USE

Field of the Invention

This invention relates to new organic compounds which have useful antitumor activity. More particularly, this invention relates to new organic polycyclic antitumor compositions derived from marine organisms, i.e., a New Zealand Tunicate, Ritterella sigillinoides

Background of the Invention

Tumors are common in a variety of mammals and the prevention, control of the growth and regression of tumors in mammals is important to man. The term tumor refers to abnormal masses of new tissue growth which is discordant with the economy of the tissue of origin or of the host's body as a whole. Various tumor related diseases and disorders inflict man and other mammals.

Tumors inflict mammals and man with a variety of disorders and conditions including various forms of cancer and resultant cancerous cachexia. Cancerous cachexia refers to the symptomatic discomfort that accompanies the infliction of a mammal with a tumor. These symptoms include weakened condition of the inflicted mammal as evidenced by, for example, weight loss. The seriousness of cancer is well known, e.g., cancer is second only to heart and vascular diseases as a cause of death in man.

Considerable research and resources have been devoted to oncology and antitumor measures including chemotherapy. While certain methods and chemical compositions have been developed which aid in inhibiting, remitting or controlling the growth of tumors additional methods and antitumor chemical compositions are needed.

A potential source for antitumor compositions is marine life and of particular interest is the New Zealand Tunicate, Ritterella sigillinoides.

Ritterella sigillinoides is a New Zealand Tunicate and occurs as a stalked, compound ascidian. This Tunicate was first collected from "coastal rocks" in Half Moon Bay, Stewart Island in November 1955 and subsequently it was taken from a 135 meter depth in waters off Otago (47° 16.5'S, 167° 48.5'E) in August 1963. It has now been collected by diving at Kaikoura on the east coast of the south island of New Zealand. Ritterella sigillinoides appears to be confined to rocks and rocky points subjected to a high degree of wave exposure; it does not appear to occur in even slightly sheltered situations. It is found between about 1-2 and 12m depth at Kaikoura with greatest densities between 4-8m depth. Colonies are confined to shaded situations where algae do not grow and where a high degree of wave-induced water movement precludes the development of erect organisms or a thick fouling community. Thus, R. sigillinoides may dominate the sessile fauna of overhangs, narrow

crevices and tunnels, especially on ridges or other points of constriction.

Observations in the field indicate that the ascidian has an annual cycle of growth through winter and spring, reproduction in early summer and a drastic die-back or senescence at about the time of highest summer water temperatures. Colonies were abundant in June, November and December but scarce in January, even in areas of known abundance. Further, the mean weights of individuals increased from 0.74 g (SD = 0.27, n =4) in June to 2.00 g (SD *= 1.63, n =5) by mid December (large colonies up to 4.55 g).

Previous work has indicated that ascidians as a Family have potential for producing metabolites that have biological activity. Of particular interest .is the work of Kenneth L. Rinehart and his co-workers as described in the following references: Rinehart, K.L. et al., "Marine Natural Products as Sources of Antiviral Antimicrobial and Antineoplastic Agents" 53 PAPCH 795 (1981); Rinehart, K.L. et al. , "Eudistomins C, E, K and L, Potent Antiviral

Compounds Containing a Novel Oxathiazepine Ring from the Caribbean Tunicate Eudistoma olivaceum" 106 JAMCS 1524 (1984); Kobayashi,J. ,Rinehart,K.L. et al. , "Eudistomins A, D, G, H, I, J, M, N, 0, P and Q Bromo, Hydroxy- Pyrrolyl- and

1-Pyrrolinyl-p-carbolines from the Antiviral Caribbean Tunicate Eudistoma olivaceum" 106 JAMCS 1526 (1984); Rinehart,K.L. Gloer, J.B. et al.,

"Structures of the Didemnins, Antiviral and Cytotoxic Depsipeptides from a Caribbean Tunicate" 103 JMACS 1857 (1981); Rinehart, K.L., "Antiviral and Antitumor Compounds from Tunicates",

