EP1216051A1 - Anti cancer agent and method of treatment of cancer - Google Patents

Anti cancer agent and method of treatment of cancer

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Publication number
EP1216051A1
EP1216051A1 EP00969057A EP00969057A EP1216051A1 EP 1216051 A1 EP1216051 A1 EP 1216051A1 EP 00969057 A EP00969057 A EP 00969057A EP 00969057 A EP00969057 A EP 00969057A EP 1216051 A1 EP1216051 A1 EP 1216051A1
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EP
European Patent Office
Prior art keywords
phomopsin
hydrogen
cancer
formula
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00969057A
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German (de)
French (fr)
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EP1216051A4 (en
Inventor
John Alexander Edgar
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Commonwealth Scientific and Industrial Research Organization CSIRO
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Commonwealth Scientific and Industrial Research Organization CSIRO
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Publication of EP1216051A1 publication Critical patent/EP1216051A1/en
Publication of EP1216051A4 publication Critical patent/EP1216051A4/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6831—Fungal toxins, e.g. alpha sarcine, mitogillin, zinniol or restrictocin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the treatment of cancer and to compositions for use in treatment of cancer.
  • phomopsin mycotoxins hereafter referred to as phomopsins
  • phomopsins phomopsin mycotoxins
  • their derivatives exhibit potent anticancer activity.
  • phomopsins may be used to provide selective activity against liver cancer. It will be appreciated that the selectivity of phomopsins in treatment of liver cancer is a significant advantage as it allows liver cancers to be targeted while minimising the effects on other tissues.
  • Phomopsins may however be utilised in treatment of cancers other than liver cancer by selecting formulations or derivatives of phomopsins which enhance selectivity of the drug for certain types of cancer cells or certain types of cancers.
  • Derivatives of phomopsins may be formed which are conjugates with monoclonal antibodies.
  • the monoclonal antibody may be produced by known methods to provide selectivity for cancer cells.
  • Phomopsins are characterised by a 13-member ring structure generally of formula I
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are optional substituents and may be independently selected from the group consisting of hydrogen, aliphatic, aromatic, peptide chains and halogen.
  • X is aliphatic, hydrogen or halogen (preferably hydrogen); and Y is aliphatic, hydrogen or halogen (preferably chlorine); where present a peptide chain may be conjugated with a monoclonal antibody (Mab).
  • the phomopsins may be derivatives of compounds of formula I such as the salts thereof.
  • R 1 , R 2 , R 3 , R 5 , R 6 and R 7 may typically be independently selected from hydrogen and aliphatic and R 4 is generally a peptide.
  • R 4 is a peptide conjugated with an antibody, particularly a monoclonal antibody (Mab). More preferably R 1 , R 2 , R 5 and R 6 are lower aliphatic and R 3 and R 7 are hydrogen. Even more preferably R 1 , R 2 and R 6 are lower alkyl and R 5 is lower alkyl or lower alkenyl. Most preferably R 1 is ethyl, R 2 is methyl, R 3 is hydrogen, R 5 is isopropyl or iso-propenyl and R 6 is methyl.
  • the terms lower aliphatic, lower alkyl and, lower alkenyl include groups containing up to six carbon atoms and most preferably up to 4 carbon atoms.
  • At least 60% by weight of the phomopsin component will have stereochemistry 1b.
  • the group R 4 is a peptide preferably a di- or tri-peptide which may optionally be bound to an antibody such as a monoclonal antibody.
  • the preferred group R 4 has the formula II and includes all stereo isomers;
  • R 8 and R 9 are independently selected from hydrogen and lower alkyl and more preferably R 8 is methyl and R 9 is ethyl and R 1 and R 10 are hydrogen or together form a double bond;
  • R 12 is selected from the group consisting of amino, mono substituted amino, disubstituted amino and an amino acid residue particularly the group of formula III:
  • R 13 and R 14 are hydrogen or together form a double bond
  • R 15 is selected from the group consisting of hydroxy, amino, substituted amino or an antibody particularly Mab.
  • R 15 is an antibody or linked to an antibody it is preferred that R 13 and R 14 form a double bond providing a dehydroaspartic acid residue.
  • the carbon-nitrogen bond in the residue of formula III is relatively weak enabling an active phomopsin of formula la (wherein in the group of formula II R 2 is amino) to be released from the MAb once it becomes bound to cancer cells.
  • a dehydroaspartic acid residue is expected to facilitate delivery of phomopsins via the Mab conjugate.
  • phomopsin A phomopsin A
  • octahydrophomopsin A iso-phomopsin A
  • phomopsinamine A phomopsinamine A
  • the invention provides a pharmaceutical composition for treatment of cancer, preferably liver cancer, containing a phomopsin compound or derivative thereof or pharmaceutically acceptable salt of the phomopsins or derivative and a pharmaceutically acceptable carrier.
  • Salts of phomopsins such as the alkaline metal salts are reasonably water soluble.
  • Aqueous solutions can be formed by dissolving the phomopsins in a dilute base such as sodium hydroxide to provide a neutral solution.
  • the invention provides a method of treatment of a patient suffering cancer including administering to the patient a phomopsin compound or derivative thereof or pharmaceutically acceptable salt of the phomopsin or derivative.
  • the phomopsin compound may be administered by a variety of methods including oral administration in the form of a syrup, capsule, tablet or the like, by injection or by intravenous infusion.
  • the compound is administered by intravenous infusion
  • the invention provides the use of a phomopsin compound as hereinbefore described for preparation of a pharmaceutical composition for treatment of cancer and in particular liver cancer.
  • Phomopsin compounds are produced by certain fungi, including Diaporte toxicus (formerly Phomopsis leptostromiformis) and Phomopsis emicis, or may be derived from these natural products.
  • phomopsins The activity of phomopsins is believed to be due in part to the strong binding of the compounds to tubulin. This may disrupt cell mitosis by inhibiting tubulin formation and cause depolymerization of formed microtubules. It may be preferred in some cases to use phomopsins in combination therapy with one or more other anticancer drugs or therapies.
  • the drugs used in combination with phomopsins may be selected to enhance results by providing complementary activity in binding to microtubules. Examples of possible drugs for use in combination with phomopsins include paclitaxel, vinblastine and vincristine.
  • the extraction process is designed to minimise difficulty and cost.
  • the fermented seed is continuously extracted with recycling 15% methanol:water through an in line XAD (styrene divinylbenzene copolymer) column.
  • XAD styrene divinylbenzene copolymer
  • the time required for adsorption of phomopsin A onto the XAD is quite lengthy, but requires minimal operator input. The timing of this step is not critical, hence can be adapted to suit operating conditions.
  • the phomopsin A has a relatively low solubility in 15% methanol.
  • the procedure relies on the adsorption of phomopsin A on the XAD resin driving the solubility equilibrium of phomopsin A in the fermented seed toward dissolution. This procedure reduces solvent usage, volumes to be handled and fiammability hazards.
  • the alternate method of extraction, without recycling would use 150+ litres of pure methanol for the initial extraction, involve a further concentration step (or dilution of the methanol extract to 900+L) then adsorption onto XAD.
