Patent Application
20200207820
The present invention relates to novel natural compounds having highly potent preventing effects for HCV, HBV and their related diseases. In particular, the present invention relates to two proteins purified from Apis mellifera royal jelly (RJ) named as major royal jelly protein 2 and its isoform X1 having potent anti-HCV and HBV effects, highly improve the liver fibrosis and are moderately cytotoxic against HepG-2 cells.
Hepatitis C virus (HCV) and hepatitis B virus (HBV) are the most common etiologies for the liver diseases in the world. Since they are the main causes of liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Globally, the prevalence rate of HCV and HBV is approximately 170 and 350 million people worldwide, respectively. More than one million patients die every year from the complications of these viruses, mainly HCC which globally extends in many countries, particularly Asia and Africa. There is no vaccine for HCV infection till now while; an effective vaccine was developed for HBV since 1976.
HCV consists of a single-stranded (+) RNA and is classified into the genus Hepacivirus of the family Flaviviridae. However, HBV is double stranded DNA virus from the genus Orthohepadnavirus of family Hepadnaviridae. Recently, HCV and HBV replication can be observed in vitro using human hepatocellular cancer cell line (HepG-2) and peripheral blood mononuclear cells (PBMCs), which facilitate the evaluation of the antiviral compounds. HCV and HBV can replicate in these cells and therefore these systems are used for identifying the compounds that inhibit the replication of these viruses.
The treatment target for HCV and HBV is to inhibit their replication, reduce inflammation in the liver, reverse fibrosis and stop the progression of cirrhosis and HCC. HCV is treated since 2002 with the interferon-free direct acting antivirals (IFN-free DAA) which are effective drugs able to cure HCV after 8-12 weeks. Sofosbuvir (Sovaldi, SOV) is one of the newest types of these drugs that used with other combinations for the treatment of HCV infection. Despite the efficiency of these drugs, they have many side effects such as osteoporosis, renal toxicity, and pulmonary hypertension. In addition, the intake of these drugs is not safe for pregnant and breast feeding women due to the association of some of them with teratogenic effects in animals. Therefore, none of these DAA has yet been approved by the U.S. Food and Drug Administration (FDA) during pregnancy and lactation. Furthermore, most of these drugs make reactivation for HBV in some HCV infected patients. On the other hand, the treatment strategies for HBV are using IFN/pegylated IFN or nucleoside analogs or their combination for long-term. But, the long-term intake of these drugs is not safe on patients and cause potential renal toxicity besides the possible development of drug resistance. Therefore, the discovery of an effective and safe therapy for HCV and HBV is of great importance and the need of the day.
RJ (bee's milk) is a creamy, whitish product secreted from mandibular and hypopharyngeal glands of nurse bees (Apis mellifera). It is the specific food for the queen bees and helps in their development from the worker bee larva with the age between 5-15 days. RJ is highly acidic substance composed mainly of water (60-70%), proteins (9-18%), sugars (7-18%), and lipids (3-8%) with other compounds. About 80% of the RJ proteins are water soluble belonging to the major RJ protein (MRJP) family, which comprises 9 members (MRJP1-MRJP9) with a molecular mass of 49-87 kDa. To date, the antiviral, antifibrotic and the anticancer activities of RJ and its components are undefined.
RJ (
The two purified MRJPs were identified using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF Ms). The novel MRJP2 isoform X1 is identified as a predicted protein in the database and thus this invention has the priority to confirm its expression from the Apis mellifera genome
The two purified MRJPs have proven their efficacy against HCV and HBV in PBMCs and HepG-2 cells as well as in CCl4-induced fibrosis in hepatocytes and have moderate, not potent anticancer effect. In addition, the two proteins prevent the HCV and HBV cellular entry. The exact anti-HCV and anti-HBV mechanism of the RJ proteins is under investigation.
The acute toxicity of the RJ on the Albino rats was tested. RI has proven its safety on the animal vital organs and interestingly multiple activities were observed like hypolipidemia and enhancing kidney function.
