodiments, Cy11 is an optionally substituted C6-10 aryl ring, or an optionally substituted 5-10 membered heteroaryl ring. When Cy11 is a bicyclic or polycyclic aryl or heteroaryl ring, L10-Cy10 and L11-W10 may be independently connected to Cy11 through any of the rings. In some embodiments, Cy11 may have a fused ring structure including an aryl or heteroaryl ring and a cycloalkyl or heterocyclic ring structure. In such embodiments, Cy11 may be connected to L10-Cy10 and L11-W10 through either of the aryl or heteroaryl ring and cycloalkyl or heterocyclic ring structure; or alternatively, one of L10-Cy10 and L11-W10 is connected to Cy11 through the aryl or heteroaryl ring and the other of L10-Cy10 and L11-W10 is connected to Cy11 through the cycloalkyl or heterocyclic ring structure.
In some embodiments, the compound of the formula II has at least one phenyl ring, which may have the following core structure as Cy10-L10-Cy11:
where Cy10 may be the left ring or the right ring in the above drawing, i.e., the drawing is not limited to a particular direction, where L11-w10 may be connected to either the left or the right ring, both of which may be optionally substituted.
In some embodiments, the compound of the formula II may have the following core structure as Cy10-L10-Cy11:
where Cy10 may be the left ring or the right ring in the above drawing, i.e., the drawing is not limited to a particular direction, where L11-w10 may be connected to either the left or the right ring, both of which may be optionally substituted.
In some embodiments, both of Cy10 and Cy11 in the formula II are an aryl or heteroaryl ring. In some embodiments, the compound of the formula II has a formula II-1:
Ar10-L10-Ar11-L11-W10 Formula II-1
where Ar10 and Ar11 are each independently an optionally substituted C6-10 aryl ring, or an optionally substituted 5-10 membered heteroaryl ring. In some embodiments, Ar10 and Ar11 in the formula II-1 are each independently an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring. In some embodiments, Ar10 and Ar11 in the formula II-1 are each independently an optionally substituted phenyl ring, an optionally substituted thienyl ring, an optionally substituted furanyl ring, an optionally substituted pyridyl ring, or an optionally substituted pyrimidinyl ring. In some embodiments, one of Ar10 and Ar11 in the formula II-1 is a bicyclic aryl or bicyclic heteroaryl ring, each of which is optionally substituted, for example, in some embodiments, Ar11 may be an optionally substituted bicyclic aryl or bicyclic heteroaryl ring.
In some embodiments, Cy11 in the formula II is a phenyl ring. In some embodiments, the compound of the formula II has a formula II-2:
where R1, R1a, R2, R2a, R2b, R3, R4, R3a, R3b, R3c, R3d, R3e, R4a, R4b, R4c, R4d, R4e, R5, R5a, and R5b are defined herein, see e.g., the formula II. It should be noted that each instance of the structural units-L11′-W10′ and -L11-W10 is independently selected and may be the same or different.
In some embodiments, Cy11 in the formula II is a benzofused ring. In some embodiments, the compound of the formula II has a formula II-3:
In some embodiments, Cy11 in the formula II is a benzofused bicyclic aryl or heteroaryl ring. For example, in some embodiments, Cy11 in the formula II may have the following core structure:
where the L10-Cy10 and L11-W10 may be independently connected to Cy11 through any of the two rings, where the phenyl ring may be optionally substituted with 1-3 R20 groups defined herein. For example, in the case of the benzothiophene ring, in some embodiments, L10-Cy10 may be attached to the thiophene ring whereas L11-W10 may be attached to the phenyl ring, or vice versa, and in some cases, both L10-Cy10 and L11-W10 may be attached to the same ring, such as the phenyl ring.
In some embodiments, the compound of the formula II may have a structure of any of the following:
where: Cy10, L10, R20, m, R21, n, R100a, L11, and W10 are defined herein, see e.g., the formula II and sub-formulae herein, such as the formula II-3. In some embodiments, Cy10 is Ar10 as defined for the formula II-3.
In some embodiments, the compound of the formula II-3 may have a formula II-4:
When X10 or X11 forms a double bond with one of the ring carbons, it cannot be CR101aR102a with both R101a and R102a present, as the valence of the carbon will exceed 4. In such cases, it should be understood that one of R101a and R102a is absent and X10 or X11 is CR101a or CR102a as defined herein. When X10 or X11 forms a double bond with one of the ring carbons, it may be NR100a with R100a typically being a lone pair.
In some embodiments, the compound of the formula II has a formula II-5:
In some embodiments, L10 in the formula II may be null, and Cy10 is directly linked with Cy11. In some embodiments, L10 in the formula II may be null, —O—, —C(O)—, —S—, —NR100—, —S(O)—, —SO2—, or —CR101R102 —. In some embodiments, L10 in the formula II may be —X1-G1- or —X2-G2-x2a—, where: X1, X2, and X2a are independently —O—, —C(O)—, —S—, —NR100a—, —S(O)—, —SO2—, or —CR101aR102a—; and G1 and G2 are independently —C(O)—, —NR100a—, —S(O)—, —SO2—, or —CR101aR102a—.
In some embodiments, L10 in the formula II may be —X12-G10-. In some embodiments, X12 is optionally substituted C2-4 alkenylene, preferably,
and G10 is —X1-G1- or —X2-G2X2a—; where: X1, X2, and X2a are independently —O—, —C(O)—, —S—, —NR100a—, —S(O)—, —SO2—, or —CR101aR102a —; and G1 and G2 are independently —C(O)—, —NR100a—. —S(O)—, —SO2—, or —CR101aR102a—.
In some preferred embodiments, L10 in the formula II may be
In some embodiments, the compound of the formula II may have the following core structure:
where L11-w10 may be attached to either of the rings, preferably to one of the two phenyl rings or the sole phenyl ring, where each of the rings may be optionally substituted with one or more suitable substituents described herein, for example, each substituent may be independently selected from F; Cl; —OH; —NH2; —SO2NH2; —SO2NH(C1-4 alkyl); —SO2NH(C1-4 alkanoyl); —COOH;
—C(OXO—C1-10 alkyl), —C(OXO—C2-10 alkenyl), —OC(O)NH2; —OC(O)NH(C1-4 alkyl)-; —O—(CO)—(C1-4 alkyl); C1-4 alkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkenyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkynyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C3-6 cycloalkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; C3-6 cycloalkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; or C1-4 alkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; optionally substituted C3-6 cycloalkyl; optionally substituted 4-10 membered heterocyclyl; optionally substituted 5-10 membered heteroaryl; or optionally substituted C6-10 aryl. For example, in some embodiments, the L11-W10 is NH2 or NH(C1-4 alkanoyl), which is connected to one of the two phenyl rings or the sole phenyl ring, whereas the other ring is optionally substituted with 1 or 2 substituents selected from methyl and methoxy.
In some particular embodiments, the compound of the formula II has a formula according to a formula II-6 or II-7:
Various W10 groups are suitable for compounds of the formula II (e.g., the formulae II-1 to II-7). In preferred embodiments, the W10 group at each occurrence is independently —OH, —NH2, —SO2NH2, —SO2NH(C1-4 alkyl):—SO2NH(C1-4 alkanoyl), —COOH,
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2, —OC(O)NH(C1-4 alkyl)-, —O—(CO)—(C1-4 alkyl), —O—(C1-4 alkyl), where each of the C1-4 alkyl is independently optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine. In some embodiments, the W10 group in the formula II is —OH, —OMe, —NH2, —SO2NH2, —SO2NH(Acetyl), —COOH,
As described herein, L11′-W10′ may in some embodiments be selected as a substituent for Cy10 or Cy11, such as for Ar10 or Ar11. When applicable, L11′ in the formula II, including any of the sub-formulae described herein, such as the Formulae II-1 to II-7, at each occurrence may be independently null, i.e., the W10′ group is directly attached to Cy10 or Cy11, such as for Ar10 or Ar11, as applicable, or C1-4 alkylene, C2-4 alkenylene, C2-4 alkynylene or C1-4 heteroalkylene. For example, the W10′ group may be attached to Cy10 or Cy11, such as for Ar10 or Ar11, as applicable, through a methylene or vinyl group. When applicable, W10′ in the formula II, including any of the sub-formulae described herein, such as the formulae II-1 to II-7, at each occurrence may be independently —OH, —NH2, —SO2NH2, —SO2NH(C1-4 alkyl), —SO2NH(C1-4 alkanoyl), —COOH,
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2, —OC(O)NH(C1-4 alkyl)-, —O—(CO)—(C1-4 alkyl), —O—(C1-4 alkyl), where each of the C1-4 alkyl is independently optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine. In some embodiments, each instance of w10′ in the formula II, when applicable, may be —OH, —OMe, —NH2, —SO2NH2, —SO2NH(Acetyl), —COOH, or —O—C(O)—CH3.
Various groups may be suitable for R20, R21, and R22 in any of the applicable formula II (e.g., the formulae II-1 to II-7, as applicable). In some embodiments, each of R20, R21, and R22 at each occurrence may be independently F; C1; —OH; —NH2; —SO2NH2; —SO2NH(C1-4 alkyl); —SO2NH(C1-4 alkanoyl); —COOH;
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2; —OC(O)NH(C1-4 alkyl)-; —O—(CO)—(C1-4 alkyl); C1-4 alkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkenyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkynyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C3-6. cycloalkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; C3-6 cycloalkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; or C1-4 alkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine. In some embodiments, each of R20, R21, and R22 at each occurrence may be independently F; Cl; —OH; —NH2, —SO2NH2, —SO2NH(C1-4 alkyl). —SO2NH(C1-4 alkanoyl), —COOH;
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2; —OC(O)NH(C1-4 alkyl)-; —O—(CO)—(C1-4 alkyl); —O—(C1-6 alkyl); —O— (C2-6 alkenyl); C1-6 alkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-6 alkoxy, —OH, —NH2, and fluorine; or C2-6 alkenyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-6 alkoxy, —OH, —NH2, and fluorine. In some embodiments, each of R20, R21, and R22 at each occurrence may be independently —OH, C1-4 alkyl, C2-6 alkenyl, or —O—(C1-4 alkyl). In some embodiments, each of R20, R21, and R22 at each occurrence may be independently —OH, —OMe, or
In some embodiments, one or more instances of R20, one or more instances of R21, and/or one or more instances of R22 may be independently selected L11′-W10′ as described herein.
Typically, m and p, as applicable, are 0, 1, 2, or 3; preferably, 1 or 2.
Typically, n, as applicable, is 0, 1, or 2; preferably, 0 or 1.
In some embodiments, the compound of the formula II may have a formula according to any of formulae II-8 to II-10:
where R20, R22, m, and p are defined herein. In some embodiments, m is 1 or 2, p is 1, 2, or 3. In some embodiments, each of R20 and R22 at each occurrence is independently F; C1; —OH; —NH2, —SO2NH2, —SO2NH(C1-4 alkyl), —SO2NH(C1-4 alkanoyl), —COOH;
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2; —OC(O)NH(C1-4 alkyl)-; —O—(CO)—(C1-4 alkyl); —O—(C1-6 alkyl); —O— (C2-6 alkenyl); C1-6 alkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-6 alkoxy, —OH, —NH2, and fluorine; or C2-6 alkenyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-6 alkoxy, —OH, —NH2, and fluorine.
In some embodiments, the structural unit
II may be selected from in any of the applicable formula
In some specific embodiments, the compound of the formula II may be:
or a pharmaceutically acceptable salt or ester thereof.
