CLONING AND EXPRESSION OF COMOSAIN'S MINIGENE AS IMMUNO-TARGET THERAPY IN TREATING AND/OR PREVENTING VARIOUS TYPES OF CANCER IN A

MAMMAL ( FINAL REVISED 3/23/2017 )

(COVER PAGE)

SPECIFICATION - -— TOTAL PAGES- 65

1. TITLE OF THE INVENTION ----- — PAGE 2

2. ABSTRACT OF THE DISCLOSURE -— PAGE 3

3. INVENTORS — - PAGE 4

4. REFERENCES CITED — -- PAGE 5

5. OTHER REFERENCES - — PAGE 6

6. CLAIMS — - — PAGE 20

7. SUMMARY OF INVENTION— - PAGE 22

8. BACKGROUND OF THE INVENTION — - PAGE 22

9. BRIEF DESCRIPTION OF THE DRAWINGS- — - PAGE 29

10. DETAILED DESCRIPTION OF THE INVENTION PAGE 50

1 1. PHARMACEUTICAL FORMULATION AND PREPARATION-— PAGE 64-66 1 2. DRAWINGS TOTAL PAGE 67-90

( )· TITLE OF THE INVENTION

The Cloning and Expression of Comosain's Minigene, and as Immuno-target Chemotherapeutic Agents in treating and or preventing various types of cancer in mammals.

INVENTORS

Liao, Benedict S., ( Los Angeles, CA ); Liao, Alex B., ( Los Angeles, CA ); Liao, Austin L., ( Los Angeles, CA ); Liao, Judy F., ( Los Angeles, CA ); Liao, Burton A., ( Los Angeles, CA )

Correspondence: Benedict, Liao, M.D.

Name and Address: 3106 East Garvey Ave. South, Suite # 1

West Covina, CA 91791, USA.

Assignee Name and Address: Benedict S. Liao, M.D.

3106 East Garvey Ave, South. Suite #1

West Covina, CA 91791 , USA

Application No.:

Serial No.:

Serial Code:

Filed: 1 1-26-2015

(o) REFERENCES CITED: (U.S. Patent Documents)

US 2421062 1947 Pulley, Von Loesecke, Ca. Fruit Growers Exchange

US 2421063 1947 Pulley, Von Loesecke, Ca. Fruit Growers Exchange

US 24421 10 1948 Baier, Ca. Fruit Growers Exchange

OTHER REFERENCES

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chemotherapy agents in treating and / or preventing various types of cancer in the mammal; US Patent US/2012/000323 , ( via PCT, Patent Cooperation Treaty ) and International Patent WO/2013/089803

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119. Keegan A. and Martini R. et al,: Ethanol -related liver injury in the rat: a model of steatosis, inflammation and pericentral fibrosis, Journal of Hepatology; 23 (5), pg. 591-600 (1995).

120. Fleming, Thomas. Chief editor, Crataegus laevigata; Physician Desk Reference for Herbal Medicine, 2nd edition, Medical Economics Co. publishing; pg.271-275(2000).

121. Lee-Huang S. et al ; Proc. Natl. Sci. USA. 81, pp 2708-2912 (1984)

122. Sanger, F. , Nicken, S. & Coulson, A.R. ; Proc. Natl. Sci. USA, 74, pp 5463-5467( 1977).

123. Okayama, H. & Berg, P. Mol. Cell. Biol. 2, pp 161-170(1982). 124. Gasser, C. S., Simonsen, C, Schilling, J.W. & Schmike, R. T.; Proc.Natl.Sci. USA 79, pp 6522-6526 (1982).

125. Woo, S. L. C. Methods Enzymol.; 68, pp 389-395 (1979)

126. Maxam A. M. & Gilgert, W. ; Mothods Enzymol. 65; pp 499-560 (1980).

127. Lin, F.K., Lai, P.H., Smalling, R., Egrie, J., Browne, J., Lin, C.H., Wang, F.F. & Goldwasser, E. ; Exp. Hematol. ; 12, pp 357 (abstr. 1984).

128. Miyake, T. Kung, C. K. H. & Goldwasser, E.; Journal Biol. Chem. 253, pp5558-5564 (1977).

129. Lawn, R.M., Fritsch, E. F., Parker, R. C, Blake, G. & Maniatis, T. Cell. 15; pp 1 157-1174 (1978).

[002] We combine this patent application with previous application of US/2012/00323 ( application no #13/374,328, filing date on 12/16/201 1 ) and WO/2013/089803 as a combined entity. Previous invention title as " Bromelainases (( Comosain )) as Chemotherapeutic agents in treating and or preventing various types of cancer in a mammal". Copy of this certificate also herein enclose for your reference and expertise consideration.

BACKGROUND OF THE INVENTION

[ 001] Cloning and expression of the gene of the human erythroblast cell has been initiated by several institutes in early 1970, Dr. Lee-Huang from University of New York in 1984 successfully cloning the erythroprotein from human erythroblast cells ( Proc. Natl. Sci. USA . 81, 2708-2712). Fu-K, Lin, ; Sidney Suggs, : Eugene Goldwasser, et al also successfully cloning the erythroprotein from human erythroblast cells and put into commercialized production.(Proc. Natl. Sci. USA. 82; 7580-7584 ). Present inventors have been cloning and expression in Comosains minigene and put it to clinical usage.

[002] Comosain represents 80 % of Bromelainases component,{ ( Dr. Henry Mauror, Harrach T. Eckert K, Schulze-Forster K., Nuck R. et al ; Isolation and partial characterization of basic proteinases from stem bromelain, Journal of protein Chemistry Volume 14, pp 41-52 (1995) :

Volume 17 ; pp 351-361 (1998), ; Volume 20 ; pp 53-64 (1997)). The other forms of bromelain proteinases also include Ananase ( Bromelain F-9a)( represent 10 %), Bromelain-F4, Bromelain-F5, Bromelain F-9b ( Comosain), and Bromelain F2, F3, F-6, F-6, F-7 and F-8( H.R. Maurer, Bromelain: Biochemistry, Pharmacology and Medical use; CMLS ,Cell. Mol. Life Sci. 58; pp 1234-1245 (2001). (ok)

[002] The Comosains gene has been isolated from a genomic phase library by using mixed 20 mer and 10 mer oligonucleotide probes. The entire coding region of the gene is contained in a 4.8- Kilobase Q9S8M1 - Blast V to Blast-G fragment. The gene contains four mtervening sequences (3026 base pairs) and four exons (1816 base pairs). It encodes 20 amino acid signal peptides with calculated Molecular mass of 23,509 to 23,569. The Comosain protein gene, when introduced into New Zealand white rabbit ovarian cells produces Comosain that is biologically active in vitro and in vivo.

[003] Administered of oral bromelain in cancer treatment in nonclinical trials has been reported as early in 1968 by Wolf M, & Ransberger k. ¹ , In vitro and animal studies have suggested of anti-metastatic effect for bromelain. Batkin &Taussig ^2,3 in 1988 reported that orally administered bromelain reduced the incidence of pulmonary metastasis in Lewis lung cancer cells in mice, In recent years, 1988 Batkin & Taussig ⁴ suggested the antitumor mechanisms are due to fibrinolytic effect in Bromelain. Taussig & Batkin in 1988 ⁵ discovered that Bromelain has anti-platelet aggregation effects. Taussig and Batkin .in 1985 ⁵ also discovered Inhibition the growth of tumor cells such as Lewis lung carcinoma, V-8 lymphoma, MCl-1 acites, KATO- gastric carcinoma cells. Maurer, & Hozumi, in Ι994 ^ΰ Discovered bromelian Induced

Differentiation in leukemic cells. Hale, & Haynes in 1992 ⁷ and Cantrell et a in 1996 have been suggested that MMAPT( Major Mitogen Activating Protein Kinase ) and TPK ( Tyrosine Phosphorylation Kinase ) inhibitors were activated by Bromelain. T-cell activation and cascade production of Interleukin II-B, 6, 8, and TNF-a (Tumor Necrotizing Factors) via CD-2, CD-3 surface antigen of WBC.

Garbin, Harrach, Eckert, & Maurer in 1994 ¹⁵ and Hale, & Haynes inl992 ⁷ also suggested that Bromelain will reduce surface antigens of CD-44, CD-44 v, CD-44s, CD45, & CD 47 in tumor cells of breast carcinoma.

From the experimental studies above, we conclude that activation of Bromelain proteinases in lymphocytes and T-cells have anti-metastatic effects both in vitro and in vivo.

[003-A] According to the recent studies and reports, various types of cancer and neoplastic diseases have been amount the number one cause of deaths in America. The search for new medicines with non-toxic, low side effects which are compatible with most other drugs have been under way since Cohen in 1964, Renzini in 1972, and Tinozzi in 1978. Bomelain was first isolated from Ananas Comosus fruit in 1891 by Marcano. (Marcano. Bull. Pharma. 5, 77, (1891). Heinecke and Gortner discovered stem bromelain, as a new proteinase preparation from Ananas Comosus plant in 1957, by precipitating with acetone and with ammonium sulfate in 1961.

Further purification of crude preparation was formulated by Gibian and Bratfish in 1960 and a patent right was granted to Pineapple Research Institute (US3002891) and to AG Schering Company (US2950227). Bayer T. , reported the anti-inflammatory effects in flavonoids in 1989 ( Phytochemistry, 28, pg. 2373-2378 (1989 ).

[004] Utilization of plant and / or fruit flavonoids such as Quercetin, Rutin ( Quercetin -3- Retinoside ), Naringenin and Hesperetin, Genistein, in inhibiting tumor growth have been reported by Saija A. , Scalese M. et al., ( Free Radical Biology and Medicine vol. 19, no.4 pg. 481-486 (1995)), Felicia, V.S.; , Najla Guthrie, et al. depicted evidence of inhibition of human breast cancer cell proliferation and delay of mammary tumor-genesis by flavonoids and citrus juices. ( Nutrition and Cancer vol.26, no. 2, pg.167-181 ( 1996 )).

In the year 2000, Revilla, E., and Ryan J.M., et al. analyzed of several phenolic compounds with potential antioxidant properties in grape extracts and wines by high-performance liquid chromatography-photodiode array detection without sample preparation. (Journal of Chromatographia; 6, 881 (1 -2); pg.461-469 (2000)). Also in 1991 by Kandaswai, C. and Perkins E. et al reported that citrus flavonoids have anti-proliferative effects on human squamous cell carcinoma in vitro: (Cancer Lett.; 56, pg. 147-152 (1991). Guthrie , N., and Moffatt, M., et al claimed Naringenin , a flavonoid from grapefruit, has anti-proliferative effect in human breast cancer cell lines. (National Forum Breast Cancer, Montreal, pg. 1 19 ( 1993 )).

[005] Bioflavonoids are group of naturally occurring compounds, which have a common flavone nucleus composed of two benzene rings linked through a heterocyclic-pyrone ring. They are found plentifully in various plants, vegetables, fruits (such as; citrus fruits, grapes), food products (such as buckwheat and oatmeal) and dyes of natural origin. Bioflavonoids exhibit various biochemical and pharmacological activities including anti-oxidative, anti-inflammatory, anti-cancer, anti-viral, and anti-platelet aggregation. (DA. Rakotoarison et al. Antioxidant activities of polyphenolic extracts from flowers of Crataegus monogyna. Pharmazie; 52: pg. 60- 64 (1997)), (Bayler, T.,et al., Phytochemistry 28, 2373-2378 (1989)), ( Goda ,Y.,et al., Chem. Pharm. Bull. 40, pg. 2455-2457(1992)), (TN. Kaul and Elliott Middleton et al., Antiviral effect of Citrus flavonoids on human viruses, Journal of medical virology : 15; pg. 71-79(1985)), (A. Saija and M. Scalese et al., four flavonoids, Quercetin, hesperetin, naringenin, and rutin, as antioxidant agents; Free radical biology and medicine, vol 19, no, 4, pg. 481-486(1995)) , (Felicia V. So. and Najla Guthrie et al., Inhibition of human breast cancer cell proliferation and delay of mammary tumor-genesis by flavonoids and citrus juices, Nutrition and cancer vol.26,no. 2, pg. 167-181(1996)), (SH. Bok and TS. Jeong et al., Flavonoids derived from citrus peel as collagen induced platelet aggregation inhibitor, U.S. Laid open patent: 6,221,357), (MG. Nair and HB. Wang et al., method of inhibiting cyclooxygenase and inflammation using cyanidin, U.S. Laid- open patent: 10,002,407) [006] Quercetin (3, 5, 7, 3', 4' penta-hydroxy flavone) also has anti-cancer activities against breast cancer, colon cancer cells, lymphoblastoid cell lines and squamous cancer cell lines and anti -viral activities against herpes simplex type I, polio virus type I, para-influenza type-3, and respiratory syncytial virus. (Scambia, G.and Ranelleti, F.O. et al., Cancer Chemotherapy, Pharmacology: 28, pg. 255-258 (1991)), (Singhal, R.L.and Yeh, Y.A. et al., Biochem. Biophys. Research Commun. 208, pg. 425-458 (1995)), (Ranelleti, F.O. and Ricci, R. et al., International J. of Cancer: 50; pg. 486-492 (1992)), (Scambia, G. and Ranelleti, F.O. et al., International J. of Cancer: 46; pg. 1 112-1 1 16 (1990)), (Castillo, MH. and Perkins, E. et al., American J. Surgery 158: pg. 351-355 (1989)), (Verma, A.K. and Johnson, J.A. et al., Cancer Research 48: pg. 5754- 5758 (1988)), (Kaul, T.N. and Middleton, E.J. et al., Journal of Medical Virology 15: pg. 71-79 (1985)), (Japanese Laid-open Patent No. 4-234320)

[007] Rutin, a glycosylated quercetin, (Quercitin-3-rutinoside) is decomposed in the intestine by microorganisms and absorbed in the intestine, Vitexin (Apigenin, Orientoside, or Flavone, 8-D- glucosyl-4', 5, 7-tri-hydroxy-), and Kaempherol (3,5,7,4' tetra-hydroxy-flavone) also have antihypertensive properties and increase coronary and cardiac perfusion. (Ammon,H.P.T., and Haendel, M. et al., Crataegus toxicology and Pharmacology, Planta medica: 43(2): pg. 105-120, 43(3): pg. 316-322, and 43(4): pg. 210-239 (1981)), (Schussler, M. and Holzl, J. increasing cardiac perfusion with quercetin, rutin and vitexin in guinea pig heart, Arzneimittelforschung, 45(8): pg. 842-845 (1995)), (Manach, C. et al., bioavailability, metabolism, and physiological impact of 4-oxo-flavonoids. Nutrition research 16: pg. 517-544(1996)). Furthermore, Hertog et al. reported that high intake of rutin and quercetin in the food product may reduce coronary heart disease related death rate of elderly patients (M.G.L. Hertog et al., dietary antioxidant flavonoids and risk of coronary heart disease, Lancet: 342: pg. 1007-101 1 (1993)). [008] The present inventors have discovered that bioflavonoids derived from hawthorn berry, and citrus fruits such as rutin, quercetin, kaempherol, vitexin, hesperetin, naringenin, genistenin are effective in treating and /or preventing various types of cancer.