All the studied activities for the purified RJ proteins were compared with Sovaldi. Although Sovaldi showed more potent anticancer effect than the RJ proteins, it exhibited less anti-HCV and less antifibrotic activities. Not only this but also SOV increases the activity of HBV and had highly toxic effects on kidney, spleen and lung and less toxicity on the liver.
As used herein, the term “fractionation” means a separation process in which a certain mixture is divided into a number of smaller quantities (fractions). The term “protein purification” as used herein refers to a technique by which a single protein type is isolated from a complex mixture. Therefore, the protein fraction may contain two (For example, PF30 and PF50) or more (For example, PF60 and the CPF) proteins. The purified protein or fraction means single protein with fewer impurities (For example MRJP2 and its isoform X1).
The term “iso form” as used herein means a protein that has the same function as the original protein but which is encoded by a different gene and may have small differences in its sequence. Here the MRJP2 and its isoform X1 are encoded by different genes and having slightly different in their sequences.
The term “predicted protein” means the protein which its functional expression is not yet shown in experimental studies and its prediction occurred by automated computational analysis for the genome of the organism.
The term “hepatocytes” refers to the epithelial parenchymatous cells of the liver which make up 70-85% of the liver's mass and responsible for most of the liver functions.
The term “collagen” refers to the main structural protein in the extracellular space in the various connective tissues in animal bodies. As the main component of connective tissue, it is the most abundant protein in mammals. Glycine, proline, hydroxyproline, and hydroxylysine are the main amino acid composition of collagen.
The term “dialysis” means the separation of substances in solution by means of their unequal diffusion through semipermeable membranes. For example, a separation of colloids from soluble substances. Here, the prepared protein fractions from RJ were dialyzed using a semipermeable membrane to remove the impurities from the isolated proteins such as ammonium sulfate, protease inhibitors, and all other associated small molecules.
The term “lyophilized” or “freeze dried” means to dry something (For example, food) in a frozen state under high vacuum especially for preservation. In this invention, the isolated fractions from RJ were freeze dried to obtain the powdered form for accurate preparation of different concentrations for the analyses.
As used herein, the term “crude protein” means all the water soluble proteins in the RJ.
The term “modified protein” refers to the proteins which subjected to the post-translational modification process through the attachment to specific non-protein group. Glycoproteins are that contain oligosaccharide chains (glycans or sugars) covalently attached to polypeptide side-chains. The process of attachment of sugars to the protein named glycosylation.
The term “proteolysis” or “proteolytic” refers to the hydrolysis of proteins or peptides with the formation of simpler and soluble products (digest). This process can be occurred enzymatically using trypsin, a serine protease that specifically cleaves proteins (enzyme substrate) at the carboxylic side of lysine and arginine residues. Here, the sequencing modified grade trypsin was used which is a modified trypsin by reductive methylation of the lysine residues in the porcine native enzyme yielding a highly active and stable molecule. The resulting enzyme is extremely resistant to autolytic digestion to provide maximum specificity for the PMF analysis by MALDI-TOF.
“PI” or “isoelectric point” of a protein means the pH at which the positive charge of the protein balances with its negative charge.
This invention provides two purified proteins from RJ (obtained from the local market, Egypt) having high potency in the prevention of HCV and HBV replication and improving their related liver diseases, fibrosis and cancer.
RJ was separated into three distinct fractions, sugars, lipids and proteins and their yields are recorded in Table 1.
For the preparation of sugar fraction, 2 g of RJ was dissolved in water/methanol mixture (3:1) and deproteinized using Carrez I (potassium hexacyanoferrate II) and Carrez II (zinc acetate) reagents. Then, lipids were removed by washing the deproteinized RJ two times with dichloromethane. The aqueous layer (sugar fraction) was filtered through 0.2 μm disposable syringe filter, quantified, lyophilized (Telstar, Terrassa, Spain) and kept at −80° C. until used.
Lipids were isolated from RJ with petroleum ether using Soxhlet apparatus for 30 mM. The organic solvent was evaporated, and then the lipid fraction was weighed and stored at −80° C.