In some embodiments, the compounds herein may be characterized by having a formula III, or a pharmaceutically acceptable salt or ester thereof:
Ar20-L20-W20 Formula III
where:
In some embodiments, Ar20 in the formula III is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring. For example, in some embodiments, Ar20 in the formula III may be an optionally substituted phenyl ring, an optionally substituted thienyl ring, an optionally substituted furanyl ring, an optionally substituted pyridyl ring, or an optionally substituted pyrimidinyl ring. In some embodiments, Ar20 in the formula III may also be an optionally substituted bicyclic aryl or bicyclic heteroaryl ring, each of which is optionally substituted. In such embodiments, L20-w20 may be attached to either of the bicyclic rings.
In some embodiments, Ar20 in the formula III may be an optionally substituted phenyl ring, where two adjacent substituents together with the carbon they are attached to form an optionally substituted cycloalkyl, heterocyclyl, aryl, or heteroaryl ring.
For example, in some embodiments, Ar20 in the formula III may be a benzofused bicyclic aryl or heteroaryl ring. For example, in some embodiments, Ar20 in the formula III may have the following structure:
where -L20-W20 may be attached to either of the two rings, where either or both of the rings may be optionally substituted.
In some embodiments, the compound of the formula III may have a formula III-1, III-2, or III-3:
where R1, R1a, R2, R2a, R2b, R3, R4, R3a, R3b, R3c, R3d, R38, R4a, R4b, R4c, R4d, R4e, R5, R5a, and R5b are defined herein, see e.g., the formula III,
When X20 or X21 forms a double bond with one of the ring carbons, it cannot be CR101aR102a with both R101a and R102a present, as the valence of the carbon will exceed 4. In such cases, it should be understood that one of R101a and R102a is absent and X20 or X21 is CR101a or CR102a as defined herein. When X20 or X21 forms a double bond with one of the ring carbons, it may be NR100a with R100a typically being a lone pair.
It should be noted that each instance of the structural unit-L20′-W20′ and -L20-W20 is independently selected and may be the same or different.
In some embodiments, the compound of the formula III may have a structure of any of the following:
Various W20 groups are suitable for compounds of the formula III (e.g., any of the sub-formulae such as the formulae III-1 to III-3). In preferred embodiments, W20 in the formula III may be —OH, —COOH,
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2, —NH2, —SO2NH2, —SO2NH(C1-4 alkyl):—SO2NH(C1-4 alkanoyl), —OC(O)NH(C1-4 alkyl)-, —O—(CO)—(C1-4 alkyl), —O—(C1-4 alkyl), where each of the C1-4 alkyl is independently optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine. In some embodiments, the W20 group in the formula III (e.g., any of the sub-formulae such as the formulae III-1 to III-3) is —OH, —NH2, —SO2NH2, —SO2NH(Acetyl).
—C(O)—(O—C8 alkyl), —COOH, or —O—C(O)—CH3.
As described herein, L20′-W20′ may in some embodiments be selected as a substituent for Ar20. When applicable, L20′ in the formula III, including any of the sub-formulae described herein, such as the formulae III-1 to III-3, at each occurrence may be independently null, i.e., the W20′ group is directly attached to Ar20, as applicable, or C1-4 alkylene, C2-4 alkenylene, C2-4 alkynylene or C1-4 heteroalkylene. For example, the W20′ group may be attached to Ar20, as applicable, through a methylene or vinyl group. When applicable, W20′ in the formula III, including any of the sub-formulae described herein, such as the formulae III-1 to III-3, at each occurrence may be independently —OH, —COOH,
—C(O)(O—C1-10 alkyl), —C(O)(O—C2-10 alkenyl), —OC(O)NH2, —NH2, —SO2NH2, —SO2NH(C1-4 alkyl):—SO2NH(C1-4 alkanoyl), —OC(O)NH(C1-4 alkyl)-, —O—(CO)—(C1-4 alkyl), —O—(C1-4 alkyl), where each of the C1-4 alkyl is independently optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine. In some embodiments, each instance of W20′ in the formula III, when applicable, may be —OH, —NH2, —SO2NH2, —SO2NH(Acetyl),
—COOH, —C(O)(O—C8 alkyl) or —O—C(O)—CH3.
Various groups may be suitable for R30 and R31 in any of the applicable formula III (e.g., any of the sub-formulae such as the formulae III-1 to III-3). In some embodiments, each of R30 and R31 at each occurrence may be independently F; Cl; —OH; —COOH; —OC(O)NH2; —OC(O)NH(C1-4 alkyl)-; —O—(CO)—(C1-4 alkyl); C1-4 alkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkenyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkynyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C3-6 cycloalkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; C3-6 cycloalkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; or C1-4 alkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine. In some embodiments, each of R30 and R31 at each occurrence may be independently —OH, C2-6 alkenyl, —O—(C1-4 alkyl), —COOH, or —C(O)(O—C1-10 alkyl). In some embodiments, each of R30 and R31 at each occurrence may be —OH or —OMe. In some embodiments, one or more instances of R30 and/or one or more instances of R31 may be independently selected from L20′_W20′ as described herein.
Typically, m is 0, 1, 2, or 3; preferably, 2 or 3. Typically, n is 1, 2 or 3.
In some embodiments, the present disclosure further provides the following compound,
or a pharmaceutically acceptable salt or ester thereof.
In some embodiments, the present disclosure further provides the following compound,
or a pharmaceutically acceptable salt or ester thereof, where q is 1, 2, 3, 4, or 5, and Glu is a residue of glucose. In some specific embodiments, the present disclosure further provides
or a pharmaceutically acceptable salt or ester thereof.
In some embodiments, the compounds herein may also be an alkaloid having antibacterial activity. As shown herein, certain indole alkaloids, such as Vinca alkaloids, tabersonine, vindoline, vinblastine, vincristine, etc., are shown to be effective in killing the microorganisms such as B. megaterium. In some embodiments, the compounds herein are characterized by a formula IV-1 or IV-2, which are tabersonine or vindoline and derivatives:
In some embodiments, the compound of the formula IV-1 or IV-2 has a formula according to one of formulae IV-3 to IV-6:
In some embodiments, R40 in any of the formulae IV-1 to IV-6 may be hydrogen, C1-4 alkyl, or C1-4 alkanoyl.
In some specific embodiments, the compound may have the following structure:
In some embodiments, the compounds herein may also be a glycoside having antibacterial activity, or a pharmaceutically acceptable salt or ester thereof. As shown herein, certain glycosides, such as ginsenosides, and gallic acid glycosides, are shown to be effective in killing the microorganisms such as B. megaterium. Other useful glycosides include any of those known in the art to have antibacterial activity, which may for example, include glycosides characterized by their corresponding aglyone being a phenolic compound, a flavonoid, a coumarin, a benzoic acid, or a sterol. Typically, the glycoside is a glucoside, although other glycosides may also be useful. In some embodiments, the glycosides may be characterized as amphiphilic, which can destroy biological membranes and confer antimicrobial activity to the glycosides. In some embodiments, the glycosides may also be characterized as a saponin, which for example, include various plant derived glycosides that can act as “surfactants” and can help to kill bacteria.
In some embodiments, the glycosides herein may be characterized by a formula V:
where
may be connected to the formula V-A via the steroid backbone or any of the R51 group(s), as valence permits,
In some embodiments, each R50 is hydrogen.
In some embodiments, one to four R50 may be -L50-D which are independently selected. When two or more-L50-D units are linked to the pyranose unit in the formula V, they are preferably the same. In some embodiments, one or more (e.g., 1 or 2) R50 may be a sugar residue which is connected to the remainder of the formula V via a glycoside bond. In some embodiments, the sugar residue is a glucose residue or a rhamnose residue.
Various residues may be used as D, which are typically residues from a phenolic compound, a coumarin, a flavonoid, or a sterol, which in some embodiments may have antibacterial activity without the glycoside unit.
In some embodiments, D may be an optionally substituted ring selected from:
where
may be connected to D via any of the available positions, and
In some embodiments, each of the ring systems of D as shown above may be optionally substituted with 1-5 substituents each independently selected from F; Cl; —OH; —COOH; —C(O)(O—C1-10 alkyl); —C(O)(O—C2-10 alkenyl); —OC(O)NH2; —OC(O)NH(C1-4 alkyl)-; —O—(CO)—(C1-4 alkyl); —NH2; —SO2NH2; —SO2NH(C1-4 alkyl); —SO2NH(C1-4 alkanoyl); C1-4 alkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl; C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkenyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C2-6 alkynyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine; C3-6 cycloalkyl optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; C3-6 cycloalkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl and fluorine; or C1-4 alkoxy optionally substituted with 1-3 substituents independently selected from C1-4 alkyl, C1-4 alkoxy, —OH, —NH2, and fluorine.
In some embodiments, D may be selected from:
where each of the phenolic OH groups is optionally connected to a sugar (such as glucose) via a glycoside bond.
In some embodiments, D is derived from a sterol. For example, in some embodiments, D is
where R52 is optionally substituted alkyl or optionally substituted alkenyl,
where each of the remaining-OH groups in D is optionally connected to a sugar via a glycoside bond.
Preferably, R52 may be
In any of the embodiments described above, the glycoside may have a formula V-1 or V-2:
In some embodiments, the glycoside may be a compound selected from:
In some embodiments, the compounds herein may be any one or more of compounds selected from benzoid acid, benzyl alcohol, coumarins, catechols, polyphenols, chalconoids (including licochalcones), etc., stilbenes such as resveratrol, isoresveratrol, etc., phenolic acids, such as p-hydroxbenzoic acid, 2,4-dihydroxbenzoid acid, protocatechuic acid, gallic acid, vanillic acid, syringic acid, cinnamic acid, coumaric acids, caffeic acids, ferulic acids, chlorogenic acid, sinapic acids etc., flavonoids such as catechin, narigenin, quercetin, rutin, chrysin, etc., tannins, such as ellagic acid, and pharmaceutically acceptable salts or esters thereof and glycosides thereof.
In an implementation of the present disclosure, the inhibitor or microbicid for the microorganism is compounds 1-8, having the following chemical structures:
Preferably, the inhibitor or microbicid for the microorganism in the present disclosure is a natural plant or a natural plant extract or a combination of the natural plant and the natural plant extract, preferably, the natural plant is selected from one or a combination of two or more of radix paeoniae alba, fructus forsythiae, fructus aurantii, rhizoma rehmanniae, dried tangerine peel, pseudo-ginseng, turtle shells, roots of kirilow rhodiola, dried rehmannia roots, coix seeds, semen cassiae and liquorice.
In a specific implementation of the present disclosure, the inhibitor or microbicid for the microorganism is a composition including turtle shells, roots of kirilow rhodiola, dried rehmannia roots, coix seeds, radix paeoniae alba, fructus forsythiae, semen cassiae and liquorice, preferably, the composition includes, by weight, 5-15 parts the turtle shells, 10-30 parts the roots of kirilow rhodiola, 5-15 parts the dried rehmannia roots, 5-15 parts the coix seeds, 8-20 parts the radix paeoniae alba, 5-15 parts the fructus forsythiae, 5-15 parts the semen cassiae and 5-15 parts the liquorice, more preferably, the composition includes 10 parts the turtle shells, 20 parts the roots of kirilow rhodiola, 10 parts the dried rehmannia roots, 10 parts the coix seeds, 12 parts the radix paeoniae alba, 10 parts the fructus forsythiae, 10 parts the semen cassiae and 9 parts the liquorice.
Preferably, administering to the eyes of the patient includes administering to aqueous humor, a suspensory ligament, a ciliary body, a ciliary body and ciliary muscle in an anterior chamber of each eye of the patient as well as to vitreous humor, a retina, a choroid membrane, an optic nerve, a crystalline lens or an iris in a posterior chamber of each eye of the patient.
Preferably, administering to the gastrointestinal tract of the patient includes administering to a stomach, a jejunum, an ileum, a cecum, a colon or a rectum of the patient.