(Fed.Proc.Fed.Am.Soc.Exp.Biol. ), 42 (1983) 87; and Rinehart, K.L., Didemnins "Antiviral and Antitumor

Depsipeptides from a Caribbean Tunicate", 212 Science 933 (1981). The entire disclosure of these references are hereby incorporated herein by reference. The eudistomins referenced in some of the above-noted articles are also the subject of a European Patent Application Publication No. 133,000 A2 published February 13, 1986 corresponding to Kenneth L. Rinehar 's European Patent Application No. 84304968.5 entitled, "Antibiotics" filed July 20,

1984. The entire disclosure of this publication is hereby incorporated herein by reference.

Summary of the Invention

It is therefore an object of the invention to provide novel antitumor compositions, antitumor methods of using and a process for producing such nove1 composi ions. Additional objects and advantages of the invention will be set forth, in part, in the description which follows and in part will be obvious from this description, or may be learned by the

6

- 5 - practice of the invention. The objects and advantages of the invention are realized and obtained by means of the compositions, processes, methods, and the combinations particularly pointed out in the appended claims.

To achieve the objects in accordance with the purposes of the invention, as embodied and fully described here, the invention comprises antitumor compositions comprising effective antitumor amounts of compositions of the general formula (I):

wherein X ¹ , and χ2 are the same or different and are an acyloxy group, halogen, hydroxy, or hydrogen, and X-3 is a hydrogen or acyl group; and a pharmaceutically acceptable carrier or diluent.

In preferred embodiments of the invention, the antitumor composition comprises effective amounts of active compositions of the formulae (II-IV):

II III IV

As embodied and fully described herein, the invention further comprises a method for inhibiting tumors comprising contacting a tumor with an effective antitumor amount of one or more compositions of formulae I-IV. A therapeutic method is also provided for treating cancerous cachexia in a host comprising contacting a tumor with an effective antitumor amount of one or more compositions of formulae I-IV. As embodied and fully described herein, the invention also comprises a process to produce the compositions of formulae I-IV. The process comprises the steps of: collecting Ritterella sigillinoides, a New Zealand Tunicate; contacting the Tunicate with a suitable organic solvent to obtain an extract of a composition of formulae I-IV; and isolating a composition according to formulae I-IV from the extract.

In preferred embodiments of the invention the suitable organic solvent is selected from the group consisting of toluene, methanol, acetone, methyl ethyl ketone, ethyl acetate, ethanol, methyl isobutyl ketone and mixtures thereof. In preferred embodiments isolation of the compositions of the invention is accomplished by partition between aqueous and organic solvents and chromatography methods.

It is to be understood that both the foregoing general and the following detailed description are exemplary and explanatory only and are not intended to be restrictive of the invention as claimed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Reference will now be made in detail to present preferred embodiments of the invention, examples of which are illustrated in the following example section.

In accordance with the invention as embodied and fully described herein, the invention comprises antitumor compositions comprising effective antitumor amounts of a composition of the general formula (I) :

(I ⁾ wherein X ¹ and X-2 are the same or different and are an acyloxy group, halogen, hydroxy, or hydrogen, X ^* 3 is hydrogen or an acyl group; and a pharmaceutically acceptable carrier or diluent.

In preferred embodiments of the invention, the invention comprises effective amounts of compositions of the formulae (II -IV):

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II III IV

In accordance with the invention, an antitumor composition is provided comprising as active ingredient an effective antitumor amount of one or more of the compositions described above and identified by formulae I-IV in a non-toxic pharmaceutically acceptable carrier or diluent. While effective amounts may vary, as conditions in which the antitumor compositions are used vary, a minimal dosage required for activity is generally between 0.01 and 100 micrograms against lO ^* **-* tumor cells. Useful examples of non-toxic pharmaceutically acceptable carriers or diluents include, but are not limited to, the following: ethanol, dimethyl sulfoxide and glycerol.

