(19)
(11)EP 3 466 426 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
04.11.2020 Bulletin 2020/45

(21)Application number: 18155997.2

(22)Date of filing:  09.02.2018
(51)International Patent Classification (IPC): 
A61K 31/4741(2006.01)
A61P 31/12(2006.01)
A61K 31/728(2006.01)

(54)

EVALUATION OF THE SEVERITY OF PATIENTS WITH FLAVIVIRUS INFECTION BY BLOOD HYALURONAN LEVELS AND THERAPEUTIC AGENTS TO BLOCK THE HYALURONAN

BEWERTUNG DES SCHWEREGRADES VON PATIENTEN MIT FLAVIVIRUS-INFEKTION DURCH HYALURONANSPIEGEL IM BLUT UND THERAPEUTISCHE MITTEL ZUR BLOCKIERUNG DER HYALURONAN

ÉVALUATION DE LA GRAVITÉ DES PATIENTS ATTEINTS D'UNE INFECTION À FLAVIVIRUS PAR LES NIVEAUX D'HYALURONANE SANGUIN ET AGENTS THÉRAPEUTIQUES POUR BLOQUER L'HYALURONANE


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 05.10.2017 TW 106134411

(43)Date of publication of application:
10.04.2019 Bulletin 2019/15

(60)Divisional application:
20154086.1 / 3689352

(73)Proprietor: Kaohsiung Medical University
Kaohsiung City 807 (TW)

(72)Inventors:
  • Chen, Yen-Hsu
    807 Kaohsiung City (TW)
  • Lin, Chun-Yu
    80767 Kaohsiung City (TW)
  • Heldin, Paraskevi
    751 24 Uppsala (SE)

(74)Representative: Isarpatent 
Patent- und Rechtsanwälte Behnisch Barth Charles Hassa Peckmann & Partner mbB Friedrichstrasse 31
80801 München
80801 München (DE)


(56)References cited: : 
  
  • HONSAWEK ET AL: "Increased levels of serum hyaluronan in patients with dengue infection.", J. OF INFECTION., vol. 54, 28 July 1995 (1995-07-28), - 28 July 2005 (2005-07-28), pages 225-229, XP002783264,
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] The present invention is related to a determination method and a pharmaceutical composition. In particular, the present invention is related to a method to determine whether a warning sign will occur in a subject being infected with a dengue fever virus and suffering from dengue fever in a febrile phase.

[0002] The viruses in the Flaviviridae family are single-stranded linear RNA viruses. Humans and other mammals are their natural hosts, and the viruses are spread widely via arthropods (e.g. ticks and mosquitoes). The Flaviviridae family is classified into four genera, including Flavivirus, Pestivirus, Hepacivirus and Pegivirus, and over 100 species. The Flavivirus genus includes the well-understood yellow fever virus, Japanese encephalitis virus (JEV), dengue virus, West Nile virus and Zika virus. The Pestivirus genus includes bovine viral diarrhea virus. The Hepacivirus genus is classified into 14 species which are abbreviated as the hepatitis viruses A, B, C... through N, wherein the well-known one is the hepatitis C virus. The Pegivirus genus is classified into 11 species which are abbreviated as the hepatotropic viruses A, B, C... through K.

[0003] Dengue fever, which mainly occurs in the subtropical and tropical countries, is an acute infectious disease caused by the dengue virus, and spread to humans via mosquitoes, e.g. Aedes aegypti and Aedes albopictus. The dengue viruses are classified into four types, i.e. I, II, III and IV, according to different virus serotypes, and each serotype has the abilities of infection and pathogenesis.

[0004] The latency period for the dengue fever is typically about 3 days to 8 days (the longest one being up to 14 days). The period between the day before illness and five days after illness is called "the communicability period" or "viremia". Dengue viruses exist in the patient's blood. Mosquitoes, A. aegypti or A. albopictus, will take up the dengue viruses if the patients are bitten by the mosquitoes during the communicability period. The dengue viruses enrich inside the mosquitoes during an 8-to-12-day multiplication, and the mosquitoes bite other people causing the dengue viruses to spread.

[0005] Some patients have mild syndromes during the dengue fever infection, and some do not even appear to show any symptoms. The typical symptoms of dengue fever include the phenomena such as a sudden fever higher than 38°C, headache, posterior eye pain, muscle soreness, arthralgia, rash, drowsiness, restlessness, liver enlargement, the increased vascular permeability, plasma leakage, bleeding, severe lethal bleeding and so on. Furthermore, if patients are infected with different dengue virus serotypes (DENV) for a consecutive two times, in particular the patients who are infected with the DENV-1 firstly followed by the DENV-2 or DENV-3 infection, or the patients who are infected with the DENV-3 firstly followed by the DENV-2 infection, the possibility of causing the dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) is significantly increased to result in more severe clinical syndromes, such as shock or severe complications. Mortality is up to more than 20% if timely treatment is absent.