  • the current procedure uses 12 L methanol, requires minimal operation input for the adsorption phase and uses far less solvent (total volume 85L instead of 900+L).
  • the elution of the concentrated phomopsin A from the column is the first step in a 3 stage isolation to produce crystalline phomopsin A of 80-90% purity.
  • phomopsin A may be eluted from the column using 100% methanol.
  • Silica gel flash column chromatography may be used for purification. The column is conditioned using 5:95 ammonia:isopropanol and the concentrate dissolved in a minimum of 20:65:15 ammonia:isopropanol:water. Phomopsin A is eluted using this 3 solvent combination. Recrystallisation from boiling glacial acetic acid provides phomopsin A in 80-90% purity.
  • the methanol eluate made up to 10mls, was analysed by HPLC and then was evaporated to dryness and purified using preparative HPLC.
  • Phomopsin A (15.3 mg) was dissolved in the minimum amount of 1 M HCI and left at room temperature for 28 hours.
  • the reaction mixture was diluted to 8 ml with water then passed through a strong anion exchange cartridge (SAX, 600 mg) to remove any unreacted phomopsin A (pH of solution expected to be about 1.52).
  • SAX strong anion exchange cartridge
  • the aqueous solution of non-adsorbed compounds, and the water washings of the SAX column, were then passed through a prepared C18 cartridge (900 mg).
  • the C18 cartridge was washed with H 2 O (10 ml) then the phomopsinamine A eluted with methanol (10 ml).
  • This method may be modified by sampling the reaction mixture after 5-6 hours, 24 hours and 28-30 hours. All washings and eluates may be assayed by HPLC to monitor the conversion of phomopsin A to phomopsinamine A.
  • the anticancer activity of phomopsin A, octahydrophomopsin A, iso-phomopsin A and phomopsinamine A was assessed against 60 human cancer cell lines in vitro.
  • the methods used to assess anticancer activity are those employed by the United States National Cancer Institute (NCI) as a primary screen for discovering compounds with anticancer potential (Boyd and Paull, Drug Development Research, 34, 91-109 1995).
  • the measured effect of the compound on the Percentage Growth (PG) of a cell line is currently calculated according to one or the other of the following two expressions:
  • PG 100 x (Mean OD tes t - Mean ODtzero)/Mean OD t zer o
  • Mean OD tze r o The average of optical density measurements of SRB-derived color just before exposure of cells to the test compound.
  • Mean OD test The average of optical density measurements of SRB- derived color after 48 hours exposure of cells to the test compound.
  • Mean OD ct ri The average of optical density measurements of SRB- derived color after 48 hours with no exposure of cells to the test compound.
  • Tables 1a to 4b The calculated PGs of each of 60 cell lines for various concentrations of the test compounds are presented in Tables 1a to 4b. Testing was conducted twice for each compound and the results of the testing of this compound phomopsin A (Tables 1a and 1b), octahydrophomopsin A (Tables 2a and 2b), isophomopsin A (Tables 3a and 3b) and phomopsinamine A (Tables 4a and 4b) and demonstrate a dose-related response of most of the cancer cell lines tested to phomopsin A, iso-phomopsin A, octahydrophomopsin A and phomopsinamine A. In particular, the data supported progression of the assessment procedure to in vivo testing. Table 1a
  • OVCAR-5 102 100 95 30 25
  • the Biological Testing Branch of the Developmental Therapeutics Program has adopted a preliminary in vivo screening tool for assessing the potential anticancer activity of compounds identified by the large scale in vitro cell screen (Hollingshead, MG etal., Life Sciences, 57, 131 - 141 , 1995).
  • human tumour cells are cultivated in polyvinylidene fluoride (PVDF) hollow fibers, and a sample of each cell line is implanted into each of two physiologic compartments (intraperitoneal and subcutaneous) in mice.
  • PVDF polyvinylidene fluoride
  • the protocol identifies compounds having moderate to prominent anti-cancer activity, and facilitates identification of sensitive tumor lines and appropriate treatment regimens for subsequent testing in standard, in vivo solid tumor models.
  • Each test mouse receives a total of 6 fibers (3 intraperitoneally and 3 subcutaneously) representing 3 distinct cancer cell lines.
  • Three mice are treated with potential antitumor compounds at each of 2 test doses by the intraperitoneal route using a QD x 4 treatment schedule.
  • Vehicle controls consist of 6 mice receiving the compound diluent only.
  • the fiber cultures are collected on the day following the last day of treatment.
  • viable cell mass is determined for each of the cell lines using a formazan dye (MTT) conversion assay. From this, the %T/C can be calculated using the average optical density of the compound-treated samples divided by the average optical density of the vehicle controls.
  • the net increase in cell mass can be determined for each sample as a sample of fiber cultures are assessed for viable cell mass on the day of implantation into mice.
  • the cytostatic and cytocidal capacities of the test compound can be assessed.
  • each compound is tested against a minimum of 12 human cancer cell lines. This represents a total of 4 experiments since each experiment contains 3 cell lines. The data are reported as %T/C for each of the 2 compound doses against each of the cell lines with separate values calculated for the intraperitoneal and subcutaneous samples. Evaluation
  • Compounds are selected for further in vivo testing in standard subcutaneous xenograft models on the basis of several hollow fiber assay criteria. These include: (1) a % T/C of 50 or less in 10 of the 48 possible test combinations (12 cell lines X 2 sites X 2 compound doses); (2) activity at a distance (intraperitoneal drug/subcutaneous culture) in a minimum of 4 of the 24 possible combinations; and/or (3) a net cell kill of 1 or more cell lines in either implant site. To simplify evaluation, a points system has been adopted which allows rapid viewing of the activity of a given compound. For this, a value of 2 is assigned for each compound dose which results in a 50% or greater reduction in viable cell mass.
  • the intraperitoneal and subcutaneous samples are scored separately so that criteria (1) and (2) can be evaluated.
  • Compounds with a combined IP+SC score > 20, a SC score > 8 or a net cell kill of one or more cell lines are referred for xenograft testing.
  • These criteria were statistically validated by comparing the activity outcomes of > 80 randomly selected compounds in the hollow fiber assay and in the xenograft testing. This comparison indicated that there was a very low probability of missing an active compound if the hollow fiber assay were used as the initial in vivo screening tool.
  • other factors e.g. unique structure, mechanism of action may result in referral of a compound for standard xenograft testing without the compound meeting these criteria.
  • MDA-MB-231 46, 72, 44, 46, >100, >100, 99, >100,
  • SW-620 >100, >100, >100, >100, >100, 78, 78, 97, 86,

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Abstract

A method of treatment of a patient suffering cancer comprising administering to the patient an effective amount of a phomopsin.

Description

ANTI CANCER AGENT AND METHOD OF TREATMENT OF CANCER
The present invention relates to the treatment of cancer and to compositions for use in treatment of cancer.
The search for anti-cancer agents has been, and remains, a major endeavour of the pharmaceutical industry, academic institutions and government agencies throughout the world. One of the significant problems with many cancer treatments is the severe adverse affects they have on the patient and non- cancerous tissues.
