RU2845031C2 - Treatment of retinal degeneration - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to the pharmaceutical industry, specifically to a method of treating bacterial-dependent hereditary retinal degeneration and to the use of an inhibitor or macrobicidal agent, which is a compound of the structure given below, for preparing a drug for treating bacterial-dependent hereditary retinal degeneration. Method of treating bacterial-dependent retinal degeneration involving administering an inhibitor or a microbicidal agent for bacteria into the patient’s eyes or gastrointestinal tract, wherein the bacteria are specified in: Anearostipes hadrus, Bifidobacterium pseudocatenulatum, sp. Is79A3, Oscillibacter valericigenes, Tatumella sp. TA1, Megamonas funiformis, Thiobacillus denitrificans and Akkermansia muciniphila; in which the inhibitor or microbicidal agent for the bacterium is a compound having the following chemical structure:

Use of an inhibitor or a microbicidal agent for a bacterium in preparing a drug for treating bacterium-dependent retinal degeneration with the bacteria specified in: Anearostipes hadrus, Bifidobacterium pseudocatenulatum, Nitrosomonas sp. Is79A3, Oscillibacter valericigenes, Tatumella sp.TA1, Megamonas funiformis, Thiobacillus denitrificans and Akkermansia muciniphila; in which the inhibitor or microbicidal agent for the bacterium is a compound having the above chemical structure.

EFFECT: use of said group of inventions provides treating hereditary retinal degeneration, as well as preparing a drug for treating hereditary retinal degeneration.

6 cl, 13 dwg, 2 tbl, 11 ex

Description

Field to which the invention relates

The present invention relates to the field of biomedicine and, in particular, concerns the treatment of retinal degeneration and the use of microbial inhibitors or bactericides in the preparation of drugs for the treatment of retinal degeneration.

General information about the invention

Inherited retinal degeneration (IRD) is a group of inherited disorders characterized by progressive loss of photoreceptor cells, including Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), early cone dystrophy, cone-rod dystrophy, congenital stationary night blindness and color blindness, and Stargardt disease (Broadgate et al., 2017). It is the most common cause of visual impairment in the working population in industrialized countries, with an estimated incidence of 1:2000 (Kutluer et al., 2020). Due to genetic and clinical heterogeneity, the treatment of IRD requires highly individualized treatment strategies. Although treatments such as neuroprotection, gene therapy, and cell replacement therapy have been proposed for the treatment of various stages of IRD, only one gene therapy (Luxturna), which corrects mutations in the RPE65 gene, has been approved by the FDA to date (Botto et al., 2021; Ikelle et al., 2020; Kutluer et al., 2020). Thus, there is an urgent need for a treatment for vision loss due to CRB1.

In a previous study PCT/CN2018/112022 (which is incorporated by reference in its entirety), the inventor found that microorganisms can activate the complement system and cause drusen lesions in macaques when administered live. In addition, intravitreal injection of antibiotics such as vancomycin into the vitreous to kill such microorganisms or inhibit their growth can result in a reduction in the size of drusen lesions in the retinal tissue of macaques compared to a control group. Drugs that kill microorganisms such as Bacillus magaterium or inhibit their growth may be used to treat age-related macular degeneration.

In previous studies PCT/CN2018/118929, PCT/CN2019/070572 and PCT/CN2019/117444 (the contents of which are incorporated by reference in their entirety herein), the inventor reported compounds/compositions that can be used to kill or inhibit the growth of microorganisms, as well as methods of using these compounds/compositions to treat microbial infections and to treat or prevent diseases or symptoms associated with such infections, such as AMD.

Based on previous research, the inventor also found that microbial inhibitors or bactericides could be used as drugs to treat retinal degenerative changes.

Brief description of the invention

The present invention first defines an eye disease model or an eye disease model carrier, especially a retinal degeneration model or a carrier model, and confirms that microbial inhibitors or bactericides can be used as drugs for treating retinal degenerative changes.

The objective of the present invention is achieved by means of the following technical solutions:

The present invention first provides a treatment for degenerative retinal changes that involves administering microbial inhibitors or bactericides to the eyes or gastrointestinal tract of a patient.

Mostly, retinal degeneration is a progressive degenerative change of the retina. Even more predominantly, retinal degeneration is a hereditary degenerative change of the retina. Much more preferably, hereditary retinal degeneration includes Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), early cone dystrophy, rod-cone dystrophy, congenital stationary night blindness and color blindness, and Stargardt disease.

Preferably, the microorganisms are one or more combinations of bacteria, archaea, protozoa, fungi or viruses. The microorganisms are bacteria selected from one or more of the following: Anearostipes, Bifidobacterium, Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillus sp. , 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 the following: Anearostipeshadrus , Bifidobacterium pseudocatenulatum , Nitrosomonas sp.Is79A3, Oscillibactervalericigenes , Tatumella sp.TA1, Megamonasfuniformis , Thiobacillus denitrificans , Akkermansiamucinphila .

Preferably, the microbial inhibitor or bactericide comprises compounds, polypeptides, polynucleotides, natural plants or essences of natural plants.

More preferably, the microbial inhibitor or bactericide is an antibiotic, for example, the antibiotic is a β-lactam antibiotic variants, aminoglycoside antibiotic variants, tetracycline antibiotic variants, chloramphenicol antibiotic variants, macrolide antibiotic variants, glycopeptide antibiotic variants, fluoroquinolone antibiotic variants, nitroimidazole antibiotic variants, rifampin antibiotics, echinocandin antibiotic variants, molyene antibiotic variants, pyrimidine antibiotic variants, allylamine antibiotic variants, azazole antibiotic variants and one or more other antibiotics.

For example, in some embodiments, antibiotics may include one or more of the following: β-lactam antibiotics, including penicillins (e.g., penicillin V), amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, pivampicillin, pivmecillinam, and ticarcillin. Cephalosporins such as cefacetril, cefadroxil, cephalexin, cephaloglycin, cephalonium, cephaloridine, cephalothin, cephapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cephradine, 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, ceftiolen, epoceline, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, cefluprenam, cefocelis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomezine, cephaloram, cefaparol, cefcanel, cefedrolor, cefampidone, cefetrizole, cefivitril, cefmatilene, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, thienamycin, 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), ribostamycin, micronomycin, azithromycin, dibekacin, sisomycin, netilmicin, paromomycin, bramycin, etc. Tetracycline antibiotics including tetracycline, oxytetracycline, aureomycin, doxycycline, etc. Chloramphenicol antibiotics including chloramphenicol, thiamethoxazole, etc. Macrolide antibiotics including erythromycin, albomycin, erythromycin estolate, acetylspiramycin, madecamycin, josamycin, azithromycin, clarithromycin, Dirithromycin, Roxithromycin, Telithromycin, etc. Glycopeptide antibiotics including Vancomycin, Norvancomycin, Teicoplanin, etc. Quinolone antibiotics including Norfloxacin, Ofloxacin, Ciprofloxacin, Pefloxacin, Gatifloxacin, Enoxacin, Lomefloxacin, Nalidixic acid, Levofloxacin, Moxifloxacin and Besifloxacin. Nitroimidazole antibiotics including Metronidazole, Tinidazole, Ornidazole, etc. Rifampin antibiotics including Rifampicin, Echinocandin antibiotics, Polyene antibiotics, Pyrimidine antibiotics, Allylamine antibiotics, Azazole antibiotics; and others: 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 antibiotics may be selected from the following drugs: amikacin, amoxicillin, ampicillin, neosalvarsan, azithromycin, azlocillin, aztreonam, bacitracin, capreomycin, carbenicillin, cefaclor, cefadroxil, cephalexin, cephalothin, cefamandole, cefazolin, cefdinir, cefditoren, cefixime, cefoperazone, cefotaxime sodium, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, chloramphenicol, cilastatin, clarithromycin, clavulanic acid, 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, mapirocin, nafcillin, neomycin, netilmicin, furantoin, oxacillin, oxytetracycline, paromomycin, penicillin G, penicillin V, piperacillin, platensimicin, polymyxin B, pyrazinamide, quinupristin, rapamycin, rifabutin, rifampin, rifampin, rifapentine, rifaximin, roxithromycin, sucfadiazine silver, spectromycin, streptomycin, sulbactam, sulfacetamide, sulfadiazine, sulfamethizole, sulfamethoxazole, sulfisoxazole, sulfasalazine, sulfafurazole, tazobactam, teicoplanin, telavancin, telithromycin, temocylin, tetracycline, thiamphenicol, ticarcillin, tigecycline, tinidazole, tobramycin, trimethoprim, triacetyloleandomycin, vancomycin, enoxacin, lomefloxacin, nalidixic acid, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, ofloxacin, Norfloxacin, Cefotetan, Cefonicid, Cephradine, Cephapirin, Cephalothin, Cefmetazole, Cefotaxime sodium, Latamoxef, Cefepime, Ceftaroline fosamil, Ceftobiprole, Dalbavancin, Demeclocycline, Metacycline, Ertapenem, Fidaxomicin, Geldanamycin, Herbimycin, Posizolid, Radezolid, Torezolid, Oritavancin, Spiramycin, Sulfamethazine, Sulfonamidochrizoidine, Gemifioxacin, Nadifloxacin, Trovafloxacin, Garenoxacin, Sparfloxacin, Temafloxacin, Teixobactin, Malacidins and their combinations.

Preferably, the microbial inhibitor or bactericide is a compound of formula I (e.g., formulas I-1, I-2, I-3, I-4, and I-5), formula II (e.g., formulas II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, and II-10), formula III (e.g., formulas III-1, III-2, and III-3), formula IV-1, or formula IV-2 (e.g., formulas IV-3, IV-4, IV-5, and IV-6), a glycoside (e.g., formula V), or a salt or ester suitable for use in the pharmaceutical industry, wherein the aglycone of the glycoside is a phenolic compound, flavonoid, coumarin, benzoic acid, or sterol, wherein formulas I, II, III, IV-1, IV-2, V, and their subformulas are defined as follows.

In some embodiments, the compounds of the present invention may be characterized by a salt or ester that is in formula I or is pharmaceutically acceptable:

For the avoidance of doubt, in formula I, the cyclic structure Cy ¹ is connected to another cyclic structure Cy ² through two linking groups L and L ^' . These two cyclic structures may be the same or different, and these two linking groups form another cyclic structure between Cy ¹ and Cy ^2. It should be understood that Cy ¹ and Cy ² are cyclic structures independent of L and L ^' , respectively.

In Formula I, Cy ¹ and Cy ² are independently and further substituted cycloalkyl rings (e.g., a C _3-7 cycloalkyl ring) and further substituted heterocycles, such as 4-7-membered heterocycles (e.g., one or two heterocyclic atoms independently substituted with N, O, and S), further substituted aryl rings (e.g., a C _6-10 aryl ring (e.g., phenyl)) or further substituted heteroaryl rings, such as further substituted 5-10-membered heteroaryl rings (e.g., 5- or 6-membered heteroaryl rings that have one or two heterocyclic atoms independently selected from N, O, and S).

L and L ^' are themselves empty or linking groups (e.g., as described in the article). The term "linking group" as used in the article is not limited to any specific type of linking group. In some embodiments, the linking group may also form a cyclic structure with one of its attached moieties, e.g., L and Cy ¹ may form a ring structure independently of Cy ² .

L ² is empty and further substituted C _1-6 alkylene, further substituted C _1-6 heteroalkylene, further substituted C _2-6 alkylenylene, further substituted C _2-6 alkynylene, further substituted C _3-6 cycloalkylene, further substituted arylene, further substituted heteroarylene or further substituted 4-7-membered heterocyclylene

W is -OR ¹ ; -COR ² ; -COOR ^1a ; -OCOOR ^1a ; -NR ³ R ⁴ ; -CONR ^3a R ^4a ; -OCONR ^3b R ^4b ; -SO ₂ NR ^3c R ^4c ; -OSO ₂ NR ^3d R ^4d ; -SR ⁵ ; -SO ₂ R ^5a ; -OCOR ^2a ; -OSO ₂ R ^5a or ;

Where:

R ¹ and R ^1a are independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocyclyl.

R ³ and R ⁴ independently represent hydrogen, -COR ^2b and -SO ₂ R ^5b , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _3-6 cycloalkyl, further substituted phenyl, further substituted 5- or 6-membered heteroaryl, or further substituted 4-7-membered heterocyclyl, or further substituted 4-7-membered heterocyclyl formed by the atoms bonded by R ³ and R ⁴ .

R ² , R ^2a , R ^2b , R ⁵ , R ^5a and R ^5b are independently hydrogen, -OH, -NR ^3e R ^4e , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _1-6 aloxy, further substituted C _3-6 cycloalkyl, further substituted C _3-6 cycloaloxy and further substituted phenyl, and further substituted 5- or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl; and

R ^3a , R ^3b , R ^3c , ^R 3d , R ^3e , R ^4a , R ^4b , R ^4c , R ^4d and R ^4e independently represent hydrogen, selectively substituted C _1-6 alkyl, selectively substituted C _2-6 alkenyl, selectively substituted C _2-6 alkynyl, selectively substituted C _1-6 alkoxy, selectively substituted C _3-6 cycloalkyl and selectively substituted C _3-6 cycloaloxy, as well as selectively substituted phenyl. An additionally substituted 5- or 6-membered heteroaryl or an additionally substituted 4-7-membered heterocyclyl, or R ^3a and R ^4a , R ^3b and R ^4b , R ^3c and R ^4c , R ^3d and R ^4d or R ^3e and R ^4e and an additionally substituted 4-7-membered heterocyclyl formed by the atoms linked thereto.

Cy ¹ and Cy ² in formula I may be aromatic or non-aromatic ring systems, and in some cases may contain heteroatoms. In a preferred embodiment, at least one of Cy ¹ and Cy ² in formula I is an aromatic or heteroaryl ring, such as an optionally substituted C _6-10 aryl ring or an optionally substituted 5-10 membered heteroaryl ring. For example, in some embodiments, Cy ¹ and Cy ² form the backbone structure of formula I, i.e., the structure , which does not contain additional substituents, or:

, where L ² -W can be attached to either the left or right ring. L and L ^' can be any of the types described in the article, with suitable substituents for the ring being described in the article.

In some embodiments, both Cy ¹ and Cy ² in formula I may be aromatic or heteroaryl rings. For example, in some embodiments, a compound of formula I may be represented by formula I-1:

In some embodiments, Ar ¹ and Ar ² of formula I-1 are independently either further substituted C _6-10 aryl rings or further substituted 5-10 membered heteroaryl rings. In some embodiments, Ar ¹ and Ar ² of formula I-1 are independently further substituted with phenyl rings or 5-6 membered and heteroaryl rings. For example, in some embodiments, Ar ¹ and Ar ² of formula I-1 are further substituted phenyl rings, further substituted thienyl rings, further substituted furyl rings, further substituted pyridinyl rings, or further substituted pyrimidinyl rings.

In general, formula I-1 has a multi-ring core structure. For example, in some embodiments, Ar ¹ and Ar ² make up the core structure of formula I-1, i.e., the substituents (additional substituents) are not shown and may be either:

where L ² -W can be attached to either the left or right ring. L and L ^' are defined in the article, with suitable substituents for the ring described in the article.

In some embodiments, the compound of formula I may be expressed by formula I-2:

Where:

m is 0, 1, 2 or 3,

In each occurrence of R ¹⁰ , independently is halogen, L ^2' -W ^' , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _1-6 aloxy, further substituted C _3-6 cycloalkyl, further substituted C _3-6 cycloaloxy and further substituted phenyl, further substituted 5- or 6-membered heteroaryl, or further substituted 4-7-membered heterocyclyl; or two adjacent R ¹⁰ , or one R ¹⁰ and L or L ^' that they join to form a further substituted cycloalkyl, heterocyclyl or heteroaryl ring.

Where -L ^2' -W ^' is independently chosen at each occurrence; and

at each occurrence of L ^2' is independently empty, further substituted C _1-6 alkylene, further substituted C _1-6 heteroalkylene, further substituted C _2-6 alkenylene, further substituted C _2-6 alkylenylene, further substituted C _3-6 cycloalkylene, further substituted arylene, further substituted heteroarylene, or further substituted 4-7-membered heterocycloalkylene; W ^' is independently -OR ¹ ; -COR ² ; -COOR ^1a ; -OCOOR ^1a ; -NR ³ R ⁴ ; -CONR ^3a R ^4a ; -OCONR ^3b R ^4b ; -SO ₂ NR ^3c R ^4c ; -OSO ₂ NR ^3d R ^4d ; -SR ⁵ ; -SO ₂ R ^5a ; -OCOR ^2a ; -OSO ₂ R ^5a or , where R ¹ , R ^1a , R ² , R 2a , R ^2b , R ³ , R ⁴ , R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^4a , R ^4b , R ^4c , R ^4d , R ^4e , ^{R 5 ,} R ^5a and R ^5b are defined in the article see, for example, formula I.

It should be noted that each instance of the structural units -L ^2' -W ^' and -L ² -W is independently selected and may be the same or different.

In some embodiments, Cy ¹ in formula I-2 is an optionally substituted phenyl ring, an optionally substituted thienyl ring, an optionally substituted furyl ring, an optionally substituted pyridinyl ring, or an optionally substituted pyrimidinyl ring. In some embodiments, Cy ¹ in formula I-2 is an optionally substituted C _3-6 cycloalkyl ring or an optionally substituted 4-7 heterocycle, and the heterocycle has 1 or 2 heteroatoms, from atoms independently selected from N, O, and S.

In some embodiments, Cy ¹ allows the base structure of formula I-2 to be any of the following:

,

where -L ² -W is attached to the right phenyl ring, L and L ^' are defined in the article, and the corresponding substituents for the ring are described in this article.

In a more preferred embodiment, Cy ¹ and Cy ² in formula I are phenyl rings. For example, in some embodiments, the compound of formula I-2 may be expressed by formula I-3:

where L, L ^' , ^L2 , W, ^R10 and m are defined in the paper as shown in formula I-2,

n is 0, 1, 2 or 3,

In each occurrence, R ¹¹ is independently halogen, -L ^2' -W ^' , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _1-6 aloxy, further substituted C _3-6 cycloalkyl, further substituted C _3-6 cycloaloxy, and further substituted phenyl, further substituted 5- or 6-membered heteroaryl, or further substituted 4-7-membered heterocyclyl; or two adjacent R ¹¹ , or one R ¹¹ and L or L ^' together with the atoms they link to form a further substituted cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring, where L ^2' and W ^' are defined in the article, see the definition in formula I-2, with -L ^2' -W ^' being selected independently in each instance.

L and L ^' in Formula I (e.g., any of Formulas I-1 through I-3) can be empty or independently bonded groups. In some embodiments, L and L ^' in Formula I are independently empty, -C(O)-, optionally substituted C _1-4 alkylene, optionally substituted C _2-4 alkenylene, -O-, -S-, -NR ¹⁰⁰ -, -S(O)-, -SO ₂ -, -X ¹ -G ¹ -, -X ² -G ² -X ^2a -, or -CR ¹⁰¹ R ¹⁰² -, respectively.

Where:

X ¹ , X ² and X ^2a independently represent additionally substituted C _1-4 alkylene, additionally substituted C _2-4 alkenylene, -O-, -C(O)-, -S-, -NR ^100a -, -S(O)-, -SO ₂ - or -CR ^101a R ^102a -;

G ¹ and G ² independently represent additionally substituted C _1-4 alkylene, additionally substituted C _2-4 alkenylene, -C(O)-, -NR ^100a -, -S(O)-, -SO ₂ - or -CR ^101a R ^102a -;

Preferably, in some embodiments, -X ¹ -G ¹ - or -X ² -G ² -X ^2a - does not contain ON, SS, SN (excluding SO ₂ -N), or -C(O)-S bonds.