In accordance with the invention, a method for inhibiting tumors in a host is provided comprising contacting a tumor with an antitumor amount of one or more compositions according to formulae I-IV. The effectiveness of the compositions of the invention for inhibiting tumors indicates their usefulness for controlling tumors in hosts including mammals and for treating cancerous cachexia.

In accordance with the invention, a process to produce a composition according to formulae I-IV comprises the steps of: collecting Ritterella sigillinoides, a New Zealand Tunicate; contacting the Tunicate with a suitable organic solvent to obtain an extract comprising the composition of formulae I-IV; and isolating a composition according to formulae I-IV from the extract.

A detailed description and explanation of a preferred embodiment of the process of the invention to produce the composition according to formulas I-IV is as follows.

A quantity of Tunicate Ritterella sigillinoides is collected by SCUBA at Kaikoura on the East Coast of the South Island of New Zealand. The Tunicate is contacted with a first organic solvent and homogenized to form an extract which is filtered. The organic solvent is removed by evaporation leaving an essentially aqueous solution (the source of the water is the natural water content of the Tunicate). The solution is then partitioned between water and a second organic solvent. The organic portion or extract, which incorporates the desired compositions, is removed and subjected to chromatography to obtain and isolate the composition according to formulas I-IV. The chromatography product is separated into fractions which contain the desired compositions. Specific compositions according to the invention are further isolated by various chromatographic techniques from the fractions obtained.

υ

While a mixture of toluene and methanol are the presently preferred choices for the first solvents, other suitable solvents may be substituted. A suitable first solvent should be capable of solubilizing the desired compositions from ^• ^ the homogenized Tunicate. Suitable first solvents which may be substituted for toluene and/or methanol include, but are not limited to, the following organic solvents: acetone; methyl ethyl ketone; ethyl acetate; ethanol; methyl isobutyl ketone and 0 mixtures thereof. Suitable second organic solvents should be capable of extracting, and separating into various fractions the various compounds of formulae I-IV from other components that may be present in the aqueous residue. Suitable second solvents which may 5 be substituted for ethyl acetate include, but are not limited to lower alcohols; toluene; methylene chloride; chloroform; 1, 2-dichloroethane/ trichloroethylene; hexane; lower alkanes; and mixtures thereof. Different ratios of solvent 0 mixtures may be used for the first and second solvents in the invention as would be known to those skilled in the art.

Any suitable fractionation and isolation techniques may be utilized in accordance with the 5 process of the invention. Suitable fractionation techniques include various chromotography techniques such as, reverse phase high pressure liquid chromatography with a suitable column as would be

0

5

known to those skilled in the art (e.g., a Whatman Partisil 100DS column (M9 50/10) eluted with a suitable solvent such as, for example, 4:1, methanol: water +0.05% trifluoroacetic acid.

It is therefore apparent that the compositions of the invention, the processes for producing the compositions of the invention and the methods for utilizing the compositions of the invention to inhibit tumors are effective for inhibiting or destroying tumors and therefore controlling diseases caused by or related to such tumors in fulfillment of the objects of the invention.

EXAMPLES

The invention will now be illustrated by examples. The examples are not intended to be limiting of the scope of the present invention. In conjunction with the detailed and general description above, the examples provide further understanding of the present invention and outline a process for producing compositions of the invention.

The following examples represent preferred embodiments of the compositions, processes and methods of the invention for satisfying the stated objects of the invention. The starting materials and reagents in the examples whose method of preparation are not indicated are commercially available from sources known to the art such as chemical supply houses.