A further solution of the present disclosure provides use of an inhibitor or microbicid for a microorganism in preparation of a drug for treating retinal degeneration.
Preferably, the retinal degeneration is progressive retinal degeneration; more preferably, the retinal degeneration is inherited retinal degeneration; more preferably, the inherited retinal degeneration includes Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), early-onset rod-cone cytodystrophy, rod-rod dystrophy, congenital stationary night blindness and colour blindness and Stargardt disease.
Preferably, the microorganism is one or a combination of two or more of bacteria, archaebacteria, protists, fungi or viruses. The microorganism is the bacteria, and the bacteria are selected from: one or two or more of Anearostipes, Bifidobacterium, Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillussp., Clostridium, Acinetobacter, Streptococcus, Mannheimia, Fibrobacter, Prevotella, Campylobacter, Actinomyces, Hymenobacter, Escherichia, Tissierella, Klebsiella, Porphyromonas, Azospira, Aquimarina, Achromobacter, Acidithiobacillus, Burkholderia, Marinobacter, Treponema, Actinosporangium, Vibrio, Ruminococcus, Methanobrevibacter, Shigella, Frankia, Streptomyces, Anaeroplasma and Coprococcus. More preferably, the bacteria are selected from: Anearostipeshadrus, Bifidobacterium pseudocatenulatum, Nitrosomonas sp.Is79A3, Oscillibactervalericigenes, Tatumella sp.TA1, Megamonasfuniformis, Thiobacillus denitrificans and Akkermansia muciniphila.
Preferably, the inhibitor or microbicid for the microorganism includes a compound, polypeptide, a polynucleotide, a natural plant or a natural plant extract.
More preferably, the inhibitor or microbicid for the microorganism is an antibiotic, for example, the antibiotic is one or two or more of a β-lactam antibiotic, an aminoglycoside antibiotic, a tetracycline antibiotic, a chloramphenicol antibiotic, a macrolide antibiotic, a glycopeptide antibiotic, a quinolone antibiotic, a nitroimidazole antibiotic, a rifamycin antibiotic, an echinocandin antibiotic, a polyene antibiotic, a pyrimidine antibiotic, an allylamine antibiotic, an azole antibiotic and other antibiotics.
For example, in some embodiments, the antibiotic may include one or more of the followings: β-lactam antibiotics, including penicillins (e.g., penicillin V), amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, pivampicillin, pivmecillinam, ticarcillin, cephalosporins such as cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, cefuzonam, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetime, ceftioxide, thienamycins, monobactams, β-lactamase inhibitors, methoxypenicillins; Aminoglycoside antibiotics: including streptomycin, gentamicin, kanamycin (e.g., kanamycin A), tobramycin, amikacin, neomycin (e.g., neomycin B, neomycin C and neomycin E), ribomycin, micronomicin, azithromycin, dibekacin, sisomicin, netilmicin, paramomycin, bramycin, etc.; Tetracycline antibiotics: including tetracycline, oxytetracycline, chlortetracycline, doxycycline, etc.; chloramphenicol antibiotics: including chloramphenicol, thiamphenicol, etc.; macrolide antibiotics: including erythromycin, leucomycin, odorless erythromycin, acetylspiramycin, medimycin, josamycin, azithromycin, clarithromycin, dirithromycin, oxithromycin, telithromycin, etc.; glycopeptide antibiotics: including vancomycin, norvancomycin, teicoplanin, etc.; quinolone antibiotics: including norfloxacin, ofloxacin, ciprofloxacin, pefloxacin, gatifloxacin, enoxacin, lomefloxacin, nalidixic acid, levofloxacin, moxifloxacin, besifloxacin; nitroimidazole antibiotics: including metronidazole, tinidazole, ornidazole, etc.; rifamycinoid antibiotics: including rifampicin; echinocandin antibiotics; polyene antibiotics; pyrimidines antibiotics; allylamine antibiotics; azole antibiotics; other antibiotics: fosfomycin, capreomycin, cycloserine, lincomycin, clindamycin, mitomycin, actinomycin D, bleomycin, doxorubicin, isoniazid, pyrazinamide, cyclosporine, polymyxin B combinations such as polymyxin B/trimethoprim, polymyxin B/bacitracin, polymyxin B/neomycin/gramicidin, etc.
In some embodiments, the antibiotic may be selected from Amikacin, Amoxicillin, Ampicillin, Arsphenamine, Azithromycin, Azlocillin, Aztreonam, Bacitracin, Capreomycin, Carbenicillin, Cefaclor, Cefadroxil, Cefalexin, Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime, Cefprozil, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefuroxime, Chloramphenicol, Cilastatin, Clarithromycin, Clavulanate, Clindamycin, Clofazimine, Cloxacillin, Colistin, Cycloserine, Dalfopristin, Dapsone, Daptomycin, Dicloxacillin, Dirithromycin, Doripenem, Doxycycline, Erythromycin, Ethambutol, Ethionamide, Flucloxacillin, Fosfomycin, Furazolidone, Fusidic acid, Gentamicin, Imipenem, Isoniazid, Kanamycin, Lincomycin, Linezolid, Loracarbef, Mafenide, Meropenem, Methicillin, Metronidazole, Mezlocillin, Minocycline, Mupirocin, Nafcillin, Neomycin, Netilmicin, Nitrofurantoin, Oxacillin, Oxytetracycline, Paromomycin, Penicillin G, Penicillin V, Piperacillin, Platensimycin, Polymyxin B, Pyrazinamide, Quinupristin, Rapamycin, Rifabutin, Rifampicin, Rifampin, Rifapentine, Rifaximin, Roxithromycin, Silver sulfadiazine, Spectinomycin, Streptomycin, Sulbactam, Sulfacetamide, Sulfadiazine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Tazobactam, Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracycline, Thiamphenicol, Ticarcillin, Tigecycline, Tinidazole, Tobramycin, Trimethoprim, Troleandomycin Vancomycin, enoxacin, lomefloxacin, nalidixic acid, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, ofloxacin, norfloxacin, Cefotetan, Cefonicid, Cephradine, Cephapirin, Cephalothin, Cefmetazole, Cefotaxime, Moxalactam, Cefepime, Ceftaroline fosamil, Ceftobiprole, Dalbavancin, Demeclocycline, Metacycline, Ertapenem, Fidaxomicin, geldanamycin, herbimycin, Posizolid, Radezolid, Torezolid, Oritavancin, Spiramycin, Sulfadimethoxine, Sulfonamidochrysoidine, Gemifloxacin Nadifloxacin Trovafloxacin Grepafloxacin Sparfloxacin Temafloxacin, Teixobactin, Malacidins, and combinations thereof.
Preferably, the inhibitor or microbicid for the microorganism is a compound of a formula I (e.g., a formula I-1, a formula I-2, a formula I-3, a formula I-4, and a formula I-5), a formula II (e.g., a formula II-1, a formula II-2, a formula II-3, a formula II-4, a formula II-5, a formula II-6, a formula II-7, a formula II-8, a formula II-9, and a formula II-10), a formula III (e.g., a formula III-1, a formula III-2, and a formula III-3), a formula IV-1 or IV-2 (e.g., a formula IV-3, a formula IV-4, a formula IV-5, and a formula IV-6), a glycoside (e.g., a formula V), or a pharmaceutically acceptable salt or ester thereof, where an aglycone of the glycoside is a phenolic compound, a flavonoid, coumarin, benzoic acid, or a sterol, where the formula I, the formula II, the formula III, the formula IV-1, the formula IV-2, the formula V, and subformulae thereof are defined as above.
In a specific implementation of the present disclosure, the inhibitor or microbicid for the microorganism is compounds 1-8, having the following chemical structures:
Preferably, the inhibitor or microbicid for the microorganism in the present disclosure is a natural plant or a natural plant extract or a combination of the natural plant and the natural plant extract, preferably, the natural plant is selected from one or a combination of two or more of radix paeoniae alba, fructus forsythiae, fructus aurantii, rhizoma rehmanniae, dried tangerine peel, pseudo-ginseng, turtle shells, roots of kirilow rhodiola, dried rehmannia roots, coix seeds, semen cassiae and liquorice.
In a specific implementation of the present disclosure, the inhibitor or microbicid for the microorganism is a composition including turtle shells, roots of kirilow rhodiola, dried rehmannia roots, coix seeds, radix paeoniae alba, fructus forsythiae, semen cassiae and liquorice, preferably, the composition includes, by weight, 5-15 parts the turtle shells, 10-30 parts the roots of kirilow rhodiola, 5-15 parts the dried rehmannia roots, 5-15 parts the coix seeds, 8-20 parts the radix paeoniae alba, 5-15 parts the fructus forsythiae, 5-15 parts the semen cassiae and 5-15 parts the liquorice, more preferably, the composition includes 10 parts the turtle shells, 20 parts the roots of kirilow rhodiola, 10 parts the dried rehmannia roots, 10 parts the coix seeds, 12 parts the radix paeoniae alba, 10 parts the fructus forsythiae, 10 parts the semen cassiae and 9 parts the liquorice.
Preferably, the drug is a preparation for ophthalmic administration or a preparation for gastrointestinal administration. More preferably, the preparation for ophthalmic administration is an eye drop, an eye ointment, an eye gel, an ocular insert, an eye solution or an intraocular injection. More preferably, the preparation for gastrointestinal administration is a tablet (including a sugar-coated tablet, a film-coated tablet, a sublingual tablet, an orally disintegrating tablet, an oral tablet, etc.), a pill, powder, a granule, a capsule (including a soft capsule and a microcapsule), a troche, syrup, a solution, an emulsion, a suspension, a controlled release preparation (for example, an instantaneous release preparation, a sustained release preparation and a sustained-release microcapsule), an aerosol or a film agent (for example, an oral disintegrating film agent and a mouth mucosa-adhesive film agent).
Specifically, the above pharmaceutically acceptable excipient may include one or more of a sweetening agent (specifically, for example, sucrose, xylitol, fructooligosaccharide, sodium cyclamate, stevia sugar, or aspartame), a flavoring agent (for example, spices, or essence), mucilage (specifically, for example, sodium alga acid, Arabic gum, gelatin, methylcellulose, or sodium carboxymethylcellulose), a clarifying agent (specifically, for example, chitosan, or gelatin), a preservative (specifically, for example, benzoic acid and salt thereof, sorbic acid and salt thereof, or nipagin series), a disintegrating agent (specifically, for example, low-substituted hydroxypropyl cellulose, crospovidone, sodium starch glycolate, croscarmellose sodium, or starch), a binder (specifically, for example, hydroxy propyl cellulose, hydroxypropyl methylcellulose, povidone, copovidone, or pregelatinized starch), a lubricant (specifically, for example, stearic acid, magnesium stearate, or sodium fumaryl stearate.), a wetting agent (specifically, for example, polyoxyethylene sorbide fatty acid ester, poloxamer, or a polyoxyethylene castor oil derivative), a suspending agent (specifically, for example, hydroxypropyl methylcellulose, hydroxy propyl cellulose, povidone, copovidone, sodium carboxymethylcellulose, or methylcellulose), a stabilizer (specifically, for example, citric acid, fumaric acid, or succinic acid), a filler (specifically, for example, starch, sucrose, lactose, or microcrystalline cellulose), a binding agent (specifically, for example, a cellulose derivative, alginate, gelatin, or polyvinylpyrrolidone), etc. The present disclosure further provides a method for establishing an eye disease model. The method includes infecting the eye disease model with a microorganism.
Preferably, the infection includes direct contact with the microorganism or indirect contact with the microorganism. In a specific implementation, the eye disease model with eyes infected with the microorganism is obtained by raising a non-human animal in an SPF environment.
Preferably, the microorganism is from intestinal bacteria of the same individual or is the same as the intestinal bacteria of the individual.