The water soluble proteins were extracted from RJ using ammonium sulfate crystals (Brixworth, Northants, UK). In brief, 1.5 g of RJ was dissolved in phosphate buffer saline (PBS, 0.1 M, pH 7) containing 1× protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo., USA) and the solution was centrifuged at 3800 g and 4° C. for 30 min. Then the water soluble proteins in the supernatant were precipitated by adding crystals of ammonium sulfate until the saturation reach to 60%. Pellet (crude protein fraction, CPF) was dissolved in PBS, dialyzed for 24 h against the same buffer and finally freeze dried to obtain the powdered fraction. The protein content in the prepared fraction was quantified using Bradford's method.
The three prepared fractions (sugars, lipids, and proteins) were tested for the anti-HCV efficacy and the results have proven the potency of the CPF only.
To know the effective anti-HCV protein in the CPF, it was subjected to ammonium sulfate precipitation using different degrees of saturation (5, 10, 15, 20, 30, 50 and 60%). The precipitated proteins were obtained by centrifugation at 3800 g (4° C.) for 30 min and the protein content was examined in each fraction using Bradford method. The fraction containing detectable protein content (30, 50 and 60) were dialyzed for 24 h against PBS, lyophilized and then analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The purification method of MRJP2 and its isoform from RJ is a novel method.
PF50 was used for purification of MRJP2 and MRJP2 isoform X1 using carboxymethyl (CM)-Sephadex ion-exchange column chromatography. The amount of PF50 that obtained from 10 g of RJ was dissolved in 20 mL of the binding buffer (20 mM phosphate buffer containing 1× protease inhibitor cocktail, pH 6.7). The protein solution then applied to CM-Sephadex column (16×2.5 cm) and left for 1 h at 4° C. The unbound protein (MRJP2 isoform X1, fraction 1) was obtained by washing the column with about 100 mL of the binding buffer. Elution of the bound protein (MRJP2, fraction 2) was achieved by a one-step gradient of about 50 mL of 0.5 M NaCl in the binding buffer. The protein content was determined in the purified fractions by UV measurement at 280 nm after dialysis for 24 h against PBS (pH 7). Both dialyzed fractions were freeze dried, then quantified by Bradford and analyzed by SDS-PAGE. The results showed that the protein content in 1 mg of the fraction 1 and fraction 2 powders were 0.95 and 0.5 mg protein, respectively. This observation indicates that nearly half the weight of fraction 2 (MRJP2) is non-protein which means that MRJP2 is a modified protein. Several studies reported the glycosylation of MRJP2 at several sites, therefore; the loss in weight of fraction 2 powder refers to the attached carbohydrates. In addition, MRJP2 isoform X1 may be unmodified or slightly modified protein.
These two proteins were identified through determination of their molecular weights by SDS-PAGE and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). In addition, peptide mass fingerprinting (PMF) after tryptic digestion was used for identification of these proteins using MALDI-TOF MS instrument.
The prepared CPF and each of its precipitated PFs were analyzed by 14% SDS-PAGE (
Matrix assisted laser desorption ionization-time of flight mass spectrometry
The MALDI-TOF Ms was used to identify the purified proteins from PF50 through their molecular weights and their PMF after tryptic digestion. In-solution digestion of the proteins was performed by dissolving 200 μg of each lyophilized PF in 100 μL of 50 mM sodium bicarbonate (pH 8.0). Cysteines were reduced by 5 μL of DTT (45 mM) and alkylated by 5 μL of iodoacetamide (IAA, 100 mM), and then another amount of DTT was added to destroy the excess of IAA. Afterward, proteins were subjected to proteolysis by trypsin modified sequencing grade at a (enzyme:substrate) ratio of (1:50) and incubated at 37° C. for 24 h. The reaction was terminated by addition of 10 μL of 10% trifluoroacetic acid (TFA). One microliter of the tryptic digest was added to the preloaded MALDI plate with 14, of the MALDI matrix (saturated solution of α-cyano-4-hydroxycinnamic acid in 2.5% TFA and 50% acetonitrile) and air dried. The masses of the tryptic peptides were determined using MALDI-TOF MS UltraFlex system (Bruker Daltonics GmbH, Bremen, Germany). The analysis was done in the linear positive ion mode in the mass/charge (m/z) range of 500-4000 Da using FlexControl software version 3. The generated spectra were compared to the database (fingerprint) using the Bruker Biotyper software (version 3.1) and a library of 5,623 entries.
The results obtained in
The present study evaluated the anti-HCV and anti-HBV effects of RJ and its isolated fractions for identification of the responsible ingredients. The anti-HCV activity of RJ and its isolated fractions include lipids, carbohydrates, CPF, PF30, PF50, PF60, MRJP2 and MRJP2 isoform X1 in comparison with SOV was examined qualitatively and quantitatively. Then, the most effective anti-HCV RJ fractions were studied for their anti-HBV effects quantitatively. The antiviral activity was evaluated in vitro using two types of host cells, PBMCs and HepG-2.
PBMCs were obtained by Ficoll-Hypaque density gradient centrifugation method as described previously. In brief, the blood samples from healthy volunteers were diluted with equal volume of PBS, carefully layered on Ficoll-Hypaque, and centrifuged at 2000 rpm, 25° C. for 30 min. Then the undisturbed PBMCs layer (interface) was carefully transferred out, washed with 40 ml RPMI-1640 medium, and centrifuged at 1650 rpm for 10 min. Finally, the supernatant was removed and the cells were suspended in 5 ml of RPMI-1640 medium containing 10% FBS and counted using trypan blue stain.
HepG-2 cells were grown in RPMI-1640 medium (HyClone) supplemented with 10% heat-inactivated FBS.
Serum samples were obtained from HCV and HBV infected Egyptian patients “A.R.” after agreement by the ethics committee. The HCV and HBV samples were positive for genotype 4a and D, respectively. The infected patients were neither under treatment prior to the study nor co-infected. The sera were kept at −80° C. until use.
The viral host cells, human PBMCs (1×106 cells) and HepG-2 (1.5×105 cells) were seeded in each well of 12-well culture plate and 6-well culture plate, respectively. All wells were incubated in the CO2 incubator (New Brunswick Scientific, Netherlands) with either HCV (2.9×105 copies/ml) or HBV (1×105 copies/ml) infected serum in RPMI-1640 medium for 2 h at 37° C., 5% CO2 and 95% humidity.
After cellular infection, the infected medium was replaced with a fresh RPMI-1640 medium containing 10% FBS for positive control wells. For treated wells, the infected medium was exchanged with RPMI-1640 medium containing 10% FBS and 200 μL of RJ (0.2 and 1 mg), or one of its fractions including carbohydrates, lipid and CPF (at a dose equivalent to their amounts in 0.2 mg RJ). Additionally, 1 mg of PF30, 50, 60, 250 μg of MRJP2 and MRJP2 X1 or 4 mg of SOV were tested. Two control cultures were included, positive (infected cells only) and negative (uninfected cells). All cells were incubated at 37° C., 5% CO2 and 95% humidity for 72 h, then the total RNA was extracted from the treated and untreated cells following the Gene JET RNA purification Kit protocol (Thermo Scientific, USA). The HCV (+) strand was detected by reverse transcription-nested polymerase chain reaction (RT-nested PCR) using the Ready-To-Go RT-PCR beads (Amersham Pharmacia Biotech, Piscataway, N.J., USA) following the instructions. The primer sequences that used in this PCR are derived from the highly conserved 5′-untranslated region (5′-UTR) of HCV genome. Finally, the PCR products were electrophoresed on 2% agarose gel and the gel image was visualized on a UV transilluminator and photographed using gel documentation system.
