GB2206883A - Rolliniastatin 1 and means and methods for inhibiting cell growth therewith - Google Patents

Description

4 2 2L 0 6 8 8 " Rolliniastatin 1 and means and methods for inhi.

cell growth therewith The present invention relates generally to a new antineoplastic substance herein denominated 11Rolliniastatin lit, its synthetic counterpart and pharmaceutically acceptable derivatives thereof and the treatment of an afflicted host therewith.

The Annonaceae family (2,100 species) is composed of shrubs and trees found mainly in tropical regions. The genus Rollinia generally occurs in Central and South America and R. mucosa (Jacq.) Vaill, also known as Annona mucosa (Jacq.), has been used for the Primitve-;r.eli.cal freatment of turnors in the West Indies and in Indonesia. To assess the potential of this plant in respect to potentially useful antineoplastic components, seeds of Rollinia mucosa were collected in French Guiana in 1979. The lipophilic extract of these seeds showed activity in the P388 lymphocytic leukemia system, and fractionation of this extract was guided by bloassay using the murine P388 system in vitro.

The present invention relates to the discovery of a new and useful chemotherapeutic agent which is extracted from the seeds of Rollinia mucosa in the manner hereinafter described in detail and which can be thereafter formulated into useful pharmaceutical preparations having demonstr'-"able and confirmed levels of anticancer activity when measured by the generally accepted protocols in use at the United States National Cancer Institute. The principal substance referred to herein has been denominated 11Rolliniastatin 111. The present invention further contemplates the preparation of a synthetic counterpart and the non-toxic pharmacologically active derivatives of Rolliniastatin 1. The principal active ingredient of the present invention, namely, Rolliniastatin 1, has the structure OH 221H and has deinonstrated unexpectedly strong activity in inhibiting the growth of NCI P388 lymphocytic leukemia cell line.

Accordingly a principal object of the present invention is to provide a new agent useful in the retardation or remission of one or more types of cancer.

Another object of the present invention is to provide methods and procedures for isolating an antineoplastic substance from plant life in a form in which it may be readily and usefully employed in the therapeutic treatment and management of one or more types of cancer which occur in human hosts.

A further object of the present invention is to provide means and methods of creating useful pharmaceutical preparations for the treatment and management of neoplastic disease which preparation 4 1 1 1 1 contain as their essential active ingredient a factor extracted from the seeds of Rollinia mucosa, its synthetic counterpart, or a non-toxic pharmacologically active derivative thereof.

These and still further objects as shall hereinafter appear are readily fulfilled by the present invention in a remarkably unexpected manner as will be readily discerned from the following detailed description of an exemplary embodiment thereof.

Extraction of Rollinia mucosa seed (600 g) with hexane.afforded 136 g of oily extract from which rollinia.statin 1 was isolated by a series of chromatographic steps. Steric exclusion ellromatography was followed by gradient elutlon chromatography on silica gel with hexane-ethyl acetate. Rolliniastatin 1 (1.75 g, 0.29% yield) was isolated as a wax, mp 81-830 C from acetone [OC1 589 + 25.20, [ a 578 + 26. 20, [ cc 1 546 + 30.10, [ 0C1436 + 48.50, [ m 365 + 76.700(t = 28 C, C 1.03, CH 2 Cl 2); UV ?\- CH OH 224 nm (loge, 3.93) (0c, 0 unsaturated max 3 carbonyl; -T (CHCl) 3580, 3460 (OH), 2928, 2855 max 3 - (C-H), and 1748 ( CC, -unsaturated -lactone) em SP-SIMS, m/z 645 [M+Nal 730 [M+Ag] and IIRMS, m/z 623.4917 (caled for C 37 H 66 0 7 + H, 623.4887). + In deuterioglycerol, SP-SIMS gave m/z 627 [M+DI indicating three exchangeable protons.

Spectral characteristics indicate that rolliniastatin 1 is a member of a new class of bioactive bis-tetrahydrofurans Clacetogeninsll) which include uvaricin, rollinicin, isorollicin, rollinone, desacetyluvaricin, cherimoline, dihydrocherimoline, and 11 1 asimicin. These compounds characteristically contain 37 carbons, and have a y- -lactone moiety (usually OCIA -unsaturated) and two tetrahydrofuran rings with a long hydrocarbon chain attached.