We have now found that phomopsin mycotoxins (hereafter referred to as phomopsins) and their derivatives exhibit potent anticancer activity. In addition, and due to the tendency of phomopsins to specifically target the liver, we believe that phomopsins may be used to provide selective activity against liver cancer. It will be appreciated that the selectivity of phomopsins in treatment of liver cancer is a significant advantage as it allows liver cancers to be targeted while minimising the effects on other tissues.
Phomopsins may however be utilised in treatment of cancers other than liver cancer by selecting formulations or derivatives of phomopsins which enhance selectivity of the drug for certain types of cancer cells or certain types of cancers. Derivatives of phomopsins may be formed which are conjugates with monoclonal antibodies. The monoclonal antibody may be produced by known methods to provide selectivity for cancer cells.
Phomopsins are characterised by a 13-member ring structure generally of formula I
wherein
R1, R2, R3, R4, R5, R6 and R7 are optional substituents and may be independently selected from the group consisting of hydrogen, aliphatic, aromatic, peptide chains and halogen.
X is aliphatic, hydrogen or halogen (preferably hydrogen); and Y is aliphatic, hydrogen or halogen (preferably chlorine); where present a peptide chain may be conjugated with a monoclonal antibody (Mab). The phomopsins may be derivatives of compounds of formula I such as the salts thereof.
The preferred phomopsins as selected from compounds containing the group of formula la:
and the derivatives thereof.
In formula I and la R1, R2, R3, R5, R6 and R7 may typically be independently selected from hydrogen and aliphatic and R4 is generally a peptide. In one embodiment R4 is a peptide conjugated with an antibody, particularly a monoclonal antibody (Mab). More preferably R1, R2, R5 and R6 are lower aliphatic and R3 and R7 are hydrogen. Even more preferably R1, R2 and R6 are lower alkyl and R5 is lower alkyl or lower alkenyl. Most preferably R1 is ethyl, R2 is methyl, R3 is hydrogen, R5 is isopropyl or iso-propenyl and R6 is methyl. Where used herein the terms lower aliphatic, lower alkyl and, lower alkenyl include groups containing up to six carbon atoms and most preferably up to 4 carbon atoms.
The preferred stereochemistry of the compounds of formula la is as shown in formula lb:
Preferably at least 60% by weight of the phomopsin component will have stereochemistry 1b.
The group R4 is a peptide preferably a di- or tri-peptide which may optionally be bound to an antibody such as a monoclonal antibody. The preferred group R4 has the formula II and includes all stereo isomers;
wherein the dotted line represents an optional double bond;
R8 and R9 are independently selected from hydrogen and lower alkyl and more preferably R8 is methyl and R9 is ethyl and R 1 and R10 are hydrogen or together form a double bond;
R12 is selected from the group consisting of amino, mono substituted amino, disubstituted amino and an amino acid residue particularly the group of formula III:
wherein R13 and R14 are hydrogen or together form a double bond and
R15 is selected from the group consisting of hydroxy, amino, substituted amino or an antibody particularly Mab.
When R15 is an antibody or linked to an antibody it is preferred that R13 and R14 form a double bond providing a dehydroaspartic acid residue. In such a case, the carbon-nitrogen bond in the residue of formula III is relatively weak enabling an active phomopsin of formula la (wherein in the group of formula II R 2 is amino) to be released from the MAb once it becomes bound to cancer cells. Thus a dehydroaspartic acid residue is expected to facilitate delivery of phomopsins via the Mab conjugate.
The most preferred phomopsin compounds are selected from phomopsin A, octahydrophomopsin A, iso-phomopsin A and phomopsinamine A. These compounds have the formula set out below:
, c , d
The patent may be treated with a mixture of phomopsins and it will be understood that the reference to phomopsin in the specification and claims includes mixtures of phomopsins.
In one aspect the invention provides a pharmaceutical composition for treatment of cancer, preferably liver cancer, containing a phomopsin compound or derivative thereof or pharmaceutically acceptable salt of the phomopsins or derivative and a pharmaceutically acceptable carrier.
Salts of phomopsins such as the alkaline metal salts are reasonably water soluble. Aqueous solutions can be formed by dissolving the phomopsins in a dilute base such as sodium hydroxide to provide a neutral solution.
In another aspect the invention provides a method of treatment of a patient suffering cancer including administering to the patient a phomopsin compound or derivative thereof or pharmaceutically acceptable salt of the phomopsin or derivative.
The phomopsin compound may be administered by a variety of methods including oral administration in the form of a syrup, capsule, tablet or the like, by injection or by intravenous infusion.
Preferably the compound is administered by intravenous infusion
In a further aspect the invention provides the use of a phomopsin compound as hereinbefore described for preparation of a pharmaceutical composition for treatment of cancer and in particular liver cancer. Phomopsin compounds are produced by certain fungi, including Diaporte toxicus (formerly Phomopsis leptostromiformis) and Phomopsis emicis, or may be derived from these natural products.
The activity of phomopsins is believed to be due in part to the strong binding of the compounds to tubulin. This may disrupt cell mitosis by inhibiting tubulin formation and cause depolymerization of formed microtubules. It may be preferred in some cases to use phomopsins in combination therapy with one or more other anticancer drugs or therapies. The drugs used in combination with phomopsins may be selected to enhance results by providing complementary activity in binding to microtubules. Examples of possible drugs for use in combination with phomopsins include paclitaxel, vinblastine and vincristine.
The invention will now be described with reference to the following examples. It is to be understood that the examples are provided by way of illustration of the invention and that they are in no way limiting to the scope of the invention.
Examples For the in vitro and in vivo assessments of anticancer activity performed by the National Cancer Institute in the USA, phomopsin A, iso-phomopsin A, phomopsinamine A and octahydrophomopsin A were obtained by the methods as described in the references by C. Culvenor, J. Edgar and M. Mackay, Tetrahedron Vol. 45, No. 8 pp 2351 (1989). and by J. Edgar, J. Frahn, P. Cockrum and J. Culvenor in the paper "Lupinosis. The Chemistry and Biochemistry of the Phomopsins" Mvcotoxins and Phvcotoxins, collection of invited papers presented at the sixth International IUPAC Symposium on Mycotoxins and Phycotoxins, Pretoria, Rep. South Africa, 22-25 July 1985, or as described herein.
ISOLATION OF PHOMOPSIN A Background:
The extraction process is designed to minimise difficulty and cost. The fermented seed is continuously extracted with recycling 15% methanol:water through an in line XAD (styrene divinylbenzene copolymer) column. The time required for adsorption of phomopsin A onto the XAD is quite lengthy, but requires minimal operator input. The timing of this step is not critical, hence can be adapted to suit operating conditions.
The phomopsin A has a relatively low solubility in 15% methanol. The procedure relies on the adsorption of phomopsin A on the XAD resin driving the solubility equilibrium of phomopsin A in the fermented seed toward dissolution. This procedure reduces solvent usage, volumes to be handled and fiammability hazards. The alternate method of extraction, without recycling would use 150+ litres of pure methanol for the initial extraction, involve a further concentration step (or dilution of the methanol extract to 900+L) then adsorption onto XAD. The current procedure uses 12 L methanol, requires minimal operation input for the adsorption phase and uses far less solvent (total volume 85L instead of 900+L).