Preferably, an eye disease includes retinal degeneration; more preferably, the retinal degeneration is progressive retinal degeneration.
Preferably, the retinal degeneration is inherited retinal degeneration (IRD).
Preferably, the eye disease includes LCA, RP, arRP, EORD, EORP, PPRPE, rettelangiectasia and/or choroideremia like fundus.
Preferably, the eye disease includes ocular inflammation, for example, uveitis, glaucoma, age-related macular degeneration (AMD), hyalitis, choroiditis, retinitis, retinal vasculitis and optic neuritis as well as uveitis, a behcet disease, a Vogt-Koyanagi-Harada syndrome, uveitis, retinopathy, sympathetic ophthalmia, cataract, conjunctivitis, or glaucoma.
Preferably, the model is a non-human animal, preferably, a monkey, a dog, a chimpanzee, a rat and a mouse.
Preferably, a model carrier is a cell, tissue or an organ, and the cell, tissue or organ is from a human or the non-human animal.
Preferably, the cell is a primary cell or cell line.
Preferably, the tissue is ocular tissue, and the organ is an ocular organ.
Preferably, the tissue or the organ is regenerated tissue or a regenerated organ.
Preferably, the model has a gene pathogenic mutation.
Preferably, a gene to which the pathogenic mutation occurs is a gene related to maintaining a retinal barrier structure, and the retinal barrier is an outer blood-retinal barrier and/or an inner blood-retinal barrier.
Preferably, the model or the model carrier has a pathogenic mutation of an eye disease related gene, and the gene to which the eye gene pathogenic mutation occurs is selected from one or a combination of two or more of the following genes: ABCA4, ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHII, AIPLI, ALMS1, AMY2B, APC, ARFGEF1, ARL13B, ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1, C2CD3, CA4, CABP4, CACNAIF, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104, CEP250, CEP290, CEP290, CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC1, CLCN7, CLN3, CLN5, CLN8, CLRNI, CLRNI, CNGA1, CNGA1, CNGA3, CNGB1, CNGB3, CNNM4, COL11A1, COL11A2, COL18A1, COL2A1, COL4A1, COL9A1, COL9A2, CP, CP, CPLANEI, CRB1, ERCC4, CSPP1, CTNNA1, CYP4V2, DHDDS, DYNC2H1, DYNC211, DYNC212, ENPPI, ERCC4, EVC2, EYS, EYS, F5, FAM161A, FBN1, FKRP, FKTN, FLG, FLVCR1, FOXE3, FUZ, GLB1, GMPPB, GNATI, GRKI, GRM6, GUCAIA, GUCAIB, GUCY2D, HADHA, HGSNAT, HPS3, HPS5, IDH3B, IFT122, IFT140, IFT140, IFT43, IFT52, IFT74, IFT80, IFT80, IFT81, IFT88, IKBKG, IMPDH1, IMPG2, INPP5E, INTU, IQCB1, IQCE, IREB2, KCNJ13, KCNQ1, KCNV2, KIAA0586, KIAA0753, KIF7, KIZ, KIZ-AS1, KLHL7, KRITI, LBR, LCA5, LOC101927157, LOC111365204, LRP2, LRP5, MAK, MAPKAPK3, MATK, MCOLNI, MERTK, MKS1, MPDZ, MT-ATP6, MT-CO3, MT-TE, MT-TL1, MTHFR, MUTYH, MYO7A, MYO7A, NMNATI, NPHP1, NR2E3, OCA2, OTX2, PANK2, PAX6, PCARE, PCDH15, PDE6A, PDE6B, PDE6B, PDE6D, PEX1, PEX1, PEX12, PEX26, PEX6, PHF3, PITPNM3, PKD2, PLA2G5, POC5, POMTI, PRCD, PRDM13, PROMI, PRPF3, PRPF31, PRPF8, PRPH2, RAD51C, RBP3, RBP4, RD3, RDH12, RDH5, RGR, RGR, RHO, RIMS1, RLBP1, ROM1, RP1, RPILI, RP2, RPE65, RPE65, RPGR, RPGRIP1, RPGRIPIL, RS1, SACS, SAG, SCAPER, SDCCAG8, SIX6, SLC19A1, SLC22A5, SLC26A4, SLC2A9, SL™, SNRNP200, SPAG17, SPATA7, SPG11, TFAP2A, TGFB2, TGFBR2, TMEM107, TMEM237, TMEM67, TOGARAMI, TOPOR5, TPP1, TRAF3IP1, TREX1, TRIM59-IFT80, TSPAN12, TTC21B, TTC21B, TTC8, TULP1, USHIC, USH2A, USH2A, USH2A, USH2A, USH2A-AS1, VAC14, VCAN, VCAN, VCAN-AS1, VHL, VPS13B, WDR19, WDR19, WDR35, WDR73, YARS1, ZFYVE26, ZFYVE26 and ZNF408.
In a specific implementation, the eye disease related gene to which the pathogenic mutation occurs in the model or the model carrier includes the CRB1 gene.
Preferably, the mutation of the CRB1 gene of the model or the model carrier includes one or two or more of the following mutations: c. 107C>G, c.111delT, c. 135C>G, c.257_258dupTG, c.258C>T, c.428_432delGATTC, c.430T>G, c.470G>C, c.481 dupG, c.482C>T, c.584G>T, c.613_619del, c.717_718insG, c.750T>G, c.915T>A, c.929G>A, c.936T>G, c.998G>A, c.1084C>T, c. 1125C>G, c. 1148G>A, c. 1208C>G, c. 1269C>A, c. 1298A>G, c.1313G>A, c.1438T>C, c. 1438T>G, c. 1576C>T, c. 1604T>C, c. 1690G>T, c. 1733T>A, c. 1750G>T, c. 1760G>A, c.1834T>C, c.1963delC, c.2025G>T, c.2042G>A, c.2128G>C, c.2129C>T, c.2185_2186insAlu, c.2219C>T, c.2222T>C, c.2234C>T, c.2245_2247del 3 bp (TCA), c.2258T>C, c.2290C>T, c.2365_2367del AAT, in frame deletion, c.2401A>T, c.2438_2439ins>100A, c.2441_2442del, c.2465G>A, c.2479G>T, c.2506C>A, c.2509G>C, c.2536G>A, c.2548_2551delGGCT, c.2548G>A, c.2555T>C, c.2611_2613insT, c.2671T>G, c.2676delG, c.2681A>G, c.2688T>A, c.2816G>A, c.2843G>A, c.2853dupT, c.2884_2886delTTA, c.2957A>T, c.2966T>C, c.2983G>T, c.3002A>T, c.3008T>C, c.3035T>C, c.3037C>T, c.3074G>A, c.3074G>T, c.3122T>C, c.3212T>C, c.3296C>A, c.3299T>C, c.3299T>G, c.3307G>A/C, c.3320T>C, c.3320T>G, c.3331G>T, c.3343_3352del, c.3347delT, c.3343_3352del, c.3347delT, c.3427delT, c.3482A>G, c.3493T>C, c.3655T>G, c.3541T>C, c.3542dupG, c.3593A>G, c.3613G>A, c.3653G>T, c.3659_3660delinsA, c.3664C>T, c.3668G>C, c.3676G>T, c.3713_3716dup, c.3879G>A, c.3914C>T, c.3949A>C, c.3961T>A, c.3988delG, c.3988G>T, c.3995G>T, c.3996C>A, c.3997G>T, c.4094C>A, c.4121_4130del, c.4142C>T, c.4148G>A, c.2128+2T>G, c.2842+5G>A, c.3878+1G>T, c.4005+1G>A, c.4005+2T>G, c.4006-2A>G, c.4006-1G>T, c.619G>A, c.614T>C, c. 1472A>T, c. 1903T>C, c.2809G>A, c.3103C>T, c.4082G>A, c.4060G>A, c.866C>T, c.1463T>C, c.2035C>G, c.2306_2307GC>AG, c.2306G>A, c.2714G>A, c.2875G>A and c.3992G>A.
Further preferably, the mutation of the CRB1 gene of the model or the model carrier includes one or two or more of the following mutations: c.4006-1G>T, c.3686G>C, (p.Cys1229Ser), c.2842+1delinsAA, c.4060G>A, (p.Ala1354Thr), c.3991C>T, (p.Arg1331Cys), c.3014A>T, (p.Asp1005Val), c.4005+1G>A, c.2680_2684del, (p.Asn894fs), c. 1733T>A, (p. Val578Glu), c.455G>A, (p.Cys152Tyr), c.3462_3463del, (p.Cys1154-Glu1155delinsTer), c.3037C>T, (p.Gln1013Ter), c.2673C>A, (p.Cys891Ter), c.2230C>T, (p.Arg744Ter), c.3676G>T, (p.Glyl226Ter), c.2842+5G>A, c.2842T>C, (p.Cys948Arg), c.3988del, (p.Glu1330fs), c.2506C>A, (p.Pro836Thr), c.2291G>A, (p.Arg764His), c. 1576C>T, (p.Arg526Ter), c.613_619del, (p.Ile205fs), c.3320T>C, (p.Leu1107Pro), c.2688T>A, (p.Cys896Ter), c.2555T>C, (p.Ile852Thr), c.2222T>C, (p.Met741Thr), c.1148G>A, (p.Cys383Tyr), c.2843G>A, (p.Cys948Tyr), c.4121_4130del, (p.Ala1374fs), c.3307G>A, (p.Glyl103Arg), c.484G>A, (p.Val162Met), c.2401A>T, (p.Lys801Ter), c.2234C>T, (p.Thr745Met), c.2290C>T, (p.Arg764Cys), c.3122T>C and (p.Met1041Thr).
Preferably, the mutation of the CRB1 gene of the model or the model carrier is the Rd8 mutation.
Preferably, the mutation is a homozygous mutation or a heterozygous mutation.
Preferably, the aforementioned gene mutation exists innately in a body of the model or the model carrier or the mutation is acquired due to a gene recombination operation.
Preferably, a humanized CRB1 gene or a human CRB1 gene exists in the body of the model or the model carrier, and an endogenous CRB1 gene is missing or not expressed.
Preferably, the non-human animal has a colonic epithelium barrier defect and/or related colonic wall inflammation.
In a specific example, protein Occludin in the model body is significantly missing. In a specific example, the protein Occludin in the model body is significantly missing and Claudin 1 expression is not significant.
The microorganism is one or a combination of two or more of the bacteria, archaebacteria, protists, fungi or viruses, preferably, the microorganism is the bacteria, and the bacteria are selected from: one or two or more of Anearostipes, Bifidobacterium, Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillussp., Clostridium, Acinetobacter, Streptococcus, Mannheimia, Fibrobacter, Prevotella, Campylobacter, Actinomyces, Hymenobacter, Escherichia, Tissierella, Klebsiella, Porphyromonas, Azospira, Aquimarina, Achromobacter, Acidithiobacillus, Burkholderia, Marinobacter, Treponema, Actinosporangium, Vibrio, Ruminococcus, Methanobrevibacter, Shigella, Frankia, Streptomyces, Anaeroplasma and Coprococcus.