13 1 The C- and H-nmr spectra shown in Tables I and II below, elaborated by COSY and heterbnuclear correlation experiments, established the cC,,9 unsaturated-y -lactone moiety of rolliniastatin 1. 13 The following features were documented by C-shifts: The carbonyl group at C-1 (6 174.5, singlet), the double bond between C-2 (d 131.1, singlet) and C-35 6151.7, doublet), a d 78.0 doublet for C-36, and a 19.1 quartet for the C- 37 methyl group. A hetero- nuclear correlation experiment connected C-35 with a downfield proton at 7.17, and C-36 with another at

5.06. Chemical shifts reminiscent of fatty acids 1 - 13 were observed: H and C-signals (Tables I and II) for C-32, C-33, and C-34, and for a number of nearly equivalent methylene groups.

TABLE I H-MfIR CHEMICAL SHIFTS AND PROTON COUPLINGS FOR ROLLINIASTATIN 1 #C Chemical Couplings Shifts(d) (J, Hz) 3a 2.50 dddd 3a,3b 15.1 3a,4 3.5 3 a, 3 5 1.5 3a,36 1.6 3b 2.36 dddd 3a,3b 15.1 5 3b,4 8.1 3 b 3 5 1.2 3 b 3 6 1.4 i i i i 1 1 1 1 1 i 1 4 3.85 dd 3 a, 4 3b,4 is #C 1 6-13 1.25 14 1.50 m 1.5 3.38 m 16 3.85 m 17 18 19 3.38 20 3.85 21 22 23 24 25 23-33 34 35 36 37 1.7-1.9 m 1.7-1.9 3.85 3.85 7.16 ddd 5.02 dddq 1.41 q 3.5 8.1 3 a 3 5 1.5 3 b 3 5 1.5 3 a, 3 6 1.6 3 b, 3 6 1.4 35,36 1.5 36,37 1.5 Chemical shifts in ppm ( &)(at 300 MHz) are reported relative to tetramethylsilane using a deuteriochloroform solution.

TABLE 11

13C-NMR CHEMICAL SHIFTS OF ROLLINIASTATIN 1 Chemical Chemical S h i f t Sh i f t ( 6) #C ( (5) 174.5 s 15 74.Otd 2 131.1 s 16 83.0 d 33.2 t 4 69. 9 d 17 18 28.7 t 27. 8 t 9C 25 26 27 31 Chemi ca 1 Sh i f t 6 3 2 8 + t 25 5 t 29. 6 t 37.4 t 19 81.1 d 32 31.9 t 6 26.0 t 20 81.0 d 33 22.6 t 7 29.5 21 27.8 t 34 14.1 q 12 29.3 22 28.4 t 35 151.7 d 13 25.7 t 23 83.0 d 36 77.9 d 14" 34.1+t 24.71.8td 37 19.1 q +, t These shifts may be interchanged. Spectra were determined in deuterlochloroform solution and chemical shifts are expressed in ppm from tetramethylsilane.

13 A DEPT nmr experiment revealed eight C-doublets associated with oxygenated carbons (d 69.9, 71.8, 74.1, 77.9, and four more d81.0-83.0). One of these in the lactone ring, and three others bear hydroxy groups that account for the three exchangeable protons. The presence of three secondary hydroxyl groups was confirmed by observing spectral changes induced by acetylation. The remaining four oxygen bearing carbons were assigned to the tetrahydrofuran rings. Methinelgroton resonances complementing each of the doublet C-shifts occurred in the region (5 3.38-3.85, except for the C-37 proton signal at 5.5.02.

A low resolution mass spectrum of rolliniastatin 1 yielded a fragmentation pattern strikingly parallel to that of rollinone, an isomeric bis-tetrahydrofuran which has a 14-oxo group and a saturated lactone moiety. Furthermore, the mass spectral fragmentation pattern allowed location of the presumptive bistetrahydrofuran system with its two CX-hydroxyl groups between C-15 and C-24. Corroborative evidence for the OChydroxyl tetrahydrofuran system was i I 1 provided by H-nmr. COSY experiments established couplings between the following sets of protons:

H-14 - H-15( C53.38 ---t 1.38), H-15 - H-16Q53.38---3.80), and H-19 - H-20 b53.78 --Y3.88). Relationships between H-23, H-24, and H-25 were discerned by the same method. The following C-13/H-1 connections were delineated by heteronuelear connectivity experiments:

C-14 H-14a + 14b (J 34. 1 1. 44 + 1. 50), C-15 - H-15 d 74. 0 -) 3. 42), C-16 H-16 (C5 83. 1 --, 3. 86), and C-19 - H-19 (5 81.0 -->3.88). Corresponding connections were noted among protons and carbons from C-20 through C-25.