R ¹⁰⁰ and R ^100a are independently lone pairs (if applicable), hydrogen, COR ^2c , -SO ₂ R ^5c , further substituted with C _1-6 alkyl, further substituted with C _2-6 alkenyl, further substituted with C _2-6 alkynyl, further substituted with C _3-6 cycloalkyl, further substituted with phenyl, further substituted with 5- or 6-membered heteroaryl, or further substituted with 4-7-membered heterocyclyl; or R ¹⁰⁰ , or R ^100a and R ¹⁰ , or R ¹¹ together form a further substituted heterocycle or heteroaryl ring.

When present, each of R ¹⁰¹ , R ^101a , R ¹⁰² and R ^102a is independently hydrogen, -OH, halogen, further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _3-6 cycloalkyl, further substituted C _1-6 alkoxy, further substituted C _3-6 cycloalkoxy, further substituted amino, further substituted phenyl, further substituted 5- or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl, or R ¹⁰¹ and R ¹⁰² or R ^101a and R ^102a together with the atoms to which they are bonded and form a further substituted 3-7-membered cycloalkyl or heterocycloalkyl ring, or one of R ¹⁰¹ and R ¹⁰² , or one element from the group R ^101a and R ^102a , and the group R ¹⁰ or R ¹¹ . which form a further substituted cycloalkyl or heterocycloalkyl ring; and

R ^2c and R ^5c are independently hydrogen, further substituted with C _1-6 alkyl, further substituted with C _2-6 alkenyl, further substituted with C _2-6 alkynyl, further substituted with C _1-6 alkoxy, further substituted with C _3-6 cycloalkylo, further substituted with C _3-6 cycloaloxy and further substituted with phenyl. Further substituted with 5- or 6-membered heteroaryl; or further substituted with 4-7-membered heterocyclyl.

When a linking group L or L ^' forms a double bond with one of the cyclic carbon atoms, the linking group cannot ^{be CR101R102} , where both ^R101 and ^R102 exist, since the valence of the carbon would exceed 4. In such a case, it should be understood that one of ^R101 and ^R102 does not exist, and L or L ^' is ^CR101 or ^CR102 , as defined in this article. When L or L ^' forms a double bond with one of the cyclic carbon atoms, it can be ^NR100 , where ^R100 is typically a single pair. Other similar situations in this disclosure should be similarly understood.

In some embodiments, L and L ^' in formula I are independently empty, -O-, -C(O)-, -S-, -NR ¹⁰⁰ -, -S(O)-, -SO ₂ -, or -CR ¹⁰¹ R ¹⁰² -. In some embodiments, the compound of formula I has a formula according to any of formulas I-4 through I-5:

Where:

each of the elements X ³ , X ⁴ and X ⁵ are independently empty, -O-, -C(O)-, -S-, -NR ^100a -, -S(O)-, -SO ₂ - or -CR ^101a R ^102a -; and

R ¹⁰ , R ¹¹ , R ^100a , R ^101a , R ^102a , W, L ² , m and n are defined in the article.

In some embodiments, the compound has the formula I-4, wherein X ³ and X ⁴ are independently -O-, -C(O)-, -S-, -NR ^100a -, or -SO ₂ -. In some embodiments, the compound has the formula I-5, wherein X ⁵ is -O-, -C(O)-, -S-, -NR ^100a -, or -SO ₂ -. In some embodiments, R ^100a is hydrogen or further replaced by C _1-4 alkyl.

L ² in formula I (e.g., any nested formulas described in the article, such as formulas I-1 through I-5) is typically empty, i.e., the W group is directly attached to Cy ² . In some embodiments, L ² in formula I may be C _1-4 alkylene, C _2-4 alkenylene, C _2-4 alkynylene, or C _1-4 heteroalkylene. For example, the W group may be attached to Cy ² through a methylene or vinyl group.

Multiple W groups are subject to a compound of formula I (e.g., any nested formula described in the article, such as formulas I-1 through I-5). In preferred embodiments, the W group is independently -OH, -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(C _1-4 alkyl), -SO ₂ NH(C _1-4 alkanoyl), -COOH, , -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ , -OC(O)NH(C _1-4 alkyl)-, -O-(CO)-(C _1-4 alkyl), -O-(C _1-4 alkyl) in each occurrence, wherein each C _1-4 alkyl is independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, and -NH ₂ and is substituted with 1-3 fluorine substitutes. In some embodiments, W in Formula I is -OH, -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(acetyl), -COOH, or -OC(O)-CH ₃ .

As described in the article, in some embodiments, ^L2'- W ^' can be selected as a replacement for ^Cy1 or ^Cy2 , such as for ^Ar1 or ^Ar2 . When applicable, L2 ^' in Formula I (including any nested formulas described in the article, such as formulas I-1 through I-5) can be independently empty at each occurrence, i.e., the W ^' group is directly attached to ^Cy1 or ^Cy2 , such as ^Ar1 or ^Ar2 (if applicable), or a _C1-4 alkylene, _C2-4 alkenylene, _C2-4 alkynylene, or _C1-4 heteroalkylene. For example, the W ^' group can be attached to ^Cy1 or ^Cy2 via a methylene or vinyl group, such as ^Ar1 or ^Ar2 . When applicable, W ^' in formula I (including any nested formulas described in the article, such as formulas I-1 through I-5) can independently be -OH, -NH ² , -SO ₂ NH ₂ , -SO ₂ NH(C _1-4 alkyl), -SO ₂ NH(C _1-4 alkanoyl), -COOH, ,-C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ , -OC(O)NH(C _1-4 alkyl)-, -O-(CO)-(C _1-4 alkyl), -O-(C _1-4 alkyl), wherein each C _1-4 alkyl is independently and further selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, and -NH ₂ and is substituted with 1-3 fluoro substituents. In some embodiments, if applicable, each occurrence of W ^' in Formula I can be -OH, -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(acetyl), -COOH, or -OC(O)-CH ₃ .

Multiple groups may be applied to R ¹⁰ and R ¹¹ in any applicable Formula I (e.g., in any nested formula described in the article, such as formulas I-2 through I-5, if applicable). In some embodiments, each of R ¹⁰ and R ¹¹ in each species may independently be F; Cl; -OH; -NH ₂ ; -SO ₂ NH ₂ ; -SO ₂ NH(C _1-4 alkyl); -SO ₂ NH(C _1-4 alkanoyl); -COOH; ; -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ ; -OC(O)NH(C _1-4 alkyl)-; -O-(CO)-(C _1-4 alkyl), additionally C _1-4 alkyl is replaced by 1-3 substituents that are independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. Additionally C _2-6 alkenyl, which is independently substituted by 1-3 substituents that are independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and a second. Additionally C _2-6 alkynyl which is substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. Additionally C _3-6 cycloalkyl which is substituted with 1-3 substituents independently selected from C _1-4 alkyl and fluoro. Additionally C _3-6 cycloalkoxy which is substituted with 1-3 substituents independently selected from C _1-4 alkyl and fluoro. Or additionally C _1-4 alkoxy which is substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. In some embodiments, R ¹⁰ and R ¹¹ at each occurrence can independently be -OH; -NH ₂ ; -SO ₂ NH ₂ ; -SO ₂ NH(C _1-4 alkyl); -SO ₂ NH(C _1-4 alkanoyl); -COOH; ; -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ ; -OC(O)NH(C _1-4 alkyl)-; -O-(CO)-(C _1-4 alkyl); C _1-4 alkyl; or C _1-4 alkoxy. In some embodiments, one or more instances of R ¹⁰ and/or one or more instances of R ¹¹ may be independently selected by L ^2' -W ^' , as described in the article.

Typically m is 0, 1 or 2, preferably 1 in appropriate cases.

Typically m is 0, 1, 2 or 3, preferably 1 or 2 in appropriate cases.

In some embodiments, the compounds in this article may be characterized by a salt or ester that is in formula II or is pharmaceutically acceptable:

Where:

Cy ¹⁰ and Cy ¹¹ are independently and further replaced by cycloalkyl rings (e.g., C _3-7 cycloalkyl ring), and are further replaced by heterocycles (e.g., 4-7 membered heterocycles), are further replaced by aryl rings (e.g., C _6-10 aryl ring), which are further replaced by a heteroaryl ring (e.g., 5-10 membered heteroaryl ring). Or are further replaced by ring structures consisting of cycloalkyl rings or heterocycles and aryl or a heteroaryl ring, where the ring may be polycyclic or connected in other ways.

L ¹⁰ is an empty or linking group.

L ¹¹ is an empty group, further substituted with C _1-6 alkylene, further substituted with C _1-6 heteroalkylene, further substituted with C _2-6 alkenylene, further substituted with C _2-6 alkynylene, further substituted with C _3-6 cycloalkylene, further substituted with arylene, further substituted with heteroarylene, or further substituted with 4-7-membered heterocyclylene,

W¹⁰represents -OR¹; -COOR^1a; -OCOOR^1a; -COR²; -NR³R⁴; -CONR^3aR^4a; -OCONR^3bR^4b;-SO₂NR^3cR^4c; -OSO₂NR^3dR^4d; -SR⁵; -SO₂R^5a; -OCOR^2a; -OSO₂R^5a; or ;

Where:

R ¹ and R ^1a are independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocyclyl.

R ³ and R ⁴ independently represent hydrogen, -COR ^2b and -SO ₂ R ^5b , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _3-6 cycloalkyl, further substituted phenyl, further substituted 5- or 6-membered heteroaryl, or further substituted 4-7-membered heterocyclyl, or further substituted 4-7-membered heterocyclyl formed by the atoms bonded by R ³ and R ⁴ .

R ² , R ^2a , R ^2b , R ⁵ , R ^5a and R ^5b are independently hydrogen, -OH, -NR ^3e R ^4e , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _1-6 aloxy, further substituted C _3-6 cycloalkyl, further substituted C _3-6 cycloaloxy and further substituted phenyl, and further substituted 5- or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl; and

R ^3a , R ^3b , R ^3c , ^R 3d , R ^3e , R ^4a , R ^4b , R ^4c , R ^4d and R ^4e independently represent hydrogen, selectively substituted C _1-6 alkyl, selectively substituted C _2-6 alkenyl, selectively substituted C _2-6 alkynyl, selectively substituted C _1-6 alkoxy, selectively substituted C _3-6 cycloalkyl and selectively substituted C _3-6 cycloaloxy, as well as selectively substituted phenyl. An additionally substituted 5- or 6-membered heteroaryl or an additionally substituted 4-7-membered heterocyclyl, or R ^3a and R ^4a , R ^3b and R ^4b , R ^3c and R ^4c , R ^3d and R ^4d or R ^3e and R ^4e and an additionally substituted 4-7-membered heterocyclyl formed by the atoms linked thereto.

In some embodiments, in formula II, at least one element of Cy ¹⁰ and Cy ¹¹ is further substituted with a C _6-10 aryl ring or further substituted with a 5-10 membered heteroaryl ring. In some embodiments, Cy ¹¹ is further substituted with a C _6-10 aryl ring or further substituted with a 5-10 membered heteroaryl ring. When Cy ¹¹ is a bicyclic or polycyclic aryl or heteroaryl ring, L ¹⁰ -Cy ¹⁰ and L ¹¹ -W ¹⁰ can be independently connected to Cy ¹¹ via any ring. In some embodiments, Cy ¹¹ can have a fused ring structure comprising an aryl or heteroaryl ring and a cycloalkyl or heterocyclic ring structure. In such embodiments, Cy ¹¹ may be attached to L ¹⁰ -Cy ¹⁰ and L ¹¹ -W ¹⁰ via any of the following: an aryl or heteroaryl ring and a cycloalkyl or heterocyclic structure. If alternatively approached, one of L ¹⁰ -Cy ¹⁰ and L ¹¹ -W ¹⁰ is attached to Cy ¹¹ via an aryl or heteroaryl ring and the other of L ¹⁰ -Cy ¹⁰ and L ¹¹ -W ¹⁰ is attached to Cy ¹¹ via a cycloalkyl or heterocyclic structure.

In some embodiments, the compound of formula II has at least one phenyl ring, which may be a basic structure such as Cy ¹⁰ -L ¹⁰ -Cy ¹¹ :

where Cy ¹⁰ can be the left ring or the right ring in the above figure. This means that the figure is not limited to a specific direction, in which L ¹¹ -W ¹⁰ can be attached to either the left ring or the right ring, and both rings can be further substituted.

In some embodiments, the compound of formula II may have a basic structure such as Cy ¹⁰ -L ¹⁰ -Cy ¹¹ :

where Cy ¹⁰ can be the left ring or the right ring in the above figure. This means that the figure is not limited to a specific direction, in which L ¹¹ -W ¹⁰ can be attached to either the left ring or the right ring, and both rings can be further substituted.

In some embodiments, Cy ¹⁰ and Cy ¹¹ in formula II are aryl or heteroaryl rings. In some embodiments, the compound of formula II may be expressed by formula II-1:

wherein Ar ¹⁰ and Ar ¹¹ are independently further substituted with C _6-10 aryl rings or further substituted with 5-10 membered heteroaryl rings. In some embodiments, Ar ¹⁰ and Ar ¹¹ of formula II-1 are independently further substituted with phenyl rings or further substituted with 5 or 6 membered heteroaryl rings. In some embodiments, Ar ¹⁰ and Ar ¹¹ of formula II-1 are further substituted phenyl rings, further substituted thienyl rings, further substituted furyl rings, further substituted pyridinyl rings, or further substituted pyrimidinyl rings. In some embodiments, one of Ar ¹⁰ and Ar ¹¹ of formula II-1 is a bicyclic aryl or a bicyclic ring heteroaryl, each of which is further substituted. For example, in some embodiments, Ar ¹¹ can be an additionally substituted bicyclic aryl or bicyclic heteroaryl ring.

In some embodiments, Cy ¹¹ in formula II is a phenyl ring. In some embodiments, the compound of formula II may be expressed by formula II-2:

where Ar ¹⁰ , L ¹⁰ , L ¹¹ and W ¹⁰ are defined in the article as shown in formula II-1,

m is 0, 1, 2 or 3,

In each instance of R ²⁰ , independently, halogen, -L ^11' -W ^10' further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _1-6 alkoxy, further substituted C _3-6 cycloalkyl, further substituted C _3-6 cycloalkoxy and further substituted phenyl. Further substituted 5- or 6-membered heteroaryl; or further substituted 4-7-membered heterocyclyl; or two adjacent R ²⁰ or one form of R ²⁰ and L ¹⁰ or L ¹¹ form a further substituted cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring with the atoms to which they are bonded.

-L^11'-W^10' is chosen independently in each case.

In each instance, L ^11' is further empty and further substituted C _1-6 alkylene, further substituted C _1-6 heteroalkylkene, further substituted C _2-6 alkenylene, further substituted C _2-6 alkynylene, further substituted C _3-6 cycloalkylene, further substituted arylene, further substituted heteroarylene, or further substituted 4-7-membered heterocyclylene. In each instance, W ^10' is independently -OR ¹ ; -COR ² ; -COOR ^1a ; -OCOOR ^1a ; -NR ³ R ⁴ ; -CONR ^3a R ^4a ; -OCONR ^3b R ^4b ; -SO ₂ NR ^3c R ^4c ; -OSO ₂ NR ^3d R ^4d ; -SR ⁵ ; -SO ₂ R ^5a ; -OCOR ^2a ; -OSO ₂ R ^5a and , where ^R1 , R1a, ^R2 , R2a, ^R2b , R3, ^R4 , R3a, ^R3b , ^R3c , ^R3d , ^R3e , ^R4a , ^R4b , ^R4c , ^R4d , ^R4e , ^R5 , ^R5a and ^R5b are defined in the article as shown in 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, Cy ¹¹ in formula II is a molten benzene ring. In some embodiments, the compound of formula II may be expressed by formula II-3:

where Ar ¹⁰ , L ¹⁰ , L ¹¹ and W ¹⁰ are defined in the article as shown in formula II-1,

m is 0, 1, 2 or 3,

In each instance of R ²⁰ , independently, halogen, -L ^11' -W ^10' further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _1-6 alkoxy, further substituted C _3-6 cycloalkyl, further substituted C _3-6 cycloalkoxy, and further substituted phenyl. Further substituted 5- or 6-membered heteroaryl; or further substituted 4-7-membered heterocyclyl; or two adjacent R ²⁰ or one form of R ²⁰ and L ¹⁰ or L ¹¹ form a further substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring with the atoms to which they are bonded.

L ^11' and W ^10' are defined in the article as shown in formula II-2. -L ^11' -W ^10' is independently selected in each case.

Ring B is a 4-7-membered cycloalkyl ring, a 4-7-membered heterocycle, a phenyl ring, a 5- or 6-membered heteroaryl ring, each of which is further substituted.

In some embodiments, Cy ¹¹ in formula II is a benzo-fused dicycloaryl or heteroaryl ring. For example, in some embodiments, Cy ¹¹ in formula II may have the following basic structure:

,

^L10 - ^Cy10 and ^L11 - ^W10 may independently attach to ^Cy11 via either of the two rings. wherein the phenyl ring may be further substituted with 1-3 ^R20 groups, as defined herein. For example, with respect to benzothiophene rings, in some embodiments, ^L10 - ^Cy10 may be attached to thiophene rings while ^L11 - ^W10 may be attached to phenyl rings and vice versa. In some cases, ^L10 - ^Cy10 and ^L11 - ^W10 may both attach to the same ring, such as phenyl rings.

In some embodiments, the compound of formula II may have any of the following structures:

where: Cy ¹⁰ , L ¹⁰ , R ²⁰ , m, R ²¹ , n, R ^100a , L ¹¹ , and W ¹⁰ are defined in the article as shown in formula II and nested formulas in the article, such as formula II-3. In some embodiments, Cy ¹⁰ is Ar ¹⁰ , as defined for formula II-3.

In some embodiments, the compound of formula II-3 may be expressed by formula II-4:

where: Ar ¹⁰ , L ¹⁰ , R ²⁰ , m, L ¹¹ and W ¹⁰ are defined in the article as shown in formula II-3.

n is 0 or 1.

In each occurrence, R ²¹ is independently halogen, oxo, -L ^11' -W ^10' , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _1-6 aloxy, further substituted C _3-6 cycloalkyl, further substituted C _3-6 cycloalkoxy, and further substituted phenyl. In addition, further substituted 5- or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl; wherein L ^11' and W ^10' are defined herein as shown in formula II-2, and -L ^11' -W ^10' is independently selected in each occurrence.

If valence permits, X ¹⁰ and X ¹¹ are independently empty, -O-, -C(O)-, -S-, -NR ^100a -, -S(O)-, -SO ₂ -, or -CR ^101a R ^102a -;

R ^100a is a lone pair (if applicable), hydrogen, COR ^2c , -SO ₂ R ^5c further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _3-6 cycloalkyl, further substituted phenyl, further substituted 5 or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl. Alternatively R ^100a and R ²⁰ or R ²¹ groups forming a further substituted heterocyclic or heteroaryl ring.

When present, R ^101a and R ^102a are independently hydrogen, -OH and halogen; further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, C _3-6 cycloalkyl, further substituted C _1-6 alkoxy, further substituted C _3-6 cycloalkoxy, further substituted amino, further substituted phenyl, further substituted 5 or 6-membered heteroaryl. Or further substituted with 4-7 membered heterocyclyl or the form R ^101a and R ^102a further substituted with 3-7 membered cycloalkyl or heterocyclic ring with the atoms to which they are attached, or one of: the form R ^101a and R ^102a further substituted with cycloalkyl or heterocyclic ring with R ²⁰ or R ²¹ groups; and

R ^2c and R ^5c are independently hydrogen, further substituted with C _1-6 alkyl, further substituted with C _2-6 alkenyl, further substituted with C _2-6 alkynyl, further substituted with C _1-6 alkoxy, further substituted with C _3-6 cycloalkylo, further substituted with C _3-6 cycloaloxy and further substituted with phenyl. Further substituted with 5- or 6-membered heteroaryl; or further substituted with 4-7-membered heterocyclyl.

or R ²⁰ or R ²¹ and L ¹⁰ , X ¹⁰ or a form of X ¹¹ that is further substituted by cycloalkyl, heterocyclyl, aryl or heteroaryl ring together with the atoms to which they are bonded.