Specifically, the bacteria are selected from: one or two or more of Anearostipeshadrus, Bifidobacterium pseudocatenulatum, Nitrosomonas sp.Is79A3, Oscillibactervalericigenes, Tatumella sp.TA1, Megamonasfuniformis, Thiobacillus denitrificans, Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Acinetobacter calcoaceticus, Acinetobacter Lwoffii, Acinetobacter baumanii, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter johnsonii, Pyogenic streptococcus, Hemolytic streptococcus, Fibrobacter succinogenes, Intestinal Bacillus filiformis, Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas gingivalis, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis, Campylobacter concisus, Campylobacter fetus, Actinomyces israelii, Actinomyces naeslundii, Actinomyces odontolyticus, Actinomyces viscosus, Actinomyces neuii, Escherichia coli, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Tissierella praeacuta, Klebsiella pneumoniae, Klebsiella ozaenae, Azospirillum brasilense, Achromobacter, Thiobacillus denitrificans, Thiobacillus ferrooxidans, Thiobacillus thiooxidans, Thiobacillus neapolitanus, Burkholderia, Mycobacterium marinum, Treponema pallidum, Treponema hyodysenteriae, Vibrio metschnikovi, Ruminococcus albus, Ruminococcus flavefaciens, Methanobrevibacter ruminantium, Shigella dysenteriae, Shigella flexneri, Shigella bogdii, Shigella sonnei, Frankia, Coprococcus eutactus, Streptomyces albus, Pseudomonas mendocina, Micrococcus sedentarius, Alicycline denitrifying bacteria, Achromobacter xylosoxidans, Sphingomonas, Mycobacterium abscessus, Arthrobacter aurescens, Prevotella, Sinorhizobium meliloti, Acid yeast, Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Pseudomonas putida, Stenotrophomonas maltophilia, Bacillus cereus, Bacillus megaterium, Lactobacillus reuteri, Haemophilus vaginalis, Bee Enterococcus faecium, Normal cytophaga hutchinsonii, Bacillus licheniformis, Xanthomonas oryzae pv. oryzae, Acinetobacter baumannii, Acinetobacter calcoaceticus, Comamonas testosteroni, Mycobacterium kansasii, Bacillus thuringiensis, Citrobacter koseri, Dyadobacter fermentans, Serratia marcescens, Sphingomonas wittichii, Klebsiella pneumoniae, Pseudomonas fluorescens, Ralstonia pickettii, Lactobacillus crispatus, Burkholderia, Lactobacillus delbrueckii, Meiothermussilvanus (D), Colon Bacillus, Micrococcus luteus, Bacillus subtilis, Corynebacterium aurimucosum and Finegoldia magna.
Preferably, the infection method includes causing the microorganism to directly or indirectly make contact with a to-be -infected part of the model carrier, and the indirect contact refers to a blood-retinal barrier existing between the microorganism and the to-be -infected part, preferably, the blood-retinal barrier is the outer blood-retinal barrier or the inner blood-retinal barrier.
In a specific implementation, the infection method includes infecting eyes with intestinal bacteria through peripheral blood. Specifically, an intestinal epithelial barrier of the non-human animal model is significantly damaged, resulting in that the intestinal bacteria enter the peripheral blood. In a model body, the retinal barrier is also significantly damaged, so the retina is infected with the intestinal bacteria entering the peripheral blood. In a specific implementation, the mutation of the CRB1 gene occurs to the non-human animal model. In a specific implementation, the mutation of the CRB1 gene is the Rd8 mutation.
Further, the present disclosure provides a method for preparing an ocular inflammation model, and the method model is infecting the non-human animal with the microorganism.
Preferably, the ocular inflammation is caused by an intestinal flora or a flora which is the same as the intestinal flora.
The non-human animal is the aforementioned animal.
The microorganism is the aforementioned microorganism.
Further, the present disclosure provides a method for preparing a retinal degeneration disease model, and the method includes infecting the non-human animal suffering from the retinal degeneration with the microorganism.
Preferably, the ocular inflammation retinal degeneration disease is the aforementioned retinal disease.
The non-human animal is the aforementioned animal.
The microorganism is the aforementioned microorganism.
Further, the present disclosure provides a model carrier suffering from the ocular inflammation, and the ocular inflammation is caused by being infected with the microorganism.
Preferably, the microorganism is from intestinal bacteria of the same individual or is the same as the intestinal bacteria of the individual.
Preferably, an eye disease includes retinal degeneration; more preferably, the retinal degeneration is progressive retinal degeneration.
Preferably, the retinal degeneration is inherited retinal degeneration (IRD).
Preferably, the retinal degeneration is an inherent disease of the model animal or the retinal degeneration from which the model animal suffers due to a gene manipulation.
Preferably, the eye disease includes LCA, RP, arRP, EORD, EORP, PPRPE, rettelangiectasia and/or choroideremia like fundus.
Preferably, the eye disease includes ocular inflammation, for example, uveitis, glaucoma, age-related macular degeneration (AMD), hyalitis, choroiditis, retinitis, retinal vasculitis and optic neuritis as well as uveitis, a behcet disease, a Vogt-Koyanagi-Harada syndrome, uveitis, retinopathy, sympathetic ophthalmia, cataract, conjunctivitis, or glaucoma.
Preferably, the model is a non-human animal, preferably, a monkey, a dog, a chimpanzee, a rat and a mouse.
Preferably, a model carrier is a cell, tissue or an organ, and the cell, tissue or organ is from a human or the non-human animal.
Preferably, the cell is a primary cell or cell line.
Preferably, the tissue is ocular tissue, and the organ is an ocular organ.
Preferably, the tissue or the organ is regenerated tissue or a regenerated organ.
Preferably, the model has a gene pathogenic mutation.
Preferably, a gene to which the pathogenic mutation occurs is a gene related to maintaining a retinal barrier structure, and the retinal barrier is the outer blood-retinal barrier and/or the inner blood-retinal barrier.
Preferably, in the model carrier, the mutation occurs to one or two or more of the following genes: one or a combination of two or more of ABCA4, ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHII, AIPLI, ALMS1, AMY2B, APC, ARFGEF1, ARL13B, ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1, C2CD3, CA4, CABP4, CACNAIF, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104, CEP250, CEP290, CEP290, CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC1, CLCN7, CLN3, CLN5, CLN8, CLRNI, CLRNI, CNGA1, CNGA1, CNGA3, CNGB1, CNGB3, CNNM4, COL11A1, COL11A2, COL18A1, COL2A1, COL4A1, COL9A1, COL9A2, CP, CP, CPLANEI, CRB1, ERCC4, CSPP1, CTNNA1, CYP4V2, DHDDS, DYNC2H1, DYNC211, DYNC212, ENPPI, ERCC4, EVC2, EYS, EYS, F5, FAM161A, FBN1, FKRP, FKTN, FLG, FLVCR1, FOXE3, FUZ, GLB1, GMPPB, GNATI, GRKI, GRM6, GUCAIA, GUCAIB, GUCY2D, HADHA, HGSNAT, HPS3, HPS5, IDH3B, IFT122, IFT140, IFT140, IFT43, IFT52, IFT74, IFT80, IFT80, IFT81, IFT88, IKBKG, IMPDH1, IMPG2, INPP5E, INTU, IQCB1, IQCE, IREB2, KCNJ13, KCNQ1, KCNV2, KIAA0586, KIAA0753, KIF7, KIZ, KIZ-AS1, KLHL7, KRITI, LBR, LCA5, LOC101927157, LOC111365204, LRP2, LRP5, MAK, MAPKAPK3, MATK, MCOLNI, MERTK, MKS1, MPDZ, MT-ATP6, MT-CO3, MT-TE, MT-TL1, MTHFR, MUTYH, MYO7A, MYO7A, NMNATI, NPHP1, NR2E3, OCA2, OTX2, PANK2, PAX6, PCARE, PCDH15, PDE6A, PDE6B, PDE6B, PDE6D, PEX1, PEX1, PEX12, PEX26, PEX6, PHF3, PITPNM3, PKD2, PLA2G5, POC5, POMTI, PRCD, PRDM13, PROMI, PRPF3, PRPF31, PRPF8, PRPH2, RAD51C, RBP3, RBP4, RD3, RDH12, RDH5, RGR, RGR, RHO, RIMS1, RLBP1, ROM1, RP1, RPILI, RP2, RPE65, RPE65, RPGR, RPGRIP1, RPGRIPIL, RS1, SACS, SAG, SCAPER, SDCCAG8, SIX6, SLC19A1, SLC22A5, SLC26A4, SLC2A9, SL™, SNRNP200, SPAG17, SPATA7, SPG11, TFAP2A, TGFB2, TGFBR2, TMEM107, TMEM237, TMEM67, TOGARAMI, TOPOR5, TPP1, TRAF3IP1, TREX1, TRIM59-IFT80, TSPAN12, TTC21B, TTC21B, TTC8, TULP1, USHIC, USH2A, USH2A, USH2A, USH2A, USH2A-AS1, VAC14, VCAN, VCAN, VCAN-AS1, VHL, VPS13B, WDR19, WDR19, WDR35, WDR73, YARS1, ZFYVE26, ZFYVE26 and ZNF408.
In a specific implementation, the eye disease related gene to which the pathogenic mutation occurs in the model carrier includes the CRB1 gene.
Preferably, the mutation of the CRB1 gene of the model carrier includes one or two or more of the following mutations: c. 107C>G, c.111delT, c.135C>G, c.257_258dupTG, c.258C>T, c.428_432delGATTC, c.430T>G, c.470G>C, c.481dupG, c.482C>T, c.584G>T, c.613_619del, c.717_718insG, c.750T>G, c.915T>A, c.929G>A, c.936T>G, c.998G>A, c. 1084C>T, c.1125C>G, c.1148G>A, c. 1208C>G, c.1269C>A, c. 1298A>G, c. 1313G>A, c. 1438T>C, c. 1438T>G, c. 1576C>T, c. 1604T>C, c. 1690G>T, c. 1733T>A, c. 1750G>T, c. 1760G>A, c. 1834T>C, c. 1963delC, c.2025G>T, c.2042G>A, c.2128G>C, c.2129C>T, c.2185_2186insAlu, c.2219C>T, c.2222T>C, c.2234C>T, c.2245_2247del 3 bp (TCA), c.2258T>C, c.2290C>T, c.2365_2367del AAT, in frame deletion, c.2401A>T, c.2438_2439ins>100A, c.2441_2442del, c.2465G>A, c.2479G>T, c.2506C>A, c.2509G>C, c.2536G>A, c.2548_2551delGGCT, c.2548G>A, c.2555T>C, c.2611_2613insT, c.2671T>G, c.2676delG, c.2681A>G, c.2688T>A, c.2816G>A, c.2843G>A, c.2853dupT, c.2884_2886delTTA, c.2957A>T, c.2966T>C, c.2983G>T, c.3002A>T, c.3008T>C, c.3035T>C, c.3037C>T, c.3074G>A, c.3074G>T, c.3122T>C, c.3212T>C, c.3296C>A, c.3299T>C, c.3299T>G, c.3307G>A/C, c.3320T>C, c.3320T>G, c.3331G>T, c.3343_3352del, c.3347delT, c.3343_3352del, c.3347delT, c.3427delT, c.3482A>G, c.3493T>C, c.3655T>G, c.3541T>C, c.3542dupG, c.3593A>G, c.3613G>A, c.3653G>T, c.3659_3660delinsA, c.3664C>T, c.3668G>C, c.3676G>T, c.3713_3716dup, c.3879G>A, c.3914C>T, c.3949A>C, c.3961T>A, c.3988delG, c.3988G>T, c.3995G>T, c.3996C>A, c.3997G>T, c.4094C>A, c.4121_4130del, c.4142C>T, c.4148G>A, c.2128+2T>G, c.2842+5G>A, c.3878+1G>T, c.4005+1G>A, c.4005+2T>G, c.4006-2A>G, c.4006-1G>T, c.619G>A, c.614T>C, c. 1472A>T, c. 1903T>C, c.2809G>A, c.3103C>T, c.4082G>A, c.4060G>A, c.866C>T, c.1463T>C, c.2035C>G, c.2306_2307GC>AG, c.2306G>A, c.2714G>A, c.2875G>A and c.3992G>A.