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Examples 1-3

Preparation of Compositions. II, III and IV;

A quantity (2.6 kg, wet weight) of

Ritterella sigillinoides was collected by SCUBA diving at Kaikoura on the East coast of the South Island of New Zealand. This material without further treatment, was blended with methanol-toluene (3:1) in a high speed homogenizer, the resultant extract was filtered and the organic solvents removed under vacuum at a temperature not greater than 40 C. This gave about 2 liters of an essentially aqueous solution which was partitioned between water and ethyl acetate. The ethyl acetate soluble material incorporated the active compounds. A 2.9 gram portion of the ethyl acetate solution residue was subjected to initial purification by low pressure reverse phase chromatography on an octadecylsilyl support (C-18). The active components were eluted using an 80/20 methanol/water (0.05% trifluoroacetic acid) mixture to yield 620 mg. of active component fractions. Final purification of

the fractions was by C-18 reverse phase high pressure liquid chromatography using the same eluting solvent with UV detection at 210 nm, yielding two partially purified fractions; the first fraction (33 mg. ) contained composition II and the second fraction (112 mg. ) contained a mixture of compositions III and IV. Further purification of the first fraction by the same method yielded composition II (14 mg.). A 60 mg. portion of the second fraction was purified to yield composition III and 33 mg of composition IV.

10 Composition IV displayed the following physical properties:

[ eC ] _D 25_ ₇₈ (0.05 (g/100ml)MeOH) ^ ^■ _max (MeOH) 229 nm ( £ 24,300) 280nm (£ 5,100)

Low resolution FABMS: 354/356 I ⁵ H n.m.r. : _< f(d CH30H) 7.53 (d, J = 1.9,

H8), 7.34 (d, J = 8.4, H5), 7.14 (dd, J = 1.9, 8.4,

H6), 4.97 (d, J = 9.0, H13), 4.89 (J ^• *_ 9.0, H13),

4.32 (bs, HI), 4.18 (bd, H10), 3.62 (ddd, J = 9.8,

4.6, 2.2, H3), 3.48 (d, J = 15.8, Hll), 3.13 (ddd, J 20 = 11.8, 9,8, 4.0, H3), 3.01 (dd, J = 15.7, 6.2, Hll),

2.95 (dddd, J = 11.8, 15.0, 4.6, 2.2, H4), 2.82 (m, J

= 15.0, 4.0, 2.2 and small, H4)

C n.m.r. : ( 4CH30H) 140.0 (C8a), 129.5

(C4b), 126.5 (C9a), 123.6 (C6); 120.7 (C5), 116.5 5 (C7), 112.4 (C4a), 71.9 (C13), 67.2 (Cl), 54.6 (C3),

51.8 (CIO), 30.5 (Cll), 21.2 (C4).

0

5

H n.m.r. : <f (CD3CN) 7.52 (d, J = 1.9, H8), 7.38 (d, J = 8.4, H5), 7.16 (dd, J = 1.9, 8.4, H6), 4.91 (d, J = 9.5, H13), 4.78 (d, J = 9.5, H13), 4.32 (m, HI), 4.32 (m, H10), 3.59 (m, H3), 3.39 (d, J = 15.6, Hll), 3.12 (m, Hll), 3.11 (m, H3), 2.82 (m, 2H, H4)

C n.m.r. : S (CD3CN) : 122.8 (C6), 120.3 (C5), 114.9 (C8), 71.3 (C13), 69.0 (Cl), 54.8 (C3), 50.5 (CIO), 29.7 (Cll), 20.6 (C4) (remaining carbons not observed).

Composition II displayed the following physical properties: f _ _ -17°(0.06 g/lOOml, CH3OH)

/max (CH3OH) 224 nm ( £ 12,500), 282nm (£ 4,700) FABMS : 370/372 (m+H+)

Observed : 370.02350 Calculated for ^c 14 ^H 17 ^N 3°2 ^SBr 370.02248

Error = -1.0 mmu H n.m.r. : ξ d4θ_30H) 7.48 (s, H5), 6.94 (s, H8), 4.88 (d, J = 8.7, H13), 4.8 (d, J = 8.7, H13), 4.29 (bs, HI), 4.12 (bd, H10), 3.61 (m, H3), 3.48 (d, J = 15.2, Hll), 3.12 (M, H3), 3.00 (dd, J = 5.8, 15.2, Hll), 2.9 (m, H4), 2.72 (m, H4)

C n.m.r. : ( 4CH ₃ 0H) 148.8 (s, C6J, 134.7 (s, C9a), 130.2 (s, C8a), 128.1 (s, C4b), 116.7 (d,. C8), 111.9 (s, C7), 107.8 (s, C4a), 104.9 (d, C5), 72.3 (t, C13), 55.0 (t, C3), 52.2 (d, CIO), 30.9 (t, Cll) 21.5 (t, C4).