[009]The study of Rakotoarison, DA. et al showed anti-oxidant activity of polyphenolic extracts from flowers of Crataegus Monogyna ( Pharmazie: 52: pg. 60-64 (1997)). Kaul, TN. and Elliott Middleton et al also reported the anti-viral effect of Citrus flavonoids on human viruses. (Journal of Medical Virology: 15; pg. 71-79 (1985)). It has also been reported that pant bioflavonoids exhibit various biochemical and pharmacological activities including anti-oxidant, anti-inflammatory, anti-cancer, anti-viral and anti-platelet aggregation.

[010] Nair, MG; and Wang, HB. et al reported that a method of inhibiting cyclooxygenase and inflammation using cyaniding, ( US patent 10,002,407)), Ranelleti, F.O., and Ricci R. et al, reported growth inhibitory effect of Quercetin and presence of type II estrogen binding sites in human colon cancer cell lines and primary colo-rectal tumors; ( International Journal of Cancer: 50; pg.486-492 ( 1992)), Scambia, G. and Ranelleti, F.O. et al ; reported that Quercetin inhibits the growth of a multi-drug-resistant estrogen receptor - negative MCF-7 human breast cancer cell line expressing type-II estrogen -binding sites: ( Cancer Chemotherapy, Pharmacology: 28, pg. 255-258 (1991 ) ) also reported in Type II estrogen binding sites in a lymphoblast cell line and growth inhibiting effect of estrogen, anti-estrogen and bioflavonoids; ( International Journal of Cancer: 46; pg. 1 1 12-1 1 16 (1990 )), Castillo, M.H. and Perkins, E., et al also reported the effect of the bioflavonoid-Quercetin on squamous cell carcinoma of head and neck origin;

(American Journal of Surgery: 158, pg. 351-355 ( 1989 )).

Zhao, J., Wang , J., et al published a study showing anti-tumor promoting activity of a polyphenolic fraction isolated from grape seeds and identification of Procyanidin B 5-3'-gallate as the most effective antioxidant constituent; (Carcinogenesis, Sept.; 20 ( 9 ); pg. 1737-1745 (1999 )).

[011] In 1995 Harrach T. , Eckert K., Schulze-Forster K. and Maurer H. Rainer et al reported isolation and partial characterization of basic proteinases from stem bromelain. ( Journal Protein Chemistry 14: pg. 41-52, 1995 ), and Again in 1997 they reported isolation and characterization of two forms of an acidic bromelain stem proteinase.( Journal Protein

Chemistry 20: pg. 53-64, 1997).

[012] In 1985 Taussig , and Batkin et al suggested that the enzyme complex from Ananas Comosus produced inhibition of tumor growth in vitro ( Planta Med. 6: 538-539, 1985 ). and outlined its clinical application,( Journal Ethnopharmacology: 22, pg. 191-203,1988 ) The same enzyme complex also produces platelet anti-aggregation , and fibrinolytic activity, ( reported by Heinecke and Yokoyama in 1957, Heinecke in 1972 ,Marz, in 1982,). In 1979 Klaue, Amen, and Roman et al reported use of bromelain as chemical debridement agent in 3 ^rd degree burn patients. ( European Surgical research , 1979, pg. 355-359 ). In same year of 1988 the same team also discovered that Bromelain has an anti-metastatic effect with or without its proteolytic and anti-coagulant activity, (Journal of Cancer Research Clinical Oncology, 1 14: pg. 507-508, 1988 ).

[013] The present inventors have also discovered that Glco-polypeptides, the extract of

Bromelain proteins which derived from the fruit and stem of Ananas Comosus. ( Comosain, Bromelain, Ananase, Inflamen, Extranase, Traumanase ) which are effective in treating and/or preventing various types of cancer and neoplastic diseases including breast, colon , lung, ovarian, cervical, and uterine cancers, and so forth, the method comprises administered an effective amount of Comosain, Bromelains, Ananase, Pepsin, Trypsin., Naringenin, Hesperetin, Genistin and / or a mixture thereof. By the following attributes:

( a ) Anti-inflammatory properties

( b ) Anti-platelet aggregation ( Mynott et al in 1998 , suggested that it changes the tumor surface antigen thus preventing tumor cells from attacking the normal tissues ),

( c ) Fibrinolytic properties,

( d ) Anti-tumor genesis, this action was probably due to release of tumor necrotizing factors ( TNFs ) in T-cells of WBC. Taussig et al in 1985 and Taussig , and Batkin et al in 1988 both indicated that Bromelain and Comosain extract can be used in inhibiting tumor growth, the T- cells , peripheral blood mononuclear lymphocytes (PBMN ) with influence of bromelain, they produce and outpour of TCRS/CD2, TCRS/CD3, Interlukin IIB, II-6, II- 8 , and TNFa. and attack the tumor surface antigens of CD44 ,CD44-v, CD44-S, CD45, CD47. These Action mechanisms are through two major pathways:

(d.-l) Major Mitogen Activating Protein Kinases (MMAP) inhibitors.

(d-.2) With the engagement of Bromelain (Antigen Presenting Polypeptide) (APP) (

presented by Major Histocompatibility Complex ( MHC ) expressed on the Antigen Presenting

Cells (APCXCantrell 1996)

(d-3) Tyrosine Phosphoration Kinase inhibitors.

(e ) Anti-dedifferentiation both in vitro and in vivo in cancer cell lines, in animal, and human experiments.

[014] The present invention and discovery relates to the methods and compositions for treating and /or preventing various types of cancer and neoplastic disorders in mammals, which comprises administration thereto of an effective amount of Glycopolypeptides (such as Comosain, Bromelainases, Ananase, Pepsin, Trypsin ) and Bioflavonoids such as Quercetin; Rutin (Quercetin-3-Rutinoside), Hesperetin, Genistein, Naringenin and/or a mixture thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects of the present invention will become increasingly apparent by reference to the following descriptions and drawings.

Assembly of expression vector for the Comosain gene and preparation of genetically modified organism (GMO)

[014] The oligo-deoxyribonucleotide probe synthesis was used, the phosphoramidite method was used for oligonucleotide synthesis. Each probe mixture contained a pool of 20-oligonucleotides sequences were:

Probe mixture: CoM-V sequence equivalent to: valine-proline-glutamine-serine-isoleucine, aspartic acid-tryptophan-arginine-asparagine-tyrosine-glycine -alanine-valine-threonine-serine- valine-lysine- asparagine-glutamine-glycine.

Probe mixture: CoM-V=

Val-Pro-Glu-Ser-Iso-Asp-Trp-Arg-Asn-Tyr-Gly-Ala-Val-Thr-Ser- Val-Lys-Asn-Glu-Gly 3' CAA, GGA, GTT, - — — -,TTG,GTT, TT5'

V D G V

P w A K

Q R V N

s N T Q

I Y s G

V=Valine, P=Proline, Q= Glutamine, S=Serine, I=Isoleucine. D=Aspartic acid, W=Tryptophan, R=Arginine, N=Asparagine, Y=Tyrosine.

G=Glycine, A=Alanine, V=Valine, T=Threonine, S=Serine.

V=Valine, K=Lysine, N=Asparagine, Q=Glutamine, G=Glycine.

The probe mixtures were labeled at the 5' end with [ r ³² p ] ATP, 7500-8000 Ci/mmol (ICN) ( 1 Ci= 37 GBq), by using T4 polynucleotide kinase.

(14- A) Hybridization procedures: phage plaques were ampilified according to the procedures of Woo, except that GeneScreen Plus filters and NZYAM plates [NACL, 5 g; MgCl ₂ -6H20, 2 g; NZ- Amine A, 10 g; yeast extract, 5 g; Casamino acids, 2 g; maltose, 2 g; and agar, 15 g (per liter) were used, Phage particles were disrupted and the DNA _S were fixed on filters (50,000 plaques per 8.4 x 8.4 cm filter). The air dried filters were baked at 80° c for 1 hour and then subjected to proteinase K digestion [ 50 ug of proteinase k per ml of buffer solution containing 0.1M Tris-HCl ( PH 8.0 ), 0.15 M NaCl, 10 mM EDTA, and 0.2% NaDodSo4 for 30 min at 55 °C. Prehybridization with a 1 M NaCl/1% NaDodSo4 solution was carried out again at 55 °C for 4 hours or longer

The hybridition buffer contained 0,025pmol/ml of each of the 20 probe sequences of 0.9M NaCl/5 mM EDTA/50 m M solution phosphate, PH 6.5 /0.5% Na Dod So4/ 100ug of yeast tRNA per ml. Hybridization was carried out at 48 °C for 20 hrs by using the ComV probe mixture, (that is 2 ⁰ C below the lowest calculated dissociation temperature ( td ) for members of The mixture. At the completion of hybridization, the filters were washed three times with 0.9 M NaCl/ 90 mM sodium citrate, pH 7.0/ 0.1%NaDodSo4 at room temperature at hybridization and 10 min per wash.

(14-B). For direct expression of the genomic Comosain gene, 4.8 kilobase (kb) of BstTy/Se- and BamAs/GL fragment of Comosain, which contains the entire gene. After converting the BstTy/Se (tyrosine— serine 10 amino acids )site into Bst As/GL( asparagine -to-glycine 20 amino acids ) site with a synthetic linker( pBR322 ori ) the fragment was insert into the unique BamAs/GL site of the expression vector pDSVL, which contains a dihydrofolate reductase (DHFR ) minigene. The resulting Plasmid DSVL-gPICOS ( gene Plant Comosain) was then used to transfect New Zealand white rabbit ovarian (NWRO) cells by the calcium phosphate microprecipitate method. The transformants were selected by the medium lacking hypoxanthine and thymidine. The culture medium used was Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine.

(14-C). Isolation of Comosain mRNA. : The 4.8 kilobase Bst ty/se— Bam As/GL restriction fragment from Comosain was inserted into shuttle vector, pSV4ST. The resulting chimeric plasmid pSV gPLComo was used to transfect Cos-1 cells by the calcium phosphate micoprecipitate method. After culture for 72 hrs, RNA was prepared from the transected cells by the guanidinium thiocyanate procedure of Chirgwin et al. and poIy(A)+ mRNA was isolated by binding to oligo- ceIlulose.( Aviv & Leder ).

(14-D). cDNA cloning.: A Comosain cDNA bank was constructed according to a modification of the general procedures of Okayama and Berg by using the poly(A)+ mRNA described above ( Mol. cell biology 2, 161-170 , 1982 ).

(14-E). DNA sequencing. : Restriction fragments were cloned into M 13 phage vectors by using Escherichia coli strains JM 103 and /or JM 109 as host. (Messing, J .of methods enzymology 1983) and were sequenced by the dideoxy method of Sanger et al. some regions were sequenced by kinase labeling or end-fill labeling of restriction fragments followed by chemical cleavage as described by Maxam and Gilbert. (J. of methods of enzymology 1980).

(14-F). The final Comosain Recombinants were collected , extracted, washed with ethyl alcohol, and purified by AKAT Prime ( GE Co. ) and / or FPLC -cation-exchange chromatography, to produce of F4, F5, and F-9a, F9b ( Comosain and Ananase ) for future use. [014-G] Table I, Overview of the Bromelains (Comosain) that can be used in inhibition of these characterized cancer cells.

[015] Six types of cancer cell lines were used in our present invention and discovery, which included breast, lung, colon, cervical, ovarian, and uterine cancers, and so forth, Most of our cancer cell lines were harvested directly from surgical specimens during surgery, and were prepared and described in detail under cell culture in Example -2.