Results revealed the anti-HCV effect of RJ at the high and low concentrations, CPF, PF30, PF50, MRJP2, MRJP2 isoform X1 and SOV and the other studied fractions had no anti-HCV activity. The positive HCV samples revealed one band with a molecular size of 174 bp [(+) strand RNA amplified products] (
Different concentrations of RJ (0.2 and 1 mg), PF50 (0.25, 0.5, 1 mg), MRJP2 and its isoform X1 (250 μg, 100, 50, 25 ng) were incubated with the HCV-infected PBMCs or HepG-2 cells. In addition, PF50 (1 mg), MRJP2 and MRJP2 isoform X1 (250, 500 μg) were incubated with HBV-infected cells. SOV at the concentration of 4 mg was incubated only with HCV or HBV-infected PBMCs due to its potent anti-cancer effect. All cells were incubated for 72 h in the CO2 incubator. Then the HCV-RNA and HBV-DNA within these cells were quantified using the fully automated Cobas Ampliprep TaqMan Analyzer (CAP-CTM). This technique is one of the most recent techniques that used for diagnosis of viral hepatitis quantitatively. All steps were automated and include the extraction of RNA or DNA on the Cobas AmpliPrep instrument and simultaneously, the PCR amplification and detection on the Cobas TaqMan analyzer. The procedure was done following the manufacturer's instructions.
To examine the effect of RJ and its protein fractions on the viral entry, 1 mg of each of PF50, MRJP2 and MRJP2 isoform X1 or 4 mg of SOV were incubated with PBMCs or HepG-2 (not used with SOV) for 10 min before infection. These cells were infected as described above with either HCV or HBV in RPMI-1640 medium for 2 h in the CO2 incubator. Then the medium was discarded and medium containing 10% FBS was added to all wells. Two control cultures were included and incubated at the same conditions, positive (infected cells only) and negative (uninfected cells). After 24 h incubation, cells were washed and the viral load within the cells was quantified by fully automated Cobas Ampliprep TaqMan Analyzer (CAP-CTM). Tables (2-5) summarize the obtained results.
The anti-fibrotic effect of RJ and its protein fractions (PF50, MRJP2, and MRJP2 isoform X1) and SOV in comparison with silymarin (SM) standard drug were studied using hepatocytes.
Hepatocytes were isolated from the liver of two male Albino rats 1 week of age. Rats were anesthetized, then liver was dissected, washed and incubated for 15 min with 10× penicillin and streptomycin. Afterward, the liver was washed using PBS, minced and incubated for 30 min with 3 U/mL of collagenase I with gentle shaking to obtain the cell suspension. The solution was centrifuged for 5 mM at 1000 rpm and the pellet (hepatocytes) was washed, suspended in William's E medium containing 10% FBS and incubated at 3TC in CO2 incubator. After 90% cell confluent, hepatocytes were passaged with trypsin-EDTA. Then cells were stained with trypan blue, checked for viability and counted using the phase contrast inverted microscope (Olympus IX 81, Tokyo, Japan).
The cytotoxicity of the studied compounds on the hepatocytes was done using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In brief, about 104 cells/well were seeded in 96-well cell culture plate and treated separately with serial dilutions of RJ, PF50, MRJP2, MRJP2 X1, SOV, and SM. In addition, the untreated cells were included as negative control. All plates were incubated in the CO2 incubator at 5% CO2, 37° C., and 90% relative humidity and after 72 h, 20 μL of 5 mg/mL MTT was added/well and incubated for further 4 h. Then plates were centrifuged at 2000 rpm for 10 min and 150 μl DMSO was added to each well after supernatant aspiration and the absorbance was read at 570 nm using ELISA reader (BMG LabTech, Germany). Cell viability was determined and the safe concentrations (EC100, 100% cell viability) were calculated (Table 6).
For induction of fibrosis, hepatocytes were incubated with various concentrations (0.13-1.30 mM) of CCl4 for 48 h. At the end of the incubation period, the cell viability was assessed and the IC50 value (half maximal inhibitory concentration of CCl4 to the hepatocytes growth) was calculated by the GraphPad Instat software version 3. This value was used for in vitro induction of fibrosis in hepatocytes (FM). After induction, Fb1 was incubated with the safe concentration of each of the studied compounds and incubated for 72 h at 5% CO2 and 37° C. The normal cells and the untreated Fb1 (Fb) were involved as negative and positive control cells, respectively.
At the end of the incubation period, media were collected and used in the assessment of the hepatocyte damage indices. However, cells were examined morphologically for identification of apoptosis.