Further preferably, the mutation of the CRB1 gene of the model or the model carrier includes one or two or more of the following mutations: c.4006-1G>T, c.3686G>C, (p.Cys1229Ser), c.2842+1delinsAA, c.4060G>A, (p.Ala1354Thr), c.3991C>T, (p.Arg1331Cys), c.3014A>T, (p.Asp1005Val), c.4005+1G>A, c.2680_2684del, (p.Asn894fs), c. 1733T>A, (p. Val578Glu), c.455G>A, (p.Cys152Tyr), c.3462_3463del, (p.Cys1154-Glu1155delinsTer), c.3037C>T, (p.Gln1013Ter), c.2673C>A, (p.Cys891Ter), c.2230C>T, (p.Arg744Ter), c.3676G>T, (p.Glyl226Ter), c.2842+5G>A, c.2842T>C, (p.Cys948Arg), c.3988del, (p.Glu1330fs), c.2506C>A, (p.Pro836Thr), c.2291G>A, (p.Arg764His), c. 1576C>T, (p.Arg526Ter), c.613_619del, (p.Ile205fs), c.3320T>C, (p.Leul107Pro), c.2688T>A, (p.Cys896Ter), c.2555T>C, (p.Ile852Thr), c.2222T>C, (p.Met741Thr), c.1148G>A, (p.Cys383Tyr), c.2843G>A, (p.Cys948Tyr), c.4121_4130del, (p.Ala1374fs), c.3307G>A, (p.Glyl103Arg), c.484G>A, (p.Val162Met), c.2401A>T, (p.Lys801Ter), c.2234C>T, (p.Thr745Met), c.2290C>T, (p.Arg764Cys), c.3122T>C and (p.Met1041Thr).
Preferably, the mutation of the CRB1 gene of the model carrier is the Rd8 mutation.
Preferably, the mutation is a homozygous mutation or a heterozygous mutation.
The aforementioned gene mutation exists innately in a body of the model carrier or the mutation is acquired due to a gene recombination manipulation.
Preferably, a humanized CRB1 gene or a human CRB1 gene exists in the body of the model carrier, and an endogenous CRB1 gene is missing or not expressed.
Preferably, the non-human animal has a colonic epithelium barrier defect and/or related colonic wall inflammation.
In a specific example, protein Occludin in the model body is significantly missing. In a specific example, the protein Occludin in the model body is significantly missing and Claudin1 expression is not significant.
The microorganism is one or a combination of two or more of the bacteria, archaebacteria, protists, fungi or viruses, preferably, the microorganism is the bacteria, and the bacteria are selected from: one or two or more of Anearostipes, Bifidobacterium, Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillussp., Clostridium, Acinetobacter, Streptococcus, Mannheimia, Fibrobacter, Prevotella, Campylobacter, Actinomyces, Hymenobacter, Escherichia, Tissierella, Klebsiella, Porphyromonas, Azospira, Aquimarina, Achromobacter, Acidithiobacillus, Burkholderia, Marinobacter, Treponema, Actinosporangium, Vibrio, Ruminococcus, Methanobrevibacter, Shigella, Frankia, Streptomyces, Anaeroplasma and Coprococcus.
Specifically, the bacteria are selected from: one or two or more of Anearostipeshadrus, Bifidobacterium pseudocatenulatum, Nitrosomonas sp.Is79A3, Oscillibactervalericigenes, Tatumella sp.TA1, Megamonasfuniformis, Thiobacillus denitrificans, Clostridium tetani, Clostridium perfringens, Clostridium botulinum, Acinetobacter calcoaceticus, Acinetobacter Lwoffii, Acinetobacter baumanii, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter johnsonii, Pyogenic streptococcus, Hemolytic streptococcus, Fibrobacter succinogenes, Intestinal Bacillus filiformis, Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas gingivalis, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis, Campylobacter concisus, Campylobacter fetus, Actinomyces israelii, Actinomyces naeslundii, Actinomyces odontolyticus, Actinomyces viscosus, Actinomyces neuii, Escherichia coli, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, Tissierella praeacuta, Klebsiella pneumoniae, Klebsiella ozaenae, Azospirillum brasilense, Achromobacter, Thiobacillus denitrificans, Thiobacillus ferrooxidans, Thiobacillus thiooxidans, Thiobacillus neapolitanus, Burkholderia, Mycobacterium marinum, Treponema pallidum, Treponema hyodysenteriae, Vibrio metschnikovi, Ruminococcus albus, Ruminococcus flavefaciens, Methanobrevibacter ruminantium, Shigella dysenteriae, Shigella flexneri, Shigella bogdii, Shigella sonnei, Frankia, Coprococcus eutactus, Streptomyces albus, Pseudomonas mendocina, Micrococcus sedentarius, Alicycline denitrifying bacteria, Achromobacter xylosoxidans, Sphingomonas, Mycobacterium abscessus, Arthrobacter aurescens, Prevotella, Sinorhizobium meliloti, Acid yeast, Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Pseudomonas putida, Stenotrophomonas maltophilia, Bacillus cereus, Bacillus megaterium, Lactobacillus reuteri, Haemophilus vaginalis, Bee Enterococcus faecium, Normal cytophaga hutchinsonii, Bacillus licheniformis, Xanthomonas oryzae pv. oryzae, Acinetobacter baumannii, Acinetobacter calcoaceticus, Comamonas testosteroni, Mycobacterium kansasii, Bacillus thuringiensis, Citrobacter koseri, Dyadobacter fermentans, Serratia marcescens, Sphingomonas wittichii, Klebsiella pneumoniae, Pseudomonas fluorescens, Ralstonia pickettii, Lactobacillus crispatus, Burkholderia, Lactobacillus delbrueckii, Meiothermussilvanus (D), Colon Bacillus, Micrococcus luteus, Bacillus subtilis, Corynebacterium aurimucosum and Finegoldia magna.
Preferably, the infection method includes causing the microorganism to directly or indirectly make contact with the to-be -infected part of the model carrier, and the indirect contact refers to the blood-retinal barrier existing between the microorganism and the to-be -infected part, preferably, the blood-retinal barrier is the outer blood-retinal barrier or the inner blood-retinal barrier.
In a specific implementation, the infection method includes infecting the eyes with the intestinal bacteria through the peripheral blood. Specifically, the intestinal epithelial barrier of the non-human animal model is significantly damaged, resulting in that the intestinal bacteria enter the peripheral blood. In a model body, the retinal barrier is also significantly damaged, so the retina is infected with the intestinal bacteria entering the peripheral blood. In a specific implementation, the mutation of the CRB1 gene occurs to the non-human animal model. In a specific implementation, the mutation of the CRB1 gene is the Rd8 mutation.
The model carrier suffering from the inflammation is from the disease model prepared through the aforementioned method, or is obtained by infecting ocular cells, tissue or organs from the aforementioned non-human animal with the microorganism.
The present disclosure provides use of the aforementioned method in evaluation of an eye disease targeted therapy therapeutic effect, and an eye disease includes the aforementioned eye diseases.
In a specific implementation, a targeted therapy for the aforementioned gene mutation is performed on the aforementioned model or model carrier, the model or the model carrier subjected to the targeted therapy or not subjected to the targeted therapy is divided into two groups, an eye disease model is established respectively according to the aforementioned method, and if by contrast, modeling is not successful for the group subjected to the targeted therapy, it indicates that the targeted therapy achieves a beneficial effect.
In a specific implementation, the targeted therapy is targeted at one or a combination of two or more of the following genes: ABCA4, ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHII, AIPLI, ALMS1, AMY2B, APC, ARFGEF1, ARL13B, ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1, C2CD3, CA4, CABP4, CACNAIF, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104, CEP250, CEP290, CEP290, CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC1, CLCN7, CLN3, CLN5, CLN8, CLRNI, CLRNI, CNGA1, CNGA1, CNGA3, CNGB1, CNGB3, CNNM4, COL11A1, COL11A2, COL18A1, COL2A1, COL4A1, COL9A1, COL9A2, CP, CP, CPLANEI, CRB1, ERCC4, CSPP1, CTNNA1, CYP4V2, DHDDS, DYNC2H1, DYNC211, DYNC212, ENPPI, ERCC4, EVC2, EYS, EYS, F5, FAM161A, FBN1, FKRP, FKTN, FLG, FLVCR1, FOXE3, FUZ, GLB1, GMPPB, GNATI, GRKI, GRM6, GUCAIA, GUCAIB, GUCY2D, HADHA, HGSNAT, HPS3, HPS5, IDH3B, IFT122, IFT140, IFT140, IFT43, IFT52, IFT74, IFT80, IFT80, IFT81, IFT88, IKBKG, IMPDH1, IMPG2, INPP5E, INTU, IQCB1, IQCE, IREB2, KCNJ13, KCNQ1, KCNV2, KIAA0586, KIAA0753, KIF7, KIZ, KIZ-AS1, KLHL7, KRITI, LBR, LCA5, LOC101927157, LOC111365204, LRP2, LRP5, MAK, MAPKAPK3, MATK, MCOLNI, MERTK, MKS1, MPDZ, MT-ATP6, MT-CO3, MT-TE, MT-TL1, MTHFR, MUTYH, MYO7A, MYO7A, NMNATI, NPHP1, NR2E3, OCA2, OTX2, PANK2, PAX6, PCARE, PCDH15, PDE6A, PDE6B, PDE6B, PDE6D, PEX1, PEX1, PEX12, PEX26, PEX6, PHF3, PITPNM3, PKD2, PLA2G5, POC5, POMTI, PRCD, PRDM13, PROMI, PRPF3, PRPF31, PRPF8, PRPH2, RAD51C, RBP3, RBP4, RD3, RDH12, RDH5, RGR, RGR, RHO, RIMS1, RLBP1, ROM1, RP1, RPILI, RP2, RPE65, RPE65, RPGR, RPGRIP1, RPGRIPIL, RS1, SACS, SAG, SCAPER, SDCCAG8, SIX6, SLC19A1, SLC22A5, SLC26A4, SLC2A9, SL™, SNRNP200, SPAG17, SPATA7, SPG11, TFAP2A, TGFB2, TGFBR2, TMEM107, TMEM237, TMEM67, TOGARAMI, TOPOR5, TPP1, TRAF3IP1, TREX1, TRIM59-IFT80, TSPAN12, TTC21B, TTC21B, TTC8, TULP1, USHIC, USH2A, USH2A, USH2A, USH2A, USH2A-AS1, VAC14, VCAN, VCAN, VCAN-AS1, VHL, VPS13B, WDR19, WDR19, WDR35, WDR73, YARS1, ZFYVE26, ZFYVE26 and ZNF408.