When X ¹⁰ or X ¹¹ forms a double bond with one of the ring carbon atoms, it cannot be CR ^101a R ^102a , in which R ^101a and R ^102a exist, because the valence of the carbon would exceed 4. In such cases, it should be understood that one of R ^101a and R ^102a does not exist, with X ¹⁰ or X ¹¹ being CR ^101a or CR ^102a , as defined in this document. When X ¹⁰ or X ¹¹ forms a double bond with one of the ring carbon atoms, it can be NR ^100a , where R ^100a is usually the only pair.

In some embodiments, the compound of formula II may be expressed by formula II-5:

where: Ar ¹⁰ , L ¹⁰ , R ²⁰ , m, R ²¹ , n, L ¹¹ and W ¹⁰ are defined in the article as shown in formula II-4.

Cy ¹⁰ and Cy ¹¹ in formula II (for example, in any of the nested formulas such as formulas II-1 and II-4 described in this article) may be linked directly or via different groups. For example, in some embodiments, L ¹⁰ in formula II (e.g., formulas II-1 through II-5) are empty, -C(O)-, further substituted with C _1-4 alkylene, further substituted with C _2-4 alkenylene, further substituted with C _3-6 cycloalkylene, further substituted with 4-7-membered heterocyclylene, further substituted with phenylene, further substituted with 5 or 6-membered heteroarylene, -O-, -S-, -NR ¹⁰⁰ -, -S(O)-, -SO ₂ -, -X ¹ -G ¹ -, -X ² -G ² -X ^2a -, -X ¹² -G ¹⁰ -, -X ¹³ -G ¹¹ -X ^13a -, or -CR ¹⁰¹ R ¹⁰² -.

Where:

X ¹ , X ² and X ^2a are independently further substituted with C _1-4 alkylene, further substituted with C _2-4 alkenylene, further substituted with C _3-6 cycloalkylene, further substituted with 4-7-membered heterocyclylene, further substituted with phenylene, further substituted with 5 or 6-membered heteroarylene, -O-, -C(O)-, -S-, -NR ^100a -, -S(O)-, -SO ₂ - or -CR ^101a R ^102a -,

G ¹ and G ² are independently further substituted with C _1-4 alkylene, further substituted with C _2-4 alkynylene, further substituted with C _3-6 cycloalkylene, further substituted with 4-7-membered heterocyclylene, further substituted with phenylene, further substituted with 5 or 6-membered heteroarylene, -C(O)-, -NR ^100a -, -S(O)-, -SO ₂ -, or -CR ^101a R ^102a -.

Preferably, in some embodiments, -X ¹ -G ¹ - or -X ² -G ² -X ^2a - does not contain ON, SS, SN (except for the SO ₂ -N bond) or the -C(O)-S bond.

X ¹² , X ¹³ and X ^13a are independently further substituted with C _1-4 alkylene, further substituted with C _2-4 alkynylene, further substituted with C _3-6 cycloalkylene, further substituted with 4-7-membered heterocyclylene, further substituted with phenylene, further substituted with 5 or 6-membered heteroarylene, -O-, -C(O)-, -S-, -NR ^100a -, -S(O)-, -SO ₂ - or -CR ^101a R ^102a -.

G ¹⁰ and G ¹¹ independent -X ¹ -G ¹ - or -X ² -G ² -X ^2a -;

In some embodiments, preferably, -X ¹² -G ¹⁰ - or -X ¹³ -G ¹¹ -X ^13a - does not contain OO, ON, SS, SN (except for the SO ₂ -N bond) or the -C(O)-S bond, or three (or more) consecutive heteroatoms in which O-SO ₂ -O, O-SO ₂ -N and N-SO ₂ -N are excluded.

R ¹⁰⁰ and R ^100a are independent single pairs (as appropriate), hydrogen, COR ^2c , -SO ₂ R ^5c , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _3-6 cycloalkyl, further substituted phenyl, further substituted 5 or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl.

R ¹⁰¹ , R ^101a , R ¹⁰² and R ^102a are independently hydrogen, -OH, halogen; further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _3-6 cycloalkyl, further substituted C _1-6 alkoxy, further substituted C _3-6 cycloalkoxy, further substituted amino, further substituted phenyl, further substituted 5 or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl; or R ¹⁰¹ and R ¹⁰² , or R ^101a and R ^102a form a further substituted 3-7-membered cycloalkyl or heterocyclyl ring together with the atoms to which they are bonded.

In some embodiments, L ¹⁰ in formula II may be empty and Cy ¹⁰ is directly bonded to Cy ¹¹ . In some embodiments, L ¹⁰ in formula II may be empty, -O-, -C(O)-, -S-, -NR ¹⁰⁰ -, -S(O)-, -SO ₂ -, or -CR ¹⁰¹ R ¹⁰² -. In some embodiments, L ¹⁰ in formula II may be -X ¹ -G ¹ - or -X ² -G ² -X ^2a -, where X ¹ , X ² and X ^2a are independently -O-, -C(O)-, -S-, -NR ^100a -, -S(O)-, -SO ₂ -, or -CR ^101a R ^102a -. G ¹ and G ² are independently -C(O)-, -NR ^100a -, -S(O)-, -SO ₂ - or -CR ^101a R ^102a -.

In some embodiments, L ¹⁰ in formula II may be -X ¹² -G ¹⁰ -. In some embodiments, X ¹² is further substituted with C _2-4 alkenylene, preferably , and G ¹⁰ is -X ¹ -G ¹ - or -X ² -G ² -X ^2a -; X ¹ , X ² and X ^2a , which are independently -O-, -C(O)-, -S-, -NR ^100a -, -S(O)-, -SO ₂ - or -CR ^101a R ^102a -; G ¹ and G ² , which are independently -C(O)-, -NR ^100a -, -S(O)-, -SO ₂ - or -CR ^101a R ^102a -.

In some preferred embodiments, L ¹⁰ in formula II may be or .

In some embodiments, the compound according to formula II may have the following structure:

L ¹¹ -W ¹⁰ may be directly attached to any ring, and is preferably attached to one or two phenyl rings or a unique phenyl ring. Each ring may be further substituted with one or more suitable substituents as described herein. For example, each substitution unit may be independently selected from F; Cl; -OH; -NH ₂ ; -SO ₂ NH ₂ ; -SO ₂ NH(C _1-4 alkyl); -SO ₂ NH(C _1-4 alkanoyl); -COOH; ; -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ ; -OC(O)NH(C _1-4 alkyl)-; -O-(CO)-(C _1-4 alkyl); C _1-4 alkyl which is further substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluorine. The C _2-6 alkenyl element is further substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluorine. The C _2-6 alkynyl unit is further substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. The C _3-6 cycloalkyl unit is further substituted with 1-3 substituents independently selected from C _1-4 alkyl and fluoro. The C _3-6 cycloalkoxy unit is further substituted with 1-3 substituents independently selected from C _1-4 alkyl and fluoro or C _1-4 alkoxy which is further substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. These elements are further substituted with C _3-6 cycloalkyl, also further substituted with 4-10 membered heterocyclyl, further substituted with 5-10 membered heteroaryl, or further substituted with C _6-10 aryl. For example, in some embodiments, L ¹¹ -W ¹⁰ are NH ₂ or NH(C _1-4 alkanoyl) that is attached to one or two phenyl rings or a unique phenyl ring, wherein the other ring is further substituted with 1 or 2 substituents that are selected from methyl and methoxy.

In some specific embodiments, the compound of formula II has a formula according to formula II-6 or II-7:

where: L ¹¹ , W ¹⁰ , R ²⁰ and m are defined in the article as shown in formula II-3.

p is 0, 1, 2, 3, or 4.

In each instance, the R ²² unit is independently a halogen, -L ^11' -W ^10' , which is further substituted with C _1-6 alkyl, further substituted with C _2-6 alkenyl, further substituted with C _2-6 alkynyl, further substituted with C _1-6 alkoxy, further substituted with C _3-6 cycloalkyl, further substituted with C _3-6 cycloalkoxy, and further substituted with phenyl. This unit is further substituted with a 5 or 6-membered heteroaryl or further substituted with a 4-7-membered heterocyclyl. Or, two adjacent R ²² form a unit that is further substituted with a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring with the atoms to which they are bonded.

If L ^11' and W ^10' are defined in the article as shown in formula II-2. -L ^11' -W ^10' is independently selected in each case.

L ¹¹ in formula II (any nested formulae II-1 through II-7) is typically empty, i.e., in applicable cases, the W ¹⁰ group is directly bonded to Cy ¹¹ . In some embodiments, L ¹¹ in formula II may be C _1-4 alkylene, C _2-4 alkenylene, C _2-4 alkynylene, or C _1-4 heteroalkylene. For example, the W ¹⁰ group may be bonded to Cy ¹¹ via a methylene or vinyl group.

Several W ¹⁰ groups are suitable for compounds of formula II (e.g., formulae II-1 through II-7). In preferred embodiments, the W ¹⁰ group is independently -OH, -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(C _1-4 alkyl), -SO ₂ NH(C _1-4 alkanoyl), -COOH, , -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ , -OC(O)NH(C _1-4 alkyl)-, -O-(CO)-(C _1-4 alkyl), -O-(C _1-4 alkyl) in each occurrence, wherein each C _1-4 alkyl is independently further substituted with 1-3 substituents that are independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. In some embodiments, the W ¹⁰ group in Formula II is -OH, -OMe, -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(acetyl), -COOH, or -OC(O)-CH ₃ .

As described in the article, L ^11' -W ^10' can be selected as substituents for Cy ¹⁰ or Cy ¹¹ , such as Ar ¹⁰ or Ar ¹¹ in some embodiments. Where appropriate, L ^11' in Formula II (including any nested formulae described in this article, such as formulae II-1 through II-7) can be independently empty at each occurrence, i.e., the W ^10' group is directly bonded to Cy ¹⁰ or Cy ¹¹ , such as for Ar ¹⁰ or Ar ¹¹ (where applicable) or a C _1-4 alkylene, C _2-4 alkynylene, C _2-4 alkynylene, or C _1-4 heteroalkylene. For example, the W ^10' group can be bonded to Cy ¹⁰ or Cy ¹¹ through a methylene or vinyl group, such as for Ar ¹⁰ or Ar ¹¹ (where applicable). If applicable, each occurrence of W ^10' in formula II (including any nested formulae described in this article, such as formulae II-1 through II-7) may independently be -OH, -NH ² , -SO ₂ NH ₂ , -SO ₂ NH(C _1-4 alkyl), -SO ₂ NH(C _1-4 alkanoyl), -COOH, ,-C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ , -OC(O)NH(C _1-4 alkyl)-, -O-(CO)-(C _1-4 alkyl), -O-(C _1-4 alkyl), wherein each C _1-4 alkyl unit is independently further substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. In some embodiments, if applicable, each occurrence of W ^10' in Formula II can be -OH, -OMe, -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(acetyl), -COOH, or -OC(O)-CH ₃ .

Several groups may be applied to ^R20 , ^R21 , and ^R22 for any suitable formula II (e.g., formulae II-1 through II-7, as appropriate). In some embodiments, ^R20 , ^R21 , and ^R22 may be independently F; Cl; -OH; _-NH2 ; _-SO2NH2 ; _-SO2NH ( _C1-4alkyl ); _{-SO2NH ( C1-4alkanoyl} ); -COOH; ; -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ ; -OC(O)NH(C _1-4 alkyl)-; -O-(CO)-(C _1-4 alkyl); C _1-4 alkyl, which are further substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluorine. The C _2-6 alkenyl elements are further substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluorine. The C _2-6 alkynyl unit is further substituted with 1-3 substituents that are independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ , and fluoro. The C _3-6 cycloalkyl unit is further substituted with 1-3 substituents that are independently selected from C _1-4 alkyl and fluoro. The C _3-6 cycloalkoxy unit is further substituted with 1-3 substituents that are independently selected from C _1-4 alkyl and fluoro, or C _1-4 alkoxy that is further substituted with 1-3 substituents that are further selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ , and fluoro, in each individual occurrence. In some embodiments, each of R ²⁰ , R ²¹ , and R ²² can independently be F; Cl; -OH; -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(C _1-4 alkyl), -SO ₂ NH(C _1-4 alkanoyl), -COOH; ; -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ ; -OC(O)NH(C _1-4 alkyl)-; -O-(CO)-(C _1-4 alkyl); -O-(C _1-6 alkyl); -O-(C _2-6 alkenyl); C _1-6 alkyl, which are further substituted by 1-3 substitutents independently selected from C _1-4 alkyl, C _1-6 alkoxy, -OH, -NH ₂ and fluoro or C _2-6 alkenyl, which is further substituted by 1-3 substitutents which are independently selected from C _1-4 alkyl, C _1-6 alkoxy, -OH, -NH ₂ and fluoro in each individual occurrence. In some embodiments, each of R ²⁰ , R ²¹ , and R ²² can independently be -OH, C _1-4 alkyl, C _2-6 alkenyl, or -O-(C _1-4 alkyl) in each occurrence. In some embodiments, each of R ²⁰ , R ²¹ , and R ²² can independently be -OH, -OMe, or in each case. In some embodiments, one or more instances of R ²⁰ , one or more instances of R ²¹ , and one or more instances of R ²² may be independently selected by L ^11' -W ^10' , as described in this article.

Typically, m and p are 0, 1, 2, or 3, with 1 or 2 being preferred where appropriate.

Typically n is 0, 1 or 2, preferably 0 or 1 in appropriate cases.

In some embodiments, the compound of formula II has a formula according to any of formulas II-8 through II-10:

where R ²⁰ , R ²² , m and p are defined herein. In some embodiments, m is 1 or 2 and p is 1, 2 or 3. In some embodiments, each of R ²⁰ and R ²² is independently F; Cl; -OH; -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(C _1-4 alkyl), -SO ₂ NH(C _1-4 alkanoyl), -COOH; ; -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ ; -OC(O)NH(C _1-4 alkyl)-; -O-(CO)-(C _1-4 alkyl); -O-(C _1-6 alkyl); -O-(C _2-6 alkenyl); additionally C _1-6 alkyl which is substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-6 alkoxy, -OH, -NH ₂ and fluorine, or additionally C _2-6 alkenyl which is substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-6 alkoxy, -OH, -NH ₂ and fluorine.

In some embodiments, the structural unit in any applicable formula II may be selected from

In some embodiments, the compound of formula II may be expressed as follows:

, ,

or ,

Either a salt or an ester that is acceptable for pharmaceutical use.

In some embodiments, the compounds in this article may be characterized by a salt or ester that is in formula III or is pharmaceutically acceptable:

where Ar ²⁰ is a further substituted aryl ring (e.g., a C _6-10 aryl ring) or a further substituted heteroaryl ring (e.g., a 5-10 membered heteroaryl ring);

L ²⁰ is an empty group which is further substituted with C _1-6 alkylene, further substituted with C _1-6 heteroalkylene, further substituted with C _2-6 alkylene, further substituted with C _2-6 alkynylene, further substituted with C _3-6 cycloalkylene, further substituted with arylene, further substituted with heteroarylene, or further substituted with 4-7-membered heterocyclylene,

W ²⁰ is -OR ¹ ; -COR ² ; -COOR ^1a ; -OCOOR ^1a ; -NR ³ R ⁴ ; -CONR ^3a R ^4a ; -OCONR ^3b R ^4b; -SO ₂ NR ^3c R ^4c ; -OSO ₂ NR ^3d R ^4d ; -SR ⁵ ; -SO ₂ R ^5a ; -OCOR ^2a ; -OSO ₂ R ^5a ; or ,

Where:

R ¹ and R ^1a are independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocyclyl.

R ³ and R ⁴ independently represent hydrogen, -COR ^2b and -SO ₂ R ^5b , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _3-6 cycloalkyl, further substituted phenyl, further substituted 5- or 6-membered heteroaryl, or further substituted 4-7-membered heterocyclyl, or further substituted 4-7-membered heterocyclyl formed by the atoms bonded by R ³ and R ⁴ .

R ² , R ^2a , R ^2b , R ⁵ , R ^5a and R ^5b are independently hydrogen, -OH, -NR ^3e R ^4e , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _1-6 aloxy, further substituted C _3-6 cycloalkyl, further substituted C _3-6 cycloaloxy and further substituted phenyl, and further substituted 5- or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl; and

R ^3a , R ^3b , R ^3c , ^R 3d , R ^3e , R ^4a , R ^4b , R ^4c , R ^4d and R ^4e independently represent hydrogen, selectively substituted C _1-6 alkyl, selectively substituted C _2-6 alkenyl, selectively substituted C _2-6 alkynyl, selectively substituted C _1-6 alkoxy, selectively substituted C _3-6 cycloalkyl and selectively substituted C _3-6 cycloaloxy, as well as selectively substituted phenyl. Further substituted 5- or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl. Or R ^3a and R ^4a , R ^3b and R ^4b , R ^3c and R ^4c , R ^3d and R ^4d or R ^3e and R ^4e and an additionally substituted 4-7-membered heterocyclyl formed by the atoms linked thereto.

In some embodiments, Ar ²⁰ in formula III is further substituted with a phenyl ring or further substituted with a 5- or 6-heteroaryl ring. In some embodiments, the element Ar ²⁰ in formula III may be further substituted with a phenyl ring, a further substituted thienyl ring, a further substituted furyl ring, a further substituted pyridinyl ring, or a further substituted pyrimidinyl ring. In some embodiments, Ar ²⁰ in formula III may also be a further substituted dicloaryl or dicloheteroaryl ring, each element being further substituted. In such embodiments, L ²⁰ -W ²⁰ may be attached to any double ring.

In some embodiments, Ar ²⁰ in formula III may be further replaced by a phenyl ring, wherein two adjacent substituents with the carbon atoms to which they are attached form a further substituted cycloalkyl, heterocyclyl, aryl, or heteroaryl ring.

For example, in some embodiments, Ar ²⁰ in formula III may be a benzo-fused dicycloaryl or heteroaryl ring. For example, in some embodiments, Ar ²⁰ in formula III may have the following structure:

where -L ²⁰ -W ²⁰ can be attached to either of the two rings, and either or both rings can be further substituted.

In some embodiments, the compound of formula III may be expressed as formula III-1, III-2, or III-3:

where L ²⁰ and W ²⁰ are defined in this article.

m is 0, 1, 2 or 3, n is 0, 1, 2 or 3.

In each occurrence, each of ^R30 and ^R31 is independently halogen, ^{-L20'- W20'} further replaced by _C1-6 alkyl further replaced by C2-6 alkenyl further replaced by _C2-6 alkynyl further replaced by _C1-6 alkoxy further replaced by _C3-6 cycloalkyl further replaced by _C3-6 cycloalkoxy further replaced by phenyl; further replaced by 5 or 6-membered heteroaryl or further replaced _by 4-7-membered heterocyclyloxy wherein ^-L20' - ^W20' are independently selected in each occurrence. Wherein L ^20' is independently an empty unit and is further substituted with C _1-6 alkylene, further substituted with C _1-6 heteroalkylene, further substituted with C _2-6 alkenylene, further substituted with C _2-6 alkynylene, further substituted with C _3-6 cycloalkylene, further substituted with arylene, further substituted with heteroarylene, or further substituted with a 4-7-membered heterocyclylene in each occurrence, wherein W ^20' is independently -OR ¹ ; -COR ² ; -COOR ^1a ; -OCOOR ^1a ; -NR ³ R ⁴ ; -CONR ^3a R ^4a ; -OCONR ^3b R ^4b ; -SO ₂ NR ^3c R ^4c ; -OSO ₂ NR ^3d R ^4d ; -SR ⁵ ; -SO ₂ R ^5a ; -OCOR ^2a ; -OSO ₂ R ^5a or in every case. Where R ¹ , R ^1a , R ² , R ^2a , R ^2b , R 3, R ⁴ , R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^4a , R ^4b , R ^4c , R ^4d , R ^4e , R ⁵ , ^{R 5a} and R ^5b are defined in this article. For example, see formula III.