[016-A]. Materials and Methods: Naringenin, Hesperetin, Genistein, Trypsin, Pepsin, &

Bromelain (Comosain ) proteinase molecules were purchased from Sigma-Aldrich Co. St. Louis, MO.( for cell cultures) ( Catalogue No# 4882 & more ), Complete Growth Medium ( Catalogue # M4655 ), Tween-20, and Tween-80 solution ( Catalogue No # P2287, # P 8192 ), Penicillin-streptomycin-Neomycin Stabilized Solution ( Catalogue No# P 4083 ), Fetal calf serum ( Catalogue No # 4762 ). Cell culture wares were from Becton-Dickinson Co , Franklin Lakes, N. J. , ( Catalogue No # 353503, ), B-D TM Cell viability kit with liquid counting beads ( Catalogue No # 349486 ), B-D FACS Array tm Bio-analyzer ( Catalogue No # 340128 ). Pathological and microscopic images were taken by Amescope Trinocular Microscope

(American Optic Co., Model No# T- 490B-10M).

[017] The complete growth medium ( CGM ) : consists of Dulbecco's modified essential medium ( Sigma-Aldrich Co., St. Louis , MO ), supplemented with 10 % heat inactivated fetal calf serum, 2 % L- glutamine, penicillin( 100 iu/ml ), streptomycin ( 5 mg/ml ), and neomycin (10 mg/ ml) ( Sigma-Aldrich Co. St. Louis, MO.). The cells were maintained in a standard tissue culture incubator at 37° C with atmosphere humidity Of 90% air, and 10 % CO 2. All cancer cell lines were initiated by seeding 5 X (10) ⁶ cells into 75 cmsq tissue culture flasks and base were coated with 0.75 % agar in CGM. The cancer cell lines were used between 5 and 7 days of culture.

[018] Figure-IA, Growth inhibition of various types of tumor cell lines in vitro.

Bromelain in medium in mg/ml in X-axis, Percentage % of cell growth VS control in Y-axis. Inhibition of tumor cell growth with increasing Bromelain concentration in all six cancer lines: breast, colon, lung, ovarian, cervical, and uterine cancers. The cells in all groups were incubated for 72 hours at 37C degree in 10% C02 in air and were counted with Coulter counter. The graph represents the six individual experiments with 6 tumor cell cultures / each tumor line in each experiment.

[019] Figure -IB,

The methods of analysis of surface antigens production of interleukin IB, 116, 118, TNF- a, in TCRS/CD2, TCRS/CD3 in T-cells, and Mononuclear Cells. In WBC cell-lines culture fluid of 5x(10) ⁶ '. At the concentration of 1 mg/ml of Bromelain ( Comosain ) installation to produce of interleukin IIB was 13,000 pgm/ml/(10 ⁶ ) WBC (increased by 400 folds), interleukin 116 was 26,000 pgm/ml/(10) ⁶ WBC (increased by 650 folds), TNF- a was 1500 pgm/ml/(10) ⁶ WBC ( increased by 42 folds ).

[020] Figure-I-C,

Depicts that of surface antigens of CD44-S, CD44-V, modulation with two different mAbs clones, L-178, 1-173. Breast cancer cells were incubated for 1 hr at 37 C with 10ug/ ml, 50 ug/m[, 75ug/ ml of Bromelain ( Comosain ) treatment^ Using radioimmuno monoclonic antibody tests). The CD44s become 35%, 10%, and 0 % of Bromelain treated cells. The CD44v become 33 %, 1 1 %, and 0 % of Bromelain treated cells [ 20-1 ] Figure-ID,

Inhibition of tumor cell growth of lung cancer by pepsin and trypsin in vitro. , The cells in these two group were incubated for 72 hours at 37 C degree in 10 % C02 in air and were counted with Coulter counter. The graph represents the two experiments with Pepsin and Trypsin.

[ 20-2 ]Figure IE,

Growth inhibition Of breast cancer cell line by Qucercetin and Naringenin in vitro.

[021] Rutin (C.sub.27.H.sub.30. O.sub.16, Molecular weight of 610.52, Glycosylated Quercetin, or Quercetin-3-rutinoside); Quercetin (C.sub.15. H. sub.10. O.sub.7, Molecular weight of 302.24, or 3, 5, 7, 3', 4' penta-hydroxy flavone); Kaempherol (C.sub.15. H. sub.10. O.sub.6, Molecular weight of 286.24, or quercetin-3-rhamnoside); Vitexin (Apigenin)(C.sub.21.H.sub.20.O.sub. l0, Molecular weight of 432.38, or 8-D-glucosyl-4', 5, 7 trihydroxy-flavone) (Merck Index 13rd Edition 2001) may be extracted from various plants, vegetables and fruits, such as citrus fruits, hawthorn berry, and also can be synthesized in accordance with the conventional process described by Seka, Prosche and Monatsh., 69,284 (1936) and Zemplen, Bognar in Ber., 1043 (1943), and EINECS 222-963-8, Journal of European Communities; June 1990.

[022] For example, rutin may be found in hawthorn berries, flowers, leaves, stems, and roots in an amount ranging from 0.2 to 5 wt. % (PDR Herbal Medicines, 2nd Edition 2000). Rutin, quercetin, kaempherol and vitexin may be extracted from hawthorn berry by using a suitable solvent, such as water or aqueous ethanol alcohol under high temperature and pressure. The other method is using aqueous solution of ½ N of Ca (OH) sub.2 or NaOH, and then the crude extract and precipitates may be collected after neutralization. Furthermore, the dry powders of hawthorn berries, leaves, stems, flowers and roots may also be used. Generally, content of rutin, quercetin, kaempherol and vitexin in the berries is 5%, 3%, 2%, and 0.5% respectively.

[023] Rutin, Quercetin, Kaempherol and Vitexin not only possess an inhibitory, but also exert a therapeutic effect on elevated plasma lipid level related diseases, such as hyperlipidemia, hypercholesterolemia, atherosclerosis, arteriosclerosis, stroke (cerebro-vascular accident), angina pectoris and hepatic disease, such as fatty liver and fatty degeneration. Furthermore, rutin, quercetin, kaempherol, and vitexin exhibit no toxicity and no adverse effects on hematopeutic, renal, hepatic systems when they are administered orally to a mouse at a dose of 1500mg/kg, 1250 mg/kg, 1000mg/kg, 500mg/kg, respectively, which corresponds to an oral administered dose of 50 to 150 gm. of hawthorn berry extract for a person weighing 50 kg.

[024] The present invention also provides a pharmaceutical composition for inhibiting the formation of fatty streak onto the arterial endothelial wall, which comprise hawthorn berry extract as an active ingredient plus pharmaceutically acceptable excipients, carriers or diluents.

[025]The hawthorn berry extract of the present invention may be prepared in accordance with any conventional method by using suitable solvents, such as water or lower alcohol (ethanol) and an aqueous alkali or alkaline earth-metal hydroxide solution such as Ca (OH) sub.2 or NaOH solution. For example, 0.5 to 1 N of 1-10 liters of a solvent is added to 1 kg of dried hawthorn berry and the mixture is kept at a temperature ranging from 25 to 70 degrees C for a period ranging from 1 to 10 hours. The resulting extract is filtrated and concentrated to a formation of a concentrated hawthorn berry extract. For instance, if an aqueous alkali or alkaline earth metal hydroxide solution is used, the filtrate is adjusted to a PH ranging from 4.0 to 7.0 by adding an acid thereto. The resulting solution is kept at a temperature ranging from 5 to 20 degrees C for a period ranging from 5 to 30 hours. The precipitate is then dried to obtain a hawthorn berry extract. On the other hand, when ethanol is used as a solvent, 1 to 10 liters of 30% to 100% of solvent are added to 1 kg of the dried hawthorn berry, and the mixture is kept at a temperature ranging from 25 to 70 degrees C for a period ranging from 1 to 10 hours, then the resulting mixture is filtrated and concentrated to obtain a hawthorn berry extract.

[026] The hawthorn berry powder may be used in the present invention in place of the hawthorn berry extract. The hawthorn berry powder may be prepared by lyophilizing or drying the solid materials from hawthorn berry according to a conventional method and powdering it to a particle size ranging from 50 to 250 mu.m.

[027] A pharmaceutical formulation may be prepared in accordance with any of the conventional methods and procedures. In preparing the formulation, the active ingredient is preferably admixed or diluted with a carrier, or enclosed within a carrier, which may be in a form of a capsule, sachet, or other container. When the carrier serves as a diluent, it may be a solid, semisolid, or liquid material acting as a vehicle, excipient or medium for the active ingredient. Thus, the formulations may be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like.

[028] The pharmaceutical examples of suitable carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, micro-crystalline cellulose, polyvinylpyrrolidone, water, methyl-hydroxy-benzoates, propyl-hydroxy-benzoates, talcum, magnesium stearate and mineral oil. The formulation may additionally include fillers, anti-agglutinating agents, flavoring agents, lubricating agents, wetting agents, emulsifiers, preservatives and the like. The pharmaceutical compositions of the invention may be formulated to provide quick, sustained or delayed release of the active ingredient after its administration to a mammal by employing any of the procedures and/or methods well known in the art.

[029] The pharmaceutical composition of the present invention contains the active ingredient in an amount ranging from 0.01 to 100mg/kg/day, but preferably from 0.1 to 50mg/kg/day. It can be administered via various routes such as oral, transdermal, subcutaneous, intramuscular, intravenous, inhalational, intraperitoneal and transmucosal introduction. A typical daily dose of bio-flavonoids in human may range from 0.1 to 500 mg/kg of body weight, but preferably from 1.0 to 100 mg/kg of body weight and may be given in a single dose or in divided doses. The actual and exact amount of the active ingredient to be administered may vary according to patient's age, sex, body weight, disease, severity of illness and route of administration.

[030] Furthermore, Quercetin, rutin, kaempherol and vitexin may be incorporated into foods and/or beverages for the purpose of preventing and/or treating elevated plasma lipid related diseases (e.g., hyperlipidemia, hypercholesterolemia, atherosclerosis, arteriosclerosis, cerebrovascular accident, angina pectoris and hepatic disease). The foods and beverages may include food products, meats, vegetable juices, fruit juices, snacks, confectionery (chocolates and pizza), gum, dairy products, soups, broth, pastes, sauces (such as ketchup), teas, alcohol beverages, carbonated beverages, vitamin complexes and various health foods.

[031]The content of rutin, quercetin, kaempherol and vitexin, or a mixture thereof in a food or beverage may range from 0.1 to 10 wt %. It is therefore comprised of 1 to 100gm of rutin, quercetin, kaempherol, vitexin or mixture thereof per 1000 ml of beverage.

EXAMPLE 1

PREPARATION AND ANALYSIS OF HAWTHORN BERRY EXTRACT. [032]The hawthorn berries (Ogden, Utah. USA) were dried at room temperature and powdered to a particle size ranging from 100 to 200 mu.m. and then 50ml of 80 % ethanol was added to 10 gm of hawthorn berry powder and extracted in a water bath at 60 degree C for 6 hours. The extract obtained was filtrated and cooled, then ethanol was added to the filtrate to a volume of 50 ml.

[033] The above extract in an amount of 3.0 mu.l. was subjected to high performance liquid chromatography (HPLC) using prostar UV-Vis spectrophotometer lichrosorb RP-8 column (5mu.m, 4 times, 250mm) was pre-calibrated with 0.1 M borate sodium duodisulfate (SDS) solution and maintained at a temperature of 30 degree C. The extract was eluted with 0.1 M of borate SDS at flow rate of 0.5 ml/min. Standards solution were prepared by dissolving rutin, quercetin, kaempherol (Aldrich-Sigma Chemical Co. St. Louis, Mo. USA) and vitexin (Indofine Chemical Co. Somerville, N.J. USA) in 0.1 M borate SDS to a final concentration of 0.1, 0.2,

0.3, 0.4, and 0.5 mg/ml respectively, and subjected to HPLC under the same condition as that of above. The elutes were detected at 254 nm. (rutin), 257nm. (quercetin), 266 nm. (Kaempherol), 270nm. (vitexin) with UV-Vis spectrophotometer and the contents of rutin, quercetin, kaempherol and vitexin were calculated by comparing by the areas of HPLC profiles of the hawthorn berry extract and standard solution. The contents (%) of rutin, quercetin, kaempherol and vitexin in hawthorn berry extracts are depicted in table 1.

1. TABLE 1 - Hawthorn berry extract contents Rutin 5.2 %, Quercetin 3.1 %, Kaempherol 2.3 % and Vitexin 0.5 %.

EXAMPLE 2

PREPARATION OF HAWTHORN BERRY EXTRACT

[034] (A) Method of using ethanol

[035] The hawthorn berries were dried at room temperature, 300ml of 80 % ethanol was added to 100 gm of dried berry, The berry were extracted at 60 degree C for 6 hours; the resulting extract was filtrated through cheese cloth and the filtrate was concentrated under vacuum to obtain 57gm of syrupy extracts. The contents of rutin, quercetin, kaempherol and vitexin were examined in accordance with the method of example 1 which contained rutin 2.90gm, quercetin 1.82gm, kaempherol 1.31gm, and vitexin 0.285gm.

[036] The composition of hawthorn berry extract was confirmed by HPLC and the result is depicted in Table II.