In a specific implementation, the targeted therapy is targeted at one or two or more of the following mutations of the CRB1 gene: c.257_258dupTG, c.258C>T, c.428_432delGATTC, c.430T>G, c.470G>C, c.481 dupG, c.482C>T, c.584G>T, c.613_619del, c.717_718insG, c.750T>G, c.915T>A, c.929G>A, c.936T>G, c.998G>A, c. 1084C>T, c. 1125C>G, c.1148G>A, c.1208C>G, c.1269C>A, c. 1298A>G, c. 1313G>A, c.1438T>C, c. 1438T>G, c.1576C>T, c. 1604T>C, c. 1690G>T, c. 1733T>A, c.1750G>T, c. 1760G>A, c. 1834T>C, c. 1963delC, c.2025G>T, c.2042G>A, c.2128G>C, c.2129C>T, c.2185_2186insAlu, c.2219C>T, c.2222T>C, c.2234C>T, c.2245_2247del 3 bp (TCA), c.2258T>C, c.2290C>T, c.2365_2367del AAT, in frame deletion, c.2401A>T, c.2438_2439ins>100A, c.2441_2442del, c.2465G>A, c.2479G>T, c.2506C>A, c.2509G>C, c.2536G>A, c.2548_2551delGGCT, c.2548G>A, c.2555T>C, c.2611_2613insT, c.2671T>G, c.2676delG, c.2681A>G, c.2688T>A, c.2816G>A, c.2843G>A, c.2853dupT, c.2884_2886delTTA, c.2957A>T, c.2966T>C, c.2983G>T, c.3002A>T, c.3008T>C, c.3035T>C, c.3037C>T, c.3074G>A, c.3074G>T, c.3122T>C, c.3212T>C, c.3296C>A, c.3299T>C, c.3299T>G, c.3307G>A/C, c.3320T>C, c.3320T>G, c.3331G>T, c.3343_3352del, c.3347delT, c.3343_3352del, c.3347delT, c.3427delT, c.3482A>G, c.3493T>C, c.3655T>G, c.3541T>C, c.3542dupG, c.3593A>G, c.3613G>A, c.3653G>T, c.3659_3660delinsA, c.3664C>T, c.3668G>C, c.3676G>T, c.3713_3716dup, c.3879G>A, c.3914C>T, c.3949A>C, c.3961T>A, c.3988delG, c.3988G>T, c.3995G>T, c.3996C>A, c.3997G>T, c.4094C>A, c.4121_4130del, c.4142C>T, c.4148G>A, c.2128+2T>G, c.2842+5G>A, c.3878+1G>T, c.4005+1G>A, c.4005+2T>G, c.4006-2A>G, c.4006-1G>T, c.619G>A, c.614T>C, c. 1472A>T, c.1903T>C, c.2809G>A, c.3103C>T, c.4082G>A, c.4060G>A, c.866C>T, c. 1463T>C, c.2035C>G, c.2306_2307GC>AG, c.2306G>A, c.2714G>A, c.2875G>A and c.3992G>A.
In a specific implementation, a targeted therapy drug includes modified cells, modified protein, RNA targeted at the aforementioned gene or at the aforementioned mutation site and/or DNA targeted at the aforementioned gene or at the aforementioned mutation site.
The present disclosure provides use of the disease model prepared by the aforementioned method in eye disease related study. The eye disease includes the aforementioned eye diseases. The study includes a mutual response between a disease related to inherited retinal degeneration and intestinal flora, and the like.
In a specific implementation, the disease model is infected with a type of intestinal bacteria, in a case that a pathogenic mutation carried thereby is subjected to a cell therapy, a therapeutic effect with drug administration and a therapeutic effect without drug administration are observed, and the drug is a small molecule drug, preferably, a broad-spectrum antibiotic or an antibiotic for infecting bacteria. In a specific implementation, the disease model is infected with a type of intestinal bacteria, in a case that a pathogenic mutation carried thereby is subjected to an RNA therapy, a therapeutic effect with drug administration and a therapeutic effect without drug administration are observed, and the drug is a small molecule drug, preferably, a broad-spectrum antibiotic or an antibiotic for infecting bacteria. In a specific implementation, the disease model is infected with two or more types of intestinal bacteria, in a case that a pathogenic mutation carried thereby is subjected to the cell therapy, a therapeutic effect with drug administration and a therapeutic effect without drug administration are observed, and the drug is a small molecule drug, preferably, a broad-spectrum antibiotic or an antibiotic for infecting bacteria. In a specific implementation, the disease model is infected with two types of intestinal bacteria, in a case that a pathogenic mutation carried thereby is subjected to the RNA therapy, a therapeutic effect with drug administration and a therapeutic effect without drug administration are observed, and the drug is a small molecule drug, preferably, a broad-spectrum antibiotic or an antibiotic for infecting bacteria.
Preferably, the pathogenic mutation occurs to the aforementioned gene, or the pathogenic mutation is the mutation of the aforementioned CRB gene.
The present disclosure provides use of the disease model carrier prepared by the aforementioned method in eye disease related study. The eye disease includes the aforementioned eye diseases. The study includes a synergistic effect between a disease related to inherited retinal degeneration and intestinal flora, and the like.
In a specific implementation, the disease model carrier is infected with a type of intestinal bacteria, in a case that a pathogenic mutation carried thereby is subjected to the cell therapy, a therapeutic effect with drug administration and a therapeutic effect without drug administration are observed, and the drug is a small molecule drug, preferably, a broad-spectrum antibiotic or an antibiotic for infecting bacteria. In a specific implementation, the disease model carrier is infected with a type of intestinal bacteria, in a case that a pathogenic mutation carried thereby is subjected to the RNA therapy, a therapeutic effect with drug administration and a therapeutic effect without drug administration are observed, and the drug is a small molecule drug, preferably, a broad-spectrum antibiotic or an antibiotic for infecting bacteria. In a specific implementation, the disease model carrier is infected with two or more types of intestinal bacteria, in a case that a pathogenic mutation carried thereby is subjected to the cell therapy, a therapeutic effect with drug administration and a therapeutic effect without drug administration are observed, and the drug is a small molecule drug, preferably, a broad-spectrum antibiotic or an antibiotic for infecting bacteria. In a specific implementation, the disease model carrier is infected with two types of intestinal bacteria, in a case that a pathogenic mutation carried thereby is subjected to the RNA therapy, a therapeutic effect with drug administration and a therapeutic effect without drug administration are observed, and the drug is a small molecule drug, preferably, a broad-spectrum antibiotic or an antibiotic for infecting bacteria.
Preferably, the pathogenic mutation occurs to the aforementioned gene, or the pathogenic mutation is the mutation of the aforementioned CRB gene.
The present disclosure provides use of the aforementioned disease model or disease model carrier in eye disease related drug screening. The drug includes one or a combination of two or more of a small molecule drug, a chemical drug, a polymer drug, a biological drug or a natural drug (for example, Chinese herbs or Chinese herb extracts), a cell drug, an RNA drug and a DNA drug.
The eye disease includes the aforementioned eye diseases.
Preferably, a small molecule compound is an antibiotic, and the antibiotic is usually a broad-spectrum antibiotic drug well known to those skilled in the art.
Preferably, the small molecule compound is a non-broad-spectrum antibiotic targeted at specific bacteria.
Preferably, the cell includes a modified immune cell, for example, one or a combination of two or more of a T-cell, a B-cell and a stem cell.
Preferably, the RNA includes mRNA, siRNA, sgRNA, miRNA, ASO and/or replicon RNA.
In a specific implementation, the targeted therapy is performed on the disease model or the disease model carrier, at the same time, the aforementioned drug is administered or not administered to the disease model or the disease model carrier subjected to the targeted therapy and the disease model or the disease model carrier not subjected to the targeted therapy, inflammatory progressions of the several groups are observed, and a drug therapeutic effect of the targeted therapy is evaluated.
In a specific implementation, the targeted therapy is performed on the disease model or the disease model carrier, at the same time, the aforementioned drug is administered or not administered to the disease model or the disease model carrier subjected to the targeted therapy and the disease model or the disease model carrier not subjected to the targeted therapy, inflammatory progressions of the several groups are observed, and a therapeutic effect of the small molecule drug is evaluated.
The technical solutions in the examples of the present disclosure will be described clearly and completely below. In the following provided examples, only a modeling method of raising a Crb1 gene mutation mouse in an SPF environment is adopted, and it is determined that a model retina is infected with bacteria by proving existence of a local inflammatory reaction of the retina and existence of the bacteria in a lesion. It is further proved that the bacteria are from an intestinal tract through the following environment. This specific example does not exclude other modeling methods, for example, raising in an environment with more complex microorganism conditions, or a microorganism from the intestinal tract or a microorganism which is the same as the intestinal microorganism is administered to ocular tissue, so the eye tissue directly or indirectly makes contact with the aforementioned microorganism, and the like.
Apparently, the described examples are merely some rather than all of the examples of the present disclosure. All other examples obtained by those ordinarily skilled in the art based on the examples of the present disclosure without making creative efforts fall within the protection scope of the present disclosure.
A C57BL/6N mouse (Crb1rd8/Rd8, named an Rd8 mouse) and a C57BL/6J mouse (Crb1wt/wt, named a wt mouse) carrying an Rd8 mutation are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and maintained in a specific pathogen free (SPF) condition of an animal facility of Zhongshan Ophthalmic center, Sun Yat-sen University. An animal facility of the First Affiliated Hospital, Sun Yat-sen University produces a germ-free (GF) RD8 mouse by using an embryo of a female RD8 mouse. The GF mouse is kept in a germ-free state, and a facility worker performs microorganism and parasite detection on a fecal sample every week so as to ensure sterility of the GF unit. Mouse genetic typing is performed as above (Mattapallil, et al., 2012). Crb1 genotypes of two mouse strains are verified (
The mouse is killed through cervical dislocation, and eye balls are removed and are immobilized in a 4° C. phosphate buffer solution (PBS) with 4% paraformaldehyde (PFA) for 24 h. A sample is washed three times with the PBS, dehydrated in a series of alcohols, dehydrated twice in xylene, then buried in paraffin, and continuously sliced in a 10 μm position with a slicer (RM 223; Leica, Wetzlar, Hesse-Darmstadt, Germany). A slice is subjected to hematoxylin and eosin (H&E) staining. An H&E image is obtained by Imager.Z2 (Zeiss).
Eyes are obtained and put in 4% PFA at a room temperature for 5 min, then dissected and fixed with an eye patch for 45 min, colon tissue is obtained, immobilized in 4% PFA at the room temperature for 4 h and flushed with the PBS, then the separated eye patch is infiltrated with 30% sucrose and stays with a colon overnight for cryopreservation, an OCT compound is embedded (Cat. 4583; SAKURA, USA), and it is stored before a −80° C. slice. The slice is cut in a 12 μm position for all immunostaining purposes.
The tissue slice is blocked with 10% donkey serum/PBST (0.1% tritonx-100/PBS) for 30 minutes and then incubated overnight with a primary antibody at 4° C. After being washed with the PBST, the slice is incubated with a secondary antibody combined with a fluorescent dye and immobilized with Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Phalloidin (A12379;Thermo-Fisher) staining is performed by using the same immunohistochemistry method, except for missing the secondary antibody. ApoTome (Zeiss) is equipped by using a Zeiss confocal microscope (Zeiss LSM880; Zeiss, Oberkochen, Germany) and Imager.Z2. Main antibodies used in this study are: anti-Crb1 (PA5-66373, ThermoFisher; 1:50), anti-Ibal (ab178846, Abcam; 1:500), anti-ZO-1 (61-7300, ThermoFisher; 1:500), anti-Occludin (OC-3F10, Invitrogen; 1:200), AlexaFluor 488 phalloidin (A12379, ThermoFisher; 1:500).
The mouse is anaesthetized and pupils are dilated. A cornea remains moist by applying hydroxypropyl methylcellulose eye drops regularly. A mouse fundus photo is obtained by using a Micron IV mouse fundus camera (Phoenix Research Laboratories, Inc., Pleasanton, CA, USA).
Total RNA is extracted from the retinal superior and inferior parts by using MasterPure™ complete DNA and RNA purification kit (epicentre). An RNA concentration is measured by using a Qbit-RNA-HS analytical kit. A sequencing library conforms to a standard agreement provided by a manufacturer and prepared by using VAHTS™ Total RNA seq (H/M/R) library preparation kit (Vazyme, China), and performs sequencing in an MGISEQ 2000RS platform.
Quality control evaluation is performed on raw reads first from FastQC (v0.11.8) and cutadapt (v1.15). Clean reads are aligned with a mouse genome (mm10) by using HISAT2 (v2.1.0). Gene expression data is introduced into a DESeq2 packet of R software (v3.6.1) for differential expression analysis. Differentially expressed genes (DEGs) are introduced into ingenuity pathway analysis (IPA) for function enrichment analysis.