The plausible assumption that rolliniastatin 1, like rollinone, has an oxygen function at C-14 proved erroneous despite their similar mass spectra. However, by raeatis of nmr experiments (including COSY, heteronuclear correlation, and simulation by PANIC) the third hydroxyl was located in relation to the lactone ring and placed at C-4. Allylic couplings (J= 1.5, 1.2 Hz) between the two nonequivalent protons, 3a and 3b (J2.50 and 2.36), and H- 35 (7.16) supported this assignment. In addition, protons 3a and 3b exhibited long range couplings (J=1.6, 1.4 Hz) with H-36 (J 5.02). Protons 3a and 3b were also found coupled (J= 3.4, 8.1 Hz) to H-4 Q5 3. 85). In turn H-4 was found connected to a carbon appearing as a doublet at 6 69.9. Thus, the 13 C and 1 H-shifts both indicated that C-4 was bonded to oxygen. Also established were appropriate conneefions between carbons and protons at C- 3 ( J2.38 + 2.51 - 33.4) and C-5 ( 6'1.5 - 37.5).

The structure shown below for rolliniastatin 1 with undefined stereochemistry also fits the isometric bis-tetrahydrofuran asimicin recently described by Rupprecht and colleagues (See: Heterocycles, 24, 1197 13 (1986) The C_ shifts for rollinlastatin 1 (Tabl II) and asimicin are somewhat similar except those for C-14, C-24, and C-25 (alternatively, C-14, C15, and C-25). These observations indicate that the two compounds are diastereomers differing in configuration either at C-15 or C-24. Asimicin's 13 C-shifts for carbons 14-19 are virtually identical with those for carbons 10-15, suggesting that asimicin is pseudosyrrinetrical about a plane perpendicular to the bond connecting C-19 and C-20.

In contrast, rolliniastatin 1 is not symmetrical in this respect. Note the EIMIS Fragmentation of rolliniastatin 1 shown below:

311(100) i22 21 is 17 (C H 2) 24 15 3 0 2 0 19 16, 14 CH3 (CH A 2111 0 M OR, 341 e CH3 Biological evaluation of rolliniastatin 1 showed 20 P388, 28% life extension; ED so 4.5 x 10 -5 Ag/mi using NA.H. standard protocols.

Biologically active glycoside derivatives of rolliniastatin 1 are made by coupling the principal with suitably protected sugars or other hydroxylated compounds using methods well known in the art [see Methods in Chemistry by R. L. Whistler and J. N. Bemiller (eds.), Academic Press, N.Y., 1972, Vol. 6, or The Carbohydrates: Chemistry and Biochemistry by W. Pigman, Academic Press, NY, 19811. These derivatives of rolliniastatin 1 are used for the same purposes as rolliniastatin 1.

Rolliniastatin 1, as demonstrated, has free hydroxyl groups available for derivatization. Thus, acyl esters of these compounds can also be prepared by rnehods livell Knowii ti thut,, skilled in the art. Acyl derivatives of the rolliniastatin 1 can be used for the same biological purposes as the parent compounds.