[0006] There is no sovereign remedy for treating dengue fever. In the prevention of dengue fever, Sanofi Pasteur (Lyon, France) developed the first dengue fever vaccine in the world (a tetravalent attenuated live vaccine) for people between the ages of 9 to 45 years old. However, this vaccine generated poor protection against DENV-2. In addition, GlaxoSmithKline (GSK, Brentford, the United Kingdom) also developed a deactivated vaccine, TDEN-PIV, that progressed to phase II clinical trials. Honsawek et al. (Increased levels of serum hyaluronan in patients with dengue infection, J. Infect. 2007. 54:225-229.) disclose that the serum hyaluronan (HA) levels were evaluated to determine their importance in dengue fever (DF)/DHF/DSS patients, and the results showed that the mean serum HA levels significantly increased in the DSS patients in the acute phase in comparison to patients with hepatitis A and healthy people, and the serum HA levels of the DSS patients rapidly decreased during the convalescent phase, but were still significantly higher than those in healthy people.

[0007] The World Health Organization (WHO) classified dengue fever patients into groups A, B and C depending on the occurrence of "warning sign" or not (Dengue Guidelines for Diagnosis, Treatment, Prevention and Control, 2009, World Health Organization, ISBN 978 92 4 154787 1), rather than the simple and traditional classification for dengue fever patients and DHF patients. The warning sign means that a dengue fever patient shows abdominal pain or abdominal tenderness, persistent vomiting, clinical fluid accumulation (e.g. ascites, pleural effusion and so on), mucosal bleed, lethargy, restlessness, the liver enlargement more than 2 cm, an increase in the hematocrit (HCT) concurrent with a rapid decrease in the platelet count, while keeping in mind co-existing conditions (e.g. diabetes, renal failure, chronic hemolytic disease, obesity, pregnancy, infancy, and old age), and adverse social circumstances (such as living alone, or living far from hospital).

[0008] Among these, patients in group A do not have neither "warning signs", co-existing conditions nor adverse social circumstances; patients in group B have "warning signs", and have the features of co-existing conditions and/or adverse social circumstances; and patients in group C have severe plasma leakage with shock and/or fluid accumulation with respiratory distress, severe bleeding, and/or organ impairment (such as liver function impairment, central nervous system impairment, heart failure, renal failure, cardiomyopathy, encephalopathy, encephalitis and so on).

[0009] In the treatment, the patients in group A are monitored at home for disease progression; the patients in group B accept hospitalization; and the patients in group C need an emergency remedy or are transferred to the hospital with good equipment and experienced physicians and nurses.

[0010] The classification of dengue cases by the WTO (2009 Edition) was established to provide appropriate triage to assist the clinical remedy, so as to study the mechanism of the disease and evaluate medical intervention (such as intravenous dehydration, new anti-virus drugs and vaccines and so on) in the future.

[0011] It is therefore the Applicant's attempt to deal with the above situation encountered in the prior art.

[0012] To assist doctors to evaluate the subsequent illness course of patients with the Flavivirus infectious illness at his/her early stage of illness, and to adopt the appropriate treatment or therapy, novel and progressive techniques are developed in the present invention.

[0013] The present invention discloses a method to determine whether a warning sign will occur in a subject with the Flavivirus infectious illness, and the method includes steps of: (a) providing a serum of the subject; (b) measuring a level of a hyaluronan in the serum; and (c) determining that the warning sign will occur in an illness course of the subject when the level is higher than or equal to 70 ng/mL.

[0014] In one embodiment, the subject is a human, and a period that the human is infected with the Flavivirus virus is at an early stage of the illness of the human. The Flavivirus virus is a a dengue virus which causes the human to suffer from dengue fever.

[0015] In one embodiment, the human with the dengue fever shows the warning sign in the illness course, and the warning sign includes an abdominal pain, an abdominal tenderness, a persistent vomiting, a clinical fluid accumulation, a mucosal bleed, a lethargy, a restlessness, a liver enlargement of more than 2 centimeters, an increase in a hematocrit (HCT) concurrent with a rapid decrease in a platelet count, and a combination thereof.

