CN109364083A - Purposes of the hydrochloric acid conivaptan in preparation treatment feline infectious peritonitis drug - Google Patents

Purposes of the hydrochloric acid conivaptan in preparation treatment feline infectious peritonitis drug Download PDF

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Publication number
CN109364083A
CN109364083A CN201811307771.XA CN201811307771A CN109364083A CN 109364083 A CN109364083 A CN 109364083A CN 201811307771 A CN201811307771 A CN 201811307771A CN 109364083 A CN109364083 A CN 109364083A
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Prior art keywords
hydrochloric acid
cell
acid conivaptan
conivaptan
drug
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CN201811307771.XA
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CN109364083B (en
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彭贵青
李慧
刘楠
刘一楠
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Wuhan duweikang Biotechnology Co.,Ltd.
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Huazhong Agricultural University
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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
    • 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

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses new application of the hydrochloric acid conivaptan in preparation treatment feline infectious peritonitis drug.Hydrochloric acid conivaptan significantly inhibits the toxic effect very little of cat normal cell to cat coronavirus, can be substantially reduced malicious valence of the virus in cell；Indirect immunofluorescene assay result shows that hydrochloric acid conivaptan can inhibit proliferation of the cat coronavirus in cell, and has dose dependent.Therefore, hydrochloric acid conivaptan can be used for preparing cat coronavirus inhibitor, for treating feline infectious peritonitis, have many advantages, such as good effect, highly-safe.