2. TABLE II - Ingredient content (%), moisture 67 %, fructose 4 %, glucose 3 %, sucrose 2.9 %, rutin 5.1 %, quercetin 3.2 %, kaempherol 2.3 %, vitexin 0.5 %, others 12.1 %.

[037] (B) Method of using NaOH

[038] The dried hawthorn berries (Ogden, Utah. USA) in an amount of 100 gm was added to ½ N 500 ml of NaOH solution, and kept at room temperature for 3 hours while stirring. The resulting extract was obtained by filtrating through a cheese cloth, then 1 N HC1 solution was added to the filtrate to adjust its PH to 4.5. The same procedure as that of above was repeated to obtain a filtrate to adjust its PH to 6.5. The resulting filtrates were kept at 6 degree C for 12 hours and then the precipitates were collected and dried to obtain 8.8 gm and 9.8 gm powders, respectively. The compositions were confirmed by HPLC analysis which showed that hawthorn berry extracts contained rutin, (4.08gm, 4.55gm), quercetin (2.56gm, 2.85gm), kaempherol (1.84gm, 2.05 gm), vitexin (0.40gm, 0.45gm) and the purity was 29.9% and 20%, respectively.

[039] (C) Method of using Ca (OH) sub.2

[040]The dried hawthorn berry (Ogden, Utah. USA) in an amount of 100 gm was added to ½ N 500 ml of Ca(OH) sub.2 solution, and kept at room temperature for 3 hours while stirring. The resulting extract obtained by filtrating through a cheese cloth, and then 1 N HC1 solution was added to the resulting filtrate to adjust its Ph to 4.5. The same procedure as that of above was repeated to obtain a filtrate and to adjust its Ph to 6.5. The resulting filtrates were kept at 6 degree C for 12 hours and then the precipitates were collected and dried to obtain lgm and 2gm powders, respectively, HPLC analysis of the powders showed that hawthorn berry extracts contained rutin ( 0.464gm, 0.928gm), quercetin (0.29gm, 0.58gm), kaempherol (0.209gm, 0.418gm), vitexin (0.455gm, 0.91 Ogm) and the purity was 60% and 63%, respectively.

EXAMPLE 3

TOXICITY OF RUTIN, QUERCETIN, KAEMPHEROL AND VITEXIN IN MICE BY ORAL ADMINISTRATION.

[041] 24 specimens of 8 week old, with specific pathogen free, ICR female mice, each weighing from 25 to 30 gm, were divided into four groups (6 mice each) and were kept in separate cages under an environment of 23+_3 degree C, relative humidity of 45+_5%, and 12 light/12 dark photoperiod, fed with Harlan Teklad-2018 global rodent diet (18% protein) (Kaytee Co. Madison, WI. USA) and water was sterilized to feed to the mice.

[042] Rutin, quercetin, kaepherol (Aldrich-Sigma Co. St. Louis, Mo. USA) and vitexin (Indofine Co. Somerville, N.J., USA) were dissolved in 0.5% of tween-80 solution to a final concentration of 150mg/ml, 12.5mg/ml, 100mg/ml and 50mg/ml respectively, and was orally fed to the 4 separate groups of mice in an amount of 0.2ml per 20gm of mouse body weight, i.e., rutin 1500mg/kg, quercetin 125mg/kg, kaempherol 1000mg/kg and vitexin 500mg/kg respectively. The solution was administered once and the mice were observed for 180 days for signs of adverse effects or death according to the following schedule: 1H, 4H, 8H, 12H, (hour) after administration and then every 12 hours thereafter. The daily weight of each mouse was recorded. On day 181 , the mice were sacrificed and the internal organs including liver, kidney, heart, lung, muscle, stomach, urinary bladder, intestines, pancreas and spleen were examined visually and microscopically.

[043] All mice were alive at day 180 and no body weight loss occurred during this period of observation. The mice did not develop any pathological abnormality either visually or microscopically. Therefore, it is concluded that the hawthorn berry extract which includes rutin, quercetin, kaempherol and vitexin is not toxic when orally administered to a mammal.

EXAMPLE 4

ADMINISTRATION OF HAWTHORN BERRY BIOFLAVONOIDS; RUTIN,

QUERCETIN, KAEMPHEROL AND VITEXIN TO RABBITS.

[044] (Step A) 36 specimens of three month old New Zealand white rabbits (Harlan Kaytee Co. San Diego, CA) each weighing 2.5-to 2.6 kg were fed under a condition of temperature of 23+_3 degree C, relative humidity of 45+_5 % and photoperiod 12 light/ 12 dark. The rabbits were divided into six groups with 6 heads each and were fed with six different diets; Harlan Taklad rabbit diet-TD-1376 (Madison, WI. USA) which contained of 1% cholesterol in control group; 1% cholesterol plus 1.5 mg/kg Simvastatin (Merck Co. N.J. USA) in comparative group; 1% cholesterol plus 0.15% rutin in rutin group; 1% cholesterol plus 0.15% quercetrin in quercetin group; 1% cholesterol plus 0.15% kaempherol in kaempherol group; 1% cholesterol plus 0.15% vitexin in vitexin group.

Harlan Taklad rabbit diet TD-1376 contains moisture 12%, crude protein 16%, crude fat 2%, crude fiber 15%, ash 8%), and nitrogen free substances 47%.

[045]The rabbits were fed for 8 weeks with free access to specific high cholesterol diets and water. Body weight was recorded every 7 days and the records were analyzed. All rabbits showed a normal growth rate and there were no significant differences among the six groups in regard to the diet ingestion amount and the body weight gain.

( Rutin, quercetin and kaempherol were purchased from Aldrich-Sigma Co. St. Louis, Mo. USA) (Vitexin was purchased from Indofine Co. Sumerville, N.J. USA)

[046] (Step B) After 8 weeks of breeding, the rabbits were anesthetized with injection of ketamine 75 mg/kg in the femoral muscle and sacrificed. Blood samples were collected from the heart of each rabbit to determine the blood analysis consisting of; complete blood count (CBC), Chemistry-7 and 24, (including Liver and renal function tests), lipid profiles, (including Total cholesterol, HDL, LDL, VLDL and triglycerides), coagulation factors consisting of;

Prothrombin time (PT), Partial thromboplastin time (PTT), and immuno-globulin-E (an anti- allergenic factor).

EXAMPLE 5

ANALYSIS OF PLASMA TOTAL CHOLESTEROL, HDL AND TRIGLYCERIDES IN RABBITS.

[047] (Step C); The effects of administering rutin, quercetin, kaempherol, and vitexin to rabbits on plasma cholesterol and triglycerides contents were determined as follows.

[048] The blood samples collected from rabbits of the six dietary groups were allowed to stand for 2 hours, then centrifuged at 4000 rpm for 10 minutes. (Megafuge, Baxter- Heraeus instrument Co. N.J., USA) The supematants were separated and stored in a deep freeze before analysis. The chemistry analysis was carried out by blood chemistry analyzer (Cobras-Integra-700, Roche Diagnostic Lab. Indiana, USA) to determine the changes in total cholesterol, HDL, LDL, triglycerides, liver function tests (such as SGOT, SGPT, G-GPT) and coagulation factors (PT, PTT) (Bayer-MLA-Electra-900 Automatic coagulation timer). The results were tested by using student t-test and Microsoft excel-7.0 program. The results are depicted in Table III.

3. TABLE III.

Blood analysis on rabbits fed on 6 different high cholesterol diets

Control group, Simvastatin group, Rutin group, Quercetin group, kaempherol group, vitexin group.

Control group; TC; (383.3+_55.2 mg/dl), TRG (165.6+_40.6mg/dl), HDL (45.6+_22.4mg/dl), SGOT (35+_6 u/1), SGPT (62.5+_6.5 u/l), GGTP (5+_2 u/1); WBC; (4.8+_2.0 k/ul), A ;

(33+_10), B ;( 3.5+_0.5).

Simvastatin group; TC;(277.3+_90.7), TRG; (141+_30), HDL; (47.5+_16.5), SGOT;

(1 14.3+_30.7), SGPT; (71.2+_3.8), GGTP; (6+_l); WBC; (3.3+_2.6 k/ul), A; (13.5+_6.5), B; (2.8+_0.3).

Rutin group; TC; (254.5+_36.5), TRG; (108+_22), HDL; (36+_6), SGOT;

(52.8+J2.2), SGPT; (33+_9), GGTP; (3+_l), WBC; (4.8+J .2), A;( 1 1.3+_6), B; (2.6+_0.4).

Quercetin group: TC; (262.3+_30.7), TRG; (105.6+J 8.4), HDL; (42+_8),

SGOT; (62+_7), SGPT; (43+_12),GGTP; (4+_l), WBC; (4.5+_1.5). A;(12.8+_5.4), B;

(2.7+_0.3).

Kaempherol group; TC; (275+_23), TRG; (130+_26), HDL; (45.3+_7.3),

SGOT(65+_8.6), SGPT; (42+_8.8), GGTP; (3.2+_1.8), WBC; (4.6+_1.8), A; (13.5+_4.6), B;

(2.8+_0.4).

Vitexin group; TC; (269.5+_38.5), TRG; (138+_ 28), HDL; (46+_12),

SGOT; (60+_12.5), SGPT; (48+_15), GGTP; (3+_1.5), WBC; (4.9+_1.7), A; (13.7+_4.3), B;

(2.9+_0.5). TC: Total Cholesterol, TRG: Triglycerides, WBC: White Blood Cell,

HDL: high density lipoprotein, A: percentage (%) proportion of fatty streak to total aortic area, B: percentage (%) proportion of fat containing cells.

[049] From the data of Table III, administration of rutin and quercetin, kaempherol and vitexin decreased plasma total cholesterol and triglycerides by 32-33 % and 45-47%, also 30-30 % and 22-17%, respectively, as compared to that of control group. Rutin, quercetin, kaempherol and vitexin are more effective in reducing plasma total cholesterol and triglycerides than Simvastatin, furthermore, liver function and WBC are not affected as that of the Simvastatin group.

[050] (Step D) Analysis for fatty streak deposition in thoracic aorta.

[051] The chest wall of each rabbit (sacrificed in Step B) was incised, a portion of thoracic aorta from 1 cm site above aortic valve downward for 5 cm was cut out. The surrounding fatty tissues were removed, the aorta was incised longitudinally and pinned to a dish. The moist aorta was then photographed, and staining of fatty streak was carried out with the method described by Esper, E. et al. (Journal of Lab. Clinical Medicine; 121, ppl03-l 10(1993)) as follows.

[052] The opened portion of aortas were pinned to a wooden tongue depressor, washed three times for 2 minutes with anhydrous propylene glycol and stained for 30 minutes with saturated solution of Oil Red O dissolved in propylene glycol. Then the aortas were washed twice for 3 minutes with 85 % propylene glycol, to remove remaining staining solution by washing with normal saline solution. The aortas were photographed, and the photographs were traced with an image analyzer (LEICA, Q-600, Germany), and the area of stained portion were fatty streak region, its proportion in percentile (%) to the total aorta area were calculated. The results were shown in Table 111. [053] FIGS. IA, IB, IC, ID, IE, IF show that the aortas of the rabbits administered with 1% cholesterol control group; 1% cholesterol plus 1.5mg/kg simvastatin in comparative group; 1% cholesterol plus 0.15% rutin group; 1% cholesterol plus 0.15% quercetin group; 1% cholesterol plus 0.15% kaempherol group; 1% cholesterol plus 0.15% vitexin group respectively. The microscopic pictures which showed a thick layer of macrophage lipid cell complex in the arota walls of IA, but no or a very thin layer of macrophage lipid cell complex in the aorta walls of IB, IC, ID, IE, IF. ( Pictures are not shown in this setting ).

[054] It is concluded that the rutin, quercetin, kaempherol and vitexin in the present invention can prevent and /or inhibit the deposition of macrophage-lipid cell complex onto arterial endothelial walls. Therefore, long term administration would prevent and inhibit atherosclerosis and arteriosclerosis induced cardiovascular diseases and the formation of fatty liver as depicted on Table III. The results were tested by using student t-test and Microsoft excel-7.0 program.

[055] (Step E) Pathological examination of rabbit organs.

[056] Internal organs from the rabbits sacrificed in Step B including aorta, lung, heart, liver, kidney, muscle, bladder and pancreas were visually examined and showed no abnormalities. One half of each organ was frozen and the other half was fixed with 10% neutral buffered formalin solution for 24 hours. Then the fixed organs were washed with tap water and stepwise dehydrated with 70%, 80%, 90%, 100% ethanol, then embedded in paraffin by using Shandon, Histocentre-2 The embedded organ blocks were sectioned in 4 mu.m thickness with a microtome (McBain, M 820, American Optical Co. USA) and stained with hematoxylin and eosin stain (H. E. stain). Then the stained specimens were made transparent with xylene, and mounted with permount on micro slides. There were no pathological abnormalities or lesions under

microscopic examination. EXAMPLE 6

INHIBITION AND PREVENTION OF FATTY DEGENERATION IN THE LIVER OF THE RABBITS.

[057] According to the report of Fogt F. and Nanji A., (Toxicology and Applied Pharmacology; 136, pp 87-93, 1996) and Keegan A. et al., (Journal of Hepatology; 23, pp 591-600, 1995). Fatty degeneration of liver can be classified into four grades based upon the abnormal fat containing cells around the central vein of liver acinus, for example: grade 1 (0-25%), grade 2 (26-50%), grade 3 (51-75%), grade4 (76-100%). The effects of rutin, quercetin, kaempherol and vitexin on liver tissue on the rabbits from Example 4 Step B were examined. The results are depicted in Table III.

