WO2022213869A1 - 通过口服给药增加病毒空衣壳蛋白治疗hbv的方法 - Google Patents
通过口服给药增加病毒空衣壳蛋白治疗hbv的方法 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
- A61K31/554—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Definitions
- the object of the present invention is to provide a method for treating HBV by increasing the viral empty capsid protein by oral administration.
- nucleic acid-free capsid As used herein, the terms “nucleic acid-free capsid”, “nucleic acid-free viral capsid”, “empty capsid”, “empty viral capsid”, “empty virus particle”, “empty virus-like particle”, “Empty VLP” is used interchangeably and refers to a viral capsid or viral particle that does not contain intact viral nucleic acid within the viral capsid or viral particle.
- an empty viral capsid does not contain viral nucleic acid inside, even if it is endocytosed by a cell, due to the lack of viral nucleic acid, the viral protein cannot be replicated in the cell, and the cell cannot be reinfected.
- the empty viral capsid has almost the same spatial structure as the intact virus, so it can stimulate the body (eg, human and non-human mammals) to generate an immune response.
- C 1 -C 8 alkylamino refers to an amine group substituted with a C 1 -C 8 alkyl group, which may be mono- or di-substituted; for example, methylamino, ethylamino, Propylamine, isopropylamine, butylamine, isobutylamine, tert-butylamine, dimethylamine, diethylamine, dipropylamine, diisopropylamine, dibutylamine, diisobutylamine, diisobutylamine tert-Butylamine, etc.
- C 1 -C 8 alkoxy refers to a straight or branched alkoxy group having 1-8 carbon atoms; eg, methoxy, ethoxy, propoxy, iso Propoxy, butoxy, isobutoxy, tert-butoxy, etc.
- halogen or halogen atom refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "Halo” means substituted with an atom selected from F, Cl, Br, and I.
- Acids suitable for forming salts include, but are not limited to, inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric, formic, acetic, propionic, oxalic, malonic, succinic, fumaric, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid.
- inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric, formic, acetic, propionic, oxalic, malonic, succinic, fumaric, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzene
- the pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable excipient or carrier.
- the "safe and effective amount” refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
- the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-200 mg of the compound of the present invention per dose.
- the "one dose” is a capsule or tablet.
- ⁇ Ct mean Ct of target gene - mean Ct of housekeeping gene.
- Immunohistochemical (IHC) staining was used to detect HBsAg and HBcAg expression in liver tissue sections.
- Mouse livers were fixed with 10% NBF, processed by conventional methods, dehydrated, paraffin embedded and sectioned. Section samples of 4 ⁇ m thickness were prepared from formalin-fixed, paraffin-embedded (FFPE) livers and subjected to deparaffinization, antigen retrieval, serum blocking, primary and secondary antibody incubation, and DAB staining as routine IHC analysis.