Ring B is a 4-7 membered cycloalkyl ring, a 4-7 membered heterocyclic ring, a phenyl ring, a 5 or 6 membered heteroaryl ring, each of which is further replaced by 1-3 R ³¹ elements that are independently selected.

If valence permits, X ²⁰ and X ²¹ are independently empty, -O-, -C(O)-, -S-, -NR ^100a -, -S(O)-, -SO ₂ -, or -CR ^101a R ^102a -;

R ^100a is a lone pair (where appropriate), hydrogen, COR ^2c , -SO ₂ R ^5c , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _3-6 cycloalkyl, further substituted phenyl, further substituted 5 or 6-membered heteroaryl, or further substituted 4 to 7-membered heterocyclyl. Or R ^100a and one of R ³⁰ or R ³¹ which form a further substituted heterocyclic or heteroaryl ring together with the atoms they bind, for example, a further substituted 5 or 6-membered heteroaryl or a further substituted 4 to 7-membered heterocyclyl;

When present, R ^101a and R ^102a are independently hydrogen, -OH and halogen; further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, C _3-6 cycloalkyl, further substituted C _1-6 alkoxy, further substituted C _3-6 cycloalkoxy, further substituted amino, further substituted phenyl, further substituted 5 or 6-membered heteroaryl. Or further substituted with 4-7 membered heterocyclyl or the form R ^101a and R ^102a further substituted with 3-7 membered cycloalkyl or heterocyclic ring with the atoms to which they are attached, or one of: the form R ^101a and R ^102a further substituted with cycloalkyl or heterocyclic ring with R ³⁰ or R ³¹ groups; and

R ^2c and R ^5c are independently hydrogen, further substituted with C _1-6 alkyl, further substituted with C _2-6 alkenyl, further substituted with C _2-6 alkynyl, further substituted with C _1-6 alkoxy, further substituted with C _3-6 cycloalkylo, further substituted with C _3-6 cycloaloxy and further substituted with phenyl. Further substituted with 5- or 6-membered heteroaryl; or further substituted with 4-7-membered heterocyclyl.

Either two adjacent R ³⁰ elements or two adjacent R ³¹ elements, or R ³⁰ or R ³¹ and X ²⁰ or X ²¹ form a further substituted cycloalkyl, heterocyclic aryl, or heteroaryl ring with the atoms to which they are bonded.

When X ²⁰ or X ²¹ forms a double bond with one of the ring carbon atoms, it cannot be CR ^101a R ^102a , in which R ^101a and R ^102a exist, because the valence of the carbon would exceed 4. In such cases, it should be understood that one of R ^101a and R ^102a does not exist, and X ²⁰ or X ²¹ is CR ^101a or CR ^102a , as defined in this document. When X ²⁰ or X ²¹ forms a double bond with one of the ring carbon atoms, it can be NR ^100a , where R ^100a is usually the only pair.

It should be noted that each instance of the structural units -L ^20' -W ^20' and -L ²⁰ -W ²⁰ is independently selected and may be the same or different.

In some embodiments, the compound of formula III may have any of the following structures:

where: R ³⁰ , m, R ³¹ , n, R ^100a , L ²⁰ , and W ²⁰ are defined in this article. For example, see formula III and nested formulas such as formulas III-1 through III-3. In the tricyclic structure, the piperidine ring or morpholine ring may be further substituted.

L ²⁰ in formula III (and any nested formulas, such as III-1 through III-3) is typically empty, i.e., the W ²⁰ group is directly bonded to Ar ²⁰ . In some embodiments, L ²⁰ in formula III may be a C _1-4 alkylene, C _2-4 alkenylene, C _2-4 alkynylene, or C _1-4 heteroalkylene. For example, the W ²⁰ group may be bonded to Ar ²⁰ through a methylene or vinyl group.

Several W ²⁰ groups are suitable for compounds for formula III (e.g., any nested formulas, such as formulas III-1 through III-3). In a preferred embodiment, the W ²⁰ element in formula III may be -OH, -COOH, , -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ , -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(C _1-4 alkyl); -SO ₂ NH(C _1-4 alkanoyl), -OC(O)NH(C _1-4 alkyl)-, -O-(CO)-(C _1-4 alkyl) or -O-(C _1-4 alkyl) and each of the following, wherein the C _1-4 alkyl is further substituted with 1 to 3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. In some embodiments, the W ²⁰ group in formula III (e.g., any of the nested formulae III-1 through III-3) may be -OH, -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(acetyl), , -C(O)-(OC ₈ alkyl), -COOH or -OC(O)-CH ₃ .

As described in this article, the element L ^20' -W ^20' can be selected as a substitute for Ar ²⁰ in some embodiments. In appropriate instances, L ^20' in Formula III (including any of the nested formulae III-1 to III-3) is independently empty at each occurrence, i.e., in appropriate instances, the W ^20' group is directly bonded to Ar ²⁰ or a C _1-4 alkylene, C _2-4 alkynylene, C _2-4 alkynylene, or C _1-4 heteroalkylene. For example, in appropriate instances, the W ^20' group can be bonded to Ar ²⁰ via a methylene or vinyl group. In appropriate instances, the W ^20' element in Formula III (including any of the nested formulae described herein, such as formulae III-1 to III-3) can independently be -OH, -COOH, , -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ , -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(C _1-4 alkyl); -SO ₂ NH(C _1-4 alkanoyl), -OC(O)NH(C _1-4 alkyl)-, -O-(CO)-(C _1-4 alkyl), or -O-(C _1-4 alkyl) in each instance, wherein each of the C _1-4 alkyl units is further substituted with 1 to 3 substituents that are independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. In some embodiments, if applicable, each occurrence of W ^20' in formula III may be -OH, -NH ₂ , -SO ₂ NH ₂ , -SO ₂ NH(acetyl), , -COOH, -C(O)(OC ₈ alkyl) or -OC(O)-CH ₃ .

Multiple groups may be applied to ^R30 and ^R31 in any applicable Formula III (e.g., any nested formula such as Formulas III-1 through III-3). In some embodiments, each of the ^R30 and ^R31 elements may independently be F; Cl; -OH; -COOH; -OC(O) _NH2 ; -OC(O)NH( _C1-4alkyl )-; -O-(CO)-( _C1-4alkyl ) in each occurrence. The _C1-4alkyl elements are further replaced with 1-3 substituents that are independently selected from _C1-4alkyl , _C1-4alkoxy , -OH, _-NH2 , and fluoro. The _C2-6alkenyl elements are further replaced with 1-3 substituents that are independently selected from _C1-4alkyl , _C1-4alkoxy , -OH, _-NH2 , and fluoro. The C _2-6 alkynyl elements are further substituted with 1-3 substituents that are independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluorine. The C _3-6 cycloalkyl element is further substituted with 1-3 substituents that are independently selected from C _1-4 alkyl and fluorine. The C _3-6 cycloalkoxy element is further substituted with 1-3 substituents that are independently selected from C _1-4 alkyl and fluorine. Or the C _1-4 alkoxy element is further substituted with 1-3 substituents that are independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluorine. In some embodiments, each of R ³⁰ and R ³¹ can independently be -OH, C _2-6 alkenyl, -O-(C _1-4 alkyl), -COOH, or -C(O)(OC _1-10 alkyl) at each occurrence. In some embodiments, each of R ³⁰ and R ³¹ can be -OH or -OMe at each occurrence. In some embodiments, one or more instances of R ³⁰ and/or one or more instances of R ³¹ can be independently selected from L ^20' -W ^20' , as described in the article.

Typically, m is 0, 1, 2, or 3, preferably 2 or 3. Typically, n is 1, 2, or 3.

In some embodiments, the following connections are also described in this disclosure: or a salt or ester that is acceptable for pharmaceutical use.

In some embodiments, the following connections are also described in this disclosure: , or a pharmaceutically acceptable salt or ester thereof, wherein q is 1, 2, 3, 4, or 5 and Glu is a glucose residue. In certain specific embodiments also described in this disclosure, or a salt or ester thereof acceptable for use in pharmaceuticals.

In some embodiments, the compounds of the present invention may be alkaloids with antimicrobial activity. As defined herein, certain indole alkaloids, such as the vinca alkaloids, tabersonine, vindoline, vincaleukoblastine and vincristine, are effective against microorganisms such as Bacillus megaterium. In some embodiments, the compounds of the present invention are characterized by IV-1 or IV-2, which is tabersonine or vindoline and derivatives thereof:

Where:

R ⁴⁰ is hydrogen, the elements -COR ² ; -COOR ^1a ; -SO ₂ R ^5a ; is further substituted by alkyl, is further substituted by alkenyl, is further substituted by alkynyl, is further substituted by cycloalkyl, is further substituted by aryl, is further substituted by heteroaryl, or is further substituted by heterocyclyl;

R ⁴¹ is -OR ¹ ; -OCOOR ^1a ; -OCONR ^3b R ^4b ; -OCOR ^2a ; or -OSO ₂ R ^5a ; n is 0 or 1;

R ⁴² , R ⁴³ and R ⁴⁴ are independently hydrogen, -OR ¹ , OCOR ^2a ; or -OSO ₂ R ^5a ;

Element L ³⁰ is empty or methylene.

W ³⁰ is -OR ¹ ; -COR ² ; -COOR ^1a ; -OCOOR ^1a ; -NR ³ R ⁴ ; -CONR ^3a R ^4a ; -OCONR ^3b R ^4b ; -OSO ₂ NR ^3d R ^4d ; -OCOR ^2a ; or -OSO ₂ R ^5a ;

Where:

R ¹ and R ^1a are independently hydrogen, further substituted alkyl, further substituted alkenyl, further substituted alkynyl, further substituted cycloalkyl, further substituted aryl, further substituted heteroaryl or further substituted heterocyclyl,

R ³ and R ⁴ independently represent hydrogen, -COR ^2b and -SO ₂ R ^5b , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _3-6 cycloalkyl, further substituted phenyl, further substituted 5- or 6-membered heteroaryl, or further substituted 4-7-membered heterocyclyl, or further substituted 4-7-membered heterocyclyl formed by the atoms bonded by R ³ and R ⁴ ;

R ² , R ^2a , R ^2b , R ⁵ , R ^5a and R ^5b are independently hydrogen, -OH, -NR ^3e R ^4e , further substituted C _1-6 alkyl, further substituted C _2-6 alkenyl, further substituted C _2-6 alkynyl, further substituted C _1-6 aloxy, further substituted C _3-6 cycloalkyl, further substituted C _3-6 cycloaloxy and further substituted phenyl, and further substituted 5- or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl; and

R ^3a , R ^3b , R ^3c , ^R 3d , R ^3e , R ^4a , R ^4b , R ^4c , R ^4d and R ^4e independently represent hydrogen, selectively substituted C _1-6 alkyl, selectively substituted C _2-6 alkenyl, selectively substituted C _2-6 alkynyl, selectively substituted C _1-6 alkoxy, selectively substituted C _3-6 cycloalkyl and selectively substituted C _3-6 cycloaloxy, as well as selectively substituted phenyl. Further substituted 5- or 6-membered heteroaryl or further substituted 4-7-membered heterocyclyl. Or R ^3a and R ^4a , R ^3b and R ^4b , R ^3c and R ^4c , R ^3d and R ^4d or R ^3e and R ^4e and an additionally substituted 4-7-membered heterocyclyl formed by the atoms linked thereto.

In some embodiments, the compound of formula IV-1 or IV-2 has a formula according to one of formulas IV-3 through IV-6:

where R ⁴⁵ is hydrogen or methyl.

In some embodiments, R ⁴⁰ is any element in formulas IV-1 through IV-6 and may be hydrogen, C _1-4 alkyl, or C _1-4 alkanoyl.

The element L ³⁰ in formulas IV-1 through IV-6 is usually empty. However, in some embodiments, L ³⁰ in formulas IV-1 through IV-6 may also be CH ₂ .

The W ³⁰ element in formulas IV-1 through IV-6 is typically a carboxylic acid derivative, an amine derivative, or an alcohol derivative that can be used in the combinations and methods described herein. Naturally occurring indole alkaloids and tabersonine contain a CO ₂ Me group that serves as W ³⁰ while the L ³⁰ element is empty. The CO ₂ Me group can be conventionally converted to the corresponding acids and amides or rearranged and converted to amines, such as by the Curtius rearrangement. In some embodiments, the W ³⁰ element in formulas IV-1 through IV-6 may be -OH, -NH ₂ , -OSO ₂ NH ₂ , -COOH, -C(O)(OC _1-10 alkyl), -C(O)(OC _2-10 alkenyl), -OC(O)NH ₂ , -OC(O)NH(C _1-4 alkenyl)-, -O-(CO)-(C _1-4 alkyl), -O-(C _1-4 alkyl), wherein each of the C _1-4 alkyl elements is independently and further substituted with 1-3 substituents that are independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluorine. In some embodiments, the W ³⁰ element in formulas IV-1 through IV-6 may be -OH, -NH ₂ , -OSO ₂ NH ₂ , -C(O)-(OC ₈ alkyl), -COOH, or -OC(O)NH ₂ .

In some embodiments, the connection may have the following structure:

.

In some embodiments, the compounds in this document may be a glycoside having antimicrobial activity, or a salt or ester thereof, which are acceptable for use in pharmaceuticals. As defined in this document, some glycosides, such as ginsenosides and gallic acid glycosides, are not effective against microorganisms such as Bacillus megaterium. Other effective glycosides include any glycosides with antimicrobial activity known in the art, which include, for example, a glycoside that is characterized by an appropriate aglycone, such as a phenolic compound, a flavonoid, a coumarin, benzoic acid, or a sterol. Typically, the glycosides are glucosides, but other glycosides can be used. In some embodiments, the glycosides can be characterized as amphipathies that can disrupt biofilm and provide the glycosides with an antimicrobial effect. In some embodiments, glycosides can also be characterized as saponins, which include, for example, various plant-derived glycosides that can serve as “surfactants” and help kill bacteria.

In some embodiments, the glycosides provided herein may be characterized by formula V:

,

where each R ⁵⁰ element is independently hydrogen, -L ⁵⁰ -D, an oxygen protecting group, or a glycosidic residue.

Element L ⁵⁰ is empty or -C(O)-;

D is further substituted with aryl (e.g. C _6-10 aryl), further substituted with heteroaryl (e.g. 5-14 membered heteroaryl), further substituted with aryl, heteroaryl, cycloalkyl or heterocyclyl containing two or more fused rings which are independently selected from them (e.g. 8-14 membered, e.g. benzo-fused cycloalkyl/heterocycloalkyl pyridino-fused cycloalkyl/heterocycloalkyl), or a steroid moiety of formula VA:

Where can be linked to the VA formula via a steroid skeleton or any R ⁵¹ group.

In each instance, R ⁵¹ is independently and further substituted by alkyl, further substituted by alkenyl, further substituted by alkynyl, -OH, oxygen or halogen, further substituted by an oxygen protecting group, further substituted by cycloalkyl, further substituted by alkoxy, further substituted by cycloalkoxy, further substituted by amino, further substituted by phenyl, further substituted by heteroaryl or further substituted by heterocyclyl, or further substituted by cycloalkyl, heterocyclyl, aryl or a heteroaryl ring formed by the two R ⁵¹ groups with the atoms to which they are bonded.

m is an integer between 1 and 8 and

Where -L ⁵⁰ -D is independently selected at each occurrence; and

In some embodiments, each element of R ⁵⁰ is hydrogen.

In some embodiments, one to four R ⁵⁰ units may be independently selected from -L ⁵⁰ -D. When two or more -L ⁵⁰ -D units are attached to a pyranose unit in formula V, they are preferably the same. In some embodiments, one or more (e.g., 1 or 2) R ⁵⁰ may be a glycosidic residue attached to the remainder of formula V via a glycosidic linkage. In some embodiments, the glycosidic residues are glucose residues or rhamnose residues.

L ⁵⁰ in formula V may be an empty group or represent a carbonyl group -C(O)-, depending on whether the linking group is a phenol-OH or a COOH group of benzoic acid or its heteroaryl analogue.

Various residues can be used as D, which are typically derived from phenolic compounds, coumarins, flavonoids or sterols, and in some embodiments they can have antimicrobial activity in the absence of glycosidic units.

In some embodiments, D may be an optionally substituted ring selected from any of the following:

Where

R ^100a is a lone pair (if applicable), hydrogen, a nitrogen protecting group, further substituted with C _1-6 alkyl, further substituted with C _2-6 alkenyl, further substituted with C _2-6 alkynyl, further substituted with C _3-6 cycloalkyl, further substituted with phenyl, further substituted with 5 or 6-membered heteroaryl, or further substituted with 4-7-membered heterocyclyl; or R ^100a , wherein the phenyl or pyridyl ring forms a further substituted heterocyclic or heteroaryl ring.

Where can attach to D through any available location, and

each D ring system is further substituted with 1-5 (e.g., 1, 2, or 3) substituents, wherein each substituent is independently selected from -OH; -COOH; -C(O)(OC _1-10 alkyl); -C(O)(OC _2-10 alkenyl); -OC(O)NH ₂ ; -OC(O)NH(C _1-4 alkyl)-; -O-(CO)-(C _1-4 alkyl); -NH ₂ ; -SO ₂ NH ₂ ; -SO ₂ NH(C _1-4 alkyl); -SO ₂ NH(C _1-4 alkanoyl); halogen; which is further substituted with C _1-6 alkyl; which is further substituted with C _2-6 alkenyl; which is further substituted with C _2-6 alkynyl; which is further substituted with C _3-6 cycloalkyl; which is further substituted with C _1-6 alkoxy; which is further substituted with C _3-6 cycloalkoxy; which is further substituted with amino; which is further substituted with phenyl; which is further substituted with 5 or 6-membered heteroaryl; or which is further substituted with 4 to 7-membered heterocyclyl.

In some embodiments, each D ring system, as shown above, may be further substituted with 1-5 substituents, wherein each substituent is independently selected from F; Cl; -OH; -COOH; -C(O)(OC _1-10 alkyl); -C(O)(OC _2-10 alkenyl); -OC(O)NH ₂ ; -OC(O)NH(C _1-4 alkyl)-; -O-(CO)-(C _1-4 alkyl); -NH ₂ ; -SO ₂ NH ₂ ; -SO ₂ NH(C _1-4 alkyl); -SO ₂ NH(C _1-4 alkanoyl); C _1-4 alkyl, which is further substituted with 1-3 substituents that are further selected from C _1-4 alkyl; C _1-4 alkoxy, -OH, -NH ₂ and fluoro. The C _2-6 alkenyl unit is further substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. The C _2-6 alkynyl unit is further substituted with 1-3 substituents independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluoro. The C _3-6 cycloalkyl unit is further substituted with 1-3 substituents independently selected from C _1-4 alkyl and fluoro. The C _3-6 cycloalkoxy unit is further substituted with 1-3 substituents independently selected from C _1-4 alkyl and fluoro. Or the C _1-4 alkoxy element is further substituted with 1-3 substitutents that are independently selected from C _1-4 alkyl, C _1-4 alkoxy, -OH, -NH ₂ and fluorine.

In some embodiments, D can be selected from:

,

In this case, each phenol-OH group is optionally attached to a glycoside (e.g., glucose) via a glycosidic bond.

In some embodiments, D is a sterol derivative. For example, in some embodiments, D is , where R ⁵² is further substituted alkyl or further substituted alkenyl, and each of the remaining -OH groups in D is further attached to the glycoside through a glycosidic linkage.

Preferably R ⁵² can be .