A retinal sample is collected, and DNA is extracted by using a MasterPure™ complete DNA and RNA purification kit (epicentre). Contents of the stomach, jejunum, ileum, cecum, colon and rectum are collected, and DNA is extracted by using a QIAampPowerFecal DNA kit (QIAGEN). After a concentration is measured, sequencing library preparation is performed on DNA by using a VAHTS™ MGI universal DNA library preparation kit (Vazyme, China) according to a standard solution provided by a manufacturer. Metagenomics sequencing is performed by using MGISEQ-2000RS. Quality filtering is performed on raw reads through Trimmomatic (v0.36) and PRINSEQ (v0.20.4). A mouse read (v0.6.1) is deleted by using KneadData (https://bitbucket.org/biobakery/kneaddata). A non-mouse-clear read is mapped into a pre-built MiniKraken database by using Kraken 2 (v2.0.9). Classification results are screened by using a confidence coefficient 0.20. A negative blank control and the sample are processed together. All species existing in the negative blank control are removed.
The in vivo progressive test is performed by using an FITC labeled dextran method for evaluating a barrier function. Food and water of the previous night are taken out, and the 8-week-old mouse of every 100 grams (weight) takes orally 50 milligrams of FITC labeled dextran (FD-70;Sigma-Aldrich). A serum is collected 5 h after drug administration, and a fluorescence intensity of each sample is measured (excitation, 492 nm; emission, 525 nm).
The WT and Rd8 mice fast overnight with intragastric administration of 1×109 E. Bacillus coli (designed as consistently expressing RFP). 6 hours after intragastric administration, euthanasia is performed on the mice, and 400 μL of peripheral blood is gently transferred to a test tube containing 4 mL of an ACK lysis buffer solution (Gibco, USA) and cultured in RT for 3-5 minutes. After being centrifuged in 300×g for 5 minutes, cells are immobilized and infiltrated (Cytofix/perm solution, BD Biosciences, USA), and the analysis is performed through the flow cytometer (MACSQuantAnalyzer 10, MiltenyiBiotec, Germany).
The colon and eyes are collected immediately after euthanasia and immobilized for 1 h at a room temperature in a phosphate buffered glutaraldehyde-paraformaldehyde solution. The colon is cut into 2 mm blocks. An anterior eye segment is cut off, and a posterior eye segment is cut into 2 mm×2 mm blocks. Tissue obtained after dissection is put in a fresh stationary liquid for 12 h, immobilized with 1% osmium tetroxide, dehydrated and then buried in epon-resin. Staining is performed with toluidine blue under an optical microscope, and a region of interest is screened in advance on a micron-thickness slice. Then an 80 nm ultrasonic image is collected, and counterstaining is performed with uranyl acetate and lead citrate. An ultrasonic slice is observed by using a transmission electron microscope.
This study uses the following oligonucleotide probe: EUB338, 5′-GCTGCCTCCGTAG-GAGT-3′ (Amann, et al., 1990). A 5′ end of the probe has a primary amino group, and Tetramethyl rhodamine isothiocyanate and the amino group are in covalent binding. A dye oligonucleotide conjugate (100 μM) is stored at −20° C.
A pre-fixed retinal slice is flushed three times with a DEPC processed PBS. After being processed with the 0.2% Triton X-100/DEPC processed PBS, the slice and the probe (500 nM) are hybridized overnight at 37° C. and mounted by using Fluoromount-G.
Fresh mouse colon tissue (˜1 cm) is subjected to quick freezing in liquid nitrogen, ground and lysed by using an RNA extraction lysis buffer solution. Total RNA is purified by using a Qiagen RNeasy Plus kit and reversely transcripted into cDNA by using a Takara PrimeScript RT kit and a gDNA eraser. qPCR detects expression levels of corresponding genes. Data is standardized as β-actin.
13 days after 2.5% DSS processing, plasma is separated from whole blood of WT and Rd8 mice. About 50 μL of plasma is used for using a MasterPure isolation total nucleic acid™ complete DNA and RNA purification kit (epicentre, USA). Settled nucleic acid is dissolved in 20 μL of nuclease-free water. qPCR analysis (ChamQ-SYBR-Color -qPCR-Master-Mix, Vazyme, China) is performed by using a LightCycler 96 system (Roche of USA). DNA concentrations in all samples are extremely low, so each sample with the equal volume is used as a template (4 μL in 20 μL). A total bacterial load is measured by using the following universal 16S rRNA gene primers: 27F 5′-AGAGTTTGATCCTGGCTCAG-3′, and 534R 5′-GCATTACCGCGGCTGCTGG-3′.
Each sample is measured directly by using 100 μL of plasma. An LPS concentration of the plasma is measured by using an enzyme linked immunosorbent kit (SEB526Ge; Cloud-Clone Corp., USA).
A fresh fecal sample is collected in a 50 ml cone-shaped tube with bacteria-free 1×PBS and rotated till being homogeneous. Contents are filtered by using a 0.22 μm filter (Millipore) to remove fecal residues, centrifuged to obtain an intestinal flora and then incubated with vanco-bodipy in RT for 30 minutes. The vanco-bodipy labeled intestinal bacteria are administered to the mouse in PBS in 1×108cfu/intragastrically. 24 hours after intragastric administration, a mouse retinal slice is observed.
A mouse intestinal inflammation is induced through long-term oral administration of 2.5% DSS (MW 36000-50000d, Yeasen, China) through drinking water. The weight is monitored every day from Day 0 to Day 13, the mouse is killed on Day 13, and a colon length is measured. For survival analysis, the mouse is allowed to drink 2.5% DSS freely in drinking water for 43 days, and a death rate state of the mouse is monitored every 24 hours during the 43 days.
A broad-spectrum antibiotic mixture of ampicillin (A; 1 g/L; Sigma), metronidazole (M; 1 g/L; neomycin (N; 1 g/L; Sigma) and vancomycin (V; 500 mg/L; a pregnant female mouse is fed with Sigma (AMNV) in drinking water, and a young mouse continues to be fed after weaning. A mouse in control is put in a conventional facility on the same rack.
Retinal microenvironment characteristics of Crb1rd8/rd8 (rd8) and Crb1wt/wt (C57BL/J, named wt) mice are observed (
Transcriptomic analysis is performed by using an RNA-seq technology for comparing a gene expression profile of superior (without lesions) and inferior (with lesions) regions of the Rd8-SPF mouse (
Metagenomic analysis is performed on retinal tissue of the WT-SPF (n=5, age=4 weeks) and Rd8-SPF (n=4, age=4 weeks) mice. It is discovered through the analysis that bacteria DNA contents in the retinae of the WT and Rd8 mice are extremely low, and after all quality control and decontamination steps, no virus and fungus DNA is detected. However, as shown in
Immunofluorescent staining data proves that CRB1 protein expression of an Rd8 retinal outer limiting membrane is reduced or missing (
All of the seven types of bacteria discovered in the Rd8 retina (
An expression of CRB 1 protein in cecum intestinal cells of a wild-type mouse is identified through immunofluorescent staining, but the expression thereof is significantly weakened in the Rd8 mouse (
Similar to the result of the cecum, it is discovered that the CRB1 protein has a significant expression in a top surface and a bottom surface of a colon intestinal epithelial cell (
An intestinal FITC-dextran permeability test is performed on the WT and Rd8 mice. As shown in
The WT and Rd8 mice are exposed in drinking water containing 1.5% dextran sodium sulfate (DSS), which causes mild colitis of the WT mouse. 13 days after DSS therapy, it is discovered that a colon length of the Rd8 mouse is significantly less than a wild-type mouse (
Though in-lesion bacteria caused by breakdown of an outer blood-retinal barrier and an intestinal epithelial barrier are discovered in the Rd8 mouse retina, whether these bacteria are a reason or an outcome of Rd8 mouse retinal degeneration is not clear yet. Thus, the Rd8 mouse is reseparated in a germ-free (GF) condition, and whether a retinal degeneration phenotype of the mouse changes is detected. As shown in
When a sample for test is prepared, 4 mL of castor oil which is already autoclaved is measured, 1.2 g of compound 6 powder is weighted, 1-2 mL of castor oil is added, full grinding is performed, a ground drug and residual castor oil are all transferred to a 50 mL tube with 36 mL ultrapure water to be oscillated and uniformly mixed for still standing overnight, so a compound 6 suspension with a concentration being 30 mg/mL is prepared, and the prepared drug liquid is labeled, saved at a normal temperature and used up within a month.
Calculation formula: theoretical weighing sample quantity (mg)=theoretical concentration (mg/mL) of the sample for testxpreparation volume (mL)
When drug administration starts, a healthy one-month-old Crb1rd8/rd8 mouse is selected for an experiment. When the animal experiment starts, check of a slit lamp is performed, a mouse having a cataract disease is not allowed in the experiment; and a fundus examination is performed, and both eyes of a mouse, one eye fundus of which does not have a light spot lesion, are not allowed in the experiment.
Animals are hierarchically divided according to weights and then randomly divided into a model control group and a compound 6 group. Design details are shown in the following table:
Information related to drug administration and dosage design
Information related to drug administration
Drug administration route and method: intragastric administration, 10.0 mL/kg weight
Drug administration frequency and time limit: once every other day, for 4 weeks
Dosage design
Dosage of the compound 6 is 0.3 g/kg weight for the mouse and is converted according to previous clinical medication.
Experiment method and steps
One-month-old Crb1rd8/rd8 mice are randomly divided into the model control group and the compound 6 drug administration group. A solvent or a compound 6 drug is administered intragastrically to the animals, a dosage of the compound 6 is 0.3 g/kg, once every other day, continuous drug administration for 4 weeks. The number of fundus light slots 2 weeks and 4 weeks after the mouse is administered intragastrically is recorded for analysis and comparison.
A change of the number of fundus light spots of the mice at each time point after drug administration is used as a parameter, and SPSS software is used for the statistic analysis. A statistical result is represented by X+s; and an inspection level=0.05, and an average of experiment data and statistically processed data remain 2 decimal places.
The mice in the model control group have fundus light spots in the beginning of drug administration, and 2 weeks and 4 weeks after drug administration, which indicates that fundus degenerative retinopathy occurs to the model animals. Compared with the model control group, the number of the light spots changes to 1.31±3.18 2 weeks after intragastric administration of the compound 6, which has no statistic difference compared with 2.88+3.41 of the change of the number of spots of the model control group. The number of fundus light spots of the C57BL/6N mouse 4 weeks after intragastric administration changes to −3.63±4.06, which has a statistic difference compared with 2.24±4.01 of the change of the number of the spots of the model control group (
In Week 2 and Week 4 of the experiment, fundus light spots of 2/18 and 1/18 eyes in the compound 6 group cannot be observed clearly due to slight decrease of crystalline lens transparency caused by anesthesia, which is not allowed for statistical count. At the same time, in Week 4 of the experiment, 1/18 eye in the model control group also has decrease of crystalline transparency, so the fundus light spots cannot be observed clearly, and correlation between slight decrease of the crystalline lens transparency and use of the compound 6 is not excluded. A document reports that after the mice are anaesthetized, transparency of a crystalline lens may be decreased, lens abnormalities are common in the Crb1rd8/rd8 mice, transparency of 67% of an anterior lens capsule is decreased, and when decrease of the transparency affects observation of the fundus, it is not allowed for statistical count.
The compound 6 can effectively inhibit the fundus degenerative retinopathy of the Crb1rd8/rd8 mice in the dosage of 0.3 g/kg.
The above examples are merely for describing the technical solutions of the present disclosure but not for limiting them. Although the present disclosure has been described in detail with reference to the examples, those ordinarily skilled in the art are to understand that changes may still be made for the technical solutions recorded in the above examples, or some or all technical features may be replaced equivalently, and these changes or replacements do not cause essence of the corresponding technical solutions to depart from the scope of the technical solutions of the examples of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111067959.3 | Sep 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/114540 | 8/24/2022 | WO |