- FFPE paraffin-embedded
- HBV capsids examined by electron microscopy showed normal capsid morphology in LW231-treated cells, also observed in AT130-treated cells, but different from the abnormal capsid morphology of GLS4-treated cells (Fig. 2B).
- LW231 enhanced the formation of a morphologically intact nucleic acid-free capsid.
- R2 i.e.
- the HBV DNA and HBV DNA produced in the nucleus will be exposed in the cytoplasm, thereby stimulating the cell's intrinsic innate immune system.
- Activation of CTLs may also be attributed to the downregulation of regulatory T cells by LW231 ( Figure 4).
- single-cell suspensions were prepared from liver tissues of mice dosed with LW231 for 1 week and 26 weeks continuously and subjected to FACS analysis.
- HBV DNA was extracted from cell culture supernatants and DNA levels were assessed by qPCR.
- EC50 values were determined from the mean inhibition value of 2 wells for each compound concentration.
- PK studies were performed in AAV-HBV mice to estimate pharmacologically active doses for in vivo efficacy studies. Serum levels of compounds were measured following oral administration of 50, 100 and 200 mg/kg of LW231. The concentration of unbound LW231 in plasma was plotted against EC50 and EC90 values derived from cell-based assays ( Figure 5).
- Example 7 Effect of treatment with preferred compounds of formula I on nucleic acid-free capsid content in hepatocytes
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- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Biotechnology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明提供了一种通过口服给药增加病毒空衣壳蛋白治疗HBV的方法。具体地,本发明提供了式I化合物,可用于:(a)增加病毒空衣壳;(b)增加肝脏中IFN-γ产生;(c)降低肝脏中CD4+Treg细胞数量;(d)激活肝组织中cGAS/STING/IRF3信号通路;和/或(e)减少病毒感染的肝细胞的数量。本发明的化合物能够将肝脏中免疫耐受性环境转变为致免疫性环境,使得病毒感染的肝细胞能得到有效清除,达到抗病毒的效果。
Description
本发明涉及生物免疫领域,具体地,涉及一种通过口服给药增加病毒空衣壳蛋白治疗HBV的方法。
慢性HBV的建立是病毒有效逃避先天免疫系统造成的。HBV病毒具有隐身性,使得病毒能够有效的逃避先天免疫系统,避免激活肝内IFN/ISG反应,在患者肝组织中建立免疫耐受。在免疫耐受致性条件下,髓样细胞(肝巨噬细胞,DC,M-MDSC)能够分泌TGF-β,促进FoxP3+调节性T细胞的分化,从而诱导T细胞衰竭和免疫耐受。在慢性HBV感染期间,髓样细胞和调节性T细胞可以产生IL-10,一方面可以抑制肝细胞促炎性细胞因子的产生,另一方面可以抑制病毒特异性T细胞的反应。HBV的免疫耐受性还归因于前核心或HBeAg蛋白,HBsAg和HBV病毒体对免疫系统的抑制。
机体清除HBV的关键在于免疫耐受性环境转变为致免疫性环境,有效清除病毒感染的肝细胞。目前本领域中,仍然缺乏有效的诱导机体清除体内HBV的技术。
发明内容
本发明的目的在于提供一种通过口服给药增加病毒空衣壳蛋白治疗HBV的方法。
在本发明的第一方面,提供了一种式I化合物或其药学上可接受的盐的用途,用于制备一制剂或组合物,所述制剂或组合物被用于:
(a)增加病毒空衣壳;
(b)增加肝脏中IFN-γ产生;
(c)降低肝脏中CD4
+T
reg细胞数量;
(d)激活肝组织中cGAS/STING/IRF3信号通路;和/或
(e)减少病毒感染的肝细胞的数量。
式中,
R
1,R
2,R
3和R
4各自独立的地选自下组:H、卤素、氰基、取代或未取代的C
3-C
4的环烷基,取代或未取代的C
1-C
4的烷基、取代或未取代的C
1-C
4的烷氧基;其中,所述的取代指基团上的氢原子被一个或多个选自下组的取代基取代:卤素、C
1-C
4的烷基(如二氟甲基、二氟乙基、单氟甲基、三氟甲基、三氟甲氧基);
R
5选自下组:H、卤素、-CN、羟基、氨基、羧基、-(C=O)-取代或未取代的C
1-C
8烷基、取代或未取代的C
1-C
8烷基、取代或未取代的C
2-C
6烯基、取代或未取代的C
2-C
6炔基、取代或未取代的C
1-C
8烷胺基、取代或未取代的C
1-C
8烷氧基、取代或未取代的C
3-C
10环烷基、取代或未取代的具有1-3个选自下组N、S和O的杂原子的3-10元杂环烷基、取代或未取代的C
6-C
10芳基、或取代或未取代的具有1-3个选自下组N、S和O的杂原子的5-10元杂芳基;
R
g选自下组:H、卤素、-CN、羟基、氨基、羧基、-(C=O)-取代或未取代的C
1-C
8烷基、取代或未取代的C
1-C
8烷基、取代或未取代的C
2-C
6烯基、取代或未取代的C
2-C