In any embodiment, as defined above, the glycoside may have the formula V-1 or V-2:

In some embodiments, the glycoside may be selected from the following compounds:

In some embodiments, the compounds of the present invention may be selected from several of the following compounds: benzoic acid, benzyl alcohol, coumarins, catechols, polyphenols, chalcones (including licorice chalcones), etc., stilbene derivatives such as resveratrol and isoresveratrol, phenolic acids such as p-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, protocatechuic acid, gallic acid, vanillic acid, syringic acid, cinnamic acid, coumaric acid, caffeic acid, ferulic acid, chlorogenic acid and sinapic acids, flavonoids such as catechin, naringenin, quercetin, rutin and chrysin, tannins such as ellagic acid, a salt or ester thereof and glycosides that are acceptable for pharmaceutical use.

In embodiments of the present invention, the microbial inhibitor or sterilizing agent is compounds 1-8 with the following chemical structure:

Preferably, the microbial inhibitor or sterilizing agent of the present invention is a natural plant or an extract thereof, and the natural plant is selected from a combination of one or more species of licorice, white peony root, forsythia, fructus auranti, rehmannia glutinosa, mandarin, pseudoginseng peel, tortoiseshell, rhodiola rosea, rehmannia shoots, coix-Job's tears, white peony root, forsythia, cassia seed and licorice, an extract of the above-mentioned natural plants, or a combination thereof.

In a specific embodiment of the present invention, the microbial inhibitor or sterilizing agent is a combination comprising tortoise shell, rosea rhodiola, rehmannia rhizome, Job's tears coix, white peony root, forsythia, cassia seed and licorice. By weight, the combination preferably comprises 5-15 tinctures of tortoise shell, 10-30 tinctures of rosea rhodiola, 5-15 tinctures of rehmannia rhizome, 5-15 tinctures of Job's tears, 8-20 tinctures of white peony root, 5-15 tinctures of forsythia, 5-15 tinctures of cassia seed and 5-15 tinctures of licorice; more preferably, the combination includes 10 tinctures of tortoise shell, 20 tinctures of Rhodiola rosea, 10 tinctures of Rehmannia rhizome, 10 tinctures of Job's tears, 12 tinctures of white peony root, 10 tinctures of Forsythia, 10 tinctures of Cassia seed, and 9 tinctures of white peony root. Licorice decoctions.

Preferably, administration to the patient's eye includes administration into the aqueous humor, suspensory ligament, ciliary body, ciliary body, and ciliary muscle in the anterior chamber, and into the vitreous humor, retina, choroid, optic nerve, lens, or iris in the posterior chamber of the patient's eye.

Preferably, administration into the gastrointestinal tract of a patient includes administration into the stomach, jejunum, ileum, cecum, colon, or rectum of a patient.

The present invention also provides for the use of a microbial inhibitor or sterilizing agent in the preparation of medicaments for the treatment of retinosis.

Mostly, retinal degeneration is a progressive degenerative change of the retina. More predominantly, retinal degeneration is a hereditary degenerative change of the retina. Much more preferably, hereditary retinal degeneration includes Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), early cone dystrophy, congenital stationary night blindness (CSNB) and color blindness, as well as Stargardt disease.

Preferably, the microorganisms are one or more combinations of bacteria, archaea, protozoa, fungi or viruses. The microorganisms are bacteria selected from one or more of the following: Anearostipes, Bifidobacterium, Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillus sp. , 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 the following: Anearostipeshadrus , Bifidobacterium pseudocatenulatum , Nitrosomonas sp.Is79A3, Oscillibactervalericigenes , Tatumella sp.TA1, Megamonasfuniformis , Thiobacillus denitrificans , Akkermansiamucinphila .

Preferably, the microbial inhibitor or bactericide comprises compounds, polypeptides, polynucleotides, natural plants or essences of natural plants.

More preferably, the microbial inhibitor or bactericide is an antibiotic, for example, the antibiotic is a β-lactam antibiotic variants, aminoglycoside antibiotic variants, tetracycline antibiotic variants, chloramphenicol antibiotic variants, macrolide antibiotic variants, glycopeptide antibiotic variants, fluoroquinolone antibiotic variants, nitroimidazole antibiotic variants, rifampin antibiotics, echinocandin antibiotic variants, molyene antibiotic variants, pyrimidine antibiotic variants, allylamine antibiotic variants, azazole antibiotic variants and one or more other antibiotics.

For example, in some embodiments, antibiotics may include one or more of the following: β-lactam antibiotics, including penicillins (e.g., penicillin V), amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, pivampicillin, pivmecillinam, and ticarcillin. Cephalosporins such as cefacetril, cefadroxil, cephalexin, cephaloglycin, cephalonium, cephaloridine, cephalothin, cephapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cephradine, 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, ceftiolen, epoceline, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, cefluprenam, cefocelis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomezine, cephaloram, cefaparol, cefcanel, cefedrolor, cefampidone, cefetrizole, cefivitril, cefmatilene, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, thienamycin, 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), ribostamycin, micronomycin, azithromycin, dibekacin, sisomycin, netilmicin, paromomycin, bramycin, etc. Tetracycline antibiotics including tetracycline, oxytetracycline, aureomycin, doxycycline, etc. Chloramphenicol antibiotics including chloramphenicol, thiamethoxazole, etc. Macrolide antibiotics including erythromycin, albomycin, erythromycin estolate, acetylspiramycin, madecamycin, josamycin, azithromycin, clarithromycin, Dirithromycin, Roxithromycin, Telithromycin, etc. Glycopeptide antibiotics including Vancomycin, Norvancomycin, Teicoplanin, etc. Quinolone antibiotics including Norfloxacin, Ofloxacin, Ciprofloxacin, Pefloxacin, Gatifloxacin, Enoxacin, Lomefloxacin, Nalidixic acid, Levofloxacin, Moxifloxacin and Besifloxacin. Nitroimidazole antibiotics including Metronidazole, Tinidazole, Ornidazole, etc. Rifampin antibiotics including Rifampicin, Echinocandin antibiotics, Polyene antibiotics, Pyrimidine antibiotics, Allylamine antibiotics, Azazole antibiotics; and others: 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 antibiotics may be selected from the following drugs: amikacin, amoxicillin, ampicillin, neosalvarsan, azithromycin, azlocillin, aztreonam, bacitracin, capreomycin, carbenicillin, cefaclor, cefadroxil, cephalexin, cephalothin, cefamandole, cefazolin, cefdinir, cefditoren, cefixime, cefoperazone, cefotaxime sodium, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, chloramphenicol, cilastatin, clarithromycin, clavulanic acid, 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, mapirocin, nafcillin, neomycin, netilmicin, furantoin, oxacillin, oxytetracycline, paromomycin, penicillin G, penicillin V, piperacillin, platensimicin, polymyxin B, pyrazinamide, quinupristin, rapamycin, rifabutin, rifampin, rifampin, rifapentine, rifaximin, roxithromycin, sucfadiazine silver, spectromycin, streptomycin, sulbactam, sulfacetamide, sulfadiazine, sulfamethizole, sulfamethoxazole, sulfisoxazole, sulfasalazine, sulfafurazole, tazobactam, teicoplanin, telavancin, telithromycin, temocylin, tetracycline, thiamphenicol, ticarcillin, tigecycline, tinidazole, tobramycin, trimethoprim, triacetyloleandomycin, vancomycin, enoxacin, lomefloxacin, nalidixic acid, ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, ofloxacin, Norfloxacin, Cefotetan, Cefonicid, Cephradine, Cephapirin, Cephalothin, Cefmetazole, Cefotaxime sodium, Latamoxef, Cefepime, Ceftaroline fosamil, Ceftobiprole, Dalbavancin, Demeclocycline, Metacycline, Ertapenem, Fidaxomicin, Geldanamycin, Herbimycin, Posizolid, Radezolid, Torezolid, Oritavancin, Spiramycin, Sulfamethazine, Sulfonamidochrizoidine, Gemifioxacin, Nadifloxacin, Trovafloxacin, Garenoxacin, Sparfloxacin, Temafloxacin, Teixobactin, Malacidins and their combinations.

Preferably, the microbial inhibitor or bactericide is a compound of formula I (e.g., formulas I-1, I-2, I-3, I-4, and I-5), formula II (e.g., formulas II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, and II-10), formula III (e.g., formulas III-1, III-2, and III-3), formula IV-1, or formula IV-2 (e.g., formulas IV-3, IV-4, IV-5, and IV-6), a glycoside (e.g., formula V), or a salt or ester suitable for use in the pharmaceutical industry, wherein the aglycone of the glycoside is a phenolic compound, flavonoid, coumarin, benzoic acid, or sterol, wherein formulas I, II, III, IV-1, IV-2, V, and their subformulas are defined as follows.

In specific embodiments of the present invention, the microbial inhibitor or bactericide is compounds 1-8 with the following chemical structure:

Preferably, the microbial inhibitor or sterilizing agent of the present invention is a natural plant or an extract thereof, and the natural plant is selected from a combination of one or more species of licorice, white peony root, forsythia, fructus auranti, rehmannia glutinosa, mandarin, pseudoginseng peel, tortoiseshell, rhodiola rosea, rehmannia shoots, coix-Job's tears, white peony root, forsythia, cassia seed and licorice, an extract of the above-mentioned natural plants, or a combination thereof.

In a specific embodiment of the present invention, the microbial inhibitor or sterilizing agent is a combination comprising tortoise shell, rosea rhodiola, rehmannia rhizome, Job's tears coix, white peony root, forsythia, cassia seed and licorice. By weight, the combination preferably comprises 5-15 tinctures of tortoise shell, 10-30 tinctures of rosea rhodiola, 5-15 tinctures of rehmannia rhizome, 5-15 tinctures of Job's tears, 8-20 tinctures of white peony root, 5-15 tinctures of forsythia, 5-15 tinctures of cassia seed and 5-15 tinctures of licorice; more preferably, the combination includes 10 tinctures of tortoise shell, 20 tinctures of Rhodiola rosea, 10 tinctures of Rehmannia rhizome, 10 tinctures of Job's tears, 12 tinctures of white peony root, 10 tinctures of Forsythia, 10 tinctures of Cassia seed, and 9 tinctures of white peony root. Licorice decoctions.

Preferably, the drug is a drug delivery preparation for the eye or gastrointestinal tract. More preferably, the drug delivery preparation for the eye is eye drops, eye ointments, eye gel, eye inserts, eye washes or intraocular injection. More preferably, the drug delivery preparation for the gastrointestinal tract is tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, fast-dissolving tablets and tablets for oral use), pills, powder, granules, capsules (including soft capsules and microcapsules), troches, syrup, solutions, emulsions, suspensions, controlled-release formulations (e.g., immediate-release preparation, sustained-release preparation and sustained-release microcapsules), aerosol or film agent (e.g., oral soluble film agent and oral mucosa - adhesive film).

In particular, such excipients acceptable for use in pharmaceuticals may include one or more types of sweeteners (such as sucrose, xylitol, fructooligosaccharide, sodium cyclamate, stevioside and aspartame), flavoring agents (such as fragrances and essences), mucilages (such as sodium alginate, gum arabic, gelatin, methylcellulose and sodium carboxymethylcellulose), brightening agents (such as chitosan and gelatin), antiseptics (such as benzoic acid and its salts, sorbic acid and its salts, and a number of parabens), disintegrants (such as β-substituted hydroxypropyl cellulose, crospovidone, sodium starch glycolate, sodium croscarmellose and starch), binders (such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone, copovidone and pregelatinized starch), lubricants (such as stearic acid, magnesium stearate and sodium fumaryl stearate), wetting agents (such as polyoxyethylene sorbitan fatty acid ester, poloxamer and polyoxyethylene castor oil derivatives), suspending agents (such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, copovidone, sodium carboxymethylcellulose and methylcellulose), stabilizers (such as citric acid, fumaric acid and succinic acid), fillers (such as starch, sucrose, lactose and microcrystalline cellulose), binders (such as cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone), etc. The present invention also provides a method for creating an eye disease model, which comprises infecting an eye disease model with microorganisms.

Preferably, the contamination includes direct or indirect contact with microorganisms. In a particular embodiment, the eye disease model is contaminated with microorganisms by keeping non-human animals in a solid-phase formation environment.

Preferably, the microorganisms are derived from the intestinal bacteria of the same individual or are identical to the intestinal bacteria of such an individual.

Preferably, the eye disorder comprises retinal degeneration. More preferably, the retinal degeneration is progressive retinal degeneration.

Most retinal degenerations are inherited retinal degenerations (IRDs).

Preferably, the ocular disease includes LCA, RP, arRP, EORD, EORP, PPRPE, rettelangiectasia and/or fundus-like choroideremia.

Preferably, the eye disease includes eye inflammations such as uveitis, glaucoma, age-related macular degeneration (AMD), hyaloiditis, choroiditis, retinitis, retinal vasculitis, optic neuritis, uveitis, Behcet, Vogt-Koyanagi-Harada syndrome, uveitis, retinopathy, sympathetic ophthalmia, cataract, conjunctivitis, and glaucoma.

Preferably, the model is a non-human animal, preferably a monkey, dog, chimpanzee, rat or mouse.

Preferably, the model carrier is a cell, tissue or organ obtained from a human or animal body.

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 organ is a regenerative tissue or organ.

Preferably, the model contains a gene with a pathogenic mutation.

Preferably, the gene with the pathogenic mutation is involved in maintaining the structure of the retinal barrier, which is the outer blood-retinal barrier and/or the inner blood-retinal barrier.

Preferably, the model or model carrier contains a pathogenic mutation of a gene associated with an eye disease, wherein the gene that causes the pathogenic mutation of the eye gene is selected from a combination of one or more of the following genes: ABCA4, ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHI1, AIPL1, ALMS1, AMY2B, APC, ARFGEF1, ARL13B, ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1, C2CD3, CA4, CABP4, CACNA1F, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104, CEP250, CEP290, CEP290, CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC1, CLCN7, CLN3, CLN5, CLN8, CLRN1, CLRN1, CNGA1, CNGA1, CNGA3, CNGB1, CNGB3, CNNM4, COL11A1, COL11A2, COL18A1, COL2A1, COL4A1, COL9A1, COL9A2, CP, CP, CPLANE1, CRB1, ERCC4, CSPP1, CTNNA1, CYP4V2, DHDDS, DYNC2H1, DYNC2I1, DYNC2I2, ENPP1, ERCC4, EVC2, EYS, EYS, F5, FAM161A, FBN1, FKRP, FKTN, FLG, FLVCR1, FOXE3, FUZ, GLB1, GMPPB, GNAT1, GRK1, GRM6, GUCA1A, GUCA1B, 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, KRIT1, LBR, LCA5, LOC101927157, LOC111365204, LRP2, LRP5, MAK, MAPKAPK3, MATK, MCOLN1, MERTK, MKS1, MPDZ, MT-ATP6, MT-CO3, MT-TE, MT-TL1, MTHFR, MUTYH, MYO7A, MYO7A, NMNAT1, NPHP1, NR2E3, OCA2, OTX2, PANK2, PAX6, PCARE, PCDH15, PDE6A, PDE6B, PDE6B, PDE6D, PEX1, PEX1, PEX12, PEX26, PEX6, PHF3, PITPNM3, PKD2, PLA2G5, POC5, POMT1, PRCD, PRDM13, PROM1, PRPF3, PRPF31, PRPF8, PRPH2, RAD51C, RBP3, RBP4, RD3, RDH12, RDH5, RGR, RGR, RHO, RIMS1, RLBP1, ROM1, RP1, RP1L1, RP2, RPE65, RPE65, RPGR, RPGRIP1, RPGRIP1L, RS1, SACS, SAG, SCAPER, SDCCAG8, SIX6, SLC19A1, SLC22A5, SLC26A4, SLC2A9, SLTM, SNRNP200, SPAG17, SPATA7, SPG11, TFAP2A, TGFB2, TGFBR2, TMEM107, TMEM237, TMEM67, TOGARAM1, TOPORS, TPP1, TRAF3IP1, TREX1, TRIM59-IFT80, TSPAN12, TTC21B, TTC21B, TTC8, TULP1, USH1C, USH2A, USH2A, USH2A, USH2A, USH2A-AS1, VAC14, VCAN, VCAN, VCAN-AS1, VHL, VPS13B, WDR19, WDR19, WDR35, WDR73, YARS1, ZFYVE26, ZFYVE26, ZNF408.

In a particular embodiment, the model or model carrier has a pathogenic mutation in a gene associated with an eye disease and containing the CRB1 gene.

Preferably, the CRB1 gene mutation of the model or model carrier comprises one 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 3bp (TCA), c.2258T>C, c.2290C>T, c.2365_2367del AAT, when deleting a frame, 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, c.3992G>A.

More preferably, the CRB1 gene mutation of the model or model carrier comprises one 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.Gly1226Ter), 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.Gly1103Arg), c.484G>A, (p.Val162Met), c.2401A>T, (p.Lys801Ter), c.2234C>T, (p.Thr745Met), c.2290C>T, (p.Arg764Cys), c.3122T>C, (p.Met1041Thr).

Preferably, the CRB1 gene mutation of the model or model carrier is the Rd8 mutation.

Preferably, the mutation is either a homozygous mutation or a heterozygous mutation.

Preferably, the model or model carrier is an inherited mutation of a gene as mentioned above, or the mutation is acquired as a result of genetic recombination.

Preferably, the model or model vehicle contains a humanized CRB1 gene or a human CRB1 gene and does not express or express the endogenous CRB1 gene.

Preferably, the animal, rather than the human, has defects in the colonic epithelial barrier and/or associated inflammation of the colonic wall.

In this particular embodiment, occludin is significantly lost in the model. In this particular embodiment, occludin is significantly lost, and claudin1 is not clearly expressed in the model.

The microorganisms are one or more combinations of bacteria, archaea, protozoa, fungi, or viruses that are selected from one or more of the following: Anearostipes, Bifidobacterium, Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillus sp. , 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 .

In particular, the bacteria are selected from one or more of the following variants: Anearostipeshadrus , Bifidobacterium pseudocatenulatum , Nitrosomonas sp.Is79A3, Oscillibactervalericigenes , Tatumella sp.TA1, Megamonasfuniformis , Thiobacillus denitrificans , Clostordium tetani, Clostordium perfringens, Clostordium botulinum, Asinetobacter acinetobacter calcoaseticus, Asinetobacter ruffiensis, Asinetobacter baumanii, Asinetobacter hemolyticus, Asinetobacter junii, Asinetobacter johnson, Streptococcus pyrogenus, Streptococcus hemolyticus, Bacillus filiformis succinogenes, Bacillus filiformis intestinalis, Porphyromonas asacarolytica, Porphyromonas endodontalis, Porphyromonas ginvinalis, Camilobacter jejuni, Camilobacter coli, Camilobacter seabirds, Camilobacter uppsala, Camilobacter consius Camilobacter foetal, Actinomyces Israelii, Actinomyces naeslundii, Actinomyces odontolyticus, actinomyces viscosus, actinomyces neuii, escherchia coli, escherchia blattae, escherchia ferguson, escherchia hermanii, escherchia vulneris, tissierella praecuta, Klebsiella pneumnie, Klebsiella ozanae, azospirillum brasilense, achromobacter, thiobacillus dentrificansthiobacillus, thiobacillus ferroxidans, thiobacillus thioxidans, thiobacillus neapolitanus, burkholderia, mycobacterium marinum, treponema pallidum, treponema chiodisetrerie, vibrio metschenicowii, ruminococcus albus, ruminococcus flavesiensis, methanobrevibacter ruminatium, shigella dysenterie, shigella flexneri, shigella bogdia, shigella phonei, francia, coprococcus eutheactus, streptomyces albus, pseudomonas mendocina, micrococcus sedentarius, alicyclic denitrifying bacterium, achromobacter silosidans, sphingomonas, mycobacterium abscessus, arthrobacter auresens, Prevotella, sinorgisobium melilotus, acid saccharomyes, Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus chemolyticus, Pseudomonas putrida, Stenotrophomonas maltoilia, Bacilus sereus, Bacilus megatherium, Lactobacillus reuteri, gardnerella vaginalis, enterococcus phesium, cytophage hutchinsonia, bacillus licheniformis, santomonas oryzae, asinetobacter baumannii, asinetobacter calcoaseticus, comamonas testosteroni, mycobacterium cansasia, basillus thuringensis, citrobacter coseri, Diadobacter zymosyte, Serratia marsesens, Sphingomonas vittisii, Klebsiella preumoniae, Pseudomonas fluoresens, Ralstonia picettiii, Lactobacillus crispatus, Burkholderia, Lactobacillus delbruecii, Meiothermussilvanus (D), Echerichia coli, Micrococcus luteus, Basillus subtilis, Corynebacterium gleum and Finegoldia magna.