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Composition III is a novel composition and displayed the following physical properties: _L CCJD -43° (0.07 g/lOOml, CH3OH) /max (CH3OH) 223nm ( £ 11,500), 272nm (£ 2,900) H n.m.r.: ^" (d4CH30H) 7.43 (d, J = 8.0, H8),

7.35 (d, J=8.0, H5), 7.13 (dd, J=8.0, 8.0, H7), 7.02 (dd, J=8.0, 8.0, H6), 4.93 (d, J=15.5, H13), 4.85 (d, J=15.5, H13), 4.35 (bs, HI), 4.16 (bd, H10), 3.63 (ddd, J=9.9, 4.7, 1.8, H3), 3.50 (d, J=15.4, Hll), 3.15 (ddd, J=11.9, 9.9, 4.0, H3), 3.01 (dd, J=15.4, 5.8, Hll), 2.96 (m, H4), 2.83 (m, H4) .

C n.m.r.: $ (d4CH30H) 138.5 (C8a), 128.6 (C9a), 127.8 (C4b), 123.6 (C5), 120.8 (C6), 119.5 (C7), 112.8 (C8), 112.5 (C4a), 72.2 (C13), 67.6 (Cl), 55.0 (C3), 52.2 (CIO), 30.8 (Cll), 21.6 (C4).

Preparation of diacetate of Composition II

acetic anhydride (200 microl) and pyridine (100 microl) . After 2 hours at room temperature the reagents were removed under high vacuum.

Purification by reverse phase HPLC (C-18 Semiprep column, 90/10 CH3OH/H2O (0.05% TFA), 3 ml/min, 210nm UV detection) gave 2.5 mg. of the diacetate. The diacetate displayed the following physical properties: [*] _D -40° (c. 0.05 g/lOOml. CHCI3)

Tmax : (CH3OH) 231 nm ( £ 12,000) 291nm ( £ 3,200) (CHC1 ₃ ) 241 nm ( £ 7 750) 291 nm (£ 3,500) Infrared (solution CHCI3) 1760, 1730, 1650, 1600 cm" ¹ )

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EIMS : 453/455 (M+)

Observed 453.03585 Calculated for ^c 18 ^H 20 ^N 3°4 ^SBr 453.03578 Error 0.1 mmu m.n.r.: (CDCI3) : 8.7 (bs, NH indole), 7.56 (s, H5), 7.15, (s, H5), 5.0 (bd H10), 4.9 (d, H13), 4.8 (d, H13), 4.1 (bs, HI), 3.6 (m, H3), 3.3 (d, Hll), 3.1 (m, H3), 2.9 (m, H4), 2.8 ( , Hll), 2.8 (m, H4), 2.35 (s, OAc), 1.80 (s, NAc).

¹³ C n.m.r. _T(CDCl3) : 115.6 (C8), 111.8 (C5), 71.0 (C13), 68.7 (Cl), 54.8 (C3), 47.0 (CIO), 32.1 (Cll), 20.5 (C4). Other carbons not observed.

Preparation of acetate of Composition IV

Composition IV (17 mg) was dissolved in pyridine (200 ul) and acetic anhydride (500 ul) and stood overnight at room temperature. Water (20ml) was added and the aqueous solution extracted with chloroform (3x10ml). The extract was dried (MgSθ4) and the solvent removed under reduced pressure to give a crude product

(15mg). This was then purified by reverse phase chromatography (C-8 semiprep column, 3 ml/min 90/10 CH3OH/H2O, UV detection at 210nm) to give 7 mg. of the acetate.