The elution of the concentrated phomopsin A from the column is the first step in a 3 stage isolation to produce crystalline phomopsin A of 80-90% purity.
After a preliminary wash with 15% methanol in water, phomopsin A may be eluted from the column using 100% methanol. Silica gel flash column chromatography may be used for purification. The column is conditioned using 5:95 ammonia:isopropanol and the concentrate dissolved in a minimum of 20:65:15 ammonia:isopropanol:water. Phomopsin A is eluted using this 3 solvent combination. Recrystallisation from boiling glacial acetic acid provides phomopsin A in 80-90% purity.
PREPARATION OF /so-PHOMOPSIN A Materials: 0.5M HgCI2: 280 mg HgCI2 dissolved in 2 ml H2O (+50 μl 10M HCI).
0.01 M Phomopsin A: 18.3 mg PhA dissolved in 2 ml H2O (with puff of
NH3).
1 M HCI
Method: 0.01 M Phomopsin A (2.0 ml) was mixed with 0.5M HgCI2 (1 ml) and 1 M HCI (200 μl), total volume 3.2 ml, and left at room temperature for 5 hours. The solution was diluted to 8 ml with water then passed through a prepared C18 Maxi-clean SPE cartridge (900 mg) and washed with 7-8 ml H2O. The adsorbed /so-phomopsin A was then eluted with 8-9 ml MeOH The aqueous eluate from the first C18 cartridge was reprocessed through a second C18 cartridge to check whether the first cartridge was overloaded. The MeOH eluate from the second cartridge had very little residue on drying and was not included in further processing.
The methanol eluate, made up to 10mls, was analysed by HPLC and then was evaporated to dryness and purified using preparative HPLC.
PREPARATION OF PHOMOPSINAMINE A Phomopsin A (15.3 mg) was dissolved in the minimum amount of 1 M HCI and left at room temperature for 28 hours. The reaction mixture was diluted to 8 ml with water then passed through a strong anion exchange cartridge (SAX, 600 mg) to remove any unreacted phomopsin A (pH of solution expected to be about 1.52). The aqueous solution of non-adsorbed compounds, and the water washings of the SAX column, were then passed through a prepared C18 cartridge (900 mg). The C18 cartridge was washed with H2O (10 ml) then the phomopsinamine A eluted with methanol (10 ml).
The methanol eluate was subjected to HPLC analysis and then evaporated to dryness and the phomopsinamine A purified using preparative HPLC.
This method may be modified by sampling the reaction mixture after 5-6 hours, 24 hours and 28-30 hours. All washings and eluates may be assayed by HPLC to monitor the conversion of phomopsin A to phomopsinamine A.
ANTICANCER ACTIVITY OF THE PHOMOPSINS In vitro Screening Assay
The anticancer activity of phomopsin A, octahydrophomopsin A, iso-phomopsin A and phomopsinamine A was assessed against 60 human cancer cell lines in vitro. The methods used to assess anticancer activity are those employed by the United States National Cancer Institute (NCI) as a primary screen for discovering compounds with anticancer potential (Boyd and Paull, Drug Development Research, 34, 91-109 1995).
The measured effect of the compound on the Percentage Growth (PG) of a cell line is currently calculated according to one or the other of the following two expressions:
If (Mean ODtest - Mean ODtzero) > 0, then PG = 100 x (Mean ODtest - Mean ODtzero)/(Mean ODctrι - Mean ODtzero)
If (Mean ODtest - Mean ODtzero) < 0, then
PG = 100 x (Mean ODtest - Mean ODtzero)/Mean ODtzero Where: Mean ODtzero = The average of optical density measurements of SRB-derived color just before exposure of cells to the test compound.
Mean ODtest = The average of optical density measurements of SRB- derived color after 48 hours exposure of cells to the test compound. Mean ODctri = The average of optical density measurements of SRB- derived color after 48 hours with no exposure of cells to the test compound. Results
The calculated PGs of each of 60 cell lines for various concentrations of the test compounds are presented in Tables 1a to 4b. Testing was conducted twice for each compound and the results of the testing of this compound phomopsin A (Tables 1a and 1b), octahydrophomopsin A (Tables 2a and 2b), isophomopsin A (Tables 3a and 3b) and phomopsinamine A (Tables 4a and 4b) and demonstrate a dose-related response of most of the cancer cell lines tested to phomopsin A, iso-phomopsin A, octahydrophomopsin A and phomopsinamine A. In particular, the data supported progression of the assessment procedure to in vivo testing. Table 1a
Compound 1 Phomopsin A ID No 9502RM16
Log 10 Co centration
Percent Growth
Leukemia
CCRF-CEM 99 106 98 34 -24
HL-60 (TB) 101 101 76 -17 -43
K-562 97 102 87 24 -23
M0LT-4 99 103 95 43 29
RPMI-8226 111 109 103 36 -5
SR 118 111 53 -16 -35
Non-small cell lung cancer
A5 9/ATCC 102 103 69 31 17
EKVX 102 106 85 40 11
HOP-62 104 106 96 72 56
HOP-92 114 116 110 91 90
NCI-H226 107 121 67 -4 -26
NCI-H23 105 102 101 74 36
NCI-H322M 102 98 94 30 8
NCI-H 60 103 111 83 16 9
NCI-H522 103 105 100 18 -21
Colon cancer
COLO 205 107 75 57 -54 -79
HCC-2998 95 95 74 4 -46
HCT-116 97 102 101 32 11
HCT-15 90 97 74 26 10
HT29 95 97 91 14 5
KM12 100 83 62 12 5
SW-620 96 107 101 62 44
CNS cancer
SF-268 101 101 89 51 36
SF-295 107 102 74 21 8
SF-539 94 94 69 -18 -54
SNB-19 93 97 92 44 22
SNB-75 93 77 48 -1 1 21
U251 100 102 89 16 4
Melanoma
LOX IMVI 90 97 82 43 20
MALME-3M 101 92 65 32 25
M1 98 83 64 0 -27
SK-MEL-2 97 95 84 32 11
SK-MEL28 94 83 68 44 37
SK-MEL-5 100 87 48 30 23
UACC-257 1 13 104 72 60 70
UACC-62 101 95 79 38 26
Ovaπaπ cancer
IGR-OV1 99 102 97 73 44
OVCAR-3 102 97 64 11 4
OVCAR-4 99 84 114 63 54
OVCAR-5 101 102 78 20 27
OVCAR-8 95 99 97 62 8
SK-OV-3 104 97 70 19 1 1
Renal cancer
786-0 105 90 86 26 18
A498 96 91 67 6 0
ACHN 101 91 89 48 36
CAKI-1 90 88 63 37 28
RXF-393 91 87 49 25 39
SN12C 104 104 92 64 38
TK-10 94 101 85 68 53
UO-31 98 94 91 64 48
Prostate cancer
PC-3 100 89 66 20 10
OU-145 108 102 66 8 -13
Breast cancer
MCF7 98 92 67 21 6
MCF7/ADR-RES 99 99 90 48 13
MDA-MB-231/AtCC 99 101 93 77 2
HS 578T 104 107 101 71 75
MDA-MB-435 98 60 16 -46 -80
MDA-N 93 71 -29 -86 -79
BT-549 100 121 1 12 60 56
T-47D 91 107 71 42 73 Table 1b
Compound 1 Phomopsin A ID No 9409SC89
Log 10 Concentration Percent Growth
Leukemia
CCRF-CEM 84 86 80 -2 -47
HL-60 (TB) 75 88 76 -31 -65
K-562 105 121 93 33 11
MOLT-4 98 93 90 28 28
RPMI-8226 103 94 87 9 -27
SR 90 88 88 25 19
Non-small cell lung cancer
A549/ATCC 107 103 82 34 25
EKVX 107 99 92 58 45
HOP-62 100 114 99 57 35
NCI-H2Σ6 87 85 96 34 -5
NCI-H23 101 101 88 20 -2
NCI-322M 93 93 80 27 44
NCI-H460 101 95 80 12
NCI-H522 102 102 93 9 -21
Colon cancer
COLO 205 99 108 69 -27 -44
HCC-2998 104 96 87 11 -37
HCT-116 102 94 93 32 15
HCT-15 102 99 103 35 15
HT29 95 95 92 -14 -52
KM12 92 87 61 -19 -52
SW-620 104 104 93 34 22
CNS cancer
SF-268 104 106 87 40 16
SF-295 100 94 74 -45 -53
SF-539 99 102 98 32 -7
SNB-19 101 98 94 56 33
SNB-75 83 55 28 15 16
UΣ51 103 98 91 26 9
Melanoma
LOX IMVI 101 108 100 46 37
M14 99 110 76 19 -31
SK-MEL-2 87 92 74 32 16
SK-MEL-28 96 98 69 37 51
SK-MEL-5 106 101 54 22 10
UACC-257 98 92 75 26 42
Ovaπan cancer
IGROV1 104 119 109 57 27
OVCAR-5 99 100 82 24 19
OVCAR-8 105 133 104 59 30
SK-OV-3 94 114 89 26 47
Renal cancer
786-0 91 97 98 38 17
A498 99 100 83 17 -5
ACHN 98 90 90 43 21
SN12C 105 100 103 59 28
TK-10 100 106 98 92 55
Prostate cancer
PC-3 99 100 86 21 15
DU-145 99 105 83 22 20
Breast cancer
MCF7 98 100 76 23 12
MCF7/ADR-RES 97 100 86
MDA-MB-231/ATCC 99 98 96 66 30
HS 578T 113 109 79 23 4
MDA-MB-435 90 81 34 1 -23
MDA-N 103 101 23 -75 -63
BT-5 9 107 110 100 65 49
T-47D 95 98 74 32 56 Table 2a
Compound 1 Octahydrophomopsin A ID No 9409SC89
Log 10 Concentration Percent Growth
Leukemia
CCRF-CEM 90 96 82 8 -3
HL-60 (TB) 106 95 88 -25 -49
K-562 100 108 92 22 14
MOLT-4 103 112 105 39 20
RPMI-8226 110 99 84 7 -33
SR 94 96 92 27 15
Non-small cell lung cancer
A549/ATCC 105 104 97 47 12
EKVX 95 97 88 62 44
HOP-62 103 96 105 72 43
NCI-H226 85 75 85 33 -22
NCI-H23 110 118 104 69 12
NCI-H322M 100 99 91 49 26
NCIH460 99 99 96 29 2
NCIH522 99 99 88 5
Colon cancer
COLO 205 97 100 70 1 -82
HCC-2998 135 -16
HCT-116 104 103 100 41 14
HCT-15 96 97 97 58 16
HT29 93 91 90 30 6
KM12 108 124 139 63 -8
SW-620 95 98 87 34 32
CNS cancer
SF-268 101 100 86 43 22
SF-295 88 88 72 -27 -65
SF-539 101 95 95 27 -32
SNB-19 100 97 99 59 38
SNB-75 90 111 89 -7 27
U251 96 99 90 20 -3
Melanoma
LOX IMVI 97 100 92 52 39
M14 101 70 94 24 -51
SK-MEL-2 111 109 106 38 60
SK-MEL-28 105 94 69 29 41
SK-MEL-5 93 105 45 3 -19
UACC-257 98 97 85 36 37
Ovaπan cancer
IGROV1 108 107 99 52 34
OVCAR-5 102 94 96 57 2
OVCAR-8 106 99 100 62 29
SK-OV-3 85 98 79 27 -11
Renal cancer
786-0 104 110 127 95 5
A498 104 100 103 49 16
ACHN 99 96 86 61 22
SN12C 99 96 92 63 28
TK-10 97 96 101 87 51
Prostate cancer
PC-3 89 100 90 37 21
DU-145 105 108 95 29 -4
Breast cancer
MCF7 115 108 108 34 26
MCF7/ADR-RES 107 106 105 66 -15
MDA-MB-231/ATCC 100 95 83 53 23
HS 578T 90 90 72 13 -10
MDA-MB-435 100 91 59 13 -14
MDA-N 101 99 51 -9 -23
BT-549 111 75 87 57 37
T-47D 95 122 89 45 48 Table 2b
Compound 1 Octahydrophomopsin A
ID No 950RM16
Log 10 Concentration
Percent Growth
Leukaemia
CCRF-CEM 99 102 93 28 -33
HL-60 (TB) 100 81 93 2 -33
K-S62 104 100 99 27 -21
MOLT-4 104 99 103 46 26
RPMI-8226 94 87 96 39 -3
SR 78 92 54 -9 -35
Non-small cell lung cancer
A5 9/ATCC 104 93 101 57 21
EKVX 105 93 93 52 23
HOP-62 89 90 85 55 21
HOP92 98 99 92 51 76
NCI-H226 108 104 102 23 -14
NCI-H23 94 96 85 57 27
NCI-H322M 99 98 99 74 17
NCI-H460 105 101 104 45 15
NCI-H522 108 103 102 27 -13
Colon cancer
COLO 205 101 94 74 13 -13
HCC-2998 97 100 104 48 -13
HCT-116 105 100 101 42 17
HCT-15 90 98 81 45 22
HT29 97 96 101 61 8
KM12 101 96 99 40 20
SW-620 100 93 92 58 36
CNS cancer
SF-268 101 98 86 52 46
SF-295 93 79 74 22 0
SF-539 83 90 87 1 -55
SNB-19 101 103 104 50 23
SNB-75 99 102 52 -11 16
U251 103 96 97 30 9
Melanoma
LOX IMVI 91 91 85 45 25
MALME-3M 99 96 84 46 27
M14 90 98 98 43 5
SK-MEL-2 101 96 93 40 12
SK-MEL-28 106 90 76 48 43
SK-MEL-5 110 107 72 36 28
UACC-257 105 105 74 65 88
UACC-62 104 96 87 31 18
Ovarian cancer
IGR-OV1 94 95 94 68 42
OVCAR-3 103 99 87 31 13
OVCAR-4 106 90 94 85 99
OVCAR-5 107 104 105 62 26
OVCAR-8 98 99 95 70 19
SK-OV-3 104 90 98 43 10
Renal cancer
786-0 113 96 90 48 33
A498 104 96 100 51 20
ACHN 100 103 104 81 52
CAKI-1 95 72 69 36 32
RXF-393 95 93 81 52 42
SN12C 91 93 92 63 40
TK-10 92 98 83 91 59
Uθ-31 100 92 99 75 52
Prostate cancer
PC-3 104 99 93 42 12
DU-145 100 97 94 17 -1
Breast cancer
MCF7 104 100 94 48 46
MCF7/ADR-RES 102 98 97 61 23
MDA-MB-231/ATCC 97 67 72 64 22
HS 578T 103 92 87 51 83
MDA-MB-435 101 89 40 -25 -66
MDA-N 85 81 11 -77 -80
BT-549 123 108 96 46 38
T-47D 99 100 86 59 77 Table 3a
Compound 1 ISO-Phomopsin A ID No 9409SC89
Log 10 Concentration Percent Growth
Cell line -6 -7 -6 -5 -4
Leukemia
CCRF-CEM 103 97 92 7 -43
HL-60 (TB) 106 98 98 -29 -59
K-562 128 123 112 25 5
MOLT-4 97 105 106 46 5
RPMI-8226 106 104 87 0 -14
SR 96 99 94 28 5
Non-small cell lung cancer
A549/ATCC 104 103 90 31 11
EKVX 103 101 98 64 58
HOP-62 95 82 79 53 21
NCI-H226 95 93 110 39 -15
NCI-H23 99 105 93 37 16
NCI-H322M 95 100 85 34 51
NCI-H460 95 96 85 7 -32
NCI-H422 100 99 95 10 -76
Colon cancer
COLO 205 102 106 76 -45 -48
HCC-2998 96 99 92 15 -35
HCT-116 100 111 99 30 6
HCT-15 100 102 102 40 16
HT29 98 98 93 -30 -26
KM12 116 98 62 -33 -69
SW-620 99 99 87 23 2
CNS cancer
SF-268 102 94 87 46 31
SF-295 100 93 88 -36 -52
SF-539 96 95 82 28 6
SNB-19 100 98 89 57 37
SNB-75 84 102 106 23 36
U251 97 93 83 15 -20
Melanoma
LOX IMVI 99 96 91 43 19
M14 78 81 46 -6 -58
SK-MEL-2 100 95 80 18 0
SK-MEL-28 93 95 78 47 43
SK-MEL-5 117 110 41 -2 1
UACC-257 98 96 85 20 27
Ovaπan cancer
IGROV1 105 106 94 49 27
OVCAR-5 102 100 95 30 25
OVCAR-8 103 107 105 66 32
SK-OV-3 105 106 86 24 35
Renal cancer
786=0 93 94 99 40 24
A498 97 93 95 21 3
ACHN 101 95 91 46 21
SN12C 102 99 101 65 24
TK 10 101 103 101 87 58
Prostate cancer
PC-3 101 106 85 28 18
DU-145 106 111 97 16 5
Breast cancer
MCF7 102 92 69 20 3
MCF7/ADR-RES 106 109 95 19 2
MDA-MB-231/ATCC 102 100 107 64 26
HS 578T 103 80 69 38 31
MDA-MB-435 98 106 58 -20 3
MDA-N 103 92 42 -7 -37
BT-549 116 108 123 71 32
T-47D 104 96 103 42 42 Table 3b
Compound 1 ISO-Phomopsin A ID No 9502RM16
Log 10 Concentration
Percent Growth
Leukemia
CCRF-CEM 103 106 97 25 -21
HL-60 (TB) 95 100 83 -19 -41
K-562 100 104 92 19 -17
MOLT-4 102 100 101 44 24
RPMI-8226 110 110 97 19 6
SR 100 96 41 -15 -22
Non-small cell lung cancer
A549/ATCC 104 100 77 33 19
EKVX 94 101 96 44 22
HOP-62 94 94 89 54 26
HOP-92 96 96 76 66 86
NCI-H226 114 110 88 -6 -27
NCI-H23 104 105 83 48 39
NCI-H322M 106 98 96 32 14
NCI-H460 93 105 79 20 4
NCI-H522 101 97 88 15 -4
Colon cancer
COLO205 90 76 44 -34 -70
HCC-2998 98 97 82 -8 -64
HCT-116 93 88 79 21 4
HCT-15 97 98 85 29 14
HT29 99 100 85 10 6
KM12 91 87 47 15 -6
SW-620 97 99 83 40 35
CNS cancer
SF-268 94 92 88 52 37
SF-295 95 100 73 12 -11
SF-539 101 97 76 -29 -68
SNB-19 97 100 89 44 20
SNB-75 97 104 84 -3 60
U251 98 96 77 11 4
Melanoma
LOX IMVI 98 100 92 45 30
MALME-3M 100 89 64 26 17
M14 101 80 69 5 -26
SK-MEL-2 94 99 75 23 17
SK-MEL-28 93 88 77 40 24
SK-MEL-5 99 87 60 29 35
UACC-257 84 92 78 46 58
UACC-62 96 96 83 45 29
Ovaπan cancer
IGR-OV1 98 97 93 58 36
OVCAR-3 97 90 46 3 -17
OVCAR-4 97 92 79 62 47
OVCAR-5 97 99 68 15 21
OVCAR-8 106 106 106 74 34
SK-OV-3 95 92 76 23 6
Renal cancer
786-0 94 84 79 32 16
A498 82 84 87 0 -16
ACHN 101 108 101 56 48
CAKI-1 90 104 82 47 46
RXF-393 100 102 75 42 35
SN12C 95 93 93 61 28
TK-10 101 105 97 96 71
UO-31 97 96 96 67 48
Prostate cancer
PC-3 100 94 68 23 23
DU-145 107 100 72 -5 -9
Breast cancer
MCF7 99 98 90 32 12
MCF7/ADR-RES 94 95 87 44 7
MDA-MB-231/ATCC 97 110 92 73 18
HS 578T 99 70 70 48 58
MDA-MB-435 103 87 34 -35 -69
DMDA-N 103 82 -16 -84 -68
BT-549 102 98 90 50 48
T-47D 84 90 76 41 52 Table 4a
Compound 1 Phomopsinamine A ID No 9409SC89
Log10 Concentration
I Percent Growth
Leukemia
CCRF-CEM 97 93 49 -13 -7
HL-60 (TB) 104 103 56 -53 -55
K-562 105 100 53 6 2
MOLT-4 95 93 91 26 17
RPMI-8226 106 103 65 9
SR 104 95 71 26 12
Non-small cell lung cancer
A549/ATCC 99 100 53 19 18
EKVX 89 96 80 44 38
HOP-62 105 101 75 29 34
NCI-H2Σ6 82 84 63 -12 -31
NCI-H23 110 112 78 11 -9
NCI-H322M 96 97 61 31 36
NCI-H460 104 111 37 4 -35
MNCI-H522 66 62 40 -42 -54
Colon cancer
COLO 205 109 103 36 -22 -72
HCC-2998 109 113 60 -16 -59
HCT-116 99 99 63 14 1
HCT-15 93 94 76 19 2
HT29 101 102 50 -53 -64
KM12 110 123 77 47 40
SW-620 93 100 59 21 24
CNS cancer
SF-268 101 100 70 32 13
SF-295 87 86 56 -22 -27
SF-539 97 102 77 -18 -44
SNB-19 86 94 65 27 20
SNB-75 63 78 36 11 22
U251 87 87 40 0 -39
Melanoma
LOX IMVI 98 101 73 38 28
SK-MEL-2 102 96 37 -1 -6
SK-MEL-28 100 93 65 42 49
SK-MEL-5 102 99 32 17 14
UACC-257 95 95 66 31 42
Ovarian cancer
IGROVI 100 98 69 37 19
OVCAR-5 102 93 60 11 15
OVCAR-8 81 103 85 55 2
SK-OV-3 101 107 59 27 14
Renal cancer
786-0 100 98 91 31 15
A498 111 101 82 2 -11
ACHN 99 100 77 41 21
SN12C 96 93 82 33 13
TK-10 98 100 91 50 31
Prostate cancer
PC-3 97 90 40 18 18
DU-145 99 99 48 10 1
Breast cancer
MCF7 114 116 38 15 10
MCF7/ADR-RES 103 99 87 -7 -48
MDA-MB-231/ATCC 95 96 86 29 15
HS 578T 87 87 50 6 -5
MDA-MB-435 126 71 -15 -55 -32
MDA-N 93 87 -6 -58 -49
BT-549 145 85 51 29 -25
T-47D 99 105 72 26 68 Table 4b
Compound 1 Phomopsinamine A Log 10 Concentration
ID No 9502RM16 Percent Growth
Leukemia
CCRF-CEM 101 99 67 -7 -34
HL-60 (TB) 106 96 26 -37 -47
K-562 100 100 63 -3 -15
MOLT-4 104 99 88 25 9
RPMI-8226 107 99 61 6 4
SR 104 68 9 -15 -21
Non-small cell lung cancer
A549/ATCC 97 81 44 21 7
EKVX 99 79 41 36
HOP-62 96 101 86 53 45
HOP-92 128 120 100 98 59
NCI-H226 114 104 47 -14 -27
NCI-H23 101 94 73 33 34
NCI-H322M 104 101 73 23 17
NCI-H460 100 102 37 12 14
NCI-H522 100 102 72 35 -24
Colon cancer
COLO 205 98 82 28 -24 -45
HCC-2998 103 43 -32 -33
HCT-116 93 93 50 16 8
HCT-15 93 59 22 6
HT29 98 101 40 4 4
KM12 96 81 26 9 1
SW-620 97 93 71 40 31
CNS cancer
SF-268 98 94 64 47 30
SF-295 103 81 24 0
SF-539 102 95 53 -45 -60
SNB-19 98 94 61 30 21
SNB-75 116 110 36 10 20
U251 104 89 41 11 12
Melanoma
LOX IMVI 95 75 37 21
MALME-3M 100 78 55 29 25
M14 99 95 56 1 -1
SK-MEL-2 99 91 70 17 16
SK-MEL-28 86 60 24 45
SK-MEL-5 99 68 38 27 19
UACC-257 103 94 73 64 73
UACC-62 97 93 53 35 37
Ovarian cancer
IGR-OV1 95 95 79 47 30
OVCAR-3 101 77 10 1 -6
OVCAR-4 98 102 89 59 45
OVCAR-5 99 105 50 25 35
OVCAR-8 102 92 42 22
SK-OV-3 100 97 51 0 4
Renal cancer
786-0 108 101 69 26 20
A498 117 61 -7 -16
ACHN 102 101 71 53 38
CAKI-1 86 82 75 45 48
RXF-393 88 85 56 11 50
SN12C 86 90 79 48 30
TK-10 100 111 73 73
UO-31 102 104 97 53 53
Prostate cancer
PC-3 102 85 38 20 10
DU-145 100 93 35 -12 1
Breast cancer
MCF7 103 106 43 27 9
MCF7/ADR-RES 97 96 85 46 21
MDA-MB-231/ATCC 96 90 76 27 2
HS 578T 102 72 70 62 77
MDA-MB-435 61 10 -42 -27
MDA-N 94 51 -58 -86 -65
BT-549 110 96 52 41 43
T-47D 93 100 77 54 78 In vivo, Hollow Fiber Screeening Assay
The Biological Testing Branch of the Developmental Therapeutics Program has adopted a preliminary in vivo screening tool for assessing the potential anticancer activity of compounds identified by the large scale in vitro cell screen (Hollingshead, MG etal., Life Sciences, 57, 131 - 141 , 1995). For these assays, human tumour cells are cultivated in polyvinylidene fluoride (PVDF) hollow fibers, and a sample of each cell line is implanted into each of two physiologic compartments (intraperitoneal and subcutaneous) in mice. The protocol identifies compounds having moderate to prominent anti-cancer activity, and facilitates identification of sensitive tumor lines and appropriate treatment regimens for subsequent testing in standard, in vivo solid tumor models.
Methodology Each test mouse receives a total of 6 fibers (3 intraperitoneally and 3 subcutaneously) representing 3 distinct cancer cell lines. Three mice are treated with potential antitumor compounds at each of 2 test doses by the intraperitoneal route using a QD x 4 treatment schedule. Vehicle controls consist of 6 mice receiving the compound diluent only. The fiber cultures are collected on the day following the last day of treatment. To assess anticancer effects, viable cell mass is determined for each of the cell lines using a formazan dye (MTT) conversion assay. From this, the %T/C can be calculated using the average optical density of the compound-treated samples divided by the average optical density of the vehicle controls. In addition, the net increase in cell mass can be determined for each sample as a sample of fiber cultures are assessed for viable cell mass on the day of implantation into mice. Thus, the cytostatic and cytocidal capacities of the test compound can be assessed.
Generally, each compound is tested against a minimum of 12 human cancer cell lines. This represents a total of 4 experiments since each experiment contains 3 cell lines. The data are reported as %T/C for each of the 2 compound doses against each of the cell lines with separate values calculated for the intraperitoneal and subcutaneous samples. Evaluation
Compounds are selected for further in vivo testing in standard subcutaneous xenograft models on the basis of several hollow fiber assay criteria. These include: (1) a % T/C of 50 or less in 10 of the 48 possible test combinations (12 cell lines X 2 sites X 2 compound doses); (2) activity at a distance (intraperitoneal drug/subcutaneous culture) in a minimum of 4 of the 24 possible combinations; and/or (3) a net cell kill of 1 or more cell lines in either implant site. To simplify evaluation, a points system has been adopted which allows rapid viewing of the activity of a given compound. For this, a value of 2 is assigned for each compound dose which results in a 50% or greater reduction in viable cell mass. The intraperitoneal and subcutaneous samples are scored separately so that criteria (1) and (2) can be evaluated. Compounds with a combined IP+SC score > 20, a SC score > 8 or a net cell kill of one or more cell lines are referred for xenograft testing. These criteria were statistically validated by comparing the activity outcomes of > 80 randomly selected compounds in the hollow fiber assay and in the xenograft testing. This comparison indicated that there was a very low probability of missing an active compound if the hollow fiber assay were used as the initial in vivo screening tool. In addition to these criteria, other factors (e.g. unique structure, mechanism of action) may result in referral of a compound for standard xenograft testing without the compound meeting these criteria.