Description

Purposes of the hydrochloric acid conivaptan in preparation treatment feline infectious peritonitis drug
Technical field
The present invention relates to the new pharmaceutical uses of hydrochloric acid conivaptan, especially treat feline infectious peritonitis drug in preparation In purposes.
Background technique
Feline infectious peritonitis is disease caused by infection cat coronavirus, is a kind of chronic, the progressive, infection of cat Property disease, mainly characterized by peritonitis, a large amount of ponding in thoracic cavity.Sick cat lethality is higher, and most drugs do not treat it Effect can only extend its life.
High agent is such as used mainly using immune and anti-inflammatory medicaments are inhibited for the treatment of feline infectious peritonitis at present The steroids of amount, or infected using broad-spectrum antibiotic and antiviral drugs preventing secondary.Not only side effect is larger for these drugs, And can only symptomatic treatment is of short duration delays disease process, the high mortality of feline infectious peritonitis cannot be reduced.
The molecular formula of hydrochloric acid conivaptan (Conivaptan hydrochkoride) is C32H27ClN4O2, it is a kind of smart ammonia The non-peptides double inhibitor of sour pitressin (AVP) V1a and V2 receptor, is mainly used for controlling for the normal hyponatremia of blood volume It treats, has no the relevant report for infecting peritonitis treatment aspect for cat at present.
Summary of the invention
It is new in preparation treatment feline infectious peritonitis drug that the object of the present invention is to provide a kind of hydrochloric acid conivaptans Purposes.
Test result shows: hydrochloric acid conivaptan has the toxic effect very little of cat normal cell to cat coronavirus There is apparent inhibiting effect, malicious valence of the virus in cell can be substantially reduced；Indirect immunofluorescene assay result shows that hydrochloric acid is examined Buddhist nun cuts down the smooth proliferation that can inhibit cat coronavirus in cell, and has dose dependent.
Therefore, hydrochloric acid conivaptan can be used for preparing cat coronavirus inhibitor, for treating feline infectious peritonitis, have It is effective in cure good, it is highly-safe the advantages that.
Detailed description of the invention
Fig. 1 is toxicity test result of the hydrochloric acid conivaptan to feline kidney cells.
Fig. 2 is inhibitory effect microphoto of the hydrochloric acid conivaptan to coronavirus in feline kidney cells.
Fig. 3 is the active TCID of the anti-cat coronavirus of hydrochloric acid conivaptan50Measurement result.
Fig. 4 is the active indirect immunofluorescence assay result of the anti-cat coronavirus of hydrochloric acid conivaptan.
Specific embodiment
The following describes the present invention in detail with reference to examples.It should be noted that the embodiment of the present invention is only limitted to The present invention will be described, without restriction effect.It is grasped involved in embodiment in relation to test method and various other experiments Make, be the ordinary skill in the art, the part being not particularly illustrated in text, those skilled in the art are referred to this Various common tool books, scientific and technical literature or relevant specification, handbook before patent application day etc. are practiced.
Toxicity test of the 1 hydrochloric acid conivaptan of embodiment to cell
1. test method
1) the good CCL-94 of growth conditions (feline kidney cells) is taken to carry out had digestive transfer culture, it is close with cell growth medium adjustment cell Degree is 1 × 105/ mL, is inoculated in 96 orifice plates, and every 100 μ L of hole is placed in 37 DEG C, 5%CO2Incubator culture 16h；
2) after 16h, 96 orifice plates are taken out, discard culture medium in hole, sterile PBS is cleaned three times, every group of first file after drying 100 μ L cell maintenance mediums are added, second and third, four, five, six column plus 50 μ L cell maintenance mediums；
3) it is marked on lid, hydrochloric acid conivaptan is added by every group of first file of flag sequence, carries out 2 times of multiple proportions Dilution under mixing gently ten with the volley of rifle fire, is sucked out 50 μ L and the second file is added, and so on, it finally mixes and discards 50 μ L.Make hydrochloric acid Conivaptan is 200 μM final concentration of, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, while the cell of drug is not added for setting Control；
4) it after 96h, usesReagent carries out cell viability detection.Calculation formula: cell survival rate (%)= Luminescence of cell value average value through drug-treated/cell controls group luminous value average value.
2. test result
The result is shown in Figure 1.Measurement cell survival rate can react hydrochloric acid conivaptan to the toxic effect of CCL-94 cell, from figure In as can be seen that hydrochloric acid conivaptan it is smaller to the toxic effect of CCL-94 cell, (< 50 μM) can under low concentration drug effect To promote cell to grow, increase cell viability.Its CC50Greater than 200 μM.
2 hydrochloric acid conivaptan of embodiment is to cat coronavirus activity TCID50Measurement
1. test method
1) CCL-94 cell is subjected to had digestive transfer culture, is 1 × 10 with cell growth medium adjustment cell density5/ mL, inoculation 12 The hole orifice plate 2mL/, 37 DEG C, 5%CO2Incubator culture 16h；
2) 12 orifice plates are taken out, marks, cell maintenance medium is mixed with hydrochloric acid conivaptan, keep hydrochloric acid conivaptan whole Concentration is respectively 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, and at least 5s is mixed on oscillator；
3) culture medium in 12 orifice bores is discarded, is cleaned three times with sterile PBS, the drug diluted is added after drying, It is placed in 37 DEG C, 5%CO2Incubator is incubated for 1h；
4) after 1h, 12 orifice plates, the cat coronavirus of every hole inoculation 0.01MOI are taken out, light 12 orifice plates that shake mix, every piece of 12 holes Virus control and cell controls are arranged in plate, in 37 DEG C, 5%CO2Incubator culture；
5) about after 12h, there is 70% lesion in virus control, and it is primary that 12 orifice plates are transferred to -80 DEG C of ultra low temperature freezer freeze thawing；
6) every boreliquid is collected into EP pipe, and 4000rpm collects the malicious valence of supernatant survey after being centrifuged 10min.
2. test result
As a result see Fig. 2 and Fig. 3.Figure it is seen that it can be observed that the cell growth of normal CCL-94 cell under mirror In good condition, cellular morphology is complete, and cell boundary is clear, and is rounded and is fallen off with the CCL-94 cell of virus infection, generates A large amount of cell fragments, no normal cell form, sick cell aggregation it is agglomerating, lesion clearly, with hydrochloric acid conivaptan and disease After poison handles cell simultaneously, cell boundary is clear, cell well-grown, and only lesion occurs for minute quantity cell.
From figure 3, it can be seen that hydrochloric acid conivaptan is at 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0 μM TCID50Respectively 10-4.50/mL、10-4.63/mL、10-5.40/mL、10-6.62/mL、10-6.74/mL、10-7.00/mL.Wherein drug Concentration can make malicious valence decline 2.5 titres at 25 μM, and 12.5 μM of whens can decline 2.37 titres, and 6.25 μM of whens reduce 1.6 titres.Show that hydrochloric acid conivaptan has good inhibiting effect to cat coronavirus.
3 hydrochloric acid conivaptan of embodiment is to the active indirect immunofluorescene assay of cat coronavirus
1. test method
1) it takes the good CCL-94 cell of growth conditions to carry out had digestive transfer culture, is 1 with cell growth medium adjustment cell density ×105/ mL is inoculated in 96 orifice plate, 100 hole μ L/, is placed in 37 DEG C, 5%CO2Incubator culture 16h；
2) cell covers with single layer after 16h, takes out 96 orifice plates and is cleaned twice with plasma-free DMEM medium, changes cell maintenance into Liquid is added compound and carries out 2 times of doubling dilutions, while 0.01MOI cat coronavirus liquid is added, and makes hydrochloric acid conivaptan final concentration For 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM are arranged virus control group and cell controls group simultaneously；
3) there is 60% lesion to cell, discard culture medium in hole, cleaned after 2 times with PBS and 4% paraformaldehyde is added fixes Cell 30min；
4) plank fixed is put into distilled water and is washed three times, the diluted primary antibody of 1:300 (N protein rabbit polyclonal is added Antibody), 37 DEG C of incubation 1h；
5) primary antibody is discarded, plate is put into distilled water to addition goat-anti rabbit fluorescence secondary antibody, 37 DEG C of incubation 1h after washing three times；
2. test result
As a result see Fig. 4.IFA detection is not it can be seen that hydrochloric acid conivaptan in 25 μM and 12.5 μM, detects cat substantially Coronavirus, with the decline of drug concentration, the virus detected is continuously increased, and at 1.5625 μM, can detecte a large amount of Cat coronavirus.