The acetate displayed the following physical and spectroscopic properties:

W _ - ⁷² ° c- °- ⁰⁵ g/100ml, CH3OH) ""max (CH3OH) 230.5nm ( £ ^• _ ^■ 34,900) 283.4nm

( £ = 7,800) Infra Red 3430, 1660, 1500, 1140 cm" ¹

E1MS : (M) 395/397

FABMS : (M+H) 396/398

H n.m.r. : (CDCI3) 8.81 (bs, H9 ), 7.45 (d, J = 1.9, H8), 7.27 (d, J = 8.5, H5 ) , 7.16 (dd, J = 8.5, 1.9 H6), 6.63 (bd, J = 9.7, NH), 5.02 (ddd, J = 9.7, 5.5, 4.7, H10), 4.96 (d, J = 8.9, H13), 4.83 (d, J = 8.9, H13), 4.13 (bs, HI), 3.61 (ddd, J = 10.2, 5.0, 1.7, H3), 3.33 (d, J » 14.6, Hll), 3.14 (ddd, J = 10.2, 11.5, 4.8, H3), 2.92 (m, J = 15.8, 11.5, 5.0, 2.4, H4), 2.78 (dd, J = 14.5, 5.5, Hll), 2.8 (m, J = 15.8, 4.8, 1.7, 0.9 H4) , 1.75 (s, CH3)

C n.m.r. : f (CDCI3) 170.4 (C=0), 137.9 (C8a), 131.2 (C9a), 125.1 (C4b), 122.7 (C5 ) , 119.1 (C6), 115.4 (C7), 114.5 (C8 ) , 109.3 (C4a), 71.0 (C13), 69.0 (Cl), 54.8 (C3), 46.7 (CIO), 32.1 (Cll), 23.3 (CH3), 20.6 (C4).

ANTITUMOR ACTIVITIES OF THE

COMPOSITIONS OF THE INVENTION

The following assay method was utilized to demonstrate the in vitro antitumor effectiveness of compositions II-IV as reported in tables 1 and 2.

P388 MOUSE LEUKEMIA CELL ASSAY

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Maintenance of Cell Line P388 mouse leukemia cells are grown in Dulbecco MEM medium with 10% horse serum, 4mM glutamine, and 20ug/ml gentamycin (Biologos, Inc.). 5 Cells are incubated in 10% CO2 and subcultured 2 times per week.

PROCEDURE

1. Add compound to each well of a 24-well plate 0 or tube and allow solvent to evaporate to dryness.

2. Add 2ml (1.2 x 10 ⁵ ) cells to each well or tube and mix.

3. Incubate in 10% CO2 at 37 for 48 hours. 5 4. Read plates with an inverted microscope, scoring activity from 1+ to 4+ as follows: ND (not detectable),>90%; 1+, 75-90%; 2+, 50-74%; 3+, 25-49%; 4+,<25% of control growth. Cell counts are performed on each 0 tube and results are reported as percent of control.

HUMAN TUMOR CELL LINE ASSAY

5 Maintenance of Cell Lines

HCT-8 human colon tumor cells are grown in RPM1 1650 medium (GIBCO). A549 human lung carcinoma- cells are cultured in Dulbecco medium (Biologos, 0

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All media are supplemented with 10% fetal bovine serum and contain 50ug/ml gentamycin. All human tumor cell lines are incubated in 5% CO2 at 37 and subcultured once a week.

PROCEDURE

1. Seed 1 ml cell (5000 HCT-8, 800 A549, 12000 MCF-7) in each well of a 24-well plate.

2. Incubate in a CO2 -incubator for 48 hours.

3. Add compound to each well and incubate for an additional 120 hours.

4. Discard medium and stain with methylene blue (HCT-8) or crystal violet (A549 and MCF-7).

5. Compare cell density of drug-treated well with that of the control (no drug added) as follows: ND (not detectable) ,>90%; 1+,

75-90%; 2+, 50-74%; 3+, 25-49%; 4+,<25% of control growth.