[058] FIGS. 2 A, 2B, 2C, 2D, 2E and 2F represent the microscopic features of the liver of the rabbits administered with 1% cholesterol; 1% cholesterol plus 1.5mg/kg Simvastatin; 1% cholesterol plus 0.15% rutin; 1% cholesterol plus 0.15% quercetin; 1% cholesterol plus 0.15%) kaempherol and 1% cholesterol plus 0.15% vitexin respectively. The features of 2A and 2B show many fat containing cells around the central vein. The other features in 2C, 2D, 2E and 2F showed that almost all of the liver cells are normal without containing fat particles. It depicts and shows that rutin, quercetin, kaempherol and vitexin can inhibit and prevent formation of fatty liver and fatty degeneration. Furthermore, one of 2B contains hepatic adenoma picture. This depicted that Simvastatin has serious side effect to the liver even with short term administration^ all the pictures are not shown in this settins )

EXAMPLE 7 HMG-CoA REDUCTASE INHIBITED BY RUTIN, QUERCETIN, KAEMPHEROL AND VITEXIN.

[059] The activity of 3 -HMG-CoA reductase was determined by modified Hulcher's method (Journal of lipid research; 14, 625-641, 1973), in which the concentration of Co-A will be produced when 3-HMG-CoA is reduced to mevalonate salt by the action of the 3-HMG-CoA reductase; then the reductase was determined by spectroscopy and the activity of the 3-HMG- CoA reductase was calculated.

060] Figure-I, Depicts the pre-treatment pathological pictures of tumor cells In various types of cancer in experimental animal, and human models:

I-A ( breast cancer); I-B ( colon cancer ); I-C (lung cancer);

I-D ( Ovarian cancer ) I-E ( Cervical cancer ) I-F ( Uterine cancer )

[061] Figure-II, Depicts the post-treatment pathological pictures of the tumor cells in various types of cancer in experimental animal models which were treated with intra-peritoneal injection of bromelain In an amount of 25 mg / twice a week for 8 weeks which all shown complete resolved of all 6 types of cancer.

[062] Figure III, Depicts the post-treatment by either CT scan report and / or pathological pictures in various types of cancer in human model which were treated with bromelain by oral and intravenous infusion. All tumors were either completed resolved or more than 50 % resolved

DETAILED DESCRIPTION OF THE INVENTION DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[063] Bromelain extract including both stem and fruit bromelains, have all the effects that natural bromelains depict including anti-inflammation, anti-platelet aggregation, fibrinolytic properties, anti-tumor growth , and differentiation effect in tumor cells. Fruit bromelain from pineapple was first isolated by

Marcano in 1891 ( Marcino Bull. Pharm. 5, 77 (1891), and from pineapple juice by precipitation with acetone and also with ammonium sulfide : Heinecke in 1961 (US 3002891 ). Bromelain has a molecular weight of 33,000. It is a glycoprotein, was purified from crude preparations by Gibian, and Bratfisch et al in 1960 ( US 2950227 ).

[064] Stem bromelain was discovered by Balls et al in 1941 , (Ind. Eng. Chem. 33; 950, 1941), both fruit and stem bromelains are acidic and basic proteins with ultrviolet light wavelength of 280 nra (A 1%/lcm 20.1 )

Preparation and analysis of Bromelain (Comosain ) extract.

Example -A

Cloning and expression of the minigene of Comosain and its extraction procedure.

Assembly of expression vector for the Comosain gene and Preparation of genetically modified organism (Gmo)

[067] The oligo-deoxyribonucleotide probe synthesis was used, the phosphoramidite method was used for oligonucleotide synthesis. Each probe mixture contained a pool of 20-oligonucleotides sequences were:

Probe mixture: CoM-V sequence equivalium to: valine-proline-glutamine-serine-isoleucine, aspartic acid-trytophan-arginine-asparagine-tyrosine-glysine -alanine-valine-threonine-serine- valine-lysine- asparagine-glutarmine-glycine.

Probe mixture: CoM-V=

Val-Pro-Glu-Ser-Iso-Asp-Trp-Arg-Asn-Tyr-Gly-Ala-Val-Thr-Ser- Val-Lys-Asn-Glu-Gly 3' CAA, GGA, GTT, — ,TTG,GTT, TT5'

V D G V

P W A K

Q R V N

s N T Q

I Y s G

V=Valine, P=Proline, Q= Glutamine, S=Serine, I=Isoleucine.

D=Aspartic acid, W=Tryptophan, R=Arginine, N=Asparagine, Y=Tyrosine.

G=Glycine, A=Alanine, V=Valine, T=Threoninie, S=Serine.

V=V aline, K=Lysine, N=Asparagine, Q=Glutamine, G=Glycine.

The probe mixtures were labeled at the 5' end with [ r ³² p ] ATP, 7500-8000 Ci/mmol (ICN) (1 Ci= 37 GBq), by using T4 polynucleotide kinase.

[068-A] Hybridization procedures: phage plaques were ampilified according to the procedures of Woo, except that GeneScreen Plus filters and NZYAM plates [NACL, 5 g; MgCl ₂ -6H20, 2 g; NZ- Amine A, 10 g; yeast extract, 5 g; Casamino acids, 2 g; maltose, 2 g; and agar, 15 g (per liter) were used, Phage particles were disrupted and the DNA _S were fixed on filters ( 50,000 plaques per 8.4 x 8.4 cm filter). The air dried filters were baked at 80° c for 1 hour and then subjected to proteinase K digestion [ 50 ug of proteinase k per ml of buffer solution containing 0.1M Tris-HCl ( PH 8.0 ), 0.15 M NaCl, 10 mM EDTA, and 0.2% NaDodSo4 for 30 min at 55 °C. Prehybridization with a 1 M NaCl/1% NaDodSo4 solution was carried out again at 55 °C for 4 hours or longer

The hybridition buffer contahined 0.025pmol/ml of each of the 20 probe sequences of 0.9M NaCl/5 mM EDTA/50 m M solution phosphate, PH 6.5 /0.5% Na Dod So4/ 100ug of yeast tRNA per ml. Hybridization was carried out at 48 °C for 20 hrs by using the ComV probe mixture, (that is 2 ⁰ C below the lowest calculated dissociation temperature ( td ) for members of The mixture. At the completion of hybridization, the filters were washed three times with 0.9 M NaCl/ 90 mM sodium citrate, pH 7.0/ 0.1%NaDodSo4 at room temperature at hybridization and 10 min per wash.

[068-B]. For direct expression of the genomic Comosain gene, 4.8 kilobase (kb) of BstTy/Se- and BamAs/GL fragment of Comosain, which contains the entire gene. After converting the BstTy/Se (tyrosine— serine 10 amino acids )site into Bst As/GL( asparagine -to-glycine 20 amino acids ) site with a synthetic linker( pBR322 ori ) the fragment was insert into the unique

BamAs/GL site of the expression vector pDSVL, which contains a dihydrofolate reductase (DHFR ) minigene. The resulting Plasmid DSVL-gPlCOS ( gene Plant Comosain) was then used to transfect New Zealand white rabbit ovarian (NWRO) cells by the calcium phosphate microprecipitate method. The transformants were selected by the medium lacking hypoxanthine and thymidine. The culture medium used was Dulbecco's modified Eagle's medium

supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine.

[068-C]. Isolation of Comosain mRNA. : The 4.8 kilobase Bst ty/se— Bam As/GL restriction fragment from Comosain was inserted into shuttle vector, pSV4ST. The resulting chimeric plasmid pSV gPLComo was used to transfect Cos-1 cells by the calcium phosphate micoprecipitate method. After culture for 72 hrs, RNA was prepared from the transected cells by the guanidinium thiocyanate procedure of Chirgwin et al. and poly(A)+ mRNA was isolated by binding to oligo-cellulose.( Aviv & Leder ).

[068-D]. cDNA cloning.: A Comosain cDNA bank was constructed according to a modification of the general procedures of Okayama and Berg by using the poly(A)+ mRNA described above ( Mol. cell biology 2, 161-170 , 1982 ).

[068-E]. DNA sequencing. : Restriction fragments were cloned into M 13 phage vectors by using Escherichia coli strains JM 103 and /or JM 109 as host. (Messing, J .of methods enzymology 1983) and were sequenced by the dideoxy method of Sanger et al. some regions were sequenced by kinase labeling or end-fill labeling of restriction fragments followed by chemical cleavage as described by Maxam and Gilbert. (J. of methods of enzymology 1980).

[068-F]. The final Comosain Recombinants were collected , extracted, washed with ethyl alcohol, and purified by AKAT Prime ( GE Co. ) and / or FPLC -cation-exchange

chromatography, to produce of F4, F5, and F-9a, F9b ( Comosain and Ananase ) for future use.

[069 ] EXAMPLE-B ( see 68-B ) Direct expression of the genomic Comosain gene.

[070 ] EXAMPLE -C ( see 68-C ) Isolation of the Comosain m-RNA

[071 ] EXAMPLE-D ( see 68-D ) Cloning of Comosain c- DNA.

[072]EXAMPLE- 1

TOXICITY OF BROMELAIN (COMOSAIN) IN MICE BY ORAL ADMINSTRATION.

[072] 30 specimens of 8 week old, with specific pathogen free, ICR female mice, each weighing 25-30 gram, were divided into five groups ( 6 mice each ) and were kept in separated cages under an environment of 23+_ 3 degree C, relative humidity of 45+_5 % , and 12 H light/ 12 H dark photoperiod, they were fed with Harlan Teklad -2018 global rodent diet ( 18 % protein ) ( Kaytee Co. Madison, WI. ) ; drinking water was sterilized.

[073] Bromelain (Comosain) was dissolved in 0.5% of tween-80 solution to a final concentration of 50 mg/ml, 100 mg/ml, 125 mg/ml and 150 mg/ml respectively, and was orally fed to the 4 separated groups of mice in an amount of 0.2 ml per 20 gram of mouse body weight, that is contains 500 mg/ kg, 1000mg/ kg, 1250 mg/ kg, and 1500 mg/ kg separately. One group of 6 mice was kept as control group and was not fed the bromelain

solution. The solution was administered once every 2 weeks, and the mice were observed for 6 months for the signs of adverse effects or death according the following schedule: 1 H, 4 H, 8 H, 12 H ( hour ), after administration and then every 12 hours thereafter. The daily weight of each mouse was recorded. On day 180, ( 6 months later ) the mice were sacrificed, and the internal organs including liver, kidney, heart, lung, muscle, stomach, urinary bladder, intestines, pancreas, and spleen were examined visually and microscopically.

[074] All mice were alive at 6 months, and no body weigh loss occurred during this period of observation. The mice did not develop any pathological abnormality either visually or microscopically. Therefore, it is concluded that Bromelain extract from pineapple is not toxic when orally administered to a mammal.

EXAMPLE- 2

BROMELAIN AND QUERCETIN INHIBIT CANCER CELL GROWTH IN VITRO

[075] Cancer cell lines were developed either from direct harvest from surgical specimens during surgery or from friends at different oncology institutes. The specimens were emulsified in normal saline and filtrated three times with the mesh permitting less than 5um particles to pass through. The supernatants were preserved in Complete Growth Medium (CGM) (Sigma-Aldrich Co. St. Louis, MO) in the 75 ml flasks at 8 degree C until they are ready to be used for seeding in a standard tissue culture. Complete Growth Medium (CGM) consisting of : Dulbecco's modified Essential medium ( Sigma- Aldrich Co. St. Louis, MO ) supplemented with 10 % heat inactivated new born calf serum, and 2 % L-glutamine, penicillin (100 IU/ ml ), streptomycin ( 5 mg / ml ) , and neomycin ( lOmg /ml ) ( all from Sigma- Aldrich Co. St. Louis, MO ) and incubated at 37 degree C with humidified atmosphere of 90 % air, 10 % C02 and cell split at 1 : 2 rate twice weekly.

[076] Six various types of cancer cell lines which included breast, lung, colon, cervical, uterine, and ovarian cancer were used to test the sensitivity of the growth inhibition by the Bromelain in different concentrations. The cells were seeded in 2x (10) ³ into 96 wells tissue culture microtiter plates (Becton-Dickinson Co. Franklin Lakes, N.J.). The cells were maintained in a standard tissue culture In Complete Growth Medium (CGM). Bromelain solutions were added to culture media in the following concentration: 0.2 mg/ml, 0.4 mg/ml, 0.6 mg/ml, and 0.8 mg/ml for 72 hours and tumor cells were counted with Coulter counter

(Model B, Beckman Coulter, Co.). The tumor cell growth inhibition percentages are depicted in Figure -2. which demonstrates that the growth of all cancer cell lines were inhibited in higher concentrations of Bromelain (Comosain).

EXAMPLE- 3

BROMELAIN AND QUERCETIN INHIBIT CANCER CELL GROWTH IN VTVO ON EXPERIMENTAL ANIMAL.

BROMELAIN AND QUERCETIN INTRAPERITONEAL ADMINISTRATION TO EXPERIMENTAL ANIMAL.

[077] 14 Experimental animals of 4 to 6 weeks old white rabbits each weighting 1 to 1 ½ pound were fed under a condition of 23+_3 degree C, relative humidity of 45+_5 % and photoperiod of 12 light/ 12 dark. The rabbits were divided into seven groups with 2 heads each and were fed with Harlan-Taklad rabbit die TD-1376 ( Madison, WI. ) containing moisture 12 %, crude protein 16 %, crude fat 2 %, crude fiber 15 % , ash 8 %, nitrogen free substances 47 %.

[078] The rabbits were fed for 3 weeks with free access to the diet and water body weight was recorded every 7 days, and records were analyzed. All rabbits showed a normal growth rate with no significant differences among the seven groups in regard to the diet ingestion amount or the body weight gain. The cancer cell lines were injected into six groups of the rabbits, that is 2 heads each group with 2 heads served as control (without tumor cells injection ). The cancer cell lines were developed from Example -2. Each head was injected 0.5 ml of different cell line fluid intraperitoneally, prefer in the peritoneum layer, then the rabbits were fed the same diet for 3-4 weeks until a tumor grew in the peritoneum. The size, and location of the tumors were recorded. When the tumors reached to 3-5 mm diameter in size. Bromelain in the amount of 25.0 mg/ ml in normal saline Quercetin 5mg/ml with 100 mg of vitamin C (to keep the solution acidified), 2 ml of Bromelain was injected into six different group of rabbits, the bromelain were given twice a week for 8 weeks.

[079] After 8 weeks of treatment, the rabbits were anesthetized with injections of ketamine 75 mg/kg in the femoral muscle and sacrificed. Blood samples were collected from the heart of each rabbit to determine the blood analysis consisting of: complete blood count ( CBC ), Chemistry-7 and 24, ( including Liver and renal function tests ), lipid profiles ( including Total cholesterol, HDL, LDL, VLDL, and triglycerides ), coagulation factors consisting of; prothrombin time ( PT ), partial thromboplastin time ( PPT ), and immune-globulin -E . All the laboratory tests were analyzed, and showed no differences among or within each groups. All laboratory blood analysis were performed on rabbits of all 7 groups, The results were tested using student t-test and Microsoft Excel-7 programs. The results are depicted in Table-II

[080] Table -II, The table presents blood analysis of the rabbits of 6 different groups which were treated with bromelain after inoculation of cancer cell lines. Control group; TC ( 183.3+_50.2 mg/dl ), TRG ( 1 10+_ 40.6 mg/dl ), HDL (45.6+_20.4 mg/dl ), SGOT ( 38.6+_ 6 .2u/l ), SGPT ( 62.5 +_ 6.5 u/1 ), GGTP ( 8+_ 2.4 u/1 ), WBC ( 6.8 +_ 2.0 k/ul ), Hb; ( 12.3+_2.2 gm/dl ). Bromelain treated groups; TC ( 175.6+_ 36.8 mg/dl ), TRG ( 92.6 +_ 38.8 mg/dl ),HDL ( 43.6 +_ 16.5 mg/ ), SGOT ( 110.8 +_ 30.7 u/1 ), SGPT ( 71.2 +_ 3.8 u/1

),GGTP ( 7+_ 1 ), WBC ( 7.3+_ 2.2 k/ul ), Hb; ( 1 1.9+_ 1.9 gm/dl ). TC; Total Cholesterol, TRG: Triglycerides, WBC: White Blood Cell, HDL; High Density Lipoprotein, SGOT; Serum Glutamo-Oxalic Transferase, SGPT; Serum Glutamo-Pyruvic Transferase, Hb ; Hemoglobin.

[081] The internal organs from the rabbits sacrificed in this sample including lung, heart, liver, kidney, muscle, omentum, intestine, stomach bladder and pancreas were visual examined and showed no abnormalities. One half of each organ was frozen and the other half was fixed with 10 % neutral buffered formalin for 24 hours. Then the fixed organs were washed with tap water and stepwise dehydrated with 70%, 80%, 90%, 100 % ethanol and then embedded in paraffin by using Shandon- Histocentre-2. The embedded organ blocks were sectioned in 4 mu.m thickness with a microtome ( McBain, M 820, American Optical Co. USA ) and stained with hematoxylin and eosin stain ( H.E. stain ). The stained specimens were made transparent with xylene, and mounted with permount on microslides. There were no pathological abnormalities or lesions under microscopic examination. All the specimens collected from six group of rabbits showed no evidence of persistent disease or cancer cells. Therefore, we conclude that Bromelain can served as a chemotherapeutic agent in various types of cancer in experimental animal without side effects.

EXAMPLE 4

BROMELAINASE (COMOSAIN) AND QUERCETIN ORAL ADMINISTRATION TO LATE STAGE CANCER PATIENTS TO INHIBIT TUMOR GROWTH.

[082] 24 volunteers were divided into six groups ( four in each group ) and 6 persons serve as control group ( no bromelain treatment ). All were in their 4th and 6th decades with various types of cancers including breast, lung, colon, ovarian, cervical and uterine origins. All were in either Stage III or Stage IV the cancers had metastased widely either to lung, liver, bladder or rectum). All had been treated with either radiation or chemotherapy after surgery but experienced no positive results. The bromelainase were administered in doses of 20-30 mg/kg ( 75 to 100 GDU / day) based on 50-60 kg of body weight. That is 1500mg-to- 2000 mg /day (or 5,000 to 6,000 GDU /day) and Quercetin in an amount of 2 mg/kg/day divided into two doses. Patients were monitored with bi-weekly blood tests consisting complete blood counts, chemistry-7, chemistry-24, kidney and liver function tests, tumor markers, coagulation factors,' and X-ray or CT scan in appropriate areas to determine the size of the tumors. There were no abnormalities in all the blood tests, no anemia, no leucopenia, no thrombocytopenia, no abnormal kidney nor liver function tests. Tumor markers decreased, tumors were shrank and decreased in size on the x-ray or CT scan measurement. Patients' lifestyle become manageable, and improved considerably. The treatment period were varied from 6 to 12 months. At this report in the treatment groups no patients expired, however, all patients in control group, who had not wish to be treated, succumbed to their related cancers in 6- 12 months.

[083] ( Step-A) After blood samples were collected and allowed to stand for 2 hours, then centrifuged at 4000rpm for 10 minutes. ( Megafuge, Baxter-Heraeus freeze before analysis. The chemistry analysis was carried out by blood chemistry analyzer ( Cobra-Integra-700 , Roche Diagnostic Lab. Indiana.) to determine the changes in total cholesterol, HDL, LDL, triglycerides, liver function tests ( such as SGOT, SGPT, G-GPT ) , renal function tests, and coagulation factors ( PT, PTT)( Bayer-MLA-Electra-900 Automatic coagulation Timer ). All results were tested with student t-test and Microsoft Excel-7.0 program. The results are depicted in

Table-Ill. Which were all within normal limits. Table-Ill, This table presents blood analysis of 24 volunteers suffering from various types of cancer with bromelain oral therapy. TC; ( 210.3+_ 30.2 mg/dl ), TRG ; ( 165.5 +_ 28.3 mg/dl ), HDL; ( 43.3 +_ 22.2 mg/dl ), SGOT ; ( 34.7+_ 6.2 u/1 ), SGPT ; ( 63.3+_ 5.6 u/1 ), GGTP; ( 7.2+_ 2.1 u/1 )WBC; ( 6.7 +_ 2.8 k/ul ), Hb; (12.3+_ 2.1 gm/dl ) Therefore, we concluded that the treatment of these various represented types of cancers with large doses and prolong periods with BROMELAIN are effective

and without side effects.

EXAMPLE 5

BROMELAIN ( COMOSAIN ) INTRAVENOUS ADMINISTRATION TO INHIBIT TUMOR GROWTH IN HUMANS

[084] In addition to a control group of two untreated individuals, tested groups were established as follows ; twelve human volunteers age from 4th through 6 decades, were divided into six groups of two , all of whom m were in Stage -3, , ovary, cervix, and uterus. In the past, all patients had chemotherapy, and /or radiation therapy after surgery without effect. They were given Bromelain (Comosain) in doses of 50GDU/kg (20 mg/ kg) body weight of 50 to 60 kg, which equivalent of 1000 mg each week/( 500 mg twice a week), Bromelain was administered intravenously in 6-8 hours period, for the 3 to 6 months. In addition to the Intravenous infusion of bromelain, all patients in the test groups also received oral bromelain in doses of 50mg/kg/day (3000mg/day) to increase therapeutic effects. Blood tests, including CBC, Chemistry-7, and 24, liver and renal function tests, lipid profile, coagulation factors, PT., PTT, IGG, tumor marks were tested every 6

weeks. And x-ray, and CT scan were performed every 3-4 months to measure the size of the tumors. The blood analysis on these groups of patients showed not affects from the bromelain infusion treatment. (The results are not depicted.) At the conclusion of the 6 months treatment period, the results are depicted as follows:

( A ) Two breast cancer patients—

( 1 ). One patient experienced left breast tumors shrinkage from 6.5 cm X 6.4 cm to 2.5 cm X 1.8 cm in size. The left axillary lymph node shrank from 2.5 cm X 1.6 cm to 0.5 cm X 0.4 cm ( Left breast ).with 3 months of intra-venous ( IV ) bromelain therapy, and achieved complete remission in 6 months

( 2 ) The second patient's right breast tumor shrank from 6.5 cm X 4.8 cm to 2.8 cm X 1.5 cm. with 4 months of intravenous bromelain therapy. And achieved complete remission in 9 months.

( B ) Two Lung cancer patients ~

( 1 ) One patient experienced the shrinkage from 4.6 cm x 5.1 cm to 1.8 cm x 2 cm with 12 weeks of intra-venous bromelain therapy. With further therapy for 4 months the tumors were completely remission.

(2) Another patient with lung tumor of 3.9 cm x 4.5 cm experienced reduction of the tumor to 1.5 cm x 1.9 cm in 3 months treatment, with 5 more months of IV bromelain therapy, Patient achieved complete remission.

( C ) Two Colon cancer patients— ( 1 ) Two patients with stage-IV disease and widely metastasis in the abdominal cavity, have been treated with intra-venous bromelain infusion for 6 -9 months, and showed no evidence of persistent disease.

( D ) Two Ovarian cancer patients—

( 1 ) Both patients suffered stage-Ill C with wide intra-abdominal metastasis, both had been treated with intra-venous bromelain infusion for 6 months, and showed no evidence of persistent disease on CT scan and Ultrasound examination.

( E ). Two patients with cervical cancers-

( 1 ) Both patients were stage-IV with rectal and urinary bladder invasions. After intense bromelain intra-venous treatment for 9 months, there was no further evidence bladder or rectal invasion. No tumors could be detected.

( F ). Two patients with Uterine cancer—

( 1) Both patients were Stage-Ill with intra-vaginal metastasis, After intra-venous bromelain treatment for 4—6 months, tumors in the vagina showed necrosis and fibrosis with no evidence of persistent disease.

( G ). Two patients comprising control group, who refused to be treated—

( 1) One patient with cervical cancer stage-IV with cervical spine metastasis, and one patient with breast cancer stage-IV with pulmonary metastasis. Both patients refused treatment, and both succumbed to their diseases with pulmonary, and septic infections in 9 to 12 months after frequent in-hospital cares and treatments.

( H ) A case of Hepatoma (hepato-cellular carcinoma ) ; this tumor when it was

discovered it was measured 15 CM x 15 CM x 10 CM in size, the surgical resection was not feasible, he received one course of chemotherapy without result and suffered from sepsis and bacteremia for 2 months, then he was given P.O. Comosain at a dose of 3000 mg/day, and IV Bromelain therapy for 7 months, patient achieved complete remission.

[085] Again, this is evidence that administration of Bromelain (Comosain) extract

(Bromelainases) in humans could treat various types of cancer and neoplastic diseases. Liver and renal function, lipid profile, white blood cells and hemoglobin were not affected. There are no side effects and toxicities.

[086] EXAMPLE -6

PHARMACEUTICAL FORMULATION AND PREPARATION

[087] Hard and/ or soft gelatin capsules are prepared with ingredients as follows:

[088] Formulation-l

Quantity (mg/capsule): Active ingredient ( 1 ) Comosain 500 , Active ingredient (2 ) Quercetin 250, Ascorbic acid 200, Starch or Lactose ( carrier ) 50, Total 1000 mg.

[089] Formulation-2

Quantity (mg/capsule) : Active ingredient ( 1 ) and ( 2 ) 1000, Ascorbic acid 300, Starch or Lactose (oarrier) 200, Total 1500 mg.

[090] FormuIation-3

Quantity (mg/capsule): Active ingredient ( 1 ) and ( 2 ) 1600, Ascorbic acid 300, Starch or Lactose (carrier) 100, Total 2000 mg.

[ 091] ] Formulation-4

Quantity (mg/ vial): Active ingredient- 1 ; Comosain 1 gram, Active ingredient-2; Quercetin 250 mg, Ascorbic acid 1000mg/ cc, Normal saline solution 3.0 ml which constitutes total volume of 5 ml for injection.

[ 092] ] Formulation-5

Quantity (mg/ vial): Active ingredient- 1 ; Comosain 2 gram, Active ingredient-2; Quercetin 500 mg, Ascorbic acid 1000mg/cc, Normal saline solution 3.0 ml which constitutes total volume of 5 ml for injection.