Preferably, the route of infection includes direct or indirect contact between the microorganism and the area of the model carrier to be infected, wherein indirect contact means that there is a blood-retinal barrier between the microorganism and the area of infection, which is preferably the barrier between the blood and the outer retina or the internal blood-retinal barrier.

In a particular embodiment, the infection route includes infection of the eye by enteric bacteria through the peripheral blood. In particular, enteric bacteria enter the peripheral blood when the intestinal epithelial barrier of the animal model is significantly damaged. In this case, the retinal barrier in the model is also significantly damaged, and enteric bacteria entering the peripheral blood infect the retina. In a particular embodiment, the animal model has a mutation of the CRB1 gene. In a particular embodiment, the mutation of the CRB1 gene is the Rd8 mutation.

Moreover, the present invention provides a method for preparing an ocular inflammation model which is an animal model infected with microorganisms.

Preferably, eye inflammation is caused by intestinal flora or the same flora as the intestinal flora.

A non-human animal is one of the animals mentioned above.

Microorganisms are the microorganisms mentioned above.

Moreover, the present invention provides a method for producing a model of a degenerative retinal disease, which comprises infecting a non-human animal suffering from a degenerative retinal disease with microorganisms.

Preferably, ocular retinal degeneration due to inflammation is a retinal disease as mentioned above.

A non-human animal is one of the animals mentioned above.

Microorganisms are the microorganisms mentioned above.

Moreover, the present invention provides a model of a host with ocular inflammation caused by a microbial infection.

Preferably, the microorganisms are derived from the intestinal bacteria of the same individual or are identical to the intestinal bacteria of such an individual.

Preferably, the eye disorder comprises retinal degeneration. More preferably, the retinal degeneration is progressive retinal degeneration.

Most retinal degenerations are inherited retinal degenerations (IRDs).

Preferably, retinal degeneration is a disease of the animal model itself or is caused by genetic manipulation.

Preferably, the ocular disease includes LCA, RP, arRP, EORD, EORP, PPRPE, rettelangiectasia and/or fundus-like choroideremia.

Preferably, the eye disease includes eye inflammations such as uveitis, glaucoma, age-related macular degeneration (AMD), hyaloiditis, choroiditis, retinitis, retinal vasculitis, optic neuritis, uveitis, Behcet, Vogt-Koyanagi-Harada syndrome, uveitis, retinopathy, sympathetic ophthalmia, cataract, conjunctivitis, and glaucoma.

Preferably, the model is a non-human animal, preferably a monkey, dog, chimpanzee, rat or mouse.

Preferably, the model carrier is a cell, tissue or organ obtained from a human or animal body.

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 organ is a regenerative tissue or organ.

Preferably, the model contains a gene with a pathogenic mutation.

Preferably, the gene with the pathogenic mutation is involved in maintaining the structure of the retinal barrier, which is the outer blood-retinal barrier and/or the inner blood-retinal barrier.

Preferably, the model carrier has a mutation in one or more of the following genes: ABCA4, ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHI1, AIPL1, ALMS1, AMY2B, APC, ARFGEF1, ARL13B, ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1, C2CD3, CA4, CABP4, CACNA1F, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104, CEP250, CEP290, CEP290, CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC1, CLCN7, CLN3, CLN5, CLN8, CLRN1, CLRN1, CNGA1, CNGA1, CNGA3, CNGB1, CNGB3, CNNM4, COL11A1, COL11A2, COL18A1, COL2A1, COL4A1, COL9A1, COL9A2, CP, CP, CPLANE1, CRB1, ERCC4, CSPP1, CTNNA1, CYP4V2, DHDDS, DYNC2H1, DYNC2I1, DYNC2I2, ENPP1, ERCC4, EVC2, EYS, EYS, F5, FAM161A, FBN1, FKRP, FKTN, FLG, FLVCR1, FOXE3, FUZ, GLB1, GMPPB, GNAT1, GRK1, GRM6, GUCA1A, GUCA1B, 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, KRIT1, LBR, LCA5, LOC101927157, LOC111365204, LRP2, LRP5, MAK, MAPKAPK3, MATK, MCOLN1, MERTK, MKS1, MPDZ, MT-ATP6, MT-CO3, MT-TE, MT-TL1, MTHFR, MUTYH, MYO7A, MYO7A, NMNAT1, NPHP1, NR2E3, OCA2, OTX2, PANK2, PAX6, PCARE, PCDH15, PDE6A, PDE6B, PDE6B, PDE6D, PEX1, PEX1, PEX12, PEX26, PEX6, PHF3, PITPNM3, PKD2, PLA2G5, POC5, POMT1, PRCD, PRDM13, PROM1, PRPF3, PRPF31, PRPF8, PRPH2, RAD51C, RBP3, RBP4, RD3, RDH12, RDH5, RGR, RGR, RHO, RIMS1, RLBP1, ROM1, RP1, RP1L1, RP2, RPE65, RPE65, RPGR, RPGRIP1, RPGRIP1L, RS1, SACS, SAG, SCAPER, SDCCAG8, SIX6, SLC19A1, SLC22A5, SLC26A4, SLC2A9, SLTM, SNRNP200, SPAG17, SPATA7, SPG11, TFAP2A, TGFB2, TGFBR2, TMEM107, TMEM237, TMEM67, TOGARAM1, TOPORS, TPP1, TRAF3IP1, TREX1, TRIM59-IFT80, TSPAN12, TTC21B, TTC21B, TTC8, TULP1, USH1C, USH2A, USH2A, USH2A, USH2A, USH2A-AS1, VAC14, VCAN, VCAN, VCAN-AS1, VHL, VPS13B, WDR19, WDR19, WDR35, WDR73, YARS1, ZFYVE26, Combination of one or more of the following: ZFYVE26 and ZNF408.

In a specific embodiment, the model carrier has a pathogenic mutation in a gene associated with an eye disease and containing the CRB1 gene.

Preferably, the CRB1 gene mutation of the model carrier comprises one 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 3bp (TCA), c.2258T>C, c.2290C>T, c.2365_2367del AAT, when deleting a frame, 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, c.3992G>A.

More preferably, the CRB1 gene mutation of the model or model carrier comprises one 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.Gly1226Ter), 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.Gly1103Arg), c.484G>A, (p.Val162Met), c.2401A>T, (p.Lys801Ter), c.2234C>T, (p.Thr745Met), c.2290C>T, (p.Arg764Cys), c.3122T>C, (p.Met1041Thr).

Preferably, the model carrier's CRB1 gene mutation is the Rd8 mutation.

Preferably, the mutation is either a homozygous mutation or a heterozygous mutation.

Preferably, the carrier of the model is an inherited mutation of the gene as mentioned above, or the mutation is acquired as a result of genetic recombination.

Preferably, the model or model vehicle contains a humanized CRB1 gene or a human CRB1 gene and does not express or lacks the endogenous CRB1 gene.

Preferably, the animal, rather than the human, has defects in the colonic epithelial barrier and/or associated inflammation of the colonic wall.

In this particular embodiment, occludin is significantly lost in the model. In this particular embodiment, occludin is significantly lost, and claudin1 is not clearly expressed in the model.

The microorganisms are one or more combinations of bacteria, archaea, protozoa, fungi, or viruses that are selected from one or more of the following: Anearostipes, Bifidobacterium, Megamonas, Nitrosomonas, Oscillibacter, Tatumella, Thiobacillus sp. , 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 .

In particular, the bacteria are selected from one or more of the following variants: Anearostipeshadrus , Bifidobacterium pseudocatenulatum , Nitrosomonas sp.Is79A3, Oscillibactervalericigenes , Tatumella sp.TA1, Megamonasfuniformis , Thiobacillus denitrificans , Clostordium tetani, Clostordium perfringens, Clostordium botulinum, Asinetobacter acinetobacter calcoaseticus, Asinetobacter ruffiensis, Asinetobacter baumanii, Asinetobacter hemolyticus, Asinetobacter junii, Asinetobacter johnson, Streptococcus pyrogenus, Streptococcus hemolyticus, Bacillus filiformis succinogenes, Bacillus filiformis intestinalis, Porphyromonas asacarolytica, Porphyromonas endodontalis, Porphyromonas ginvinalis, Camilobacter jejuni, Camilobacter coli, Camilobacter seabirds, Camilobacter uppsala, Camilobacter consius Camilobacter foetal, Actinomyces Israelii, Actinomyces naeslundii, Actinomyces odontolyticus, actinomyces viscosus, actinomyces neuii, escherchia coli, escherchia blattae, escherchia ferguson, escherchia hermanii, escherchia vulneris, tissierella praecuta, Klebsiella pneumnie, Klebsiella ozanae, azospirillum brasilense, achromobacter, thiobacillus dentrificansthiobacillus, thiobacillus ferroxidans, thiobacillus thioxidans, thiobacillus neapolitanus, burkholderia, mycobacterium marinum, treponema pallidum, treponema chiodisetrerie, vibrio metschenicowii, ruminococcus albus, ruminococcus flavesiensis, methanobrevibacter ruminatium, shigella dysenterie, shigella flexneri, shigella bogdia, shigella phonei, francia, coprococcus eutheactus, streptomyces albus, pseudomonas mendocina, micrococcus sedentarius, alicyclic denitrifying bacterium, achromobacter silosidans, sphingomonas, mycobacterium abscessus, arthrobacter auresens, Prevotella, sinorgisobium melilotus, acid saccharomyes, Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus chemolyticus, Pseudomonas putrida, Stenotrophomonas maltoilia, Bacilus sereus, Bacilus megatherium, Lactobacillus reuteri, gardnerella vaginalis, enterococcus phesium, cytophage hutchinsonia, bacillus licheniformis, santomonas oryzae, asinetobacter baumannii, asinetobacter calcoaseticus, comamonas testosteroni, mycobacterium cansasia, basillus thuringensis, citrobacter coseri, Diadobacter zymosyte, Serratia marsesens, Sphingomonas vittisii, Klebsiella preumoniae, Pseudomonas fluoresens, Ralstonia picettiii, Lactobacillus crispatus, Burkholderia, Lactobacillus delbruecii, Meiothermussilvanus (D), Echerichia coli, Micrococcus luteus, Basillus subtilis, Corynebacterium gleum and Finegoldia magna.

Preferably, the route of infection involves direct or indirect contact between the microorganism and the area of the model carrier to be infected, wherein indirect contact means that there is a blood-retinal barrier between the microorganism and the area of infection, which is preferably the external blood-retinal barrier or the internal blood-retinal barrier.

In a particular embodiment, the infection route includes infection of the eye with enteric bacteria via peripheral blood. In particular, enteric bacteria enter the peripheral blood when the intestinal epithelial barrier of the animal model is significantly damaged. In this case, the retinal barrier in the model is also significantly damaged, and enteric bacteria entering the peripheral blood infect the retina. In a particular embodiment, the animal model has a mutation of the CRB1 gene. In a particular embodiment, the mutation of the CRB1 gene is the Rd8 mutation.

The inflammatory model carrier is derived from a disease model developed in the manner described previously or is obtained by infecting three-dimensional cells, tissues or organs of the animal eye with microorganisms as described previously.

The present invention provides the use of the above-described method for assessing the therapeutic effect of targeted therapy for eye diseases, wherein the eye diseases include the eye diseases described previously.

In a specific embodiment, the targeted therapy of gene mutations as described above is performed on a model or a model vector as described above. The model or model carrier that has undergone the targeted therapy and the models or model carriers that have/have not received the targeted therapy are divided into two groups, and eye disease models are established according to the method described above. If the group that has received the targeted therapy cannot be successfully modeled for comparison, this indicates that the targeted therapy has achieved positive results.

In a specific embodiment, the targeted therapy is directed to one or more combinations of two or more genes as follows: ABCA4, ABCC6, ABCC9, ACBD5, ACO2, ACO2, ACTG1, ADGRV1, AHI1, AIPL1, ALMS1, AMY2B, APC, ARFGEF1, ARL13B, ARL13B, ARL6, ARMC9, ATOH7, B9D1, BAG3, BBS1, BBS1, BBS2, BBS5, BEST1, C2CD3, CA4, CABP4, CACNA1F, CBS, CC2D2A, CDH23, CDH23, CDHR1, CEMIP2, CEP104, CEP250, CEP290, CEP290, CEP41, CEP78, CERKL, CFAP410, CFAP418, CHM, CLCC1, CLCN7, CLN3, CLN5, CLN8, CLRN1, CLRN1, CNGA1, CNGA1, CNGA3, CNGB1, CNGB3, CNNM4, COL11A1, COL11A2, COL18A1, COL2A1, COL4A1, COL9A1, COL9A2, CP, CP, CPLANE1, CRB1, ERCC4, CSPP1, CTNNA1, CYP4V2, DHDDS, DYNC2H1, DYNC2I1, DYNC2I2, ENPP1, ERCC4, EVC2, EYS, EYS, F5, FAM161A, FBN1, FKRP, FKTN, FLG, FLVCR1, FOXE3, FUZ, GLB1, GMPPB, GNAT1, GRK1, GRM6, GUCA1A, GUCA1B, 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, KRIT1, LBR, LCA5, LOC101927157, LOC111365204, LRP2, LRP5, MAK, MAPKAPK3, MATK, MCOLN1, MERTK, MKS1, MPDZ, MT-ATP6, MT-CO3, MT-TE, MT-TL1, MTHFR, MUTYH, MYO7A, MYO7A, NMNAT1, NPHP1, NR2E3, OCA2, OTX2, PANK2, PAX6, PCARE, PCDH15, PDE6A, PDE6B, PDE6B, PDE6D, PEX1, PEX1, PEX12, PEX26, PEX6, PHF3, PITPNM3, PKD2, PLA2G5, POC5, POMT1, PRCD, PRDM13, PROM1, PRPF3, PRPF31, PRPF8, PRPH2, RAD51C, RBP3, RBP4, RD3, RDH12, RDH5, RGR, RGR, RHO, RIMS1, RLBP1, ROM1, RP1, RP1L1, RP2, RPE65, RPE65, RPGR, RPGRIP1, RPGRIP1L, RS1, SACS, SAG, SCAPER, SDCCAG8, SIX6, SLC19A1, SLC22A5, SLC26A4, SLC2A9, SLTM, SNRNP200, SPAG17, SPATA7, SPG11, TFAP2A, TGFB2, TGFBR2, TMEM107, TMEM237, TMEM67, TOGARAM1, TOPORS, TPP1, TRAF3IP1, TREX1, TRIM59-IFT80, TSPAN12, TTC21B, TTC21B, TTC8, TULP1, USH1C, USH2A, USH2A, USH2A, USH2A, USH2A-AS1, VAC14, VCAN, VCAN, VCAN-AS1, VHL, VPS13B, WDR19, WDR19, WDR35, WDR73, YARS1, ZFYVE26, ZFYVE26, ZNF408.

In a specific embodiment, targeted therapy is aimed at one 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.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 3bp (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, c.3992G>A.

In a specific embodiment, the targeted therapeutic drug comprises modified cells, modified proteins, target genes as described previously, or RNA at target sites of mutations as described previously, and/or target genes as described previously, or DNA at target sites of mutations as described previously.

The present invention provides the use of a disease model obtained by the method described above in studies related to eye diseases. Eye diseases include eye diseases described above. The described study includes a cross-reaction between diseases associated with hereditary retinal degeneration and intestinal microbiota.

In a specific embodiment, the disease model is infected with intestinal bacteria, and when the pathogenic mutation carrying the disease model is subjected to cell therapy, therapeutic results of administration and non-administration are observed. The drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic affecting the infected bacteria. In a specific embodiment, the disease model is infected with intestinal bacteria, and when the pathogenic mutation carrying the disease model is subjected to RNA cell therapy, therapeutic results of administration and non-administration are observed. The drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic affecting the infected bacteria. In a specific embodiment, the disease model is infected with more than two intestinal bacteria, and when the pathogenic mutation carrying the disease model is subjected to cell therapy, therapeutic results of administration and non-administration are observed. The drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic affecting the infected bacteria. In a specific embodiment of the invention, the disease model is infected with two intestinal bacteria. When the pathogenic mutation carried by the disease model is subjected to RNA therapy, a therapeutic effect of administration and non-administration is observed. The drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic targeted to the infected.

Preferably, the pathogenic mutation occurs in a gene as described previously, or it is a mutation in the CRB gene as described previously.

The present invention provides the use of a disease model carrier obtained by the method described above in studies related to eye diseases. Eye diseases include eye diseases described above. The described study includes a synergistic effect between diseases associated with hereditary retinal degeneration and intestinal microbiota.

In a specific embodiment, the disease model is infected with intestinal bacteria, and when the pathogenic mutation carrying the disease model is subjected to cell therapy, therapeutic results of administration and non-administration are observed. The drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic affecting the infected bacteria. In a specific embodiment, the disease model is infected with intestinal bacteria, and when the pathogenic mutation carrying the disease model is subjected to RNA cell therapy, therapeutic results of administration and non-administration are observed. The drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic affecting the infected bacteria. In a specific embodiment, the disease model is infected with more than two intestinal bacteria, and when the pathogenic mutation carrying the disease model is subjected to cell therapy, therapeutic results of administration and non-administration are observed. The drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic affecting the infected bacteria. In a specific embodiment, the disease model is infected with two intestinal bacteria, and when the pathogenic mutation carrying the disease model is subjected to RNA cell therapy, therapeutic results of administration and non-administration are observed. The drug is a small molecule drug, preferably a broad-spectrum antibiotic or an antibiotic that acts on infected bacteria.

Preferably, the pathogenic mutation occurs in a gene as described previously, or it is a mutation in the CRB gene as described previously.

The present invention provides the use of a disease model or a disease model carrier as described previously in the analysis of drugs associated with eye diseases. The drugs include one or more combinations of small molecule drugs, chemical drugs, polymeric drugs, biological drugs or natural drugs (such as traditional Chinese medicine drugs or Chinese herbal extracts), cell drugs, RNA drugs and DNA drugs.

Eye diseases include the eye diseases described previously.

Preferably, the low molecular weight compound is an antibiotic, wherein the antibiotic is a broad spectrum antibiotic widely known to those skilled in the art.

Preferably, the small molecular weight compound is a narrow spectrum antibiotic with activity directed towards specific bacteria.

Preferably, the cells comprise one or more combinations of modified immune cells, such as T cells, B cells, or stem cells.

Preferably, the RNA comprises mRNA, siRNA, sgRNA, miRNA, ASO and/or replicon RNA.

In a specific embodiment, the target therapy is applied to a disease model or a carrier of the disease model, and at the same time, the drugs described above are applied or not applied to the disease model or the carrier of the disease model subjected to the target therapy, and the disease model or the carrier of the disease model that has not been subjected to the target therapy. The progression of inflammation in different groups is observed to assess the drug therapeutic effect of the target therapy.

In a specific embodiment, the target therapy is applied to a disease model or a carrier of the disease model, and at the same time, the drugs described above are applied or not applied to the disease model or the carrier of the disease model subjected to the target therapy, and the disease model or the carrier of the disease model that has not been subjected to the target therapy. The progression of inflammation in different groups is observed to assess the effectiveness of small molecule drugs.