Table 1 Antitumor In vitro assay Results Composition IV Concentration L1210

1 ug/ml 4+

0.1 3+

0.01 1+

0.001 ND

0.0001 ND

P388

25 ug/ml 3-4+

12.5 2+

2.5 2+

0.25 2+

0.025 +/ ^■ 0.005 ND

0.001 ND

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- 20 - Composition IV Concentration A549 HCT-8

50 ug/ml 4+ 20 4+ 4+

10 4+ 4+

5 4+ 4+

1 4+ 4+

0.5 - 4+

0.1 4+

Table 2

Cytotoxicity to tumor cells - antineoplastic activity against leukemia L1210 cells

Day 0 Day 3 mg/ml Vol (ul) No. cells No. cells Difference

Composition

IV 0.1 10 70 125 55

0.1 5 70 200 130

0.1 5 70 205 135

0.01 10 70 220 150

0.01 5 70 255 185

0.01 5 70 250- 180

Control 70 250 180

Control 70 250 180

The following assay method was utilized to demonstrate the in vivo antitumor effectiveness of a mixture of compositions II-IV of the invention in an enriched extract, designated "extract RL, " comprising a concentrated mixture of compositions II - IV. The results are shown in table 3.

P388 leukemia obtained from DBA/2 mice was inoculated ip into CD2F1 mice. The inoculum level was 10° cells in 0.1 ml. Mice were randomized on day 1 (24 hrs post-inoculation) into groups of six mice

if bacteriological check of tumor was negative. Test materials were dissolved or suspended in sterile 0.9% NaCl solution and administered ip, qDl-5, in a volume of 0.5 ml/mouse. Mice were weighed on days 1 and 5 to provide evidence of toxicity and deaths were recorded daily. Each experiment included appropriate numbers of untreated control mice, two-dose levels of the positive reference compound 5-fluorouracil, vehicle controls, and four-dose levels of test materials. Test materials were prepared daily and administered immediately after preparation. Doses were DiQ/ °- ^{5 LD} 10/ 0.25 obtained from prior single treatment acute toxicity assay. The endpoints for therapeutic evaluation were median survival time and long-term survivors on day 30. Percent increase in life span (% ILS) greater than 25% was considered evidence of significant activity.

Table 3 In vivo antitumor activity against

P388 leukemia in mice:

Dose % Increase

(ip) of Extract RL in Life Span

244 mg/kg 40--50%

122 mg/kg 30%

6 mg/kg 20%

Discussion of Antitumor Assay Results

Tables 1 and 2 show that composition IV was very effective in vitro at inhibiting growth of

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L1210, P388, A549 and HCT-8 tumor cells at varying concentrations. These results indicate that effective amounts of the compositions of the invention are useful for inhibiting the growth of a wide range of tumor cells. Table 3 indicates the in vivo antitumor effectiveness of compositions II-IV of the invention against leukemia cells.

These results indicate that the antitumor compositions of the invention are effective to inhibit a wide range of tumors and tumor cells. This activity is indicative of the usefulness of the compositions of the invention for various methods of inhibiting tumor cell growth and treating various tumor related disorders including cancerous cachexia. The scope of the present invention is not limited by the description, examples, and suggested uses herein and modifications can be made without departing from the spirit of the invention. For example, it may be noted that other derivatives of the compositions of the examples such as a fluorinated derivative may possess antitumor activity analogous to those preferred embodiments described above. Further, the compositions described herein may have other useful applications such as, for example, analgesic applications.

Application of the compositions of the present invention can be accomplished by any suitable therapeutic method and technique as is presently or

prospectively known to those skilled in the art. Thus, it is intended that the present invention cover the modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents.