Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to polypeptide-containing compound(s) (hereinafter "PROTEINA” ) , composition (s) comprising such compound (s) along with divalent metal ions and/or a carbohydrate moiety (hereinafter "ALICIN”), their antiviral and anti-hepatoma activities, and the enhancement of activity of antiviral polypeptide-containing agents by their combination with a carbohydrate and at least one divalent metal ion.
• the carbohydrate is a polysaccharide that consists essentially of arabinose and galactose and, independently, the at least one divalent metal ion consists essentially of magnesium and zinc.
• the carbohydrate and divalent metal ion can be present in the form of a complex.
• the invention relates to a composition
• a composition comprising an amino acid-containing compound having the formula :
• each R is independently at least one amino acid or analog thereof.
• the compound has the formula:
• the above composition (s) further include a carbohydrate .
• the carbohydrate can be a monosaccharide, i.e. (CH 2 0) , where n is at least 3, preferably hexoses such as glucose, mannose, galactose and pentoses such as arabinose and the like or one or more of the known disaccharides .
• the carbohydrate can also be a homo- or hetero- polysaccharide. When the carbohydrate is a polysaccharide, it is preferably a homo- or hetero- polymer of glucose, mannose, arabinose, galactose or monosaccharide amine .
• a particularly preferred polysaccharide is one that consists essentially of arabinose and galactose, particularly in a weight ratio of about 1:0.05, respectively.
• the composition further comprises at least one divalent metal ion.
• the divalent metal ion is selected from the group consisting of magnesium and zinc. It is particularly preferred that they be present in a weight percent of about 4 - 20 and 2 - 10, respectively, of the total composition. Range of ratios between Mg and Zn is generally 1:1 to 3:1, preferably 2:1, and their over all weight percentage in the composition is 6 - 30 weight percent of the composition.
• Another aspect of the invention relates to a method for treating a viral infection in an individual in need thereof which comprises administering a therapeutically effective amount of the composition (s) .
• Particularly contemplated viral pathogens intended for treatment include Hepatitis A virus, Epstein-Barr and Influenza A viruses .
• Another aspect of the invention relates to a method for treating a hepatoma in an individual in need thereof which comprises administering a therapeutically effective amount of the composition (s) .
• compositions which comprises the composition of an antiviral polypeptide-containing agent, such as one or more of the interferons, trichosanthin and others, in combination with a carbohydrate, particularly a carbohydrate such as those described above, and at least one divalent metal ion, particularly such as those described above.
• carbohydrate is a polysaccharide that consists essentially of arabinose and galactose and, independently, the at least one divalent metal ion consists essentially of magnesium and zinc.
• substantially homologous means that a particular subject sequence, for example, a sequence of amino acid analogs, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between reference and subject sequences.
• sequences having greater than 90 percent homology, equivalent biological activity are considered substantially homologous.
• Sequences having lesser degrees of homology, but comparable bioactivity, are considered equivalents .
• the PROTEINA polypeptide-containing portion of the A ICIN composition (s) , its fragments or other derivatives, or analoqs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
• These antibodies can be, for example, polyclonal, monoclonal, chimeric, single chain, Fab fragments, or an Fab expression library, various procedures known in the art may be used for the production of polyclonal antibodies .
• any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature 256 :495-497) , the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al . , 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce specific single chain antibodies.
• the antibodies can be used in methods relating to the localization and activity of the protein sequences of the invention, e.g., for imaging these proteins,, measuring levels thereof in appropriate physiological samples and the like.
• Modes of administration of the compound (s) and composition (s) include but are not limited to intravenous, intramuscular and subcutaneous routes as well as by suppository.
• the compounds may be administered by any convenient route, for example by infusion or bolus injection and may be administered together with other biologically active agents .
• Administration is preferably systemic .
• compositions comprise a therapeutically effective amount of the, and a pharmaceutically acceptable carrier or excipient .
• a pharmaceutically acceptable carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
• the formulation should suit the mode of administration.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection.
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• compositions can also often be administered rectally, using suppositories, particularly for sustained-release administration or administration to very young, old, infirm or those for whom other routes of administration present unusual obstacles.
• Suppository formulations include an appropriate amount of the compound (s) /composition (s) of the invention in a suppository base.
• Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
• gelatin rectal capsules can be used whose base or excipient includes liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons .
• the therapeutics of the invention can be formulated as neutral or salt forms.
• Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids , etc . , and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine , 2-ethylamino ethanol, histidine, procaine, etc.