[0016] In one embodiment, the step (b) is performed in a well of a microplate where an aggrecan is coated thereon already, and the step (b) further includes: (b1) adding the serum to cause the hyaluronan which may exist in the serum to be bound to the aggrecan; (b2) adding a biotinylated-proteoglycan to be bound to the hyaluronan; (b3) adding a streptavidin-conjugated enzyme to be bound to the biotinylated-proteoglycan; (b4) adding a substrate to react with the streptavidin-conjugated enzyme to generate a chemiluminescent reaction; and (b5) determining an optical density at 450 nm to calculate the level of hyaluronan.

[0017] The present invention further discloses a pharmaceutical composition for blocking a hyaluronan in a serum of a subject to prevent an inflammation thereof or treating the subject suffering from the Flavivirus infection, wherein the subject was identified to suffer from the Flavivirus infection. The pharmaceutical composition includes: a component being one selected from the group consisting of a first pharmaceutically effective amount of a 4-methyl umbelliferone sodium salt, a second pharmaceutically effective amount of a CD44 small interfering RNA (siRNA), a third pharmaceutically effective amount of an anti-CD44 antibody, a fourth pharmaceutically effective amount of a hyaluronidase and a combination thereof.

[0018] In one embodiment, the hyaluronan has a level in the serum of the subject higher than or equal to 70 ng/mL to indicate that a warning sign will occur in an illness course of the subject. In one embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, an excipient, a diluents or an adjuvant. In one embodiment, the subject is a human.

[0019] In one embodiment, the first pharmaceutically effective amount is a first effective blood concentration of the 4-methyl umbelliferone sodium salt of the human being in a first range from 0.05 mM to 5 mM, the second pharmaceutically effective amount is a second effective blood concentration of the CD44 siRNA of the human being in a second range from 1 nM to 100 nM, the third pharmaceutically effective amount is a third effective blood concentration of the anti-CD44 antibody of the human being in a third range from 5 µg/mL to 500 µg/mL, and the fourth pharmaceutically effective amount is a fourth effective blood concentration of the hyaluronidase of the human being in a fourth range from 0.5 unit/mL to 50 units/mL.

[0020] In one embodiment, the Flavivirus infection causes the subject to have an illness including yellow fever, Japanese encephalitis, dengue fever, West Nile fever, or a Zika virus infection.

[0021] The present invention further discloses a usage of a 4-methyl umbelliferone sodium salt, a CD44 siRNA, an anti-CD44 antibody or a hyaluronidase to be prepared as a pharmaceutical composition for treating a subject suffering from a Flavivirus infection to have an illness, and the Flavivirus and the illness correlated to the Flavivirus are described above.

[0022] The objectives and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings.

Fig. 1 illustrates a diagram showing a receiver operating characteristic (ROC) curve for the level of serum hyaluronan (SA) of the dengue fever patients at the early stage of the illness in the present invention.

Fig. 2 illustrates a diagram showing the ratio of the warning signs (WS)-positive to the WS-negative in the dengue fever patients during their whole illness courses in the present invention.

Fig. 3 illustrates a diagram showing the platelet (PLT) nadir (smaller than or not smaller than 50,000/µl) of the dengue fever patients during their whole illness courses in the present invention.

Fig. 4 illustrates a diagram showing the blood concentration phenomenon for the increased hematocrit (HCT) level (>20%) appearing in the dengue fever patients during their whole illness courses in the present invention.

Fig. 5 illustrates a diagram showing the Akt phosphorylation in the human vascular endothelial cells being subjected to the dengue virus (DENV) nonstructural protein 1 (NS1) or the pharmaceutical composition of the present invention.

Fig. 6 illustrates a diagram showing the transwell permeability assay of human microvascular endothelial cells.



[0023] The present invention will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of the preferred embodiments of this invention are presented herein for purpose of illustration and description only; they are not intended to be exhaustive or to be limited to the precise form disclosed.

Definition:



[0024] The term "Flavivirus" herein refers to virus.

[0025] The illnesses of the dengue fever patients herein are classified into "dengue fever", "severe dengue", "dengue hemorrhagic fever (DHF)", "dengue shock syndrome (DSS)" and so on according to the clinical manifestations or examination results.

[0026] The definition of the term "severe dengue" herein conforms to one or more items as follows: severe plasma leakage with shock, severe plasma leakage with fluid accumulation and respiratory distress, severe bleeding evaluation, severe organ impairment, glutamic oxaloacetic transaminase (GOT) or glutamic pyruvic transaminase (GPT) ≥ 1,000 IU/L in the liver, consciousness impairment in the central nervous system, heart failure or others.

[0027] According to the classification of the dengue cases by the WHO (2009 Edition), the term "warning sign" herein is defined as that dengue fever patients show abdominal pain or abdominal tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleeding, lethargy, restlessness, liver enlargement of more than 2 cm, and an increase in the hematocrit (HCT) concurrent with a rapid decrease in the platelet count.

[0028] In the embodiment of the present invention, the patients who are infected with dengue virus and identified to have dengue fever are the population, and the levels of hyaluronan in the sera of the patients are measured and statistically calculated to define that the warning sign will occur in the illness course of the patients when the level is higher than or equal to 70 ng/mL.

Embodiment 1:


Experimental methods:



[0029] To determine whether a warning sign will occur in a patient with a Flavivirus infectious illness, the level of SA in the patient was determined using the Hyaluronan DuoSet® ELISA development system (Cat. No. DY3614-05, R&D systems, Inc., U.S.A.). The skilled person in the art can implement Embodiment 1 in view of the manufacturer's instructions of the Hyaluronan DuoSet® ELISA development system, or perform the experiments by preparing the materials and reagents by himself/herself and using the same experimental methods.

[0030] First, a 96-well microplate for the enzyme-linked immunosorbent assay (ELISA) was prepared as follows. The hyaluronan capture reagent (i.e. recombinant human aggrecan) was diluted to a working concentration in the phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2-7.4, 0.2 µm filtered) without carrier protein. The 96-well microplate with 100 µL per well of the diluted hyaluronan capture reagent was immediately coated. The microplate was sealed and incubated overnight at room temperature.

[0031] Each well was aspirated and washed with a wash buffer (0.05% Tween® 20 in PBS, pH 7.2-7.4), the process was repeated two times for a total of three washes. In detail, each well was washed by filling with the wash buffer (400 µL) using a squirt bottle, a manifold dispenser or an autowasher. Complete removal of liquid at each step was essential for good performance. After the last wash, any remaining wash buffer was removed by aspirating or by inverting the microplate and blotting it against clean paper towels. The microplate was blocked by adding 300 µL of a reagent diluent (5% Tween® 20 in PBS, pH 7.2-7.4, 0.2 µm filtered) to each well. The microplate was incubated at room temperature for a minimum of 1 hour. The aspiration/wash was repeated. The microplate was now ready for the sample addition and the ELISA experiment.

[0032] A 100-µL sample or hyaluronan standards in the reagent diluent (or an appropriate diluent) per well was added to the well. An adhesive strip was covered on the microplate, and the microplate was incubated for 2 hours at room temperature. The aspiration/wash was repeated. A 100-µL biotinylated detection reagent (i.e. the biotinylated recombinant human proteoglycan) which was diluted in the reagent diluent was added to each well. A new adhesive strip was covered on the microplate, which was further incubated for 2 hours at room temperature. A 100-µL working dilution of streptavidin-horseradish peroxidase (HRP) was added to each well. The microplate was covered and incubated for 20 minutes at room temperature. Placing the microplate in direct light was avoided. The aspiration/wash was repeated. A 100-µL substrate solution (1:1 (v/v) mixture of color reagent A (H2O2) and color reagent B (tetramethylbenzidine)) was added to each well, and the microplate was incubated for 20 minutes at room temperature. Placing the microplate in direct light was avoided. A 50-µL stop solution (2 N H2SO4) was added to each well. The microplate was gently tapped to ensure thorough mixing. The optical density of each well was determined immediately using a microplate reader set to 450 nm.

[0033] A six point standard curve of the hyaluronan standard using 3-fold serial dilutions in the reagent diluent was recommended. Thus, the concentration of the hyaluronan standard was 90, 30, 10, 3.33, 1.11 and 0.370 ng/mL. A standard curve was plotted based on the concentration of the hyaluronan standard and its average optical density.

Experimental results:



[0034] Please refer to Fig. 1, which illustrates a diagram showing a receiver operating characteristic (ROC) curve for the level of SA in the dengue fever patients at the early stage of the illness in the present invention. When the serum sample of each dengue fever patient (n = 108) was measured, a critical value whether the warning sign will occur was precisely determined by the ROC curve depending on whether the patients show the warning sign. As shown in Fig. 1, an area under curve (AUC) is 0.681 (95% confidence interval (C.I.) = 0.58-0.78, p value = 0.002), the best critical value is 70.06 ng/mL, the sensitivity is 0.758, and the 1-specificity is 0.548. Therefore, in the subsequent analysis, the level of SA which is higher than or equal to 70 ng/mL is the critical value for determining whether the warning sign will occur in the whole illness course of the patient.

[0035] Please refer to Table 1, which is the univariate analysis of factors associated with the dengue fever patients presented with warning signs (WS) throughout the illnesses (n = 108). The levels of hyaluronan in the sera of the dengue fever patients (n = 108) were measured during the febrile phase (at early illness stage), and their illness progression was traced until recovery. If the level of SA in the dengue fever patient is higher than or equal to 70 ng/mL during the febrile phase, the possibility that they show "the warning sign" in whole illness course is 3.78 times higher than the patients with the level lower than 70 ng/mL (p = 0.003, 95% C.I. = 1.65-8.66). The results of the univariate analysis in Table 1 also showed that the age ≥ 65 years, the secondary dengue viral infection, and the level of SA ≥ 70 ng/mL (at the febrile phase) are prediction factors for predicting whether the dengue fever patients belong to the "WS-positive" group. Furthermore, important prediction factors (i.e. patient's age, and secondary infection) were calibrated using multivariate analysis, and the results still showed that the level of SA ≥ 70 ng/mL in the dengue fever patient at the early stage (the febrile phase) is an independent prediction factor for predicting whether the warning sign will occur in the illness course of the patient (referring to Table 2). Therefore, depending on the level of SA (≥ or < 70 ng/mL) in the dengue fever patient at the early illness stage, doctors can determine whether the warning sign will occur in his/her illness course, and further evaluate the patient's subsequent conditions so as to provide him/her with the appropriate remedy or treatment.
Table 1. Univariate analysis of factors associated with dengue fever patients presented with warning signs (WS) throughout the illnesses (n = 108)
VariablesNo. (%) of patientsOdds ratio (95% Confidence interval)p-value
WS-positive (n = 66)WS-negative (n = 42)
Demographic characteristics    
  Gender, female 35 (53.0) 19 (45.2) 1.37 (0.63-2.97) 0.554
  Age ≥ 65 years 34 (51.5) 10 (23.8) 3.40 (1.44-8.02) 0.008
  Secondary infection 43 (65.2) 17 (40.5) 2.75 (1.24-6.10) 0.020
Comorbidities    
  Diabetes 16 (24.2) 7 (16.7) 1.60 (0.60-4.30) 0.486
Laboratory data    
  Thrombocytopenia (first sampling) 26 (39.4) 11 (26.2) 1.83 (0.79-4.27) 0.230
  SA (febrile phase) ≥ 70 ng/ml 50 (75.8) 19 (45.2) 3.78 (1.65-8.66) 0.003
Table 2. Multivariate analysis of the serum samples of the dengue fever patients (n = 108)
VariableAdjusted odds ratio (95% CI)p-value
Age ≥ 65 years 2.01 (0.78-5.21) 0.151
Secondary infection 2.00 (0.84-4.76) 0.118
Serum HA (febrile phase) >70 ng/ml 2.80 (1.15-6.80) 0.023


[0036] Please refer to Fig. 2 to Fig. 4, which illustrates diagrams showing (a) the ratio of the warning signs (WS)-positive to the WS-negative, (b) the platelet (PLT) nadir (< or ≥ 50,000/µl), and (c) the blood concentration phenomenon for the increased hematocrit (HCT) level (>20%) in the dengue fever patients during their whole illness course in the present invention. These 108 dengue fever patients were classified into four groups (i.e. lowest quarter, middle lower quarter, middle higher quarter, and highest quarter) depending on the levels of SA at their early illness stage (the febrile stage). It can be known from Figs. 2 to 4 that, compared to the group with the lower level of SA, the group with the higher level of SA has a high possibility of having the warning sign in the whole illness course, and the PLT nadir < 50,000/µl and the increased hematocrit (HCT) level (>20%).

Embodiment 2:



[0037] Based on whether the warning sign will occur in the illness course of the patent with the level of SA ≥ 70 ng/mL, a pharmaceutical composition for blocking the SA in the patient to prevent inflammation is disclosed in this Embodiment. The pharmaceutical composition includes a pharmaceutically effective amount of 4-methyl umbelliferone sodium salt, wherein the therapeutically effective amount is an effective blood concentration of 4-methyl umbelliferone sodium salt of the patient being in a range from 0.05 mM to 5 mM. The pharmaceutical composition may further include a pharmaceutically acceptable carrier, an excipient, a diluent, or an adjuvant. Please refer to Fig. 5, which illustrates a diagram showing the Akt phosphorylation in the human vascular endothelial cells being subjected to the dengue virus (DENV) nonstructural protein 1 (NS1) or the pharmaceutical composition of the present invention. In Fig. 5, the human vascular endothelial cells (4 × 105 cells cultivated in a 60-mm dish) were treated with a nonstructural protein 1 (3 µg/ml, Cat. No. ENZ-PRT105-0100, Enzo Life Sciences, Inc., NY, U.S.A.), and the Akt phosphorylation was significantly induced in the cells, indicating that the dengue virus results in inflammation in the human vascular endothelial cells. In addition, the human vascular endothelial cells were treated with 4-methyl umbelliferone sodium salt for 12 hours followed by the DENV NS1 treatment. Subsequently, the cellular proteins were extracted and subjected to the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins according to their molecular weights. Next, the proteins in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane, and the phospho-Akt (p-Akt) signal on the PVDF membrane was detected using the Western blotting and the p-Akt antibody. The results showed that a range from 0.05 mM to 5 mM is the effective blood concentration of 4-methyl umbelliferone sodium salt in the dengue fever patient, indicating that 4-methyl umbelliferone sodium salt can be used to inhibit the inflammation of the human vascular endothelial cells caused by the dengue virus (referring to Fig. 5).