[093] Formulation -6

Quantity (mg/ vial): Active ingredient- 1 ; Comosain 3 gram, Active ingredient-2; Quercetin 750 mg, Ascorbic acid 1000mg/cc, Normal saline solution 6.0 ml which constitutes total volume of 10 ml for injection.

[094] While the present invention and discovery has been described with respect

to the above specific descriptions and embodiments, it should be recognized that

various modifications and changes may be made to the present invention by

those skills in the art which also fall within the scope of the invention as defined

by the appended claims and their legal equivalents

FORM FDA 1572-Page-l . (CONTINUATION PAGE) Page-1-2

ITEM-3

2). Name of Research facility : For Analysis of InterLuken-IB, II-6, II-8.& TNF.

Dr. Horvaweths, Professor

Department of Molecular Bio-Chemistry

California Technological Institute

Pasadena, California.

3). Name of Research Facility : For Pathological slide Examination

Dr. & Professor Michael Fishbein

Department of Pathology

UCLA Medical Center and Hospital

Los Angeles, California

Item-4: Name of Clinical Laboratories Facility to be used

2). Garfield Medical Center and Hospital

525 N. Garfield avenue,

Monterey Park, California 91754

1 J.American Bio-Clinical Laboratories,

2730 N. Main Street

Los Angeles, California 90031

Reference 10: 3501908 you shourd submit 7-day reports by a rapid means of comm facsimile or email. You should address each submission to ft and/or to the Chief. Project Management Staff; if you intend to submit 7-day reports by email, you should obi with FDA (see information at the end of this letter); if you also send copies of tbesc reports to your IND, the sul same date as your facsimile or email submission and be cle Reporting any (1) serious, unexpected suspected adverse reacti clinical, animal, or m-vitro studies that suggest significant hum important increase in the rate of a serious suspected adverse re; ail investigators no later than 15 calendar days after determmin qualifies for reporting [21 CFfc 312.32(c)(1)]. If your WD is i 15-day reports to EDA electronically in eCTD format. If your you may submit 15-day reports in paper format; and

Submitting annual progress reports within 60 days of the anniv IND became active (the date clinical studies were permitted to

Secure email between CDER and sponsors is useful for informal cot confidential information may be included in the message (for examp information). If you have not already established secure email with set it up. send an email request to SecureEmaiI(¾fda.hhs.gov. Pleasi not be used for formal regulatory submissions to applications (excep INDs not in eCTD format).

The FDA Electronic Submissions Gateway (ESG) is the central tran information electronically to the FDA and enables the secure submh information for review, if your IND is in eCTD formal, you should additional information, see

Tf you have any questions, call Cliarlcne Wheeler, Regulatory Projec (301) 795-1141.

Sincerely,

{See appended electronic



- -

- -

- -

2. Secondary Outcome Measure:

Analysis Population Description

[Not Specified]

Measured Values

- -

- -

- -

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Bromelain Proteinases ( Comosain ) in treating of various types of late stage refractory solid carcinoma in humans - A double blind study- Complete Study Report of Phase II Clinical Trial-Revised 6/20/2015

Benedict S. Liao, M.D.; Alex B. Liao; Austin L. Liao; Burton A. Liao.; Justin Liao, M.S., M.D.; Edward Liao, M.D.; Jack Liao, M.D., Peter Liao, M.D., PH.D.

From Department of molecular biology, biochemistry, Surgical Oncology, Garfield Medical Group, Oeyama-Moto Cancer Research Foundation, and King /Drew Medical University, Los Angeles, California

This research was supported in part by a grant from The Oeyama-Moto Cancer Research Foundation Grant # (OMCR2002) [Abstract]

1. PURPOSE

a. Bromelain induced Leucocyte binding ability to tumor surface antigens, such as interleukin 2, 6, 8, and TNFs, is known as an Immuno-target therapy. b. Using different concentration of Bromelain proteinases in 6 types of cancer cell, it was studied and result in hydrolysis, fibrinolysis, necrosis, and anti- metastatic effects in tumor cells.

c. Using different concentration of Bromelain proteinases in double blind phase II clinical trial and to find out efficacy of Bromelain in various types of cancer.

2. PATIENTS AND METHODS

a. A total number of 83 patients with 3 ^rd and 4 ^th stage of refractory solid tumors were enrolled, whom at least previously failed on two regimens of chemotherapy and /or failed on radiation therapy.

b. Investigation of anti-metastatic effects in Bromelain were carried out in a double blind study: low dose cohort was on 10 mg/kg/day, and high dose cohort was on 50 mg/kg/day for a period of more than six months.

3. 3. RESULTS

a. The rates of complete response (C R) and partial responses (P R) in high dose cohort are astonishing with 52 % and 27 % respectively.

4. 4. CONCLUSION

a. Anti-cancer effect were achieved in carcinoma of lung, breast, colon, ovary, cervix, and uterus.

b. The implications and results of the findings are discussed with in view of the reported anti-metastatic activity of orally administrated Bromelain.

[Introduction]

Administered of bromelain in cancer treatment in nonclinical trials has been reported as early as in 1968 by Wolf M, & Ransberger k.(l). Both in vitro and animal studies have suggested of anti-metastatic effect for bromelain. Batkin & Taussig (2, 3) in 1988 reported that orally administered bromelain reduced the incidence of pulmonary metastasis in Lewis lung cancer cells in mice. In recent years, Batkin & Taussig(1988) (4) suggested the antitumor mechanisms are due to fibrinolytic effect in Bromelain. Taussig & Batkin in 1988 (5) discovered that Bromelain has anti-Platelet aggregation effects. Taussig and Batkin in 1985 (5) also discovered the inhibition growth of tumor cells such as Lewis lung carcinoma, V-8 lymphoma, MCl-1 acites, KATO-gastric carcinoma cells. Maurer, & Hozumi, in 1994 (6) discovered bromelian Induced Differentiation in leukemic cells. Hale, & Haynes in 1992 (7) and Cantrell (5) et al in 1996 have suggested that MMAPT( Major Mitogen Activating Protein Kinase ) and TPK ( Tyrosine Phosphorylation Kinase ) inhibitors were activated by Bromelain. T-cell activation and cascade production of Interleukin II-B, 6, 8, and TNF-a (Tumor Necrotizing Factors) via CD-2, CD-3 surface antigen of WBC. Garbin, Harrach, Eckert, & Maurer in 1994 (15) and Hale, & Haynes in 1992 (7) also suggested that Bromelain will reduce surface antigens of CD-44, CD- 44 v, CD-44s, CD45, & CD 47 in tumor cells of breast carcinoma.

From the experimental studies above, we conclude that activation of Bromelain proteinases in lymphocytes and T-cells have anti-metastatic effects both in vitro and in vivo.

In the present study, we compared the modulation of low dose cohort and high dose cohort of Bromelain administration to the patients with stage 3 and stage 4 refractory solid tumors, which include various types of carcinoma of lung, breast, colon, ovarian, cervix, uterine, prostate, melanoma, lymphoma, and gastrointestinal origins etc. All patients have previously failed on at least two regimens of chemotherapy and /or failed on radiation therapy. The treatment were carried out for at least 24 to 30 weeks, the complete blood count, liver, renal function, hematopoetic elements, tumor markers were evaluated at an interval of every 4 to 6 weeks. The computerized tomography scan were performed at an interval of every 3 to 4 months. The size of tumors were measured, the tumor markers were recorded for the evaluation of complete response (CR), partial response, ( PR), stable disease( SD), and progressive

disease(PD) according to the Standard Response Criteria of National Cancer Institute ( NCI ). The Common toxicity were recorded by using NCI's Standard Toxicity Criteria. The results of CR and PR were promising and astonishing when Bromelain were administered in high dose cohort patients.

[Materials and Methods]

Bromelain were purchased from Natural Organics Laboratories, Amityville, N.Y., Capsules to contain the Bromelian were purchased from Capusugel Co. Greenwood, North Carolina. Bromelain were analyzed by using SDS-Polyacryl-Amide Gel Electrophoresis ( SDS- PAGE ), Cation Exchange Chromatography ( CEC ), and /or Multicathodal Polyacrylamide Gel Electrophoresis( MC-PAGE ), and Florescence High Performance Liquid Chromatography ( FPLC ) to determinate the purity and separation of bromelain fraction of Fl, F2, F3, F4, F5, F6, F9 in stem Bromelin ( Harrach et al 1994 (10) ) that were detected by Amperometric detection. Monosaccharides fraction are L-fucose, D-galactosamine, D-glucosamine, D-xylose, D-mannose, D-glucose, D-galactose, D-fructose, and Deoxyribose.

Clinical Application and Study protocol:

The Phase II Clinical Study will investigate the efficacy of low dose and high dose cohort Bromelain (Comosain) in human subjects diagnosed with advanced late stage refractory cancers. The Bromelain (Comosain) extract derived from the stem and fruit of Ananas Comosus will be administered orally each day.

1. Patient Eligibility and Selection

(I) Patients suffering from late stage solid cancer of breast, lung, colon, cervical, ovarian, and uterine, prostatic, lymphoma, bladder etc. In stages III and TV with tissue proof of well-documented malignancies, whether by tissue biopsies and/or laparatomy or thoracotomy and have not been helped by conventional radiation therapy and/or chemotherapies for at least two separated regimens are eligible for this study.

(II) Other eligibility requirements to state that patients must have no available therapy known to provide clinical benefit. For example, the breast cancer patients must have failed at least 2 chemotherapy regiments in the metastatic setting, and, if their tumors are HER2+ or hormone receptor (ER, PR) positive, respectively, they must also have failed several anti-HER2 targeted therapies and no longer be eligible for hormonal therapy.

(III) Additionally, the following conditions must be met: a. Patient's age is between 18 and 85+ years. b. Patient is not taking anticoagulants or on antiplatelet therapy. c. Patient does not have a history of abdominal fistula, gastro-enteral perforation, peptic ulcer diseases, or intra-abdominal abscess within 4 months prior to study enrollment. d. Patient has not had major surgery within 4 weeks prior to study enrollment. Patients who have not recovered from adverse events due to surgery performed more than 4 weeks earlier are not eligible for this study. e. Patient does not currently have uncontrolled hypertension, diabetes, or clinically significant cardiac arrhythmia. f. Patient does not have an allergic reaction to Bromelain or Bromelain-containing products. g. Female patients should not be pregnant or breastfeeding. h. Patient's platelet counts must be greater than 100,000/uL. i. Patient's hemoglobin must be greater than 9.0 g/dL. j. Patient does not have significant abnormal hepatic and/or renal function. k. Patient's tumors are measurable; between 0.2 - 10 cm in size and number between 1- 15. All measurable tumors that have spread to the bones, liver, lung, kidney, and abdomen will be included in the data analysis.

(IV) . Patients with following conditions will be excluded from the study: a. Hemoglobulin less than 9 g/dL and WBC less than 4.0 b. Platelet count less than 100,000/μί. c. INR greater than 1.5 d. Patient currently taking therapeutic doses of warfarin or anti-platelet agents. e. Patient has a history of abdominal fistula, gastrointestinal perforation, peptic ulcer disease, or intra-abdominal abscess within 4 months prior to study enrollment. f. Patient currently has uncontrolled hypertension, diabetes, or clinically significant cardiac arrhythmia. g. Patient who had major surgery performed within 4 weeks prior to entering the study; and patients who have not recovered from adverse events due to surgery performed more than 4 weeks earlier. h. Patient with a history of allergic reaction to Bromelain or pineapple-containing products. i. Female patients who are pregnant or breastfeeding. j. Patient with tumors that are widely spread in the chest and abdomen that cannot be measured by CT scan.

Patients who are eligible for this study will be randomly assigned to either the low dose group or the high dose group by a coin toss. Each study subject will be assigned a patient number for the purpose of this study.

Drug Dosage and Schedule

The Dose of Bromelain at 50 mg (125 GDU )/kg/day is extrapolated from in vivo animal studies, and determined to be safe by a safety study on healthy human subjects (see Section VII-A and HR Maurer's study (42) in 3000 patients; Bromelain

Complimentary Tumor Therapy: Journal of Oncology, 31; p.66— 73, 1989).

For this clinical investigation, the high dose group will be given Bromelain at 50 mg /kg/day (at a body weight of 50- 60 kg) to a maximum of 2400 mg (5000 GDU ) /day and divided into 2 doses/day of 1200 mg/dose. In both high dose group and low dose group, the number of patients suffering from well-documented refractory solid malignancies will be at least 60 and 30

respectively to be assigned to each group. All patients are diagnosed with different types of carcinoma. For example: breast, lung, colon, ovarian, cervical, bladder, prostatic and uterine origin, etc. In high dose group, patients will be given Bromelain at 5000 GDU (2400 mg) / day divided into two doses of 1200 mg /dose and taken with meals. In low dose group, patients will be given Bromelain at 1250 GDU (500 mg) / day divided into two doses of 250 mg /dose and taken with meals.

1. Duration and Route of Administration

Study subjects will be provided with Bromelain for oral administration. The containers will be clearly labeled (see Section V-E). Bromelain will be taken orally twice daily with meals. On their bi-weekly visits to the doctor's office, the study patients will be provided with enough doses for two weeks. The study patients are required to keep a journal of the daily doses they take and any side effects they experience.

The study patients will be evaluated using blood tests and/or CT scans at the end of each cycle (i.e., 6 weeks) and at six months for signs of disease progression. If the disease did not progress, then treatment will continue and the patient will be evaluated every six months thereafter until the investigator determines otherwise. If the disease did progress, then the patient will be taken off the study. On the humanitarian base, the low dose group patients will be transferred to the high dose group due to lack of efficacy in the treatment.