Alanine and aspartate aminotransferases (ALT and AST), and albumin were determined using available commercial kits (RAM, Egypt). TNF-α (ELISA kit, RayBiotech, USA) and collagen IV (ELISA kit, Kamiya Biomedical) were determined following the manufacturer's instructions. Nitric oxide (NO) level was assessed by measurement of the nitrite using the Griess reaction, which produced colored azo dye with a maximum absorbance at 490 nm. Hydroxyproline content in the medium was measured using chloramines T and dimethylaminoborane solutions. The absorbance of the produced color was read spectrophotometrically at 560 nm and the concentration was determined from the hydroxyproline calibration curve.
Apoptosis was investigated by double staining cells after washing with PBS with 100 μg/mL of ethidium bromide (EB) and acridine orange (AO) as described previously and then visualized under the fluorescent phase contrast microscope.
As shown in
The cytotoxic effect of RJ and its isolated PFs against HepG-2 cell line were evaluated. The cell viability was determined by MTT assay to investigate the cytotoxicity. Cells (3×103/well) were seeded in 96-well plate and left to attach for overnight. Serial concentrations (6.25-100 μM) of each of the tested compounds were added to the attached cells and incubated for 72 h at 37° C. in an atmosphere of 5% CO2 and the untreated cells were included as negative control. Then, 20 μl of MTT solution (0.5 mg/ml) was added to each well and the plate was incubated at 37° C. and 5% CO2 for 4 h. The medium with MTT was replaced by 150 μl of DMSO and the absorbance of the produced color was read at 570 nm by the microplate reader. The concentration of each of the studied compounds that inhibit the cancer cell growth by 50% (IC50 values) was determined from the dose-response curves. Additionally, the morphological changes of the tumor cells were examined using the phase-contrast microscope.
The results in
Karber's method was used to study the acute toxicity effect of RJ in comparison with SOV using male Albino rats and the median lethal dosage (LD50) was calculated.
Fifty-five male Albino rats (80-120 g) were purchased from MISR University for Science and Technology (animal welfare assurance number A5865-01). Animals were housed in metal cages and allowed free access to a standard commercial diet and tap water. Rats were kept under conventional conditions of temperature, humidity and twelve hours light/dark cycle. All rats were acclimatized to the laboratory environment for one week prior to handling and were observed daily for abnormal signs. Handling of animals complied with the international guide for the care and use of laboratory animals (National Research Council, 1996).
Animals were divided into eleven groups (five animals in each) starting from group I to group X beside the control (received only water as the vehicle). Groups from (I) to (V) were administered RJ intraperitoneally, while groups from (VI) to (X) were orally received SOV using the gavage tube. RJ and SOV were given to the animals at various doses (140, 350, 700, 2500 and 5000 mg/kg bw), one dose for each group of animals (group I and VI received the lowest dose).
All animals were left for 24 h after RJ or SOV administration, and then the number of mortality in each group was recorded for calculation of the LD50 value. Also, the weights of animals in each group were recorded before sacrificing by decapitation. Blood samples were collected in heparin tubes by cardiac puncture for assessment of the erythrocytes, hematocrit (HCT), hematimetric indices, hemoglobin (Hb), platelets, and leukocytes (full and differential counts). In addition, ALT and AST, total proteins, albumin, urea, creatinine, sodium, potassium, triglycerides (TG) and cholesterol were determined using available commercial kits (RAM, Egypt). The vital organs including liver, lung, kidney, and spleen were carefully excised, weighed and examined.
The following results were obtained:
In summary, RJ has been shown to be safe at a dose higher than 5 g/kg in rats. However, SOV is toxic at all the studied doses starting from the dose of 0.14 g/kg of rat bw and the most affecting organs by it are kidney, spleen, and lung while the liver is the less affecting one.
Data were expressed as mean±SE and were analyzed by SPSS version 16. The mean values were compared using one-way analysis of variance (ANOVA) by Duncan's test and significance was determined at P<0.05. IC50 and EC100 values were calculated by the GraphPad Instat software version 3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20170701196 | Jul 2017 | EG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EG2017/000022 | 8/7/2017 | WO | 00 |