Embodiment 3:



[0038] The CD44 antigen, plays important roles in many biological functions (such as inflammation), is a surface glycoprotein on mammalian cells, and hyaluronan is a main molecule to bound with the CD44 antigen. Thus, an abundance of hyaluronan would bind to CD44 to influence CD44's biological functions when the level of SA in the dengue fever patient is too high. In this Embodiment, CD44 small interfering RNA (siRNA) was used to inhibit the CD44 expression of the vascular endothelial cells of the dengue fever patients, and block the combination between hyaluronan and CD44. In this Embodiment, the experimental method for inhibiting cellular protein expression using siRNA was known to the skilled person in the art, and the CD44 siRNAs were the artificially synthesized SEQ ID NO:1 (5'-GAAUAUAACC UGCCGCUUU-3'), SEQ ID NO: 2 (5'-CAAGUGGACU CAACGGAGA-3'), SEQ ID NO: 3 (5'-CGAAGAAGGU GUGGGCAGA-3') and SEQ ID NO: 4 (5'-GAUCAACAGU GGCAAUGGA-3'). Furthermore, two, three or four siRNAs in the SEQ ID NOs: 1 to 4 may be combined to inhibit the CD44 expression of the vascular endothelial cells (4 × 105 cells cultivated in a 60-mm dish) of the dengue fever patient. First, in view of the manufacturer's instructions of siLentFect™ Lipid Reagent (Bio-Rad laboratories AB, Sweden), the CD44 siRNA was mixed with siLentFect™ Lipid Reagent, the mixture was transfected into the human vascular endothelial cells for 24 hours followed by the DENV NS1 treatment (3 µg/ml, Cat. No. ENZ-PRT105-0100, Enzo Life Sciences, Inc., NY, U.S.A.). Subsequently, the cellular proteins were extracted and subjected to the SDS-PAGE to separate the proteins according to their molecular weights. Next, the proteins in the gel were transferred to a PVDF membrane, and the p-Akt signal on the PVDF membrane was detected using the Western blotting and the p-Akt antibody. The results showed that a range from 1 nM to 100 nM is an effective blood concentration of CD44 siRNA (SEQ ID NOs: 1 to 4) in the dengue fever patient, indicating that the CD44 siRNA can be used to inhibit the inflammation of the vascular endothelial cells in humans caused by the dengue virus (referring to Fig. 5). Therefore, the CD44 siRNA (SEQ ID NOs: 1 to 4) of the present invention can be used to prepare a pharmaceutical composition for treating inflammation in dengue fever patients.

Embodiment 4:



[0039] In this Embodiment, an anti-CD44 antibody (MA4400, Invitrogen, CA, U.S.A.) of 5 µg/ml to 500 µg/ml was used to inhibit the CD44 surface antigen of the vascular endothelial cells of the dengue fever patients. First, the human vascular endothelial cells (4 × 105 cells cultivated in a 60-mm dish) were treated with the anti-CD44 antibody for 12 hours followed by the treatment of DENV NS1 (3 µg/ml, Cat. No. ENZ-PRT105-0100, Enzo Life Sciences, Inc., NY, U.S.A.). Subsequently, the cellular proteins were extracted and subjected to the SDS-PAGE to separate the proteins according to their molecular weights. Next, the proteins in the gel were transferred to a PVDF membrane, and the p-Akt signal on the PVDF membrane was detected using Western blotting and the p-Akt antibody. The results showed that a range from 5 µg/ml to 500 µg/ml is an effective blood concentration of anti-CD44 antibody in the dengue fever patient to inhibit p-Akt in the vascular endothelial cells of the dengue fever patients, indicating that the anti-CD44 antibody can be used to inhibit the inflammation of the vascular endothelial cells in humans caused by the dengue virus (referring to Fig. 5). Therefore, the anti-CD44 antibody of the present invention can be used to prepare a pharmaceutical composition for treating inflammation in dengue fever patients.

Embodiment 5:



[0040] Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus, an in vitro endothelial permeability assay will provide insight into the establishment or progression of such diseases. The transwell permeability assays directly detect the penetration of a specific molecule, which can be detected by a spectrometer-based absorbance reader.

[0041] The permeability of human microvascular endothelial cells treated or not with DENV NS1 (3 µg/ml, Cat. No. ENZ-PRT105-0100, Enzo Life Sciences, Inc., NY, U.S.A.), was measured by an in vitro transwell permeability assay that mimics human endothelium in vivo (referring to http://www.bio-protocol.org/e2273). In brief, human microvascular endothelial cells (2 × 105) were grown in a 24-Transwell® (pore size: 0.4 µm, Cat. No. 3414; Corning, Kennebunk, ME, U.S.A.) for 5 days until a confluent monolayer was formed. Next, some of the samples were treated for 6 hours with DENV NS1 protein in the absence or presence of exogenously added hyaluronidase (0.5 unit/mL to 50 units/mL), media was aspirated, and 200 µL fresh serum-free medium containing 3 µL of streptavidin-horseradish peroxidase (HRP) (GE Healthcare, Cat. No. RPN1231, UK) was added. To measure the HRP that had leaked through the endothelial layer, the inserts were moved to a new 24-well plate with 500 µl of serum-free medium, covered and placed in a 37°C incubator for 5 minutes. Twenty microliters of medium, from each lower chamber, was transferred to a 96-well plate and analyzed for HRP activity by adding 100 µL 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, Cat. No. T4444, St. Louis, MO, U.S.A.). Color development was detected by an EnSpire Multimode Reader (PerkinElmer, Upplands Väsby, Sweden) at 450 nm. The results showed that the concomitantly added hyaluronidase could reverse the DENV NS1-induced endothelial hyper-permeability significantly (referring to Fig. 6).

SEQUENCE LISTING



[0042] 

<110> Kaohsiung Medical University

<120> Evaluation of the Severity of Patients with Flavivirus Infection by
Blood Hyaluronan Levels and Therapeutic Agents to Block the Hyaluronan

<130> P45738-EP

<150> 106134411
<151> 2017-10-05

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<213> Artificial sequence

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<223> CD44 siRNA 1

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GAAUAUAACC UGCCGCUUU   19

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<212> RNA
<213> Artificial sequence

<220>
<223> CD44 siRNA 2

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CAAGUGGACU CAACGGAGA   19

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<213> Artificial sequence

<220>
<223> CD44 siRNA 3

<400> 1
CGAAGAAGGU GUGGGCAGA   19

<210> 4
<211> 19
<212> DNA
<213> Artificial sequence

<220>
<223> CD44 siRNA 4

<400> 1
GAUCAACAGU GGCAAUGGA   19




Claims

1. A method to determine whether a warning sign will occur in a subject being infected with a dengue virus and suffering from dengue fever, the method characterized by comprising steps of:

(a) providing a serum of the subject in a febrile phase of the illness of the subject;

(b) measuring a level of a hyaluronan in the serum; and

(c) determining that the warning sign will occur in an illness course of the subject when the level is higher than or equal to 70 ng/mL.


 
2. The method according to Claim 1, characterized in that the subject is a human, and the human is infected with the dengue virus in the febrile phase of the illness of the human.
 
3. The method according to Claim 2, characterized in that the human with the dengue fever shows the warning sign in the illness course, and the warning sign is one selected from the group consisting of an abdominal pain, an abdominal tenderness, a persistent vomiting, a clinical fluid accumulation, a mucosal bleed, a lethargy, a restlessness, a liver enlargement of more than 2 centimeters, an increase in a hematocrit (HCT) concurrent with a rapid decrease in a platelet count, and a combination thereof.
 
4. The method according to Claim 1, characterized in that the step (b) is performed in a well of a microplate where an aggrecan is coated thereon already, and the step (b) further comprises:

(b1) adding the serum to cause the hyaluronan which may exist in the serum to be bound to the aggrecan;

(b2) adding a biotinylated-proteoglycan to be bound to the hyaluronan;

(b3) adding a streptavidin-conjugated enzyme to be bound to the biotinylated-proteoglycan;

(b4) adding a substrate to react with the streptavidin-conjugated enzyme to generate a chemiluminescent reaction; and

(b5) determining an optical density at 450 nm to calculate the level of hyaluronan.


