AU732816B2 - Immunomodulation compositions from bile - Google Patents

Description

AUS TRALITA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT 0 00 00 @00.

0 000.

0@ S 0 00 00 0 000 *0S.

0* 0 Lmus Trh urics ttr- Applicant(s): Invention Title: IMVMUNOMODULATION COMPOSITIONS FROM BILE The following statement is a full description of this invention, including the best method of performing it known to me/us: 0 @0 0 S *0 0000 o 00 00 0 0 0 @0

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*0000~ 0

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0 b000.~ 1A IMMUNOMOULATING COMPOSITIONS FROM BILE.

yIILD OF THE INVmTIOM The present invention relates to immunomodulating compositions, pharmaceutical agents containing the compositions, and the use of the compositions and agents in the treatment of animals.

BACKGROUND OF TrE INVmETIOi Therapies are continuously being developed for the prophylaxis and treatment of cancer and infectious diseases, such as Acquired Immunodeficiency Syndrome (AIDS). Some of these therapies attempt to use the immune system therapeutically. One approach is based on the antigen specific elements of the immune system, namely antibodies and T cells. For example, research has been 15 aimed at developing vaccines against foreign agents, or against certain endogenous chemical messengers, such as interleukins, to suppress antibody reactions. A second approach is based on the isolation, cloning, expression and production of peptides and proteins from the non-antigen specific parts of the immune system. For example, proteins, such as cytokines, which comprise the interleukins produced by white blood cells, and interferons which stimulate lymphocytes and scavengers cells that S* digest foreign antigens, offer possibilities for therapies.

25 The treatment of cancer could be greatly enhanced if the early immune response to a tumor could be augmented so that the tumor does not reach a critical size. Strategies which have been suggested to augment the immune response to a tumor include vaccines specific for tumor-associated 30 antigens; the use of monoclonal antibodies against antigens on the surface of tumor cells such as against the interleukin-2 receptor; the use of bispecific molecules containing antitumor antibodies and superantigens.

Relatively recently, the role of the physiologically active polypeptide, known as tumor necrosis factor ("TNF") has been studied, particularly with respect to its ability 2 to induce necrosis of tumors, with no effect upon the normal tissues of the living body. The amino acid sequence of TNF, as well as the base sequence of the DNA coding for TNF has been disclosed in U.S. Patent No. 4,879,226.

Because TNF has been shown to have a role in inducing necrosis of tumors, any agent that can stimulate the production or bioavailability of TNF in vivo has potential utility as a treatment for various tumorous conditions.

Additionally, any agent that can stimulate human monocytes and macrophages to produce TNF in vitro, is useful as a means for providing a source of TNF for therapeutic administration, as well as for analytical and diagnostic purposes.

Bile, which is secreted by the liver and stored in the gall bladder, has been investigated for various purposes, including the use of bile extracts to enhance bioavailability of drugs that are readily metabolized by normal liver function (Ase WO 90/12583) and to inhibit leucocytosis promotion in a mammal (see Shinoda et al., 20 Chem. Pharm. Bull., 0, 4429-4434 (1982)). However, bile has never been considered to be a source of therapeutically useful compositions with respect to neoplastic or infectious diseases. Interestingly, in accordance with *o British Patent No. 337,797, it was suggested to use the 25 gall bladder itself as a potential source of anti-cancer agents, but only after the bile had been removed from the gall bladder, and the gall bladder thoroughly washed.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

SUMMARY OF THE INVENTION It has now been discovered that bile is an important source of a composition that can stimulate TNF production both in vitro and in vivo and is effective in treating various carcinomas, especially pancreatic cancer.

The bile composition of use is obtained by extraction of bile with a water soluble or miscible solvent. The \\melbfiles\home$\WendyS\Keep\species\9724298 Lorus.doc 8/02/01 3 excracc so obtained may be further processed to remove unnecessary or undesirable components therefrom.

The product obtained by the process of extracting bile disclosed in further detail hereinbelow has been found to have TNF stimulating activity and is believed -o have anci-cancer activiy, especially against pancreatic and other cancers. Obviously, the entire composition so obtained may not be necessary to obtain such activity.

Accordingly, it is possible to further separate, fractionate, or otherwise process the product thus obtained, and still retain the desired TNF stimulatory and anti-cancer activity. Moreover, it is envisioned that it is possible to obtain synthetically a product with the same or similar TNF stimulatory and anti-cancer activity. Thus, 15 it is envisioned that the components of the product may be analyzed as to the components, or combination thereof, that are responsible for the desired activity, and a synthetic S.:o product made, based on such analysis.

In one aspect, the present invention relates to a medicament for use as an immunomodulator comprising small molecular weight components of less than 3000 daltons, and having one or more of the following properties; a) is extractable from bile of animals; b) is capable of stimulating monocytes and S. 25 macrophages in vitro; c) is capable of modulating tumor necrosis factor production; d) contains no measurable level of IL-la, IL-13, TNF, IL-6, IL-8, IL-4, GN-CSF or IFN-gamma; 30 e) has an anti-proliferative effect in a malignant mouse hybridoma cell line; f) shows no cytotoxicity to human peripheral blood mononuclear cells; and g) is not an endotoxin.