From description to drawings

Fig. 1: Genotype and phenotype of Crb1 ^rd8/rd8 (rd8)-SPF mice. Where, Fig. 1A is the gel electropherogram of Crb1 ^rd8/rd8 (rd8) genotype and Crb1 ^wt/wt (C57BL/J, short wt), Fig. 1B is the representative fundus image of Rd8-SPF and WT-SPF mice, typical white spots appear in the inferior nasal quadrant of the retina of Rd8-SPF mice, and Fig. 1C shows the formation of H&E stains of pathological changes in both eyes at the detection of E18 Rd8-SPF mice.

Fig. 2: Potential mechanisms of retinal abnormalities and retinopathy in Rd8-SPF mice. Figure 2A shows the pathological changes in both eyes of Rd8-SPF and WT-SPF mice detected by H&E staining at E18, P12 (before eye opening), P15 (after eye opening) and 8 weeks (8W). Rd8 mice exhibit typical retinal abnormalities including progressive retinal dysplasia (folds and false garlands) and degeneration, whereas WT mice exhibit normal retinal structures; Figure 2B shows the statistical data of Rd8- and WT-SPF mice with and without eye damage at E18, P12, P15 and 8W. Typical lesions can be seen in all retinas of Rd8 mice at P12, P15 and 8W, but none were observed in WT-SPF mice. WT-SPF mice: at E18, P12, P15 and 8W, n=6 for each group. Rd8-SPF mice: n=32 at E18, n=6 at P12, n=8 at P15. Figure 2C shows the analysis of DEGs in the superior (non-lesion) and inferior (lesion) retinas of Rd8 mice. Slamf1 (sigmoid lymphocyte-activated molecule family member 1) and Ncf4 (neutrophil cytoplasmic factor) are the two genes with the highest proliferation rate in Rd8-SPF mouse retinopathy. Figure 2D shows the functional analysis of DEGs by IPA; and Figure 2E shows the abundance of IBA1+ microglia (red) in the lesion area of Rd8 mice retina by immunofluorescence staining.

Fig. 3: RNA-seq analysis and comparison of gene expression profiles in the superior and inferior retinas of Rd8-SPF mice. Where Fig. 3A shows the superior (non-lesioned) and inferior (lesioned) regions of Rd8-SPF mice. Fig. 3B shows that RNA-seq analysis revealed at least 2-fold differences in expression (P<0.05) between the superior and inferior retinas of Rd8-SPF mice, while Fig. 3C shows the expression patterns of 179 DEGs in the superior and inferior retinas of Rd8- and WT-SPF mice.

Fig. 4: Identification of bacteria in Rd8-SPF mouse retinopathy. Where Fig. 4A is the principal coordinate analysis (PCoA) and shows the significant differences between the bacterial composition in the retinal tissue of Rd8 mice and WT mice; Fig. 4B shows the identification of the microbial species composition in the upper (S) and lower (I) retina of Rd8 mice by metagenomic sequencing (n=4). Fig. 4C shows the detection of 16srDNA and vanco-body bacteria in the retinal tissues of Rd8- and WT-SPF mice (4 weeks old) by fluorescence in situ hybridization. Fig. 4D shows the observation of the bacterial distribution in Rd8 mouse retinopathy by transmission electron microscope (TEM), with the “colay” group as the positive control.

Fig. 5: Disruption of retinal adherens junction in Rd8-SPF mice. Fig. 5A shows the immunofluorescence staining of CRB1 in the retina (red) of Rd8- and WT-SPF mice. BM refers to Bruch's membrane, CC refers to chorionic capillaries, and BL refers to the basement membrane; and Fig. 5B shows the transmission electron microscopic observation of adherens junction of the outer retinal membrane of Rd8- and WT-SPF mice. The red arrow refers to adherens junction, AJ refers to adherens junction, OLM refers to outer membrane, ONL refers to outer nuclear layer, and IS refers to inner segment. Figure 5C shows the transmission electron microscopic observation of the dissolution of retinal pigment epithelium (RPE) basal layer adhesion, distortion of chorionic capillary basal layer, and disruption of collagen layer in Rd8-SPF mice. CC refers to the chorionic capillaries, CH refers to the choroid, and BL refers to the basement membrane; Fig. 5D shows the statistical analysis of Bruch's membrane thickness ****P<0.0001 in Rd8- and WT-SPF mice.

Fig. 6A: Detachment of adherens junction (AJ) and outer nuclear layer (ONL) in the outer membrane (OLM) of Rd8-SPF mice is shown by transmission electron microscopy (TEM).

Fig. 6B: Branch membrane thickness of Rd8-SPF mice and WT-SPF mice is shown using transmission electron microscope (TEM).

Fig. 7: Shows colonic epithelial barrier defects and associated inflammation in Rd8-SPF mice. Where Fig. 7A shows the relative abundance of bacteria in Rd8 retinas in different parts of the gastrointestinal tract of Rd8 mice and WT mice (n=5). Fig. 7B-D show immunofluorescence staining of CRB1 protein (B), phalloidin protein (C), and occlusion protein (D) in colonic cells of Rd8- and WT-SPF mice. Fig. 7E shows statistical analysis of the relative strengths of occlusion proteins in Rd8- and WT-SPF mice. Fig. 7F shows Claudin1 expression in colonic epithelial cells of Rd8- and WT-SPF mice detected by Western blotting. Fig. Figure 7G shows the statistical analysis of the relative intensity of Claudin1 in Rd8- and WT-SPF mice. Figure 7H shows the adherent and tight junctions between the colonic epithelium of Rd8- and WT-SPF mice observed by transmission electron microscopy. Figures 7I-L show the statistical analysis of the number (I), length (J), width (K), and total number of intact adhesions (L) of epithelial microvilli in Rd8- and WT-SPF mice; and Figures 7M-N compare the mRNA expression levels of Tnfa, Il1b, Il12a, (M), and Mucin2 (N) in Rd8- and WT-SPF mice.

Fig. 8: Microbial composition of bacterial colonies in different gastrointestinal tract regions of Rd8-SPF mice is shown. Where Fig. 8A–F show the principal coordinate analysis (PCoA) of microbial species composition in gastrointestinal tract samples of WT (n=5) and Rd8 (n=5) mice based on the Bray-Curtis distance, including stomach (Fig. 8A), small intestine (Fig. 8B), ileum (Fig. 8C), cecum (Fig. 8D), colon (Fig. 8E), and rectum (Fig. 8F). Fig. 8G shows the microbial composition (H–J) of the lower gastrointestinal tract regions (cecum, colon, and rectum) of Rd8 and WT-SPF mice differentiated by PCoA. The relative abundance of Akkermansia mucinophila in the cecum (H), colon (I) and rectum (J) of Rd8 mice and WT mice is compared. The data show that the mean is ± SEM*P<0.05.

Fig. 9: Shows tight junctions and adherens junctions in the cecum of Rd8 mice. Where Fig. 9A shows immunofluorescence staining of CRB1 protein (green) in cecal cells of Rd8- and WT-SPF mice. Fig. 9B shows immunofluorescence staining of occludin (red) in the cecum of Rd8- and WT-SPF mice. Fig. 9C shows the statistical analysis *P<0.05 of the relative intensities of occludin proteins in Rd8- and WT-SPF mice. Fig. 9D shows the expression of Claudin1 in the cecum of Rd8- and WT-SPF mice detected by Western blot. Fig. 9E shows the statistical analysis of the relative intensities of Claudin1 in Rd8- and WT-SPF mice, with NS indicating non-significance. Figure 9F shows normal tight junction and adherens junction on the cecal epithelial barrier as observed by transmission electron microscope, MV refers to microvesicles and MC refers to mitochondria.

Fig. 10: Fig. 10A shows the adherens junction (AJ) between colonic epithelium and tight junction (TJ) of Rd8- and WT-SPF mice observed by transmission electron microscope, MV refers to microvesicles and MC refers to mitochondria. Meanwhile, Fig. 10B shows the frequency of fluorescent+ bacteria/cells in the peripheral blood of Rd8 and WT mice detected by flow cytometer.

Fig. 11: Shows the impaired intestinal epithelial barrier function in Rd8-SPF mice. Moreover, Fig. 11A shows that the fluorescence intensity of Rd8 mice serum was significantly higher than that of WT mice ***P<0.001 in the FITC glucan intestinal permeability test; Fig. 11B shows the comparison of the percentage *P<0.05 fluorescent+ bacteria/cells in the peripheral blood of Rd8 mice and WT mice using the Vanco-bodyPI-labeled faecal microbiota transplantation method. Fig. 11C-D show that both fluorescence microscopy (C) and immunofluorescence staining (D) can detect Vanco-bodyPI+ bacteria in the retinopathy of Rd8 mice. Fig. 11F-G compare the colon length, serum 16srrna bacteria (F), and bacterial LPS level (G) ****P<0.0001 between Rd8 mice and WT mice treated with DSS for 13d. Fig. 11H shows the body weight of Rd8 (n=20) and WT (n=19) mice treated with 2.5% DSS monitored daily, while Fig. 11I shows the Kaplan-Meier survival curves of Rd8 (n=20) and WT (n=19) mice treated with 2.5% DSS.

Fig. 12: Retinal phenotypes of Rd8 GF and Rd8 GF SPF mice are shown. Where Fig. 12A shows representative fundus images of Rd8-SPF and Rd8 GF mice at 4, 8, 12 and 16 weeks. Fig. 12B shows histological examination of the retina of both eyes of Rd8-GF mice at E18, P12, P15 and 8W. Fig. 12C shows fluorescent in situ hybridization staining of 16srDNA bacteria in the retina and vanco-body of Rd8-GF mice. Fig. 12D shows immunofluorescent staining of IBA1 (red) in the retina of Rd8-SPF, Rd8 GF and WT-SPF mice. Fig. 12E shows the percentage ***P<0.001 of IBA1+ microglia in the ONL of Rd8-SPF, Rd8 GF, and WT-SPF mice. NS indicates non-significant effect. Fig. 12F shows immunofluorescence staining of ZO-1 (red) and phallodin (green) proteins in the retina of Rd8-SPF, Rd8-GF, and WT-SPF mice. Fig. 12G shows the detection of retinopathy by H&E staining in Rd8-GF mice (Rd8-GF-SPF mice) at P15 and 8W raised in SPF medium.

Figure 13 shows the changes in the number of light spots in the fundus of Crb1 ^rd8/rd8 mice with degenerative retinal retinopathy treated with octyl gallate (compound 6) by gavage after 2 and 4 weeks of gavage.

Design

A clear and complete description of the technical solution in embodiments of the present invention will be presented as follows. In the presented embodiments, only the method of modeling feeding of mice with a mutated Crb1 gene in an SPF medium was used. By checking the presence of local inflammation of the retina and the presence of bacteria at the site of injury, it was found that the model retina was infected with bacteria. Further experiments will be carried out to confirm that the bacteria originate from the intestinal tract. This specific embodiment does not exclude other modeling methods, such as feeding mice in environments with more complex microbial conditions or applying microorganisms from the intestinal tract or the same microorganisms as intestinal tract microorganisms to eye tissue, which allows the eye tissue to directly or indirectly come into contact with these microorganisms, as described earlier.

It is obvious that the described embodiments are only partial embodiments, and not all embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by specialists in the field of technology without creative input fall within the scope of protection of the present invention.

1. Mice

C57BL/6N (Crb1 ^rd8/Rd8 , named Rd8 mice) and C57BL/6J (Crb1 ^wt/wt , named wt mice) mice carrying Rd8 mutations were purchased from Beijing White River Laboratory Animal Technology Co. Ltd. and maintained under specific pathogen-free conditions. Animals were kept under SPF conditions at the Zhongshan Ophthalmology Center, Sun Yat-sen University. Germ-free (GF) RD8 mice are produced at the Animal Breeding Facility of the First Affiliated Hospital of Sun Yat-sen University using embryos of RD8 female mice. GF mice are kept germ-free, and fecal samples are subject to weekly microbiological and parasite testing by the facility staff to ensure the sterility of GF specimens. Genotyping of mice was performed as described previously in this work (Mattapallil et al., 2012). The Crb1 genotypes of the two mouse species were confirmed (Fig. 4A). All animal experiments were approved by the Animal Care and Use Committee of Zhongshan Eye Center, Sun Yat-sen University and complied with the ARVO Animal Use Statement of the Association for Research in Vision and Ophthalmology.

2. Method

2.1 Immunohistochemistry

The mouse was cervically displaced, euthanized, the eyeballs were removed, and the mouse was fixed in phosphate buffered saline (PBS) at 4℃ with 4% paraformaldehyde (PFA) for 24 h. The specimen was washed three times with PBS, dehydrated in an alcohol series, dehydrated twice in xylene, then embedded in paraffin, and continuously sliced into 10 μm thick pieces using a slicer (RM 223; Leica, Wetzlar, Hesse-Darmstadt, Germany). The pieces were stained with hematoxylin and eosin (H&E). H&E images were generated using a Z2 imager (Zeiss).

The eyes should be collected and placed in 4% PFA at room temperature for 5 minutes, then the eyes should be dissected and the eye cells should be fixed for 45 minutes. Then the colon tissue should be collected and fixed in 4% PFA at room temperature for 4 hours. After washing with PBS, use 30% sucrose to soak the separated eye and colon cells, cryoprotect overnight, embed the compounds with OCT (Cat. 4583; SAKURA, USA), then store at -80℃ before dissection. Sections of 12 μm thickness are used for all immunostaining purposes.

Block tissue sections with 10% donkey serum/PBST (0.1% Triton X-100/PBS) for 30 min, and then incubate tissue sections overnight with primary antibodies at 4℃. After washing with PBST, sections were incubated with secondary antibodies coupled to fluorescent dyes and fixed with Fluoromount-G (Southern Biotech, Birmingham, AL, USA). Use the same immunohistochemical method for phalloidin (A12379; Thermo Fisher) staining except excluding secondary antibodies. Use a Zeiss confocal microscope (Zeiss LSM880; Zeiss, Oberkochen, Germany) and Imager.Z2 equipped with ApoTome (Zeiss). The primary antibodies used in this study included: anti-Crb1 (PA5-66373, ThermoFisher; 1:50), anti-Iba1 (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).

2.2 Photograph of the fundus

The mouse was anesthetized and the pupils were dilated. Apply hydroxypropyl methylcellulose eye drops regularly to maintain corneal moisture. Obtain a fundus photograph of the mouse using a Micron IV mouse fundus camera (Phoenix Research Laboratories, Inc., Pleasanton, CA, USA).

2.3 RNA-seq analysis of superior and inferior retina

Total RNA was extracted from the upper and lower retinas using MasterPure ^™ Whole DNA and RNA (Epicenter) Purification Kit. The RNA concentration was measured using Qbit-RNA-HS Assay Kit. The sequence library was prepared using VAHTS ^™ Total RNA seq (H/M/R) Library Preparation Kit (Vazyme, China) according to the standard protocol provided by the manufacturer, and the sequence was determined on the MGISEQ 2000RS platform.

Quality control and assessment of the raw reads were first performed using FastQC (v0.11.8) and cutadapt (v1.15). Align the clean read with the mouse genome (mm10) using HISAT2 (v2.1.0). Import gene expression data into the DESeq2 package of R software (v3.6.1) for differential expression analysis. Import differentially expressed genes (DEGs) into pathway mining analysis (IPA) for functional enrichment analysis.

2.4 Macrogenomic sequencing

Retinal samples were collected and DNA was extracted using MasterPure ^™ Whole DNA and RNA Purification Kit (Epicenter). Collect the contents of stomach, small intestine, ileum, cecum, colon and rectum and extract DNA using DNA QIAampPowerFecal Kit (QIAGEN). After concentration measurement, prepare DNA sequence libraries using MGI VAHTS ^™ Universal DNA Library Prep Kit (Vazyme, China) according to the standard protocol provided by the manufacturer. Perform metagenomic sequencing using MGISEQ-2000RS. Perform quality filtering of raw reads using Trimmomatic (v0.36) and PRINSEQ (v0.20.4). Delete reads with a computer mouse using KneadData (v0.6.1) (https://bitbucket.org/biobakery/kneaddata). Compare the non-mouse-cleaned readings with the pre-created MiniKraken database using Kraken 2 (v2.0.9). The classification results are checked with a confidence level of 0.20. The negative blank control is processed together with the sample. All species present in the negative blank control group are removed.

2.5 Measurement of intestinal permeability in vivo using FITC-glucan

Measure permeability in vivo using the FITC-labeled glucan assay to assess barrier function. Remove food and water overnight. Eight-week-old mice were orally administered 50 mg/100 g (weight) of FITC-labeled glucan (FD-70; Sigma-Aldrich). Collect serum 5 h after administration and measure the fluorescence intensity of each sample (excitation 492 nm; emission 525 nm).

2.6 Flow cytometry

WT and Rd8 mice were fasted overnight and gavaged with 1×109 E. coli bacteria (designed for sequential RFP expression). At 6 h post-gavage, mice were euthanized by carefully transferring 400 μl of peripheral blood into a tube containing 4 ml of ACK lysis buffer (Gibco, USA) and incubated at room temperature for 3-5 min. After centrifugation at 300×g for 5 min, cells were fixed and permeabilized (Cytofix/perm solution, BD Biosciences, USA) and then analyzed using a flow cytometer (MACSQuantAnalyzer 10, MilteNYBiotec, Germany).

2.7. Preparing tissue for electron microscopy

After euthanasia, immediately collect the colon and eyes and fix them in phosphate-buffered glutaraldehyde-paraformaldehyde at room temperature for 1 hour. Cut the colon into 2 mm long slices. Excise the anterior segment of the eye and cut the posterior segment into slices (2 mm × 2 mm). Place the dissected tissue in fresh fixative for 12 h, fix with 1% osmium tetroxide and embed in epon resin after dehydration. Stain with toluidine blue under an optical microscope and pre-view the region of interest on 1-micrometer sections. Then collect ultrasound images at 80 nm and restain with uranyl acetate and lead citrate. Observe the ultrasound sections under a transmission electron microscope.

2.8 Fluorescence in situ hybridization (FISH)

The following oligonucleotide probes are used in the assay: EUB338, 5'-GCTGCCTCCGTAG-GAGT-3' (Amann et al., 1990). Only the 5' end of the probe contains a primary amino group covalently linked to TRITC. Store dye-oligonucleotide conjugates (100 μM) at -20°C.

Wash pre-fixed retinal sections 3 times with DEPC-treated PBS. After PBS treatment with 0.2% Triton X-100/DEPC, hybridize sections and probes (500 nm) overnight at 37°C and mount them with Fluoromount-G.

2.9 Quantitative real-time PCR for determining gene expression levels in colon tissue

Quickly freeze fresh mouse colon tissues (approximately 1 cm) in liquid nitrogen, mince them and lyse them with RNA extraction lysis buffer. Then purify total RNA using the Qiagen RNeasy Plus kit and reverse transcribe it into cDNA using the Takara PrimeScript RT kit and gDNA eraser. Determine the expression level of the corresponding genes by qPCR. Data normalization is β-actin.

2.10 Quantification of 16srrna gene levels in plasma by QPCR

After 13 days of treatment with 2.5% DSS, isolate plasma from whole blood of WT mice and Rd8 mice. Use about 50 µl of plasma to separate Complete DNA and RNA Purification Kits from Total Nucleic Acids™ using MasterPure (Epicenter, USA). Dissolve the precipitated nucleic acid in 20 µl of nuclease-free water. Perform qPCR analysis (ChamQ-SYBR-Color-qPCR-Master-Mix, Vazyme, China) using LightCycler 96 System (Roche, USA). Due to the extremely low DNA concentration in all samples, use each sample in equal volumes as templates (4 µl in 20 µl). Measure total bacterial load using the following universal 16S rRNA gene primers: 27F 5’- AGAGTTTGATCCTGGCTCAG-3’, 534R 5’- GCATTACCGCGGCTGCTGG-3’.

2.11 Enzyme-linked immunosorbent assay (ELISA)

Measure each sample with 100 μl of plasma. Measure the LPS concentration in plasma using an enzyme immunoassay kit (SEB526Ge; Cloud-Clone Corp., USA).

2.12 Colonization of gut bacteria labeled with the fluorophore vanco-bodypi in Rd8 mice

Collect fresh fecal samples in a 50 ml conical tube with sterile 1x PBS and vortex until smooth. Filter the contents with a 0.22 μm filter (Millipore) to remove residual feces, centrifuge to obtain gut microbiota, and incubate with Vanko-bodypi at room temperature for 30 min. Gavage mice at ^1x108 CFU/mouse in PBS with Vanko-bodypi-labeled gut bacteria. After 24 h of gavage, collect mouse retina sections for observation.

2.13 Experimental study of dextran sodium sulfate (DSS)-induced colitis

Induce intestinal inflammation in mice by long-term oral administration of 2.5% DSS (MW 36000-50000d, Yeasen, China) in drinking water. Monitor the weight daily from day 0 to day 13, euthanize the mice on day 13, and measure the colon length. For survival analysis, allow the mice to freely drink 2.5% DSS in drinking water for 43 days and monitor their mortality status every 24 hours for 43 days.

2.14 Depletion of intestinal symbiotes

A broad-spectrum antibiotic combination consisting of ampicillin (A; 1 g/L; Sigma), metronidazole (M; 1 g/L; neomycin (N; 1 g/L; Sigma), and vancomycin (V; 500 mg/L) is available. Feed pregnant female rats Sigma (AMNV) in their drinking water and feed it continuously to the pups after weaning. Place control mice in a normal device on the same rack.

3. Design

Design 1: Modeling

Observe the retinal microenvironment characteristics of Crb1 ^rd8/rd8 (rd8) and Crb1 ^wt/wt (C57BL/J, named wt) mice (Figure 1A). When their phenotype was observed, Rd8 mice raised in specific pathogen-free medium (Rd8-SPF) exhibited all typical retinal degenerations including white spots (Figure 1B) in the nasal quadrant on fundoscopy, progressive retinal dysplasia (folds and false garlands), and histological retinal degeneration (H&E staining) (Figure 2A). The results showed that 7 out of 32 Rd8 mice had mild retinal dysplasia before birth (E18) (Figures 2A and 2B). In addition, all abnormalities at E18 were unilateral (Figs. 2A and 1C), while typical lesions such as retinal folds and false garlands were present in the retinas of all Rd8 mice at P12 (before eye opening), P15 (after eye opening) and 8-week-old (8 weeks), but none of them were present in WT-SPF mice (Figs. 2A and 2B). The data showed that retinal abnormalities in Rd8 mice arise already at embryonic stage E18, and the intraocular environment can control the retinal phenotype under the genetic influence of Crb1 mutations.

Design 2: Detection of Pathogenic Pathogens and Corresponding Local Immune Responses in the Retina of Model Mice (Rd8-SPF)

Perform transcriptome analysis using RNA-seq technology and compare the gene expression profiles of the superior (non-lesion) and inferior (lesion) regions in Rd8-SPF mice (Figure 3A). After removing genes with a differential expression pattern (2-fold change and P<0.05) between the superior and inferior retinas in WT-SPF mice, 179 genes (DEGs) had at least a 2-fold difference in expression (P<0.05) between the superior and inferior retinas in Rd8-SPF mice (Figures 3B and 2C). In Rd8-SPF mice, most of the 179 DEGs were highly expressed in the inferior half of the lesion region (Figure 3C). In functional analysis of these DEGs using the Ingenuity Pathway Analysis (IPA) tool, genes involved in bacterial and viral recognition were significantly enriched in all DEGs highly expressed in Rd8-SPF mouse retinopathy (Figure 2D). Importantly, the top two genes (Ncf4 and Slamf1) with the greatest increase in lesion size were determinants of the antibacterial response of phagocytic cells. The data showed pathological changes and corresponding local immune responses in the retina of Rd8 mice, which were combined with the enriched expression (Figure 2D) of genes with proinflammatory functions and IBA1+ microglia infiltration at the retinopathy site (Figure 2E).

Design 3: Detection occurs as a result of retinopathy in model mice.

Perform metagenomic analysis of retinal tissues from WT-SPF (n=5, age=4 weeks) and Rd8-SPF (n=4, age=4 weeks) mice. When analyzed, the bacterial DNA content in the retinas of WT and Rd8 mice was extremely low, and no viral or fungal DNA was detected after all quality control and sterilization steps. As shown in Fig. 4A, principal coordinate analysis (PCoA) reveals significant differences in the bacterial composition of the retina between WT-SPF mice and Rd8-SPF mice. Importantly, further metagenomic analysis of the superior and inferior retinas of Rd8-SPF mice (n=4, age=4 weeks) shows that seven bacterial phyla are significantly enriched in the inferior retina, including Hadrus, Bifidobacterium pseudocatenatum, Megamonas funiformis, Is79A3, Oscillibacter, Tatumella sp.TA1, and Thiobacillus denitrificans (Fig. 4B). To directly observe bacteria in retinopathy, perform fluorescence in situ hybridization (FISH) and Vanco-Body staining analysis on Rd8-SPF mice (4 weeks old), especially staining the cell walls of Gram-positive bacteria. As shown in Fig. 4C, bacteria can only be observed at the lesion site and cannot be observed in the normal retina. Then, the bacteria in the lesion are examined using a transmission electron microscope (TEM) (Fig. 4D). The data showed that bacteria can be observed in Rd8-SPF mouse retinopathy.

Design 5: Confirm that the major retinal defect of Rd8-SPF mice is the external blood-retinal barrier.

Reduced or absent expression of CRB1 protein in the outer retinal membrane of Rd8 mice was confirmed by immunofluorescence staining data (Fig. 5A). Based on the data, it was confirmed that weak expression of CRB1 protein could be observed on Bruch's membrane (Fig. 5A). The adhesive junction on the outer membrane was disrupted at the site of retinopathy of Rd8-SPF mice by transmission electron microscopy, which was associated with the externalization of the outer membrane (Figs. 5B and 6A). No dissolution adhesions and tortuous basement membrane of chorionic capillaries on the basement membrane of the retinal pigment epithelium (RPE) were observed by transmission electron microscopy in the retina of Rd8-SPF mice, and no disruption of the collagen layer between them was observed in any WT-SPF mice (Figs. 5C and 6B). This situation was associated with the rupture of Bruce membrane and a significant decrease in the thickness of Bruce membrane in Rd8-SPF mice (Figs. 5C, 6B, and 5D). However, no change in the tight junction between retinal endothelial cells and retinal pigment epithelial cells was detected when transmission microscopy was performed. Thus, the results indicated that the main defect in the retina of Rd8-SPF mice is the outer blood-retinal barrier, not the inner blood-retinal barrier.

Design 6: Metagenomic analysis of microbiota from different parts of the intestine

All seven bacteria (Fig. 4B) detected in the retina of Rd8 mice were previously located in the gastrointestinal tract (GI). Therefore, we performed metagenomic analysis of the microbiota of different sites (including stomach, small intestine, ileum, cecum, colon, and rectum) of the GI tract in WT-SPF (n=5) and Rd8-SPF (n=5) mice (Fig. 8A–F). The microbial components in the lower GI tract of Rd8-SPF mice were significantly different from those of WT-SPF mice (Fig. 8G). In the intestinal microbiota of WT and Rd8 mice, Akkermansiamucinphila was identified as the most diverse phylum of bacteria in the cecum (Fig. 8H), colon (Fig. 8I), and rectum (Fig. 8J), although it was not detected in the retina of Rd8 mice (Fig. 4B). In fact, all seven phyla of bacteria in the retina of Rd8 mice were observed in the lower gastrointestinal tract of WT and Rd8 mice, with the highest abundance of bacteria in the cecum (Fig. 7A). However, they were virtually undetectable in the upper gastrointestinal tract of Rd8 mice. These data indicated that these retinal bacteria exist in the gastrointestinal tract microbiota of Rd8-SPF mice but not WT-SPF mice.

CRB1 protein expression in wild-type mouse cecum was identified by immunofluorescence staining, and its expression was significantly reduced in Rd8 mice (Fig. 9A). In addition, a significant loss of occludin expression (Fig. 9B-C) was observed in the cecum of Rd8 mice, whereas Claudin1 expression (Fig. 9D-E) was not significant. Upon further examination by transmission electron microscopy, normal tight and adherens junctions were observed in the cecal epithelial barrier (Fig. 9F).

Design 7: Testing colon barrier defects in Rd8 mice

Similar to the cecum results, CRB1 protein was significantly expressed on the upper and lower surfaces of colonic epithelial cells (Fig. 7B). Loss of CRB1 protein was significantly associated with decreased expression of phalloidin (Fig. 7C) and occludin (Figs. 7D and 4E), but not with Claudin1 protein expression (Figs. 7F and 4G). Importantly, transmission electron microscopy revealed that adherens junctions between colonic epithelia could not be localized in most cells, while tight junctions in Rd8 mice were normal (Figs. 7H and S6A). In addition to junctional changes, mitochondrial vacuolation was also observed in the colonic epithelium of Rd8 mice. Although the number of epithelial microvilli was unchanged (Fig. 7I), microvilli of Rd8 mice were longer (Fig. 7J) and thinner (Fig. 7K). The total number of intact adhesions was significantly reduced compared to WT mice (Fig. 7L). The ultra-structural changes in junctions and microvilli were also accompanied by a significant increase in Il12a expression in the colonic wall (Fig. 7M), while Tnfa, Il1b and mucin 2 expression remained unchanged (Fig. 7N). Thus, the data showed that Rd8 mice have defects in the colonic epithelial barrier and corresponding colonic wall inflammation.

Design 8: It was examined whether defects in the intestinal epithelial barrier could cause alterations in intestinal permeability and transport of surrounding bloodstream and retinal tissue microbiota in Rd8 mice.

Perform intestinal FITC-dextran permeability measurements in WT and Rd8 mice. As shown in Fig. 11A, compared with the fluorescence intensity in the blood of WT mice, the fluorescence in the peripheral blood of Rd8 mice was significantly increased 5 h after FITC-dextran administration. Additionally, in the Vancompy-labeled fecal microbiota transfer assay, the fluorescence+ bacteria/cells in the peripheral blood of Rd8 mice were significantly increased compared with WT mice 24 h after fecal transfer (Figs. 10B and 11B). Importantly, Vancomopy+ bacteria were detected in Rd8 retinopathy mice (Fig. 11C) but not in WT retinopathy (data not shown), which was further confirmed by immunofluorescence staining of the retinal tissue of Rd8 mice (Fig. 11D). These data showed that intestinal permeability to polysaccharide molecules and bacterial cells increases, causing the passage of bacteria from the intestinal lumen into the bloodstream and a state of retinopathy.

Design 9: Intestinal stress response experiment in Rd8 mice with increased intestinal permeability

Exposure of WT and Rd8 mice to drinking water containing 1.5% dextran sodium sulfate (DSS) induced mild colitis in WT mice. After 13 days of DSS treatment, the colon length of Rd8 mice was significantly shorter than that of wild-type mice (Fig. 11E). Rd8 mice had more bacteria in their blood than wild-type mice, as evidenced by serum levels of bacterial 16S rRNA (Fig. 11F) and bacterial LPS (Fig. 11G).

Design 10: Reversal of bacterial effects on Rd8 mouse retinal phenotypes

Although bacteria were found in the retina of Rd8 mice in the retinopathy caused by the breakdown of the outer blood-retinal barrier and the intestinal epithelial barrier, it is unclear whether these bacteria are the cause or the consequence of retinal degeneration in Rd8 mice. Therefore, germ-free (GF) Rd8 mice were re-isolated to test whether the retinal degeneration phenotype was reversed. As shown in Fig. 12A, the retinopathy (leukoplakia) found in Rd8-SPF mice was almost absent in germ-free Rd8 mice (Rd8 GF). Retinal histology of Rd8-GF mice showed that the normally developed retinal tissue (Fig. 12B) did not have the typical retinopathy found in Rd8-SPF mice (Figs. 2A and 1C), proving the absence of retinal degeneration in Rd8-GF mice. The fact that retinal bacteria were not detected in Rd8 GF mice (Fig. 12C) was related to the dramatic reduction in the number of microglia in the outer retinal membrane (ONL) in Rd8 GF mice (Figs. 12D and 12E), although ZO-1 and phalloidin protein staining showed outer membrane disruption, which was also detected in Rd8-SPF and Rd8 GF mice (Fig. 12F). Furthermore, when Rd8 GF mice (Rd8 GF SPF mice) were raised in SPF medium after birth, retinopathy was again detected (Fig. 12G). These data confirmed that retinal degeneration in Rd8 mice is bacteria-dependent.

Design 11: Pharmacodynamic evaluation of octyl gallate (compound 6) in the treatment of Crb1 murine fundus degenerative retinopathy ^rd8/rd8 using a probe probe.

1.1 Prototype

Name of the prototype Source Item No. Batch No. Physical and chemical properties Storage conditions Composition 6 Sigma-Aldrich 48700 STBJ0496 White powder Room temperature

When preparing the test sample, weigh and take 4 ml of sterilized castor oil, 1.2 g of compound 6 powder, add 1-2 ml of castor oil and then grind completely, then transfer all the ground preparations and the remaining castor oil into a 50 ml test tube with 36 ml of ultrapure water, shake and mix evenly and leave overnight to prepare a suspension of compound 6 with a concentration of 30 mg / ml. Label the prepared liquid, store it at room temperature and use within one month.

Calculation formula: theoretical sample mass (mg) = theoretical concentration of test sample (mg/ml) × volume of preparation (ml).

1.2 Information about experimental animals

Species and types: Crb1 ^rd8/rd8 mice

Degree: SPF Degree

Number and gender 18 mice, male or female

Weight range: 16-24g

Supplier: GemPharmatek Co. Ltd.

1.3 Selection of experimental animals

At the beginning of the administration, healthy Crb1 ^rd8/rd8 mice aged 1 month were selected for the test. At the beginning of the test, the animals were examined using a slit lamp and mice with cataract lesions were excluded from the test. Then, the fundus was examined and both eyes of mice with one eye without light spots in the test were excluded.

1.4 Construction of experience

Grouping and Dosage

Animals were randomly divided into model control group and compound 6 group by weight. For detailed description of the design, see the table below:

Group Dosage (g/kg, eg) Number of eyes control group 0 18 Composition 6 0.3 18

Directions for use and dosage regimen

Application information

Route and method of administration: through a tube, 10.0 ml/kg.

Frequency and duration of administration: once every other day for 4 weeks.

Dose composition

The dosage of compound 6 for mice is 0.3 g/kg. The dosage is calculated according to previous clinical administration.

Experimental method and steps

One-month-old Crb1 ^rd8/rd8 mice were randomly divided into a model control group and a compound 6 group. The vehicle or compound 6 was administered to the animals by gavage, and the dose of compound 6 was 0.3 g/kg once every other day for 4 weeks. The number of light spots on the fundus of mice was recorded and analyzed after 2 and 4 weeks of feeding.

1.5 Data processing and statistical analysis of data

Take the changes in the number of light spots in the eye fundus of mice at each time point after treatment as parameters and perform statistical analysis using SPSS software. The statistical results are expressed as ±s . The verification level = 0.05, and the average value of the test data and the data after statistical processing is displayed with 2 decimal places.

1.6 Test results

Light spots were found on the fundus of the mice in the model control group at the beginning of the treatment, as well as after 2 weeks and 4 weeks of administration, indicating the presence of degenerative retinopathy of the fundus in the experimental animals. Compared with the control group of the model, the number of light spots changed to 1.31±3.18 after 2 weeks of feeding with compound 6, while there was no statistical difference in the number of spots (2.88±3.41) compared with the control group of the model. After 4 weeks of feeding, the number of light spots on the fundus of C57BL/6N mice changed to -3.63±4.06, which was statistically different in the number of spots 2.24±4.01 compared with the control group of the model (Fig. 13). For detailed description of the results, see the table below:

Grouping Number of eyes Change in spots after 2 weeks of administration Change in spots after 4 weeks of administration Control group of the model 18 2.88 ± 3.41 2.24 ± 4.01 Composition 6 18 1.31 ± 3.18 ^* -3.63 ± 4.06 ^**

Note: Compared with the control group, the model *P=0.1518, **P=0.0005.

In the 2nd and 4th weeks of the test, the bright spots could not be clearly seen in 2/18 eyes and 1/18 eyes in the compound 6 group due to a slight decrease in crystal transparency after anesthesia, so they could not be counted statistically. In addition, in the 4th week of the experiment, the bright spots were also not clearly seen in the fundus of 1/18 eyes in the control group of the model due to a slight decrease in crystal transparency. A correlation between the slight decrease in crystal transparency and the use of compound 6 cannot be ruled out. It has been reported in the literature that crystal transparency decreases in mice after anesthesia. Crystal abnormalities are more common in Crb1 ^rd8/rd8 mice, and the transparency of the anterior phacocyst in 67% of mice is decreased. When the transparency decreases, which affects the observation of the fundus, it cannot be counted statistically.

Compound 6 is able to effectively inhibit degenerative retinopathy of the fundus in Crb1 ^rd8/rd8 mice at a dose of 0.3 g/kg.

The above embodiments are used only to explain the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technical personnel in this field will know that the technical solutions described in the previous embodiments can still be changed or some or all technical characteristics can be changed or replaced with equivalents. However, these modifications or replacements will not lead to a deviation of the corresponding technical solutions from the scope of the technical solutions of the various embodiments of the present invention.

Claims (8)

1. A method of treating bacterial-related hereditary retinal degeneration, comprising administering an inhibitor or microbicide for the bacterium to the eye or gastrointestinal tract of a patient; wherein the bacteria are selected from: Anearostipes hadrus , Bifidobacterium pseudocatenulatum , Nitrosomonas sp. Is79A3, Oscillibacter valericigenes , Tatumella sp. TA1, Megamonas funiformis , Thiobacillus denitrificans and Akkermansia muciniphila ; wherein the inhibitor or microbicide for the bacterium is a compound having the following chemical structure: 2. The method of treatment according to claim 1, wherein the bacterial-dependent hereditary retinal degeneration includes Leber's congenital amaurosis (LCA), retinitis pigmentosa (RP), early-onset rod-cone cytodystrophy, rod dystrophy, congenital stationary night blindness and color blindness, and Stargardt disease. 3. The method of treatment according to claim 1, wherein administration into the gastrointestinal tract of the patient comprises administration into the stomach, jejunum, ileum, cecum, colon or rectum of the patient. 4. The use of an inhibitor or microbicide for a bacterium in the preparation of a medicament for the treatment of a bacterium-dependent hereditary retinal degeneration; wherein the bacteria are selected from: Anearostipes hadrus , Bifidobacterium pseudocatenulatum , Nitrosomonas sp. Is79A3, Oscillibacter valericigenes , Tatumella sp.TA1, Megamonas funiformis , Thiobacillus denitrificans and Akkermansia muciniphila ; wherein the inhibitor or microbicide for the bacterium is a compound having the following chemical structure: 5. The use according to claim 4, wherein the bacterial-dependent hereditary retinal degeneration comprises Leber's congenital amaurosis (LCA), retinitis pigmentosa (RP), early-onset rod-cone cytodystrophy, rod dystrophy, congenital stationary night blindness and color blindness, and Stargardt disease. 6. The use according to claim 4, wherein the medicinal product is a preparation for ophthalmic administration or a preparation for administration into the gastrointestinal tract, which is a tablet, pill, powder, granule, capsule, lozenge, syrup, solution, emulsion, suspension, controlled release preparation, aerosol or film product.