 


Ansprüche

1. Verfahren zur Bestimmung, ob ein Warnzeichen bei einem Subjekt auftritt, der mit einem Dengue-Virus infiziert ist und an Dengue-Fieber leidet, wobei das Verfahren die folgenden Schritte umfasst:

(a) Bereitstellen eines Serums des Subjekts in einer fieberhaften Phase der Krankheit des Subjekts;

(b) Messen eines Hyaluronsäurespiegels im Serum; und

(c) Bestimmen, dass das Warnzeichen in einem Krankheitsverlauf des Subjekts auftritt, wenn der Spiegel höher oder gleich 70 ng/ml ist.


 
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass das Subjekt ein Mensch ist und der Mensch mit dem Dengue-Virus infiziert ist und sich in der fieberhaften Phase der Krankheit des Menschen befindet.
 
3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, dass der Mensch mit dem Dengue-Fieber das Warnzeichen im Krankheitsverlauf zeigt und das Warnzeichen aus der Gruppe ausgewählt ist, die aus Bauchschmerzen, Empfindlichkeit im Bauchraum, anhaltendem Erbrechen, einer klinischen Flüssigkeitsansammlung, einer Schleimhautblutung, einer Lethargie, einer Unruhe, einer Lebervergrößerung um mehr als 2 Zentimeter, einer Zunahme eines Hämatokrits (HCT) gleichzeitig mit einer raschen Abnahme der Thrombozytenzahl und einer Kombination davon besteht.
 
4. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass der Schritt (b) in einer Vertiefung einer Mikroplatte durchgeführt wird, auf der bereits ein Aggrecan beschichtet ist, und der Schritt (b) ferner umfasst:

(b1) Hinzufügen des Serums, um zu bewirken, dass die im Serum vorhandene Hyaluronsäure an das Aggrecan gebunden wird;

(b2) Hinzufügen eines biotinylierten Proteoglykans, das an die Hyaluronsäure gebunden werden soll;

(b3) Hinzufügen eines Streptavidin-konjugierten Enzyms, das an das biotinylierte Proteoglycan gebunden werden soll;

(b4) Hinzufügen eines Substrats zur Reaktion mit dem Streptavidin-konjugierten Enzym, um eine Chemilumineszenzreaktion zu erzeugen; und

(b5) Bestimmen einer optischen Dichte bei 450 nm, um den Hyaluronsäurespiegel zu berechnen.


 


Revendications

1. Procédé pour déterminer si un signe d'avertissement se produira chez un sujet étant infecté avec un virus de la dengue et souffrant de la fièvre de la dengue, le procédé étant caractérisé comme comprenant les étapes de :

(a) fournir un sérum du sujet dans une phase fébrile de la maladie du sujet ;

(b) mesurer un niveau d'un hyaluronane dans le sérum ; et

(c) déterminer qu'un signe d'avertissement se produira dans un cours de la maladie du sujet lorsque le niveau est supérieur ou égal à 70 ng/mL.


 
2. Procédé selon la revendication 1, caractérisé en ce que le sujet est un être humain, et que l'être humain est infecté avec le virus de la dengue dans la phase fébrile de la maladie de l'être humain.
 
3. Procédé selon la revendication 2, caractérisé en ce que l'être humain avec la fièvre de la dengue montre le signe d'avertissement dans le cours de la maladie, et le signe d'avertissement est un choisi dans le groupe constitué par une douleur abdominale, une sensibilité abdominale, un vomissement persistant, une accumulation de fluide clinique, un saignement mucosal, une léthargie, une absence de repos, un élargissement du foie de plus de 2 centimètres, une augmentation dans un hématocrite (HCT) simultanée avec une diminution rapide d'un nombre de plaquettes, et une de leurs combinaisons.
 
4. Procédé selon la revendication 1, caractérisé en ce que l'étape (b) est effectuée dans un puits d'une microplaque où un aggrécane est déjà revêtu dessus, et l'étape (b) comprend en outre :

(b1) l'addition du sérum pour entraîner le hyaluronane qui peut exister dans le sérum à être lié à l'aggrécane ;

(b2) l'addition d'un protéoglycane biotinylé pour être lié au hyaluronane ;

(b3) l'addition d'une enzyme conjuguée à la streptavidine pour être liée au protéoglycane biotinylé ;

(b4) l'addition d'un substrat pour réagir avec l'enzyme conjuguée à la streptavidine pour produire une réaction chimioluminescente ; et

(b5) la détermination d'une densité optique à 450 nm pour calculer le niveau de hyaluronane.


 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description