2. Evaluations to be conducted

(A) . Blood and laboratory tests schedule: Blood tests will be conducted every 4-6 weeks, Blood tests include CBC, Chemistry-7, Chemistry-24, liver and renal function , CEA, CA125, CA153, CA199, PSA,TSH, alfa-Feto-Protein and other tumor markers, The test results will be recorded for discussion and evaluation.

(B) . Radiological tests schedule will be also assessed every 3 months for the result of CT scan and/ or PET scan,

Each Study Subject will be also assessed every four weeks for any side effects that they may have experienced during the previous four weeks. These side effects will be recorded for evaluation.

(C) . Use of standard toxicity criteria. (NCI Common Toxicity Criteria Manual Page-1 ~

20). Grade I toxicity: WBO 3000/mm ³ (3 k/ ul), Hb> 10 gm/dl- Platelets > 75,000/mm ³ (75 k/ul )No dehydration, No infection, No transfusion, No renal and liver function impairments. Temperature and Fever: 38-39° c.

Grade II toxicity: WBC 2000-3000/ mm ³ , Hb 8-10 gram/dl., platelets 50.000- 75,000/mm ³ , No infection, No transfusion, Mild to Moderate Diarrhea and Dehydration, and requires IV Hydration, Temperature and Fever :39-40°c.

Grade III toxicity: WBC 1000-2000 /mm ³ , Hb 6.5- 8.0 gram/dl, Platelets 10,000 - 50,000/ mm ³ , Has infection, Need Transfusion, Moderate Dehydration from diarrhea Need parenteral hydration .Temperature and Fever: > 40 ° C for less than 24Hrs.

Grade IV toxicity: WBC < 1000/ mm ³ , Hb < 6.5 gm/ dl, Platelets < 10,000/ mm ³ , when WBC <l/ul (1000/ mm ³ ), Life threatening Infection (Sepsis), Need Transfusion, Need ICU Care. Fever and Temperature: > 40 ⁰ C for more than 24 Hrs.

(D) Adverse events and serious adverse events and reporting information:

The NCI listed Adverse Events in CMC (Common Toxicity Criteria) is based on pathological (Allergy/Immunology) and anatomical (Dermatology /Skin) categories to facilitate location of related adverse events.

(E) Grades of Adverse Events

For each adverse event, grades are assigned and defined using a scale from 0 to 5. With 0 representing no adverse event within normal limits and 5 representing death related to an adverse event.

(F) Documenting Related Adverse Events

3. Study Endpoints

At the end of six months, an assessment of the therapy results for each study patient will be performed to determine whether or not to continue with this therapy. Individual data sets will be combined to assess the efficacy of the therapy for the cancers studied. The study endpoints for the both groups are:

3-a.Use of Standard response Criteria: (NCI Chapter Standard Response Criteria 11.1.1 through 11.1.7 and 11.2, 11.3,).

I) . Evaluation of Target Lesions

(A) Complete Response (C R): Disappearance of all Target lesions. Any pathological lymph nodes must have reduction in short axis to < 10mm.

(B) Partial Response (P R): At least a 30 % decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters.

(C) Progressive Disease (P D): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the sum to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions)

(D) Stable Disease (S D): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference to the smallest sum diameters while on study.

II) . Evaluation of Non-Target Lesions

(A) Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (< 10mm short axis) (if tumor makers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response.

(B) Non-CR/ Non-PD: Persistence of one or more non-target lesion(s) and /or

maintenance of tumor marker level above the normal limits.

(C) . Progressive Disease (PD): Appearance of one or more new lesions and/ or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase.

III. Evaluation of Best Overall Response A). For Patients with Measurable (Target Disease) Disease

B) For Patients with Non-Measurable Disease (No-Target Disease)

4. Data Analysis

Data collected from all patients will be analyzed to determine overall efficacy of Bromelain to treat advanced cancers. Statistical analysis such as Student t-test, will be used. The results of the ongoing analyses will be reported to the FDA in annual report.

(4-a) Adverse events are mild to moderate anemia, leukopenia, thrombo -cytopenia and / or liver and renal impairment.

(4-b) Severe and serious adverse events are Liver and / or Renal damage or failure, anaphylactic reaction.

All serious adverse events will be reported to the FDA. 5. Results and Conclusions

The results of the study will be reported as required to the FDA in annual report and now report as following:

1. Age distribution: both in high dose group and low dose group patients are mainly in their 6 decade and above, 68 % and 81 % respectively (see table I). And the male gender are 75 % and 67 % respectively.

2. The disease classification and distribution are as following: breast carcinoma account for 25 % in high dose group and 28 % in the low dose group, in lung carcinoma the incidence are 9.9 %, and 19 % respectively, in colon & G-I carcinoma the incidence are 3.7 % and 7.1 % respectively, in ovarian carcinoma the incidence are 8.6 % and 9.5 % respectively. The uterine and cervical carcinoma in high dose group is about 13 % etc.

Please see table II for Overall disease distribution. Table II showed breast cancer incidence in low dose group and in high dose group are 28.6% and 25% respectively. In lung cancer, the incidence are 19% and 10% respectively. In colon cancer, the incidence are 7.1% and 3.7% respectively. In Ovarian, uterine, and cervix cancer, the incidence are 9.5% and 30.2% respectively. In bladder and prostate cancer, the incidence are 14.4% and 16% respectively. The incidence of melanoma cancer are both 2.5%. In the incidence of liver cancer are 2.4% and 1.2% respectively. In the incidence of lymphoma are 9.5% and 5% respectively. In the incidence of thyroid cancer and sarcoma cancer, both are 2.4% and 1.23% respectively.

The overall clinical response rate in high dose group patient and low dose group patient are as following: the Complete Response (CR) rate are 52 % and 0 % respectively, the Partial Response (P R) rate are 27 % and 0 % respectively. In the patients of low dose group there were no stable disease (SD) and in the patients of high dose group is 13.6 %. The progressive disease (PD) in the high dose group is 9 %, and in the low dose group is 100 %.( Please see Table VI.)

The overall adverse effects and toxicities are shown in Table III-A to Table III-D which all concluded that there were no hematological, renal, and hepatic toxicities in patients of all group.

The primary target lesion size is less than or equal to 2 cm in low dose group and high dose group are 36 % and 38 % respectively. The lesion size between 2-5 cm are 38 % and 39.5 % respectively, the lesion size between 5-10 cm are 28.6 % and 25 % respectively ( Please see table VII).

The tumor markers such as CEA,CA-125,CA-153, CA-199, PSA, TSH, and alpha-feto- protein are being monitored, their value corresponds to the tumor masses, they return to normal value when tumor have been complete responded (CR), and when the tumor progress the tumor marker value are elevated (Please see Table VIII). The serious adverse effect in toxicity in the both low dose group and high dose group are not observed as seen in Table III-A to Table III-D. There were no serious hematopoietic toxicity, no hepato-renal toxicity, no anaphylactic reaction and life threaten events. There were very rarely minor or non-serious side effects such as nausea, vomiting, diarrhea, palpitation, headache, insomnia, pruritus, urticaria and skin rash. We conclude that Bromelain administered in an amount of 2500 to 3000 mg/ day to the patients with average body weight are effective and non-toxic (Please see Table Villi and Table X).

The following figures are self- explanatory for the above results.

Table I. Baseline Characteristics - Age, Gender

Table III. Lab Test WBC, Hb, Platelets, Survey, Labs Test TableIII-A Breast Ca. Patients Number: 20/81 (25%)

Table ΙΠ-Β Uterine Ca., Ovarian Ca. and Cervical Ca. Patients Number: 25/81 (31%)

Table IH-C Lung and Colon Ca. Patients Number: 11/81 (14%) Tumor Measurement in Phase -2 Clinical Trial Study

A. Low dose cohort with measurable disease were either by direct measurement, X-ray, CT scans and /or PET scans (See Table IV). Table IV showed tumors progressions in all low dose group patients without exception.

## Special Note :##Due to the non-efficacy in this low dose treatment, all patients in this cohort were transferred to the high dose cohort to continuing therapy for humanitarian reason.

TablelV. Tumor Measurement In The Low Dose Group

High dose cohort measurable disease were either by direct measurement, x-ray, CT scans and / or PET scans (see Table V). Table V showed tumor regression in 80% of patients in the high dose group.

Table V. Tumor Measurement of The High Dose Group

Table VII. Outcome Measurement- Primary Target Lesion Size

Discussion

In summary, throughout the 6 to 10 months course of double bind study of Bromelain administration for high and low dose group, only the high dose group patients of 50 mg/kg/day regimen showed effectiveness, and the low dose group patients showed no efficacy at all. Both groups did not show serious adverse effects such as leukopenia, anemia, hepato-renal toxicity, anaphylactic reaction, and life-threaten events. Minor adverse effects such as nausea, vomiting, diarrhea, urticaria, insomnia, palpitation, pruritus, and headache occurred rarely. The glycopetitdes of sten Bromelai was obtained from proteolytic digestion with pronase as described by Murachi et al in 1967, and later found that there were four kinds of

glycopeptides, that only differed from each other in the peptide part (Ishihara et al 1979).The amount of the glycopeptide was calculated from its content of glutaminic acid as determinate by amino acid analysis. The average molecular weight was assumed to be 1.5xlO ^A 3 DA. Bromelain contains nine different glyco-polypeptides. Each polypeptides contains amino acids in double benzene ring structure and one of twelve different monosaccharides fraction (Harrach et al 1994). Specifically, breakthrough fraction such as Comosain (F9) account for 80%, ananase account for 10%, the rest of 10% were derived from Bromelain Fl, F2, F3, F5, F6, and so forth(Batkin et al 1988). They mainly comprise of glycosylated multiple enzyme species of the papain superfamily with different proteolytic activities, molecular masses between 20 to 31 kDa, and isoelectric points>10 and 4, 8 respectively. Two major basic proteinase, F4 and F5, were further characterized and shown to have molecular masses of 24397 Da and 24472 DA, respectively(Harrach and Haynes et al 1994&1989). Napper and Bennett et al in 1994 further purified and characterized multiple forms of bromelainases derived from cysteine proteinases Ananain and Comosain. Lee and Albee in 197 postulated the complete amino acids sequence of Ananian and comparison with Bromelain and other plant cysteine proteinases. They all have protein electronic density between 272 to 282 mu.

The remarkable cancercidal effects(Cantrell et al- 5) (Mott-57) probably due to massive production of Interleukin-II, VI, VIII, and tumor necrotizing factors (TNF) (Wajant - 58) from CD-2, CD-3 (Cell Device 2 & 3) in monocytes and lymphocytes (T- cells). The fibrinolitic effects on tumor surface antigens of CD-44, CD-44V, CD-44S, CD-45, and CD-47 (Denning-8) (Eckert & Maurer-12) (Harrach & Maurer - 21) (Hoffman-28) (Matsumoto-41), which induce dehydration, necrosis, and possible calcification in the tumor cells. This action mechanism of Bromelain is mainly due to the inhibition of following two kinases (1) MMAPK (Major Mitogen Activating Protein Kinases) (Cantrell et al-5) (2) TPK (Tyrosine Phosphorylation

Kinases)(Cantrell et al -5). In the WBC culture test that with the concentration of Bromelain in an amount of 1 mg/ml will increase the production of the Interleukin II by 400 times/10 ⁶ WBC, Interleukin-6 by 650 time/10 ⁶ WBC, and the TNF by 42 times / 10 ⁶ WBC (Barnes et al - 2) (Desser et al - 9) (Garbin & Maurer et al - 17).

{Figure l-A} Growth inhibition of various types of tumor cell lines in vitro

{ Figure-IB }: Depicts that detection of the CD44s modulation with two different mAbs clones, L-178, J- 173. Breast carcinoma cells were incubated for 1 hr at 37 C with 10, 50, 75 ug/ml of Bromelain ( Comosain ) treatment. The CD44s become 35 %, 10 %, and 1% of Bromelain (Comosain) treated cells(Birch et al - 4).

According to the outcome measure, the results in the patients in the high dose group showed remarkable complete response rates of 52%, partial response rates of 27%, stable disease of 13%, progressive diseases of 8% in these late stage refractory solid carcinomas by using student T statistical analysis P<0.05 which showed statistically significant. Dr. HR Maurer (42) in his complimentary tumor therapy, he employed more than 3000 patients and treated with bromelain in an amount between 1000-to-3000 mg/day for the period of 1 to 3 years, he did not discover of any severe side effects nor had any life threaten events.

In the present investigation we conclude that high dose of Bromelain therapy (Comosain) in an amount of 2500 mg to 3000 mg is a lifesaving regimen and hoping to save thousands of lives in the future.

Acknowledgement

The authors sincerely thank Dr., Harvowitz of Molecular Biochemistry Department at California Institute of Technology for her expertise assistance in molecular protein sequence analysis and assay in SDS-PAGE mass spectroscopy. Dr. Michael Fishbein of Department of Pathology at University of California in Los Angeles for his expertise and kindness assistance in Pathology. Editorial assistance of Miss Beth Shirokawa, Miss Margaret Cheng, Miss Janet Shih and Miss Oceania Wang are gratefully acknowledged.

( finish ).

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