6炔基、取代或未取代的C
1-C
8烷胺基、取代或未取代的C
1-C
8烷氧基、取代或未取代的C
3-C
10环烷基、取代或未取代的具有1-3个选自下组N、S和O的杂原子的3-10元杂环烷基、取代或未取代的C
6-C
10芳基、或取代或未取代的具有1-3个选自下组N、S和O的杂原子的5-10元杂芳基;
除非特别说明,所述的“取代”是指被选自下组的一个或多个(例如2个、3个、4个等)取代基所取代:卤素、C1-C6烷基、卤代的C1-C6烷基、C1-C6烷氧基、卤代的C1-C6烷氧基、C3-C8环烷基、卤代的C3-C8环烷基、氧代、-CN、羟基、氨基、羧基、未取代或被一个或多个选自下组的取代基取代的选自下组的基团:C6-C10芳基、卤代的C6-C10芳基、具有1-3个选自N、S和O的杂原子的5-10元杂芳基、卤代的具有1-3个选自N、S和O的杂原子的5-10元杂芳基;所述的取代基选自下组:卤素、C1-C6烷氧基。
在另一优选例中,所述的增加病毒空衣壳包括:
(a1)提高空衣壳的数量E1与有核酸病毒的正常衣壳的数量E0之比R1(即E1/E0);
(a2)提高空衣壳的数量或水平;
(a3)促进空衣壳的组装;和/或
(a4)降低衣壳内的病毒核酸数量C1与病毒衣壳数量C0之比R2(即C1/C0),其中,将一个完整病毒的衣壳内的病毒核酸数量记为1个病毒核酸。
在另一优选例中,所述的R1为≥10,较佳地≥50,更佳地≥100。
在另一优选例中,所述的R2为≤1/10,较佳地≤1/50,更佳地≤1/100。
在另一优选例中,所述的病毒核酸包括病毒RNA或病毒DNA。
在另一优选例中,所述病毒空衣壳蛋白为无病毒核酸的空衣壳蛋白。
在另一优选例中,所述病毒选自乙肝病毒、丁肝病毒和/或丙肝病毒。
在另一优选例中,所述病毒选自不同基因型的HBV,例如A、B、C、D、E、F、G、H、I、J基因型的HBV。
在另一优选例中,所述的制剂或组合物还被用于:
(f)增加肝巨噬细胞(库普弗细胞和单核细胞),树突状细胞和M-MDSC亚群数量;
(g)促进肝巨噬细胞的吞噬活性;
(h)促进CD8
+T细胞的成熟。
在另一优选例中,所述的“抑制CD4
+T
reg细胞”指降低CD4
+T
reg细胞在CD4
+T细胞中的比例,较佳地所述的“降低”的幅度≥20%,更佳地≥30%,最佳地≥40%,如30%-50%。
在另一优选例中,所述的“抑制CD4
+T
reg细胞”指用于降低免疫受抑制的病灶部位(如肿瘤部位)中CD4
+T
reg细胞在CD4
+T细胞中的比例。
在另一优选例中,所述制剂或组合物被用于施用于一对象并:
(a)增加病毒空衣壳;
(b)增加肝脏中IFN-γ产生;
(c)降低肝脏中CD4
+T
reg细胞数量;
(d)激活肝组织中cGAS/STING/IRF3信号通路;和/或
(e)减少病毒感染的肝细胞的数量;
(f)增加肝巨噬细胞(库普弗细胞和单核细胞),树突状细胞和M-MDSC亚群数量;
(g)促进肝巨噬细胞的吞噬活性。
(h)促进CD8
+T细胞的成熟。
在另一优选例中,所述的抑制CD4
+T
reg细胞包括抑制CD4
+T
reg细胞功能、和/或减少CD4
+T
reg细胞的数量或水平。
在另一优选例中,所述的对象包括人和非人哺乳动物。
在另一优选例中,所述的式I化合物选自表1所示的化合物或其药学上可接受的盐。
在另一优选例中,所述式I化合物选自:10a1、10b1、10u1、10v1、20a1、20b1、20u1、20v1、20u2、100a01、100a03、100a05、100u01、100a07、100b01、100b05。
在本发明的第二方面,提供了一种用于人或动物的药物组合物,所述药物组合物含有:
(i)如式I所示的化合物,或其药学上可接受的盐,和
(ii)药学上可接受的载体;
所述药物组合物用于选自下组的一种或多种应用:
(a)增加病毒空衣壳;
(b)减少病毒感染的肝细胞的数量;
(c)增加肝脏中IFN-γ产生;
(d)降低肝脏中CD4
+T
reg细胞数量;和/或
(d)增强免疫功能;
其中,式I化合物如本发明的第一方面所述。
在另一优选例中,所述的药物组合物还包括:(iii)额外的抗肿瘤药物和/或抗病毒药物。
在另一优选例中,所述药物组合物中含有0.001-99wt%,较佳地0.1-90wt%,更佳地1-80wt%的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐,按组合物的总重量计。
在另一优选例中,所述抗肿瘤药物选自下组:奥沙利铂、紫杉醇、多西他赛、卡培他滨、利妥昔单抗、吉非替尼、阿西替尼、瑞格非尼、卡博替尼、乐伐替尼、阿帕替尼、索拉非尼、纳武单抗、派姆单抗、阿特朱单抗、或伊匹单抗、阿韦鲁单抗、度伐鲁单抗或其组合。
在另一优选例中,所述抗病毒药物选自下组:阿昔洛韦、替比夫定、齐多 夫定、恩替卡韦(ETV)、替诺福韦酯、韦立得。
在本发明的第三方面,提供了一种抗病毒的方法,包括步骤:给需要的对象施用式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐,其中,式I化合物如本发明的第一方面所述。
在另一优选例中,所述的对象包括人或非人哺乳动物(如啮齿动物)。
另一优选例中,所述病毒选自乙肝病毒、丁肝病毒和/或丙肝病毒。
在另一优选例中,所述病毒选自不同基因型的HBV,例如A、B、C、D、E、F、G、H、I、J基因型的HBV。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
图1显示了LW231清除HBV感染的作用机制(MOA)。
图2显示了HBV衣壳的Western印迹检测,其中A,用LW231和其他参考化合物按所示浓度处理HepG2.2.15细胞6天。将细胞裂解物在非变性琼脂糖凝胶中进行电泳。用多克隆兔抗HBV核心抗体通过Western印迹检测完整的HBV衣壳。通过Southern和Northern印迹分别检测衣壳化的HBV DNA和RNA。通过SDS-PAGE用单克隆小鼠抗HBV核心抗体检测核心蛋白。β-肌动蛋白用作上样对照。B,通过电子显微镜评估LW231处理对HBV衣壳形态的影响。分别显示DMSO对照组、LW231以及参考化合物AT130和GLS4处理组。通常以110000倍的放大倍率获取图像。
图3显示了LW231增加肝脏CTL中的IFN-γ产生。其中,vehicle为溶媒,wk(s)为周数。
图4显示了LW231下调调节性T细胞(T-reg)的量。wk(s)为周数。
图5显示了LW231的PK/PD相关性。AAV-HBV小鼠分为3组(n=3),并口服LW231。在指定的时间点收集血液样本,并分析LW231血浆浓度。未结合的浓度从总浓度中转换为13.6%的调整因子,该调整因子来自血浆蛋白结合测定法。EC
50,EC
90和CC
50的浓度是根据体外实验估算的。