In accordance with a.preferred embodiment the medicament is extracted from the bile of bovines and is H:\annK.,ep\Rep~es\76489-24.doc 4/12198 4 capable of stimulating the release of -umor necrosis raccor.

The medicament of the invention may be prepared by mixing bile from an animal, preferably a bovine, with a solvent that is soluble or miscible with .waer, preferably an alcohol, and preferably with an equal volume of an alcohol, to produce a bile/alcohol solution; (b) separating the solution which preferably is an alcohol soluble fraction, and isolating therefrom a solution substantially free of alcohol, as by removing mose of the alcohol, such as the use of heat; removing bile pigments from the solution to obtain a colorless liquid; optionally treating the colorless liauid co substantially remove any residual alcohol; removing 15 fatty organic materials, as by extracting the colorless liquid with.ether and isolating the aqueous phase; and (f) optionally removing residual ether from the aqueous phase.

The composition may be used without further Smodification by simply packaging it in vials and sterilizing. The composition may be also be used in a concentrated form. A preferred concentrated form is prepared as follows. Prior to step the colorless liquid may optionally be concentrated to about one eighth of the volume of the bile/alcohol solution and after step 25 the aqueous phase may be concentrated so that it is one a etenth of the volume of the bile/ethanol solution.

The invention also relates to a pharmaceutical agent comprising the novel composition of the invention.

The invention further relates to a method of 1 30 treating a patient comprising administering to said patient an effective amount of a medicament of the invention. The invention still further relates to the use of a composition of the invention in the prophylaxis and treatment of diseases and conditions requiring modulation of the immune response; preferably infectious diseases and neoplasias.

Throughout the description and claims of this specification, the word "comprise" and variations of the H:\anna\VEeep\P~et,,-pes 7idd9- 4.oc 4/12/9R 4a word, such as "comprising" and "comprises", means "including but not limited to" and is not intended to exclude other additives, components, integers or sceps.

The word "medicament" is to be understood to comprise one or more active agents, and a mixture of active agents is within the scope of this term. Unless specifically stated, the medicament of the invention -ay have immunomodulatory activity, but does not necessaril have such activity.

M. Thirlwell et Journal of Cancer Research and Clinical Oncology Supplement, 1990 116 Page 51, Abstract A1.240.01 and E. Warner et al, Clinical and Investigative Medicine, 1992 15 A89 Abstract 546, describe a product referred to as "Virulizin". Thirlwell et-ac e rp 0.0: 15 stated that "Virulizin is purified animal organ extract", while Warner et al stated that Virulizin is a "novel biological response modifier extracted from bovine ereticular endothelial tissue.[and that] studies to identify the active agent(s) in Virulizin are currently under way.

Neither of these publications in any way discloses or suggests that an immunomodulatory agent could be extracted from animal bile. It is merely proposed that the product disclosed might be useful for treatment of cancer. In particular, neither reference in any way discloses or 0 25 suggests that it might be possible to obtain a lowmolecular weight agent or agents from animal bile which is capable of modulating the production of tumour necrosis Sfactor.

0i0 04b H:\Kanna\Kdp\ P.etypes\7 ;54139-94.doc 1i2/9.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, reference is made herein to various publications, which are hereby incorporated by reference in their entirety.

RIZ' DESCRIPTION OF =ER DRAWINGS Further details of the invention are described below with the help of the examples accompanying drawings in which: Figure 1 is an HPLC profile composition of the invention; Figure 2 is an HPLC profile composition of the invention; Figure 3 is an HPLC profile composition of the invention; SFigure 4 is an HPLC' prof ile for invention; Figure 5 is an HPLC profile for invention; Figure 6 is an HPLC profile for invention; illustrated in the for a concentrated for. a concentrated for a concentrated a composition of the a composition of the a composition of the **Figure 7 is an HPLC prof ile for a, composition of the invention; Figure 8 is a graph showing the effect of the composition on LPS-inducsd release of TNT by PBMN; .Figure 9 is a bar graph showing the eff ect of the composition on LPS-induced release of TNF by PEEN; Figure 10 shows the C 18 RP-HPLC profile of a N composition of the invention; 30 Figure 11 shows the RP-HPLC analysis of precipitated fractions of the composition of the invention; Figure 12 shows the RP-HPLC analysis of soluble *fractions of the composition of the invention.

Figure 13 is a graph showing survival taken f rom diagnosis of pancreatic cancer patients treated with the composition of the invention; Figure 14 is a graph showing survival taken f rom treatment of pancreatic cancer patients treated with the composition of the invention; Figure 15 is a graph shoving survival of all melanoma patients treated with the composition of the invention; Figure 16 is a graph showing survival of melanoma patients with two or more tumor sites treated with the composition of the invention; Figure 17 is a graph showing survival of melanoma patients with three or more tumor sites treated with the composition of the invention; Figure 18 is a graph showing the RP-HPLC .profile of whole composition of the invention; .*Figure 19 is a graph showing the RP-HPLC profile of a precipitate of the composition of the invention; 0:Figure 20 is a graph showing the EP-HPLC profile of a supernatant of the composition of the invention; *Figure 21 is an SDS gel of th e composition of the invention; Figure 22 shows the conditions and times of elution of the composition of the invention on hydrophilic HPLC (a) and the, elution profile for a supernatant of the* composition of the invention Figure 23 shows the elution of a precipitate of the composition of the invention on hydrophilic HPLC; :Figure 24 is a graph showing dose response of the composition of the invention in stimulating peripheral S blood monocyte function; and Figure 25 is a photograph of a malignant melanoma before (25a) and after (25b) treatment with the composition of the invention.