Acids which can be used in the acylation of a rolliniastatin 1 include: (a) saturated or unsaturated, straight or branched chain aliphatic carboxylic acids, for example, acetic, propionic, butyric, isobutyric, tert-butylacetic, valeric, isovaleric, caproic. caprylic, decanoic, dodecanoic, lauric, tridecanoic, myristic, pentadecanoic, palmitic, margaric, stearic, acrylic, crotonle, undecylenic, oleic, hexynoic, heptynoic, octynoic acids, and the like; (b) saturated or unsaturated, alicyclic carboxylic acids, for example, cyclobutanecarboxylic acid, cyclopentanecarboxylic acid, cyclopentenecarboxylic acid, methyleyclopentenecarboxylic acid, cyclohexanecarboxylic acid, dimethyleyclohexanecarboxylic acid, dipropyleyclohexanecarboxylic acid, and the like; (c) saturated or unsaturated, alicyclic aliphatic carboxylic acids, for example, cyclopentaneacetic acid, cyclopentanepropionic acid, cyclohexaneacetic 5 acid, cyclohexanebutyric acid, methylcyclohexaneacetic acid, and the like; (d) aromatic carboxylic acids, for example, benzbic acid, toluic acid, naphthoic, acid, ethylbenzole acid, isobutylbenzolc acid, methylbutylbenzoic acid, and the like; and (e) aromatic-aliphatic carboxylic acids, for example, phenylacetic acid, phenylpropionic acid, phenylvaleric acid, cinnamic acid, phenylpropiolic acid and naphthylacetic acid, and the like. Suitable halo-, nitro-, hydroxy-, keto-, amino-, cyano-, thiocyano-, and lower alkoxyhydrocarbon carboxylic acids include hydrocarboncar-boxylic acids as given above which are substituted by one or more of halogen, nitro, hydroxy, keto, amino, cyano, or thiocyano, or loweralkoxy, advantageously loweralkoxy of not more than six carbon atoms, for example, methoxy, ethoxy, propoxy, butoxy, amyloxy, hexyloxy, and isomeric forms thereof. Examples of such substituted hydrocarbon carboxylic acids are: mono-, di-, and trichloroacetic acid;6C- and,6 -chloropropionic acid; OCC- andYbromobutyric acid;CX and d-iodovaleric acid; mevalonic acid; 2- and 4-chlorocyclohexanecarboxylic acid; shikimic acid; 2-nitro-1-methylcyclo- butanecarboxylic acid; 1, 2, 3, 4, 5, 6-hexachlorocyclohexanecarboxylic acid; 3-brorno2methylcyclohexanecarboxylic acid; 4- and 5-bromo2methyleyclohexanecarboxylle acid; 5- and 6-bromo-2methyleyclohexanecarboxylic acid; 2, 3-dibromo-2methyleyclohexanecarboxylic acid; 1 i 1 1 1 1 1 1 i 1 i i j 2,5-dibromo-2-methyleyclohexanecarboxylic acid; 4,5-dibromo-2methyleyclohexanecarboxylic acid; 5, 6-dibromo-2methyleyclohexanecarboxylic acid; 3-bromo-3-methylcyclohexanecarboxylle acid; 6-bromo-3-methyleyclohexanecarboxylic acid; 1,6-dibromo-3methyleyclohexanecarboxylic acid; 2-brorr!c-4-methylc, 7clocohexanecarboxylic acid; 1,2-dibromo-4-methyleyclohexanecarboxylic acid; 3-bromo-2, 2, 3-tri- methyl-cyclopentanecarboxylic acid; 1-bromo-3,5-dimethyl-cyclohexanecarboxylic acid; homogentisic acid, o-, m-, and p-chlorobenzoic acid; anisic acid; salicylic acid; phydroxybenzoic acid; b-resorcylic acid; gallic acid; veratric acid; trimethoxybenzoic acid; trimethoxycinnamic acid; 4,41-dichlorobenzilic acid; o-, m-, and p-nitrobenzo;.-- acid; cyaroaciz- t!2 acid, 3,4- aiid 3,5=-dinitrobenzoic acid; 2,4,6-trinitrobdnzoic acid; thlocyanoacetic acid; cyanoproplonic acid; lactic acid; ethoxyformic acid (ethyl hydrogen carbonate); malic acid; citric acid; isocitric acid; 6-methylsalicylic acid; mandelic acid; levulinic acid; pyruvic acid; glycine; alanine; valine; isoleucine; leucine; phenylalanine; proline; serine; threonine; tyrosine; hydroxyproline; ornithine; lysine; arginine; histidine; hydroxylysine; phenyIglycine; p-aminobenzoic acid; m-aminobenzoic acid; anthranilic acid; aspartic acid; glutamic acid; aminoadipic acid; glutamine; asparagine; and the like.

The administration of rolliniastatin 1 and its pharmacologically active physiologically compatible derivatives is useful for treating animals or humans bearing a neoplastic disease, for example, acute myelocytic leukemia, acute lymphocytic leukemia, malignant melanoma, adenocarcinoma of lung, neuroblastoma, small cell carcinoma of lung, breast carcinoma, colon carcinoma, ovarian carcinoma, bladder carcinoma, and the like.