• the compound and composition are used in at least 5 ⁇ g/kg body weight and most generally need not be more than 500 ⁇ g/kg. Preferably, it is at least about 20 ⁇ g/kg and usually need not be more than about 100 ⁇ g/kg.
• the compound is typically administered for a period of at least about 7 days but generally not to exceed 30 days, with a typical therapeutic treatment period of 7 to 14 days . It will preferably be administered rectally by suppository, one to three times per day, and will be adjusted to meet optimal efficacy and pharmacological dosing.
• the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
• Associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products,, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• Fresh root tubers (1-0 kg) of the Zei-Bai plant (ZB) was obtained from local vendors of produce and farmers in the He-Nan province, PRC- After the skin of the tuber was removed, its juice (approximately 200 ml) was extracted using a Panasonic Juice Extractor.
• the extract were chilled to 4° C on an ice bath and adjusted to pH 4 by dropwise addition of 2M hydrochoric acid. While precipitates occured, slowly added 0.8 volume of the iced acetone to the chilled extracts . Centrifuge and discard the precipitates using Beckman Centrifuge at 4° C and 3000 rpm for 20 minutes.
• SDS-Page revealed one large protein band (85%) and three other minor bands .
• the large band has a molecular weight approximately 21 kD.
• the above prepared lyophilisate was reconstituted to about 200 ml and maintained at 4° C overnight observed precipitate was removed by centrifugation at 3000 rpm for 30 minutes at 4° C.
• the supernantant was loaded over a Ricinus communis Agglutinin affinity column (Sepharose 4B, 2.5x58 cm) which was prepared following the procedures of Dulaney (Mol . Cell. Biochem. 21, 43-63, 1978) .
• the glycoprotein was displaced from the column with a carbohydrate buffer (0.1 M phosphate and 0.2 M galactose, pH 7.2) . After lyophilization, about 1.0 q of the glycoprotein was obtained.
• ALICIN was dissociated into the polypeptide component, PROTEINA, as follows.
• ALICIN (1-0 g) prepared as described above was dissolved in 20 ml solution composed of 8M urea and 0.1M phosphate buffer (pH 7.2). Centrifuge and discard the undissolved solid with the Beckman Centrifuge at 4° C, 3000 rpm for 20 minutes. Pass the supernantant through a CM Sepharose C-50 colum (5 x 53 cm) , eluted the protein by applying 2L buffer of 8M urea and O.IM phosphate at pH 7.2 and followed at pH 9 respectively. The flow rate was 36 ml/hr with each collected fraction of 1ml per tube. The protein content was detected by 280 nm absorption, one large peak of the Protein was observed. The protein solation from each tube was polled and dialyzed against double distilled water for 48 hours at 4° C with several buffer changes.
• PROTEINA (20 mg) , prepared as described in Example 2, was dissolved in 70% formic acid (1 ml) . An excess amount (50 times) of cyanogen bromide solution was added and mixed continuously with the protein solution at 20° C in the dark for 24 hours. During this time, nitrogen was also continuously added to the mixture . At the end of this protocol, the protein was broken down to consitutent fractions . The hydrolyzed peptide fractions were diluted with ten volumes of d.d. water and freeze-dried.
• the hydrolyzed peptide fractions (5 mg) were dissolved together in 0.3 ml of elution solution (8 M urea and 10% HoAc) and passed through a Sephadex G 25 column to achieve homogeneity and remove cyanogen bromide .
• the column flow rate was 4 Ml/h.
• the peptide fractions (0.7 ml/tube) were pooled and processed for amino acid sequence analysis using a Beckman 890C amino acid sequence autoanalyzer . The results obtained by computer analysis are shown in Table 1.
• This protein showed a negative content of carbohydrate based on the periodic acid test.
• the atomic emission analysis of the protein indicated the absence of Mg and Zn ( ⁇ 0.001%) .
• ALICIN (1.0 g) , purified as described in Example 1 (>95%) in double distilled water (10 ml) .
• the resulting protein solution was boiled on a steam bath for 20 minutes to denature the protein.
• Carbohydrate was separated from the denatured protein by the method of Sevag (Staub, A.M. , Removal of Proteins from polvsaccarides , Methods in Carbohydrate Chem. 1965, 5:5). Approximately 217 mg of polysacchride was detected and determined.
• a portion of the polysacchride (100 mg) from step 1 was further purified using Sephadex G-75 column (2 x 100 cm) and eluted with d.d. water. Fractions were collected and showed a positive absorption at 620 nm when reacted with Anthrone Reagent. Purified polysacchride (about 55 mg) was obtained.
• a portion of the purified Polysacchride (10 mg) from step 2 was hydrolysed to monosacchrides by mixture with 2 ml concentrated sulfuric acid (2 ml) at 100° C for 3 hours. Saturated Ba(OH), was added to neutralize the acid solution. BAi30, was removed by centrifugation (3000 rpm for 20 minutes) . The supernantant was vacuum dried by rotary evaporator. Hydrolysed monosacchrides (about 9 mg) were obtained.
• a portion of the hydrolyzed monosacchrides (5 mg) from step 3 was analyzed for sugar 6ontent by gas chromatography (Gas Chromatogram-model 103 , Shanghai Analytical Instruments Corp.) .
• DC-200 column (i.d. 3 mm, length 2 m) and FID detector were used for analysis. Arabinose and galactose were identified as the sugar components present, and were observed in a weight ratio of 1:0.05.
• ALICIN 500 mg
• d.d water 10 ml
• dialyzed in 20 volume of d.d. (20 volumes) water at 4° C for 24 hours with several water changes. Collect the protein solution inside the dialysis bag, and the dialysate solution outside the bag separately and evaporating each to 1 ml volume. Add 1 ml concentrated acid solution (nitric acid:hydrochloric acid 1:1) to each of the tube to hydrolize the protein. Dilute each of the solution with d.d. water to 6 ml .
• HAV suspension was purchased from the Center of China Preventive medicine in Shanghai . It was diluted with Hank's solution (Sigma, St. Louis, MO) to give a final concentration of 200TCD50 per 0.1 ml .
• Preparation of human serum samples containing HAV Prepare six samples of 0.5 ml heat (56°C) inactivated healthy human serum. Each sample has the following dilutions with the Hank's solution: 1:4,1:8,1:16,1:32,1:64,1:128. Add 0.5 ml of HAV suspension from step 1 to each sample and vortex thoroughly. Incubate the sample mixtures at 37°C for 1 hour .
• Hepatic cell line (BEL-7405) was purchased from Shanghai Cell Biology Institute. Prepare 24 samples of 0.8 ml cell suspension (BEL-7405), each sample contains a cell concentration of 3xl0 6 . Add 0.2 ml each of thd six HAV- serum samples to 6 BEL-7405 cell suspensions respectively. Repeat this protocol 4 times . A group of four identical samples composed of HAV-serum- hepatic cell were thus prepared at each serum dilution.
• step 4 Incubate all 24 samples from step 3 in an incubator at 37°C until ready for experiments. Examine and count the number of infected cells in each tube daily for ten days . Determine the serum dilution which leads to a 50% cell infection. This serum dilution was found to be 1:45 and was used as the control sample and for all other samples prepared below.
• step 5 Prepare 48 samples containing HAV-serum-hepatic cells according to the protocols of step 3 except that the serum dilution used is determined at step 4 (i.e. 1:45). The samples were divided into 6 groups, and each group had 8 identical tubes. The six groups are defined below:
• Group 1 control samples .
• Group 2 control samples added with 400 ug of 85% pure ALICIN.
• Group 3 control samples added with 400 ug of pure
• PROTEINA Control samples added with 400 ug of combination (200 ug pure PROTEINA, 104 ug polysacchrides (prepared in example 4) and 96 ug divalent metals prepared in example 5) .
• Group 5 control samples added with 96 ug of divalent metals .
• Group 6 control sarnies added with 104 ug of polysacchrides .
• Table 2 clearly shows that all 5 formulations inhibit the growth of HAV.
• PROTEINA combination formula can irreversibly neutralize the virus completely in-vitro at 400 ug.
• the ALICIN (85%) at the same concentration can neutralize virus up to 83% and the PROTEINA at the same dosage can neutralize virus up to 66%.
• the white cell suspension was transfered to a glass dish and incubated on water bath at 37°C for 40 minutes. Most of the monocytes were adhered to the glass wall. Remove lymphocyte cells . Twenty test tubes containing lymphocyte cells were prepared. Each tube had 1 ml of cell solution and a cell concentration of 2xl0 6 /ml.
• EBV suspension 200TCD50 was purchased from Center of China Preventive Medicine. 0.1 ml of EBV suspension was added to the above prepared 20 lymphocyte tubes (step 1) .
• group 2 Irradiation of EBV with X-ray (4000 R) for 1 minute before added to lymphocyte tubes .
• group 3 200 ug of the combined formula of PROTEINA + polysacchride + divalent metals was added to lymphocyte tubes .
• group 4 400 ug of the combined formulation of PROTEINA + polysaccharide + divalent metals was added to lymphocyte tubes.
• Human hepatoma cell line (BEL-7402) was purchased from Shanghai Cell Biology Institute. Cells were removed from the medium in which they were provided, suspended in solution A (2.0 ml; 0.25% trypsin, 0.02% EDTA, 8 g/L of NaCl, 0.2 g/L KC1, 1.56 g/L Na,HPO,-H,0, 0.2 g/L KH,P0,), maintaimed at room temperature for 1 minute, transferred to culture solution B, mixed thoroughly for 2 minutes and adjusted as necessary to suspension concentration oflo4 cells/ml.
• the composition of culture solution B was as follows: DMEM cell culture solution (Siqma, St. Louis, MO), 15% fetal calf serum, 50 u streptomycin, 50 u penicillin. This suspension was used as the control .
• ALICIN >95 % pure
• PROTEINA Trichosanthin
• Trichosanthin has abortifacient , antitumor, ribosome inactivation, anti-HIV, immunomodulatory and insulin-like activities.
• Trichosanthin is a nonmetallo- and nonglyco- protein of 234 amino acids with an N-terminal aspartate and an carboxy terminal alanine-
• a selected partial amino acid sequence of trichosanthin starting from 108 (arginine) to 158 (leucine) and from 160 (valine) to 232 (asparagine) are homologous to the major part of PROTEINA: from amino acid 1 (arginine) to 51 (leucine) and 53 (valine) to 125 (asparagine) .
• HAV-serum-hepatic cells (BEL-7405) were prepared according to the protocol of Example 7. They were divided into 7 groups of 10 samples each as follows .
• Group 1 control group (untreated cells)
• Group 2 sample plus 300 ug of ALICIN (>95%) ;
• Group 3 sample plus 200-300 ug of PROTEINA (>99%) ;
• Group 4 sample plus 200-300 ug of trichosanthin
• Group 5 sample plus 100-300 ug of zinc glucose
• Group 6 sample plus 200 ug of PROTEINA and 100 ug of zinc glucose
• Group 7 sample plus 200 ug of trichosanthin and 100 ug of zinc glucose
• Group 1 control group, abdominal injection of 0.5 ml of saline solution (0.9% NaCl) 2 hours prior to tail vein injection of IAV; Group 2: same as 1, except 4 hour prior to injection of
• IAV IAV.
• Group 3 treated group, abdominal injection of 0.5 ml of the Proteina Combination Formula (400 ug) 2 hours prior to tail vein injection of IAV.
• Group 4 Same as 3, except 4 hours prior to injection of IAV.
• Group 5 Same as 3, except 6 hours prior to injection of IAV.
• Group 6 Same as 3, except 10 hours prior to injection of IAV.
• Group 7 Same as 3, except 18 hours prior to injection of IAV.
• Group 8 Same as 3, except 24 hours prior to injection of IAV.
• Group 9 Same as 3, except 48 hours prior to injection of IAV.
• Influenza A3 Virus (Jinfong-75-39) was purchased from Center of China Preventive Medicine. It was multiplied in chick embryo (10 days old) at 37°C for 40 hours. The allantoic fluid containing 10-0 titer of the virus was harvested from the chick embryo.
• the obtained allantoic fluid was centrifuged at 4000 rpm for 30 minutes to remove large particulates.
• the supernatant was mixed with a final concentration of 3.5 % chick red blood cells (prefixed by formaldehyde) and stored at 4°C overnight. After centrifugation at 2000 rpm for 10 minutes , the precipitated RBCs and viruses were washed twice with cold (0°C) saline solution and centrifuged. Appropriate amount (1/5 volume of the original allantoic fluid) of 0.01 M phosphate buffer (pH 7.8) was added to RBCs and viruses and incubated at 37°C water bath for 3 hours before centrifugation (2000 rpm for 10 min) . Save the supernantant which contained the viruses.
• phosphate buffer 100 volume (of the original allantoic fluid) of phosphate buffer to RBCs and incubated at 37°C for 2 hours. Centrifuged again. Combined the two supernantant which contained viruses and recentrifuged at 4000 rpm for 30 minutes to remove residual RBCS. Applied 20 ml of the supernantant to a Sephadex G200 column (2.5 cmx60 cm, flow rate 40 ml/hr) preequilibrated with the phosphate buffer (0.01M) . Centrifuged the virus solution (collected from the column) at 2800 rpm for 60 minutes. Discarded the supernantant. Added 2 ml of the phosphate buffer to the virus precipitate and dispersed the virus suspension.
• mice Observed and recorded the number of mice died in 5 days after the virus injection.

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  • 1997-07-16 WO PCT/US1997/011997 patent/WO1999003492A1/en not_active Application Discontinuation
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