Claims (2)

1. purposes of the hydrochloric acid conivaptan in preparation cat coronavirus inhibitor medicaments.
2. purposes of the hydrochloric acid conivaptan in preparation treatment feline infectious peritonitis drug.
CN201811307771.XA 2018-11-05 2018-11-05 Application of conivaptan hydrochloride in preparation of medicine for treating feline infectious peritonitis Active CN109364083B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113679716A (en) * 2021-10-13 2021-11-23 山东领海生物科技有限公司 Application of bromophenol-pyrazoline compounds in treatment of feline coronavirus diseases

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992008487A1 (en) * 1990-11-14 1992-05-29 Smithkline Beecham Corporation Recombinant feline coronavirus s proteins
CN102885892A (en) * 2012-10-30 2013-01-23 天津集合科技有限公司 Traditional Chinese medicine composition for treating feline infectious peritonitis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992008487A1 (en) * 1990-11-14 1992-05-29 Smithkline Beecham Corporation Recombinant feline coronavirus s proteins
CN102885892A (en) * 2012-10-30 2013-01-23 天津集合科技有限公司 Traditional Chinese medicine composition for treating feline infectious peritonitis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PAUL GAGLIO•KWAKU MARFO•JOSEPH CHIODO III: "Hyponatremia in Cirrhosis and End-Stage Liver Disease:Treatment with the Vasopressin V2-Receptor AntagonistTolvaptan", 《DIG DIS SCI》 *
SAEED SHARIF ET AL.: "Diagnostic Methods for Feline Coronavirus: A Review", 《VETERINARY MEDICINE INTERNATIONAL 》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113679716A (en) * 2021-10-13 2021-11-23 山东领海生物科技有限公司 Application of bromophenol-pyrazoline compounds in treatment of feline coronavirus diseases
CN113679716B (en) * 2021-10-13 2024-03-26 史大永 Use of bromophenol-pyrazoline compounds for treating feline coronavirus diseases

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