The dosage administered will be dependent upon the identity of the neoplastic disease; the type of host involved, including its age, health and weight; the kind of concurrent treatment, if any; the frequency of treatment and therapeutic ratio.

Illustratively, dosage levels of the administered active ingredients are: intravenous, 0.1 to about 20 mg/kg; intramuscular, 1 to about 50 mg/kg; orally, 5 to about-100 mglkg; intranasal instillation, 5 to about 100 mg/kg; and aerosol, 5 to about 100 mg/kg of host body weight.

Expressed in terms of concentration, an active ingredient can be present in the compositions of the present invention for localized use about the cutis, intranasally, pharyngolaryngeally, bronchially, intravaginally, rectally, or ocularly in a concentration of from about 0.01 to about 50% w/w of the composition; preferably about 1 to about 20%-w/w of the composition; and for parenteral use in a concentration of from about 0.05 to about 50% w/v of the composition and preferably from about 5 to about 20% w/v.

The compositions of the present invention are preferably presented for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, suppositories, 1 i 1 1 1 i 1 1 1 1 1 sterile parenteral solutions or suspensions, sterile non-parenteral solutions or suspensions, and oral solutions or suspensions and the like, containing suitable quantities of an active ingredient.

For oral administration either solid or fluid unit dosige forris can be prepared.

Powders are prepared quite simply by comminuting the.

active ingredient to a suitably fine size and mixing with a similarly conininuted diluent. The diluent can be an edible carbohydrate material such as lactose or starch. Advantageously, a sweetening agent or sugar is prese,nt as well as a flavoring oil.

Capsules are produced by pi.eparing a powder mixture as hereinbefore described and filling into formed gelatin sheaths. Advantageously, as an adjuvant to the filling operation, a lubricant such as a tale, magnesium stearate, calcium stearate and the like is added to the powder mixture before the filling operation.

Soft gelatin capsules are prepared by machine encapsulation of a slurry of active ingredients with an acceptable vegetable oil, light liquid petrolatum or other inert oil or triglyceride.

Tablets are made by preparing a pow-der mixtureT gran- ulating or slugging, adding a lubricant and pressing into tablets. The powder mixture is prepared by mixing an active ingredient, suitably conTninuted, with a diluent or base such as starch, lactose, kaolin, dicalcium phosphate and the like. The powder 1 1 mixture can be granulated by wetting with a binder such as corn syrup, gelatin solution, methyleellulose solution or acacia mucilage and forcing through a screen. As an alternative to granulating, the powder mixture can be slugged, i.e., run through the tablet machine and the resulting imperfectly formed tablets broken into pieces (slugs). The slugs can be lubricated to prevent sticking to the tablet-forming dies by means of the addition of stearic acid, a stearic salt, tale or mineral oil. The lubricated mixture is then compressed into tablets.

Advantageously the tablet can be provided with a protective coating consisting of a sealing coat or enteric coat of shellac, a coating of sugar and methyleellulose and polish coating of carnauba wax.

Fluid unit dosage forms for oral administration such as syrups, elixirs and suspensions can be prepared wherein each teaspoonful of composition contains a predetermined amount of active ingredient for administation. The water-soluble forms can be dissolved in an aqueous vehicle together with sugar, flavoring agents and preservatives to form a syrup.

An elixir is prepared by using a hydroalcoholic vehicle with suitable sweeteners together with a flavoring agent. Suspensions can be prepared of the insoluble forms with a suitable vehicle with the aid of a suspending agent such as acacia, tragacanth, methyleellulose and the like.

For parenteral administration, fluid unit dosage forms are prepared utilizing an active ingredient and a sterile vehicle, water being preferred. The active 1 i i i i 1 1 1 1 1 ingredient, depending on the form and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions the watersoluble active ingredient can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampule and sealing. Advantageously, adjuvans such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle. Parenteral suspensions are prepared in substantially the same manner except that an active ingredient is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The active ingredient can be sterilized by expos.ure to ethylene oxide before suspending in jr, the sterile vehicle. Advantageously, a surfactant or wetting agent is included 1In the composition to facilitate uniform distribution of the active ingredient.

In addition to oral and parenteral administration, the rectal and vaginal routes can be utilized. An active ingredient can be administered by means of a suppository. A vehicle which has a melting point at about body temperature or one that is readily so.luble can be utilized. For example, cocoa butter and various polyethylene glycols (Carbowaxes) can serve as the vehicle.

For intranasal instillation, a fluid unit dosage form is prepared utilizing an active ingredient and a suitable pharmaceutical vehicle, preferably pyrogen free (11P.F.'1) water. A dry powder can be formulated when insufflation is the administration of choice.

For use as aerosols, the active ingredients can be packaged in a pressurized aerosol container together with a gaseous or liquefied propellant, for example, dichlorodifluoromethane, carbon dioxide, nitrogen, propane, and the like, with the usual adjuvants such as cosolvents and wetting agents, as may be necessary or desirable.

The term "unit dosage form" as used in the specl- fication and claims refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical diluent, carrier or vehicle. The specifications for the novel unit dosage forms of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitation inherent in the art of compounding such an active material for therapeutic use in humans, as disclosed in this specification, these being features of the present invention. Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, troches, suppositories, powder packets, wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampules, vials, segregated multiples of any of the foregoing, and other forms as herein described.

The active ingredients to be employed as antineoplastic agents can be easily prepared in such unit dosage form with the employment of pharmaceutical materials which themselves are available in the art 1 i i i i 1 i i and can be prepared by established procedures. The following preparations are illustrative of the preparation of the unit dosage forms of the present invention, and not as a limitation thereof.

EXAMPLE 1

Several dosage forms were prepared embodying the present invention. They are shown in the following examples in which the notation "active ingredient" signifies rolliniastatin 1, its synthetic counterpart and the non-toxic pharmaceutically active derivatives thereof.

is COMPOSITION "All Ilard-C-eiatin Capsules One thousand two-piece hard gelatin capsules for oral use, each capsule containing 20 mg of an active ingredient are prepared from the following types and amounts of ingredients:

Active ingredient, micronized 20 gm gm Talc 20 gm, Magnesium stearate 2 gm The active ingredient, finely divided by means of an air micronizer, is added to the other finely powdered ingredients, mixed thoroughly and then encapsulated in the usual manner.

Corn Starch The foregoing capsules are useful for treating a neoplastic disease by the oral administration of one or two capsules one to four times a day.

Using the procedure above, capsules are similarly prepared containing an active ingredient in 5, 25 and 50 mg amounts by substituting 5 gm, 25 gm and 50 gm of an active ingredient for the 20 gm used above.

COMPOSITION "B" Soft Gelatin Capsules One-piece soft gelatin capsules for oral use, each containing 20 mg of an active ingredient (finely divided by means of an air micronizer), are prepared by first suspending the compound in 0.5 mI of polysorbate 80 to render the material capsulatable and then encapsulating in the above manner.

The foregoing capsules are useful for treating a neoplastic disease by the oral administration of one or two capsules one to four times a day.

COMPOSITION "C" Tablets One thousand tablets, each containing 20 mg of an active ingredient are prepared from the following types and amounts of ingredients:

Active ingredient micronized Lactose Corn starch Magnesium stearate Light liquid petrolatum gm 300 gm 50 gm 4 gm 5 gm The active ingredient finely divided by means of an air micronizer, is added to the other ingredients and i i 1 1 1 j 1 1 1 1 i i then thoroughly mixed and slugged. The slugs are broken down by forcing through a Number Sixteen screen. The resulting granules are then compressed into tablets, each tablet containing 20 mg of the active ingredient.

The fer-going tablets are useful for treating a neoplastic Gisease by the oral administration of one or two tablets one to four times a day.

Using the procedure above, tablets are similarly prepared containing an active ingredient in 25 mg and 10 ing amount's by substituting 25 gm and 10 gm of an active'ingredient for the 20 gm used above.

COMPOSITION 11W Oral Suspension One thousand ml of an aqueous suspension for oral use, containing in each teaspoonful (5 ml) dose, 5 mg of an active ingredient, is prepared from the following types and amounts of ingredients:

Active ingredient micronized Citric acid Benzoic acid Sucrose Tragacanth Lemon Oil Deionized water, q.s. 1000 ml.

The citric acid, benzoic acid, sucrose, tragacanth and lemon oil are dispersed in sufficient water to make 850 ml of suspension. The active ingredient 1 gm 2 gm 1 gm 790 gm 5 gm 2 gm finely divided by means of an air micronizer, is stirred into the syrup until uniformly distributed. Sufficient water is added to make 1000 ml.

The composition so prepared is useful for treating a neoplastic disease at a dose of 1 tablespoonful. (15 ml) three times a day.

COMPOSITION 1IE" Parenteral Product A sterile aqueous suspension for parenteral injection, containing in 1 mI 30 mg of an active ingredient for treating a neoplastic disease, is prepared from the following types and amounts of ingredients: Active ingredient, micronized Polysorbate 80 Methylparaben Propylparaben Water for injection, q.s. 1000 ml.

3 gm 5 gm 2. 5 gm 0. 17 gm All the ingredients, except the active ingredient, are dissolved in the water and the solution sterilized by filtration. To the sterile solution is added the sterilized active ingredient, finely divided by means of an air micronizer, and the final suspension is filled into sterile vials and the vials sealed.

The composition so prepared is useful for treating a neoplastic disease at a dose of 1 milliliter (1 M) three times a day.

1 i i i I 1 i j i I I 1 CONTOSITION "F" Suppository, Rectal and Vaginal One thousand suppositories, each weighing 2.5 gm and containing 20 mg of an active ingredient are prepared from the following types and amounts of ingredients:

Active ingredient, micronized.1.5 gm Propylene glycol 150 gm Polyethylene glycol #4000, q.s. 2,500 gm The active ingredient is finely divided by means of an air micronizer and added to the propylene glycol and the mixture passed through a colloid mill until uniformly dispersed. The polyethylene glycol is melted and the propylene glycol dispersion added slowly with stirring. The suspension is poured into ijrclilil-ip.d nicIds a 4V C. The composition is allowed to cool and solidify and then removed from the mold and each suppository foil wrapped.

The foregoing suppositories are inserted reetally or vaginally for treating a neoplastic disease.

COMPOSITION "G" Intranasal Suspension One thousand mI of a sterile aqueous suspension for intranasal instillation, containing in each mI 20 mg of an active ingredient, is prepared from the following types and amounts of ingredients:

Active ingredient, micronized 1.5 gm Polysorbate 80 Methylparaben Propylparaben Deionized water, q.s. 1000 ml.

gm 2.5 gm 0.17 gm All the ingredients, except the active ingredient, are dissolved in the water and the solution sterilized by filtration. To the sterile solution is added the sterilized active ingredient, finely divided by means of an air micronizer, and the final suspension is aseptically filled into sterile containers. The composition so prepared is useful for treating a neoplastic disease,

by -intranasal instillation of 0.2 to 0.5 mI given on. e to four times per day.

An active ingredient can also be present in the undiluted pure form for use locally about the cutis, intranasally, pharyngolaryngeally, bronchially, or orally.

COMPOSITION "H" Powder Five grams of an active ingredient in bulk form is finely divided by means of an air micronizer. The micronized powder is placed in a shaker- type container.

The foregoing composition is useful for treating a neoplastic disease, at localized sites by applying a powder one to four times per day.

COMPOSITION "I" Oral Powder Ten grams of an active ingredient in bulk form is 1 1 1 i 1 i 1 1.

finely divided by means of an air micronizer. The micronized powder is divided into individual doses of 20 mg and packaged.

The foregoing powders are useful for treating a neoplastic disease, by the oral administration of -one or two powders suspended in a glass of water, one to four times per day.

COMPOSITION 11J11 Insufflation Ten grams of an active ingredient in bulk form is finely divided by means of an air micronizer.

The foregoing composition is useful for treating a neoplastic disease, by. the inhalation 01 30 mg oile to four times per day.

COMPOSITION 'W' Hard Gelatin Capsules One hundred two-piece hard gelatin capsules for oral use, each capsule containing 20 mg of an active ingredient.

The active ingredient is finely divided by means of an air micronizer and encapsulated in the usual manner.

The foregoing capsules are useful for treating a neoplastic disease, by the oral administration of one or two capsules, one to four times a day.

Using the procedure above, capsules are similarly 1 prepared containing active ingredient in 5, 25-and 50 mg amounts by substituting 5 gm, 25 gm and 50 gm of the active ingredient for the 20 gm used above.

EXAMPLE 2

Unit dosage forms of rollin iastatin 1 prepared according to selected compositions described in Example 1 were screened utilizing Protocol 1,200 described in Cancer Chemotherapy Reports, part 3, Vol. 3, No. 2, September 1972, pp 9 et seq for lymphocytic leukemia P388. Rolliniastatin 1 provided a 28-40% life extension at 0.75-2.5 mg/Kg host body weight against the murine P388 lymphocytic leukemia.

Rolliniastatin 1 also markedly inhibited growth of 5 the P388 in vitro cell line (ED 50= 4.5x10 lj-glml).

EXAMPLE 3

Unit dosage forms of rolliniastatin 1 were prepared according to Example 1 and were screened using accepted protocols of the National Cancer Institute.

The preparation obtained up to 40% life extension at 2 mg per kg host body weight against murine P388 -5 lympocytic leukemia and an ED 50 of 10 micrograms per mI against the P388 cell line.

From the foregoing it becomes readily apparent that a new and useful antineoplastic factor and new and useful antineoplastic preparations have been herein described and illustrated which fulfill all of the aforestated objectives in a remarkably unexpected fashion. It is of course understood that such modifications, alterations and adaptations as will 1 readily occur to the artisan confronted with this disclosure are intended within the spirit of the present invention which is limited only by the scope of the claims appended hereto.

r 1 i 1

Claims (14)

1. CLAIMS 1. A compound of formula: DH T7 ti bW 0 Cha 22 21 CH3

   physiologically functional equivalents thereof and salt and esters of either.
2. 2. A compound having the following 13C-MR chemical shifts:

   Chemical Chemical Chemical Shift Shift Shift C c C ( &) Ir ( 6) it ( 8) 1 174.5 s 15 74.0+d 25 32.8+t 2 131.1 s 16 83.0 d 26 25.5 t
3. 3 33.2 t 17 28.7 t 27 29.6 t 4 69.9 d 18 27.8 t 31 371. 4 t 19 81.1 d 3 22 31.9 t 6 26.0 t 20 81.0 d 33 26.6 t 7 29.5 21 27.8 t 34 14.1 q 12 29.3 22 28.4 35 151.7 d 13 25.7 t 23 83.0 d 36 77.9 d 14 34.1+t 24 71.8.1d 37 19.1 q or a salt or ester thereof.

   t k I 1 3. A compound according to Claim 1 or 2 for use medicine.
4. 4. A compound according to Claim 1 or 2 f or use in the treatment of a neoplastic disease.
5. 5. A compound according to Claim 4 in which the neoplastic disease is lymphocytic leukemia P388.
6. 6. A compound according to Claim 3, 4 or 5 in which said compound is presented for administration intravenously at a dosage level of from 0.1 to 20 mg per kilogram of host body weight.
7. 7. A compound according to Claim 3, 4 or 5 in which said compound is presented for administration subcutaneously at a dosage level of from 1 to 50 mg per kilogram of host body weight.
8. 8. A compound according to Claim 3, 4 or 5 in which said compoiln(i. is presented for administration orally at a dosage level of from 5 to 100 mg per kilogram of host body weight.
9. 9. A compound according to any one of Claims 3 to 5 in which said compound is presented for administration at a dosage level of from 0.75 to 2.5 mg per kilogram of host body weight.
10. 10. A pharmaceutical composition comprising a compound according to Claim 1 or 2 and a pharmaceutically acceptable carrier.
11. 11. A process for preparing a compound according to Claim 1 or 2 by (a) solvent extraction from Rollinia muc-osa followed by optional derivatisation or salt formation or (b) synthesis.
12. 12. A compound substantially as described herein.
13. 13. A process for preparing a compound according to Claim 1 or 2 substantially as described herein.

    1 i 1
14. 14. A pharmaceutical preparation substantially as described herein.

    Published 1988 at The Patent Office. Stae House. 66 71 High Holborn. London WC1R 4TP F=ther copies mkv be obtained frorn The Patent Office. Sales Branch. St Mar:, Cray. Orpingtor.. Ken. BM 3RD Printed by Multiplex techniques It-d. St Mary Cray. Kent. Con 1 S-