Results The data acquired for phomopsin A demonstrated significant cell growth inhibition and cytocidal activity as demonstrated by the %T/C results shown for various cell lines in Table 5. Table 5
Hollow fibre assay (%test/control, %T/C) for Phomopsin A
Cell line 30mg/kg/dose 20mg/kg/dose 45mg/kg/dose 30mg/kg/dose
IP sc IP SC IP SC IP SC
Expt591 ExptδδO
LOX IMVI >100, >100, 37, >100, 98, 80, 88, 85,
>100 >100 29 >100 98 84 90 88
COLO 205 67, 49, 58, 85, >100, 64, 58, 86,
59 34 48 81 >100 72 67 89
OVCAR-3 35, 79, 36, >100, 61 , >100, 25, 37,
-18 22 -15 >100 79 >100 60 66
Expt590 Expt579
NCI-H23 81 , 96, >100, >100, 44, -41 , 60, 21 ,
76 94 >100 >100 68 38 77 65
MDA-MB-231 46, 72, 44, 46, >100, >100, 99, >100,
30 63 28 29 >100 >100 99 >100
SW-620 >100, >100, >100, >100, 78, 78, 97, 86,
>100 >100 >100 >100 82 83 98 89
Expt581
NCI-H522 85, 96, 70, >100, 59 91 18 >100
UACC-62 100, 92, 97, 90, 100 81 95 79
U251 >100, 95, 90, 99, >100 91 83 98
Expt582
MDA-MB-435 71 , 69, 81 , 87, 62 58 74 82
OVCAR-5 51 , 92, 89, 95, 38 90 87 94
SF-295 89, >100, >100, >100, 68 >100 >100 >100
Data are results from duplicate assessments against implanted cell lines IP = intraperitoneal SC = subcutaneous
Finally, it is to be understood that various alterations, modifications and/or additions may be introduced into the composition and/or arrangement of steps previously described without departing from the spirit or ambit of the invention

Claims

Claims
1. A method of treatment of a patient suffering cancer comprising administering to the patient an effective amount of a phomopsin.
2. A method according to claim 1 wherein the patient is treated with a compound selected from compounds of formula I and derivates thereof
wherein:
R1, R2, R3, R4, R5, R6 and R7 are optional substituents X is selected from the group consisting of aliphatic, hydrogen and halogen; and Y is selected from the group consisting of aliphatic, hydrogen and halogen.
3. A method according to claim 2 wherein the patient is treated with a compound selected from compounds of formula I and derivatives and salts thereof wherein in said compound of formula I the substituent X is hydrogen, Y is chlorine and R1, R2, R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen, aliphatic, aromatic, peptide chains and halogen and wherein a conjugate may be formed between a peptide chain and a monoclonal antibody.
4. A method according to claim 1 wherein the patient is treated with an effective amount of a compound of formula la or derivative or salt thereof
wherein R1, R2, R3, R4, R5, R6 and R7 are independently selected from 0 hydrogen and aliphatic and R4 is a peptide optionally conjugated with an antibody.
5. A method according to claim 4 wherein R1, R2, R5 and R6 are lower aliphatic and R3 and R7 are hydrogen.
15
6. A method according to claim 4 wherein R1 is ethyl, R2 is methyl, R3 is hydrogen, R5 is isopropyl or iso-propenyl and R6 is methyl and R7 is hydrogen.
7. A method according to any one of claims 4 to 6 wherein the phomopsin 20 of formula la comprises compounds of the stereochemistry lb
8. A method according to claim 7 wherein at least 60% by weight of phomopsins present are stereochemistry Ib.
30
9. A method according to any one of claims 2 to 7 wherein R4 is a di or tripeptide optionally bound to an antibody.
10. A method according to claim 8 wherein R4 has the formula II with all possible stereochemical permutations
wherein the dotted lines represents an optional double bond; R8 and R9 are independently selected from hydrogen and lower alkyl; and R10 and R11 are hydrogen, or together make a double bond andR12 is selected from the group consisting of amino, mono substituted amino, disubstituted amino and an amino acid residue.
11. A method according to claim 10 wherein R12 is of formula III
wherein R13 and R14 are hydrogen or together form a double bond and
R15 is selected from the group consisting of hydroxy, amino, substituted amino and a monoclonal antibody.
12. A method according to claim 1 wherein the patient is treated with a phomopsin selected from the group consisting of phomopsin A, octahydrophomopsin A, iso-phomopsin A, phomopsinamine A, salts thereof and mixtures of two or more thereof.
13. A method according to any one of claims 1 to 12 wherein the patient is suffering from liver cancer.
14. A method according to any one of claims 1 to 13 wherein said phomopsin or derivative thereof is administered in a pharmaceutical composition with a pharmaceutically acceptable carrier.
15. A method according to any one of claims 1 to 14 wherein the patient is also treated with one or more other anticancer drugs in combination with phomopsin.
16. A method according to any one of claims 1 to 15 wherein the administration of phomopsin is at a dosage to effect anticancer activity without adverse cytotoxic effects on normal cells.
17. A pharmaceutical composition for treatment of cancer comprising a compound of formula I or derivative thereof and a pharmaceutically acceptable carrier therefore
wherein
R1, R2, R3, R4, R5, R6 and R7 are optional substituents X is selected from the group consisting of aliphatic, hydrogen and halogen; and
Y is selected from the group consisting of aliphatic, hydrogen and halogen.
18. A pharmaceutical composition for treatment of cancer comprising a compound of formula la or salt thereof
wherein R1, R2, R3, R4, R5, R6 and R7 are independently selected from hydrogen and aliphatic and R4 is a peptide optionally conjugated with an antibody.
19. A pharmaceutical composition according to claim 17 wherein the phomopsin or derivative thereof is selected from the group consisting of phomopsin A, octahydrophomopsin A, isophomopsin A, phomopsinamine A, salts thereof and mixtures of two or more thereof.
EP00969057A 1999-09-29 2000-09-29 Anti cancer agent and method of treatment of cancer Withdrawn EP1216051A4 (en)

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AUPQ314899 1999-09-29
AUPQ3148A AUPQ314899A0 (en) 1999-09-29 1999-09-29 Anti cancer agent and method of treatment of cancer
PCT/AU2000/001193 WO2001022986A1 (en) 1999-09-29 2000-09-29 Anti cancer agent and method of treatment of cancer

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NZ517539A (en) 2004-11-26
CA2385252A1 (en) 2001-04-05
AUPQ314899A0 (en) 1999-10-21
WO2001022986A1 (en) 2001-04-05
CN1376071A (en) 2002-10-23
EP1216051A4 (en) 2003-02-19
JP2003510287A (en) 2003-03-18

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