图6显示了LW231对血清HBV病毒指标的抑制作用。根据血清HBsAg,HBeAg和HBV DNA水平将AAV-HBV小鼠随机分组,并以50、100和200mg/kg bid的剂量用LW231治疗。经恩替卡韦ETV(0.1mg/kg,q.d.)处理的小鼠或溶媒(vehicle)分别用作阳性对照和阴性对照。还测试了100mg/kg的LW231与ETV的组合。每周监测所有三个病毒指标,并在对数刻度上绘制出给药前水平的变化。A.血清HBV DNA的变化。B.血清HBsAg的变化。C.血清HBeAg的变化。
本发明人经过广泛而深入地研究,经过大量筛选,获得了一类结构新颖的式I化合物(以LW231化合物为代表),这些化合物能够增加病毒空衣壳,促进无病毒核酸的空衣壳的组装,导致HBV病毒DNA在细胞质中积累,进而激发细胞内在的先天免疫系统,激活肝巨噬细胞和树突状细胞,将肝脏中免疫耐受性环境转变为致免疫性环境,最终导致HBV感染的肝脏细胞的清除。在此基础上完成了本发明。
术语
如本文所用,术语“无核酸衣壳”、“无核酸的病毒衣壳”、“空衣壳”、“空的病毒衣壳”、“空的病毒颗粒”、“空的病毒样颗粒”、“空VLP”可互换使用,指在病毒衣壳或病毒颗粒内部不包含完整的病毒核酸的病毒衣壳或病毒颗粒。在本发明中,由于空的病毒衣壳在其内部不含有病毒核酸,因此即使被细胞内吞后因缺乏病毒核酸,因此无法在该细胞内复制病毒蛋白,从而无法再次感染细胞。此外,该空的病毒衣壳具有与完整病毒几乎一致的空间结构,因此可以激发机体(如人和非人哺乳动物)产生免疫应答。
如本文所用,术语“烷基”包括直链或支链的烷基。例如C
1-C
8烷基表示具有1-8个碳原子的直链或支链的烷基,例如甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基等。
如本文所用,术语“烯基”包括直链或支链的烯基。例如C
2-C
6烯基指具有2-6个碳原子的直链或支链的烯基,例如乙烯基、烯丙基、1-丙烯基、异丙烯基、1-丁烯基、2-丁烯基、或类似基团。
如本文所用,术语“炔基”包括直链或支链的炔基。例如C
2-C
6炔基是指具有2-6个碳原子的直链或支链的炔基,例如乙炔基、丙炔基、丁炔基、或类似基团。
如本文所用,术语“C
3-C
10环烷基”指具有3-10个碳原子的环烷基。其可以是单环,例如环丙基、环丁基、环戊基、环己基、或类似基团。也可以是双环形式,例如桥环或螺环形式。
如本文所用,术语“C
1-C
8烷胺基”是指被C
1-C
8烷基所取代的胺基,可以是单取代或双取代的;例如,甲胺基、乙胺基、丙胺基、异丙胺基、丁胺基、异丁胺基、叔丁胺基、二甲胺基、二乙胺基、二丙胺基、二异丙胺基、二丁胺基、二异丁胺基、二叔丁胺基等。
如本文所用,术语“C
1-C
8烷氧基”是指具有1-8个碳原子的直链或支链的烷氧基;例如,甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、叔丁氧基等。
如本文所用,术语“具有1-3个选自下组N、S和O的杂原子的3-10元杂环烷基”是指具有3-10个原子的且其中1-3个原子为选自下组N、S和O的杂原子的饱和或部分饱和的环状基团。其可以是单环,也可以是双环形式,例如桥环或螺环形式。具体的实例可以为氧杂环丁烷、氮杂环丁烷、四氢-2H-吡喃基、哌啶基、四氢呋喃基、吗啉基和吡咯烷基等。
如本文所用,术语“C
6-C
10芳基”是指具有6-10个碳原子的芳基,例如,苯基或萘基等类似基团。
如本文所用,术语“具有1-3个选自下组N、S和O的杂原子的5-10元杂芳基”指具有5-10个原子的且其中1-3个原子为选自下组N、S和O的杂原子的环状芳香基团。其可以是单环,也可以是稠环形式。具体的实例可以为吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、吡咯基、吡唑基、咪唑基、(1,2,3)-三唑基以及(1,2,4)-三唑基、四唑基、呋喃基、噻吩基、异恶唑基、噻唑基、恶唑基等。
本发明所述的基团除非特别说明是“取代的或未取代的”,否则本发明的基团均可被选自下组的取代基所取代:卤素、腈基、硝基、羟基、氨基、C
1-C
6烷基-胺基、C
1-C
6烷基、C
2-C
6烯基、C
2-C
6炔基、C
1-C
6烷氧基、卤代C
1-C
6烷基、卤代C
2-C
6烯基、卤代C
2-C
6炔基、卤代C
1-C
6烷氧基、烯丙基、苄基、C
6-C
12芳基、C
1-C
6烷氧基-C
1-C
6烷基、C
1-C
6烷氧基-羰基、苯氧羰基、C
2-C
6炔基-羰基、C
2-C
6烯基-羰基、C
3-C
6环烷基-羰基、C
1-C
6烷基-磺酰基等。
如本文所用,“卤素”或“卤原子”指F、Cl、Br、和I。更佳地,卤素或卤原子选自F、Cl和Br。“卤代的”是指被选自F、Cl、Br、和I的原子所取代。
除非特别说明,本发明所描述的结构式意在包括所有的同分异构形式(如对映异构,非对映异构和几何异构体(或构象异构体)):例如含有不对称中心的R、 S构型,双键的(Z)、(E)异构体等。因此,本发明化合物的单个立体化学异构体或其对映异构体、非对映异构体或几何异构体(或构象异构体)的混合物都属于本发明的范围。
如本文所用,术语“互变异构体”表示具有不同能量的结构同分异构体可以超过低能垒,从而互相转化。比如,质子互变异构体(即质子移变)包括通过质子迁移进行互变,如1H-吲唑与2H-吲唑。化合价互变异构体包括通过一些成键电子重组而进行互变。
如本文所用,术语“溶剂合物”是指本发明化合物与溶剂分子配位形成特定比例的配合物。
如本文所用,术语“水合物”是指本发明化合物与水进行配位形成的配合物。
活性成分
如本文所用,“本发明化合物”指式I所示的化合物,并且还包括及式I化合物的各种晶型形式、药学上可接受的盐、水合物或溶剂合物:
如本文所用,“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于:盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸,甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸,苯磺酸等有机酸;以及天冬氨酸、谷氨酸等酸性氨基酸。
在另一优选例中,所述的R
1、R
2、R
3、R
4、R
5、R
g各自独立地为表1中各个化合物所对应的基团。
优选的本发明化合物如表1所示:
表1
在另一优选例中,本发明的优选化合物是表1中的化合物10a1、10b1、10u1、10v1、20a1、20b1、20u1、20u2、20v1、100a01、100a03、100a05、100u01、100a07、100b01、100b05或其药学上可接受的盐。
药物组合物和施用方法
由于本发明化合物具有优异的提高对象的免疫功能的活性,例如增加先天免疫系统功能,包括细胞内cGAG/STING先天免疫系统信号通道的激活,先天性免疫系统细胞的增加、抗原呈递细胞的增加以及活化、CD8
+T细胞活性的增加,T
reg细胞数量的减少,因此本发明化合物及其各种晶型,药学上可接受的无机或有机盐,水合物或溶剂合物,以及含有本发明化合物为主要活性成分的药物组合物可用于预防和/或治疗(稳定、减轻或治愈)肿瘤、或病毒感染的疾病。
在另一优选例中,所述的肿瘤选自下组:胰腺癌、膀胱癌、结肠直肠癌、乳腺癌、前列腺癌、肾癌、肝细胞癌、肺癌、卵巢癌、宫颈癌、胃癌、食道癌、黑色素瘤、神经内分泌癌、中枢神经系统癌、脑癌、骨癌、软组织肉瘤、非小细胞肺癌癌症、小细胞肺癌或结肠癌、皮肤癌、肺癌、泌尿系肿瘤、血液肿瘤、胶质瘤、消化系统肿瘤、生殖系统肿瘤、淋巴瘤、神经系统肿瘤、脑瘤、头颈癌。
在另一优选例中,所述的病毒选自下组:乙肝病毒、麻疹病毒、腮腺炎病毒、狂犬病毒、流感病毒、EB病毒、丙肝病毒、丁肝病毒、禽流感病毒。
本发明的药物组合物包含安全有效量范围内的本发明化合物及药学上可以接受的赋形剂或载体。其中“安全有效量”指的是:化合物的量足以明显改善病情,而不至于产生严重的副作用。通常,药物组合物含有1-2000mg本发明化合物/剂,更佳地,含有10-200mg本发明化合物/剂。较佳地,所述的“一剂”为一个胶囊或药片。
“药学上可接受的载体”指的是:一种或多种相容性固体或液体填料或凝胶物质,它们适合于人使用,而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明化合物以及它们之间相互掺和,而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制,代表性的施用方式包括(但并不限于):口服、肠胃外(静脉内、肌肉内或皮下)。
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中,活性化合物与至少一种常规惰性赋形剂(或载体)混合,如柠檬酸钠或磷酸二钙,或与下述成分混合:(a)填料或增容剂,例如,淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸;(b)粘合剂,例如,羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶;(c)保湿剂,例如,甘油;(d)崩解剂,例如,琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠;(e)缓溶剂,例如石蜡;(f)吸收加速剂,例如,季胺化合物;(g)润湿剂,例如鲸蜡醇和单硬脂酸甘油酯;(h)吸附剂,例如,高岭土;和(i)润滑剂,例如,滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠,或 其混合物。胶囊剂、片剂和丸剂中,剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备,如肠衣和其它本领域公知的材料。它们可包含不透明剂,并且,这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时,活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外,液体剂型可包含本领域中常规采用的惰性稀释剂,如水或其它溶剂,增溶剂和乳化剂,例知,乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油,特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外,组合物也可包含助剂,如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性化合物外,悬浮液可包含悬浮剂,例如,乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液,和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明化合物可以单独给药,或者与其他药学上可接受的化合物(例如抗-HBV剂)联合给药。
联合给药时,所述药物组合物还包括与一种或多种(2种,3种,4种,或更多种)其他药学上可接受的化合物(例如抗-HBV剂)。该其他药学上可接受的化合物(例如抗-HBV剂)中的一种或多种(2种,3种,4种,或更多种)可与本发明的化合物同时、分开或顺序地用于预防和/或治疗HBV感染或HBV相关疾病。
使用药物组合物时,是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人),其中施用时剂量为药学上认为的有效给药剂量,对于60kg体重的人而言,日给药剂量通常为1~2000mg,优选20~500mg。当然,具体剂量还应考虑给药途径、病人健康状况等因素,这些都是熟练医师技能范围之内的。
本发明清除HBV的机理
LW231能够增强空衣壳的组装(图1),这将导致HBV病毒DNA在细胞质中积累。因此,先天免疫系统的cGAS/STING信号通道被细胞质中的裸露HBV病毒DNA激活,导致下游转录因子IRF3的磷酸化增加,后者进入细胞核并激活I型干扰素的转录表达。释放的I型干扰素可激发一连串的免疫反应,包括自分泌式刺激JAK/STAT信号通道,诱导干扰素刺激基因的表达。肝细胞释放的I型干扰素还可以激发肝巨噬细胞。研究证明,活化的肝巨噬细胞能够通过直接吞噬作用清除感染的肝细胞,并通过释放有毒分子(如颗粒酶B和穿孔素)诱导肝细胞凋亡(Boltjes等,2014)。
在慢性感染阶段,肝HBV DNA既可以cDNA的形式存在,也可以染色体整合的形式存在于细胞核中。HBV DNA的转录产生pgRNA等4个mRNA,分别为0.7、2.1、2.4、3.5kb。通过这些mRNA,可以产生包括核心蛋白(HBcAg),表面蛋白(HBsAg)和聚合酶等病毒蛋白。pgRNA负责聚合酶连接,形成复合物,然后被核心蛋白包裹,形成pgRNA衣壳,衣壳被表面蛋白进一步包裹以形成病毒体,然后释放到循环中。在pgRNA衣壳内,pgRNA被逆转录为单链DNA,然后再转录为rcDNA。rcDNA衣壳也被表面蛋白进一步包裹并释放到循环中。另外,病毒感染的细胞会形成大量没有包含病毒RNA/DNA的空衣壳,并且在表面蛋白包封后从细胞中释放出来。在感染了HBV的病人的血液中,空衣壳的数量大约是病毒粒子的100倍。此外,感染细胞还分泌出比病毒粒子多1000至10,000倍的空包膜颗粒或细丝(Tang等人,2017)。
在HBV感染的肝脏细胞,LW231能够增强空衣壳的组装(Zhang和Wang2020),进而使得含乙肝病毒核酸的病毒粒子的形成大大降低,导致病毒DNA在细胞质的积累,激活和先天免疫的cGAS/STING信号通道。随后,下游转录因子IRF3的磷酸化增加。磷酸化的IRF3进入细胞核并激活I型IFN的转录。I型干扰素可以通过自分泌方式刺激JAK1/Tyk2/STAT信号通路(González-Navajas等,2012),磷酸化STAT1同型二聚体和STAT1/STAT2异二聚体,增加干扰素刺激基因(ISG)的转录。治疗后不久,从感染的肝细胞释放的I型干扰素和其他促炎细胞因子会增加肝巨噬细胞(库普弗细胞和单核细胞),树突状细胞和M-MDSCs亚群。LW231治疗促进了肝巨噬细胞的吞噬活性,导致感染肝细胞的清除。此外,活化的肝巨噬细胞和树突状细胞可能增强CD8
+T细胞的成熟,分化成细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL),增进IFN-γ的产生。此外,活化的肝巨噬细胞和CTL可分泌抑制病毒繁殖的细胞因子IL-1、IL-18和IFN-γ(Boltjes等,2014)。
在本发明中,从LW231给药小鼠所分离出肝脏细胞,对数千个肝脏细胞进行了单细胞RNA测序分析,并对肝巨噬细胞的转录组进行了分析,发现LW231给药后可增强细胞因子的产生和吞噬作用相关的生物学过程,证明了肝巨噬细胞在清除HBV感染的肝细胞中具有重要作用。另一方面,肝树突状细胞显示出对免疫原性过程的负调控,这可能是保护肝脏组织免受肝巨噬细胞免疫原性损伤的重要保护机制。文献显示,活化的巨噬细胞以及树突状细胞能够刺激肝脏浸润的CD8
+T细胞分化成CTL,从而导致IFN-γ的形成(Crispe,2009;Eckert等,2015;Link,A.等,2011)。
本发明证明,LW231治疗可以增加肝脏CTL中的IFN-γ产生(图3),表明LW231可以同时激活先天免疫和适应性免疫系统,是导致AAV-HBV感染清除的主要原因。CTL的激活可能归因于LW231对调节性T细胞的下调(图4)。
本发明证明,LW231治疗不仅增加肝巨噬细胞等髓性细胞亚群,而且还增强了这些效应功能,特别是增强了肝巨噬细胞的吞噬活性。
因此,本发明揭示了LW231治疗对巨噬细胞和CTL免疫功能的激活导致了病毒感染的肝细胞的清除,并且随着病毒在肝组织中的清除,树突状细胞对免疫原性进行了负调控。同时,LW231治疗增加的肝组织中M-MDSC亚群。这些细胞可能在维持肝细胞稳态中起作用。
本发明揭示了LW231引起肝脏AAV-HBV清除的作用机制。通过激活先天性cGAS/TING途径,LW231在AAV-HBV感染的肝脏中把免疫耐受性环境转变为致免疫性环境,其中诱导了肝脏巨噬细胞,树突状细胞和其他免疫细胞的数量和效应功能,导致病毒感染的肝细胞的清除。
本发明中,还提供了一种LW231治疗在AAV-HBV模型中引起HBV清除的作用机制(MOA),并揭示了LW231对先天免疫cGAS/STING信号通道的诱导以及对肝巨噬细胞和树突状细胞的激活是HBV清除的主要作用机制。
根据本发明的结果,LW231清除HBV感染的MOA如图1所示。
本发明的主要优点包括:
(1)本发明的LW231将AAV-HBV感染的肝脏中免疫耐受性环境转变为致免疫性环境,导致病毒感染的肝细胞能得到有效清除;
(2)本发明化合物具有良好的成药性;
(3)本发明化合物以及含有本发明化合物为主要成分的药物组合物可用于乙肝等相关疾病的治疗。
(4)本发明的化合物能够同时激活先天免疫系统功能和获得性免疫系统功能,提高对机体的抗病毒和抗癌能力。
下面结合具体实施例,进一步陈述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
通用方法
LW231对乙肝病毒衣壳装配的影响
HepG2.2.15细胞在补充有2%胎牛血清(FBS,HyClone)的DMEM/F12培养基中培养。测试化合物6天。用NP-40裂解细胞。通过非变性琼脂糖凝胶电泳评估裂解液。用多克隆兔抗HBV核心抗体通过Western印迹检测HBV衣壳。通过Southern印迹和Northern印迹分析检测衣壳化的HBV DNA和RNA。用单克隆小鼠抗HBV核心抗体通过蛋白质印迹分析评估细胞内HBV核心蛋白。通过蛋白质印迹分析评估了β-肌动蛋白,并将其用作内部对照。目测观察蛋白质印迹分析的结果。
肝脏HBV DNA的定量
用Qiagen TissueLyser匀浆肝组织,然后用蛋白酶K消化。收集水相并用RNase A(Sigma,R4642)消化。消化混合物用苯酚:氯仿:异戊醇萃取两次。DNA用异丙醇沉淀并溶于H
2O HBV DNA被通过TaqMan实时PCR测定法量化。
肝脏3.5kb HBV RNA的定量
用Qiagen TissueLyser和TRIzol(Life Technologies)将肝组织匀浆。收集水相,并用氯仿萃取两次。RNA用异丙醇沉淀,并溶于DEPC处理的H
2O。根据手册,使用FastQuant RT试剂盒(带有gDNase)通过逆转录合成cDNA。通过TaqMan实时PCR测定法对获得的cDNA进行定量,使用FastStart通用探针(ROX)(Roche,04914058001)补充了3.5kb HBV RNA特异性探针和引物(3.5kb HBV RNA特异性引物F和3.5kb HBV RNA特异性引物(R)。作为参考基因,使用来自ABI的ID为Mm99999915的特异性引物确定了GAPDH的表达水平。使用比较Ct(ΔΔCt)方 法通过相对定量(RQ)确定每个测试样品中靶基因的表达。该方法确定了目标基因(3.5-kb HBV RNA)和管家基因(GAPDH)之间的Ct差异(ΔCt),然后将经过化合物处理的样品的ΔCt值与载体组的ΔCt值进行了比较。
使用了以下公式:
ΔCt=目标基因的Ct平均值-管家基因的Ct平均值。
ΔΔCt=经过化合物处理的样品的ΔCt–载体组的ΔCt。
血清HBV RNA定量
根据手册,使用PureLink
TMPro 96病毒RNA/DNA试剂盒提取血清RNA。用DNase酶切DNA后,使用含有HBV特异性序列,锚定序列和poly T序列的3'RACE引物将HBV RNA反转录为cDNA。使用HBV X基因特异性引物,荧光团标记的TaqMan探针与锚定序列引物结合,通过实时PCR扩增获得的cDNA。
免疫组织化学
免疫组织化学(IHC)染色用于检测肝组织切片中的HBsAg和HBcAg表达。小鼠肝脏用10%NBF固定,通过常规方法进行处理,脱水,石蜡包埋和切片。从福尔马林固定,石蜡包埋(FFPE)的肝脏中制备厚度为4μm的切片样品,并将其进行脱蜡,抗原回收,血清阻断,一级和二级抗体孵育以及DAB着色作为常规IHC分析。
用柠檬酸抗原回收溶液(MXB Biotechnologies,MVS-0100)和3%H
2O
2处理肝组织,并用山羊血清封闭。使用多克隆马抗HBsAg抗体(Abcam,ab9193)和兔抗马IgG H&L(HRP)(Abcam,ab6921)检测肝内HBsAg。使用多克隆兔抗HBsAg抗体(Abcam,ab115992)和驴抗兔IgG H&L(HRP)(Abcam,ab6802)检测肝内HBcAg。阳性肝细胞用DAB(Vector Laboratories,SK-4105)染色并染成棕色;用苏木精(Harris,BASO,BA4041)对细胞核进行染色,并染成蓝色。HBcAg
+肝细胞的定量用自动玻片扫描仪(Aperio VERSA 200Brightfield&Fluorescence)进行。
HepG2.2.15细胞的抗病毒测定
HepG2.2.15细胞在补充有2%胎牛血清(FBS;HyClone)的DMEM/F12培养基中培养。以1:3的系列稀释液以8个浓度一式三份测试化合物。细胞用化合物处理6天。使用QIAamp 96DNA Blood Kit(Qiagen)从细胞培养上清液中提取 HBV DNA。通过TaqMan实时PCR分析定量HBV DNA。HBV DNA拷贝数由每个样品的Ct值和标准曲线确定。抑制百分比通过以下公式计算:%Inh.=(1-样本中的HBV数量/DMSO对照中的HBV数量)×100。EC
50和EC
90值使用GraphPad Prism使用“四参数对数方程式”绘制。使用CellTiter-Glo(Promega)测量化合物的细胞毒性,并计算CC
50和CC
90值。SI计算为在HepG2.2.15细胞中测得的LW231的平均CC
50或CC
90值与在HepG2.2.15细胞中测得的LW231的平均EC
50或EC
90值之比。
AAV-HBV小鼠的功效研究
4~5周龄的雄性C57BL/6小鼠购自上海灵昌生物技术有限公司(中国上海)。经过7天的适应期后,通过尾部静脉向小鼠静脉注射1×10
11rAAV8-1.3HBV(批号:2019032703,基因型D,血清型ayw)购自北京FivePlus分子医学研究所(中国北京),在200μL磷酸盐缓冲溶液(PBS)中进行模型诱导。感染后五周,选择合格的AAV-HBV-感染小鼠进行治疗。根据血清HBsAb,HBeAb和HBV DNA水平随机化。然后给小鼠口服LW231bid,分别为50、100和150mg/kg,并每周监测所有三种病毒指标。ETV被用作0.1mg/s q的阳性对照治疗。还施用了100mg/kg的LW231与ETV的组合。治疗六周后,只有选定的组以延长的给药期继续进行,直到第112天,最后一次给药后4小时将一半的动物处死以收集血清和肝组织以进行进一步分析。
化合物制备
本发明中的化合物制备方法参见中国申请CN2019100275736实施例中的制备方法。
测试化合物LW231为本发明优选化合物10a1、10b1、10u1、10v1、20a1、20b1、20u1、20u2、20v1、100a01、100a03、100a05、100u01、100a07、100b01、100b05中的一个优选化合物。
实施例1.LW231增强HBV感染的HepG2.2.15肝细胞内的无核酸衣壳含量
实验将HBV感染的HepG2.2.15肝细胞中加入10、100、1000nM终浓度的LW231,培养6天,然后将细胞裂解液在非变性琼脂糖凝胶中电泳,通过Western印迹检测完整的HBV衣壳。
如图2A所示,LW231增加了核衣壳的水平,并且呈现出了浓度相关性。 LW231在100nM及1000nM终浓度条件下,并没有促进细胞内核衣壳蛋白单体的表达。核衣壳的增加(图2A的iv)是由于促进核衣壳蛋白聚合所致。与DMSO阴性对照相比,100nM及1000nM终浓度的LW231完全抑制了完整HBV衣壳装载的通过Southern印迹检测到的HBV DNA(图2A的ii)和Northern印迹检测的HBV RNA(图2A的iii)。
从100nM开始的LW231增强了无HBV DNA或RNA的空衣壳的形成。参比化合物AT130也仅在1000nM时增强了无核酸衣壳的形成。相反,CpAM型参考化合物GLS4导致衣壳的异常组装。因此,在处理过的细胞中不能检测到完整的衣壳。如所预期的,用ETV处理抑制了DNA的形成。
通过电子显微镜检查的HBV衣壳在LW231处理的细胞中显示了正常的衣壳形态,在AT130处理的细胞中也观察到了,但是不同于GLS4处理的细胞的异常衣壳形态(图2B)。因此,LW231可增强形态上完整的无核酸衣壳的形成。从图中可以看出,在100nM本发明化合物的情况下,在核衣壳内几乎无病毒核酸,这提示,衣壳内的病毒核酸数量C1与病毒衣壳数量C0之比R2(即C1/C0)接近于0,即远远小于1/100(其中,将一个完整病毒的衣壳内的病毒核酸数量记为1个病毒核酸)。换言之,空衣壳数量E1与正常衣壳的数量E0之比R1(即E1/E0)远大于100。
此外,没有核衣壳的装载,细胞核产生的HBV DNA和HBV DNA将会裸露在细胞质中,进而激发细胞内在的先天免疫系统。
实施例2.LW231的治疗增加肝脏CTL中的IFN-γ
用单细胞RNA测序鉴定的总肝细胞中CTL的百分比不受LW231处理的影响。对CTL中IFN-γ表达的分析表明,LW231治疗后第1周表达IFN-γ的CTL百分比显着增加,并且在26周时不再出现这种增加(图3)。
结果表明,LW231治疗可以增加肝脏CTL中的IFN-γ产生,表明LW231可以同时激活先天免疫和适应性免疫系统,是导致AAV-HBV感染清除的主要原因。
实施例3.LW231的治疗下调T
reg细胞(调节性T细胞)
CTL的激活也可能归因于LW231对调节性T细胞的下调(图4)。
为了探究LW231治疗如何影响肝免疫系统以导致AAV-HBV感染的清除,从LW231连续给药1周和26周的小鼠肝脏组织中制备了单细胞悬浮液,并对其进行FACS分析。
通过FACS方法分析的肝脏组织中的CD4
+细胞的数量在LW231连续给药后出现显著降低。对CD4
+细胞表达FoxP3的亚群细胞进一步分析,结果表明,FoxP3阳性的T细胞(即T
reg细胞)在CD45
+细胞(髓性白细胞)中的比例显著下降,在第1周和第26周时分别从基线水平的1.0%降至0.6%(p≤0.05)和0.6%(p≤0.05),下降幅度达40%。这表明,LW231给药可显著降低CD4
+T
reg细胞数量(图4)。
实施例4.LW231对不同基因型HBV的抗HBV活性
在被HBV基因型A到J的DNA瞬时转染的HepG.2细胞中,测试了LW231对不同HBV基因型的抗病毒活性。从细胞培养上清液中提取了HBV DNA,并通过qPCR评估了DNA水平。在每个实验中,EC50值是根据每个化合物浓度的2个孔的平均抑制值确定的。
细胞培养上清液中的HBV DNA的qPCR分析显示,从所有测得的基因型中,LW231可以有效抑制LW231,EC50值为8-60nM或更低(表2)。对于全球优势基因型A,B和C,测得的EC50值约为10nM。
表2.LW231对不同基因型HBV的EC50
基因型 | GeneBank ID | LW231(nM) | ETV(nM) |
A | HE974371 | 9.86 | 4.68 |
B | JN406371 | 10.13 | 3.12 |
C | AB246346 | 9.02 | 3.07 |
D | U95551 | 29.23 | 6.69 |
E | HE974380 | 58.31 | 2.92 |
F | HE974369 | 22.50 | 3.74 |
G | AB064315 | 17.67 | 3.78 |
H | AB179747 | 22.31 | 4.35 |
I | FJ023673 | 8.27 | 5.08 |
J | AB486012 | 7.52 | 5.90 |
实施例5.LW231的药代动力学表征
在AAV-HBV小鼠中进行了药代动力学(PK)研究,以估计体内功效研究的药理学活性剂量。在口服给予50、100和200mg/kg的LW231之后,测量化合物的血清水平。将血浆中未结合的LW231的浓度与基于细胞测定得出的EC
50和EC
90值作图(图5)。
结果表明,在100和200mg/kg的剂量下,未结合的LW231的血浆浓度在超过12小时的时间内保持在EC
90值20.12ng/ml以上,从而支持了LW231的功效研究中b.i.d.(一日两次)给药方案。
实施例6.在稳定感染AAV-HBV的小鼠中LW231的处理可基本完全清除HBV
评估稳定感染AAV-HBV的小鼠中LW231的抗病毒活性。该研究分为两个阶段。第一阶段,持续6周,重点是评估LW231的最佳抗病毒剂量。如图6所示,LW231治疗导致血清HBV DNA水平快速且剂量依赖性降低(图6A)。到第42天,与分别以50、100和200mg/kg LW231给药的小鼠的预处理水平相比,HBV DNA水平降低了0.24、2.15和2.25log。值得注意的是,在100mg/kg治疗组的小鼠中,LW231治疗使HBsAg和HBeAg的血清水平分别降低了0.82log和1.11log(图6B,C)。出乎意料的是,在200mg/kg治疗组中的小鼠与治疗前相比,HBsAg(-0.48log)和HBeAg(-0.73log)的降低幅度较小,将100mg/kg作为抗病毒活性的LW231最佳剂量。
相比之下,尽管ETV治疗有效抑制了HBV DNA(-3.82log),但对HBsAg和HBeAg水平没有影响。ETV与LW231(100mg/kg)的联合治疗可将HBV DNA水平协同降低5.08log,但也降低了LW231对HBsAg(p<0.05)和HBeAg(p<0.001)的抑制作用。
实施例7.式I优选化合物治疗后对肝细胞内的无核酸衣壳含量的影响
在本实施例中,采用实施例1的方法,测试了式I优选化合物中的另一化合物对肝细胞内的无核酸衣壳含量的影响。
结果表明,该化合物可增强形态上完整的无核酸衣壳的形成。在100nM该化合物的情况下,在核衣壳内几乎无病毒核酸。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (10)
- 一种式I化合物或其药学上可接受的盐的用途,其特征在于,用于制备一制剂或组合物,所述制剂或组合物被用于:(a)增加病毒空衣壳;(b)增加肝脏中IFN-γ产生;(c)降低肝脏中CD4 +T reg细胞数量;(d)激活肝组织中cGAS/STING/IRF3信号通路;和/或(e)减少病毒感染的肝细胞的数量;式中,R 1,R 2,R 3和R 4各自独立的地选自下组:H、卤素、氰基、取代或未取代的C 3-C 4的环烷基,取代或未取代的C 1-C 4的烷基、取代或未取代的C 1-C 4的烷氧基;其中,所述的取代指基团上的氢原子被一个或多个选自下组的取代基取代:卤素、C 1-C 4的烷基(如二氟甲基、二氟乙基、单氟甲基、三氟甲基、三氟甲氧基);R 5选自下组:H、卤素、-CN、羟基、氨基、羧基、-(C=O)-取代或未取代的C 1-C 8烷基、取代或未取代的C 1-C 8烷基、取代或未取代的C 2-C 6烯基、取代或未取代的C 2-C 6炔基、取代或未取代的C 1-C 8烷胺基、取代或未取代的C 1-C 8烷氧基、取代或未取代的C 3-C 10环烷基、取代或未取代的具有1-3个选自下组N、S和O的杂原子的3-10元杂环烷基、取代或未取代的C 6-C 10芳基、或取代或未取代的具有1-3个选自下组N、S和O的杂原子的5-10元杂芳基;R g选自下组:H、卤素、-CN、羟基、氨基、羧基、-(C=O)-取代或未取代的C 1-C 8烷基、取代或未取代的C 1-C 8烷基、取代或未取代的C 2-C 6烯基、取代或未取代的C 2-C 6炔基、取代或未取代的C 1-C 8烷胺基、取代或未取代的C 1-C 8烷氧基、取代或未取代的C 3-C 10环烷基、取代或未取代的具有1-3个选自下组N、S和O的杂原子的3-10元杂环烷基、取代或未取代的C 6-C 10芳基、或取代或未取代的具有1-3个选自下组N、S和O的杂原子的5-10元杂芳基;除非特别说明,所述的“取代”是指被选自下组的一个或多个(例如2个、3个、4个等)取代基所取代:卤素、C1-C6烷基、卤代的C1-C6烷基、C1-C6烷氧基、卤代的C1-C6烷氧基、C3-C8环烷基、卤代的C3-C8环烷基、氧代、-CN、羟基、氨基、羧基、未取代或被一个或多个选自下组的取代基取代的选自下组的基团:C6-C10芳基、卤代的C6-C10芳基、具有1-3个选自N、S和O的杂原子的5-10元杂芳基、卤代的具有1-3个选自N、S和O的杂原子的5-10元杂芳基;所述的取代基选自下组:卤素、C1-C6烷氧基。
- 如权利要求1所述的用途,其特征在于,所述的增加病毒空衣壳包括:(a1)提高空衣壳的数量E1与有核酸病毒的正常衣壳的数量E0之比R1(即E1/E0);(a2)提高空衣壳的数量或水平;(a3)促进空衣壳的组装;和/或(a4)降低衣壳内的病毒核酸数量C1与病毒衣壳数量C0之比R2(即C1/C0),其中,将一个完整病毒的衣壳内的病毒核酸数量记为1个病毒核酸。
- 如权利要求1所述的用途,其特征在于,所述制剂或组合物被用于施用于一对象并:(a)增加病毒空衣壳;(b)增加肝脏中IFN-γ产生;(c)降低肝脏中CD4 +T reg细胞数量;(d)激活肝组织中cGAS/STING/IRF3信号通路;和/或(e)减少病毒感染的肝细胞的数量;(f)增加肝巨噬细胞(库普弗细胞和单核细胞),树突状细胞和M-MDSC亚群数量;(g)促进肝巨噬细胞的吞噬活性;(h)促进CD8 +T细胞的成熟。
- 如权利要求1所述的用途,其特征在于,所述的式I化合物选自表1所示的化合物或其药学上可接受的盐。
- 如权利要求4所述的用途,其特征在于,所述式I化合物选自:10a1、10b1、10u1、10v1、20a1、20b1、20u1、20v1、20u2、100a01、100a03、100a05、100u01、100a07、100b01、100b05。
- 一种用于人或动物的药物组合物,其特征在于,所述药物组合物含有:(i)如式I所示的化合物,或其药学上可接受的盐,和(ii)药学上可接受的载体;所述药物组合物用于选自下组的一种或多种应用:(a)增加病毒空衣壳;(b)减少病毒感染的肝细胞的数量;(c)增加肝脏中IFN-γ产生;(d)降低肝脏中CD4 +T reg细胞数量;和/或(d)增强免疫功能;其中,式I化合物如权利要求1中所述。
- 如权利要求6所述的药物组合物,其特征在于,所述的药物组合物还包括:(iii)额外的抗肿瘤药物和/或抗病毒药物。
- 如权利要求7所述的药物组合物,其特征在于,所述抗肿瘤药物选自下组:奥沙利铂、紫杉醇、多西他赛、卡培他滨、利妥昔单抗、吉非替尼、阿西替尼、瑞格非尼、卡博替尼、乐伐替尼、阿帕替尼、索拉非尼、纳武单抗、派姆单抗、阿特朱单抗、或伊匹单抗、阿韦鲁单抗、度伐鲁单抗或其组合。
- 如权利要求7所述的药物组合物,其特征在于,所述抗病毒药物选自下组:阿昔洛韦、替比夫定、齐多夫定、恩替卡韦(ETV)、替诺福韦酯、韦立得。
- 一种抗病毒的方法,其特征在于,包括步骤:给需要的对象施用式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐,其中,式I化合物如权利要求1中所述。
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