DETAILED DESCRIPTION OF TE INYMUTION As hereinbefore mentioned, the present invention relates to a composition for use as an immunomodulator comprising small molecular weight components of less than 3000 daltons, and having the following properties: a) is extractable from bile of animals; b) is capable of stimulating monocytes and macrophages in vitro; c) is capable of modulating tumor necrosis factor production; d) contains no measurable level of IL-la, IL-1l, TNF, IL-6, IL-8, IL-4, GN-CSF or IFN-gamma; e) has an anti-proliferative effect in a malignant mouse hybridoma cell line; f) shows no cytotoxicity to human peripheral blood mononuclear cells; and g) is not an endotoxin.

More particularly, investigations have shown that the at least some of the compositions of the invention will 15 stimulate normal monocytes to effect cytotoxicity towards the Chang hepatoma cell line, which is used to measure monocyte toxicity. Monocytes and macrophages from cancer patients (cervical and ovarian cancer) also have been reported to be stimulated by the composition to attack and destroy their own particular tumor cells.

The composition of the invention can modulate tumor necrosis factor (TNF) production. A preferred composition of the invention isolated from bile from bovines, promotes the release of TNF from human peripheral blood mononuclear cells in what appears to be physiological quantities.

Because TNF is known to initiate a cascade of inflammatory and antitumor cytokine effects, the preferred composition could exert its antineoplastic effect by stimulating human leucocytes to release TNF (and possibly other cytokines).

Accordingly, the present invention also may enhance lymphocyte and macrophage cytotoxicity towards tumor cells.

The composition of the invention has also been found to inhibit the growth of cells of mouse hybridoma cell line The inhibitory effect of the composition in the mouse hybridoma cells suggests antiproliferative activity.

The effect of the composition on the survival of human peripheral blood mononuclear cells (PBMN) was also examined. The composition was found to be non-cytotoxic to human PBMN.

As further exemplified below, the composition of the present invention has, among others, the following characteristics: 1) The component or components responsible for TNFrelease from PBMN eluted early from a C 18 RP-HPLC column.

2) The TNF-releasing component(s) is (are) precipitated, in part, by 80% acetonitrile.

3) The material unprecipitated by 80% acetonitrile retains some TNF-releasing activity.

4) The TNF-releasing activity in both the 15 acetonitrile precipitate and supernatant fractions eluted at the same early time from RP-HPLC. The results suggest that active TNF-releasing components in the composition

C..

belong to the same molecular family with perhaps some g0 subtle molecular differences that account for solubility differences.

The composition causes the release of and the component responsible for the IL-10 release elutes early from RP-HPLC, suggesting S0 00 that it is likely the same substance(s) that releases TNF.

6) The composition also causes the release of low quantities of interleukin-2 (IL-2).

7) The composition causes the release of granulocyte C Smacrophage colony stimulating factor (GM-CSF); the N acetonitrile precipitate fraction is more active than the supernatant fraction.

The ratio of TNF to GM-CSF release is about 2:1.

9) The 80% acetonitrile precipitate fraction contains component(s) that release about 3 fold more TNF and GM-CSF than component(s) in the supernatant fraction.

10) Analysis of the aforementioned precipitates and supernatant fractions separated by RP-HPLC shows that releasing activity for TNF, IL-10 and GM-CSF elutes early for both the precipitate and supernatant. However, in the supernatant, some IL-10 activity elutes late.

11) It is likely that the same molecule(s), i.e., component(s), in the composition are responsible for releasing TNF, IL-1 and GM-CSF. It is possible that the composition acts to stimulate the release of multiple different cytokines, or alternatively, the composition triggers the production and release of one cytokine that in turn stimulates production and release of other cytokines.

12) Physicochemical analysis of the composition, including the precipitates and supernatants thereof, by SDS gel electrophoresis and molecular sieve HPLC indicates that the principal components are less than 2500 daltons.

15 13) Further physicochemical separation by hydrophilic (polyhydroxyethyl) molecular sieve HPLC confirms the small V molecular weight of the components in the composition.

SPoo 14) Amino acid analysis before and after acid hydrolysis suggest the presence of peptide bonds, indicating the presence of peptides.

Amino acid content of the active fraction from RP-HPLC shows high levels of glutamate/glutamine and glycine. In addition, residues of asparagine, threonine, serine and alanine were detected.

16) There are some unidentified ninhydrin positive residues that are likely free amino acids.

As hereinbefore mentioned, the composition of the invention may be prepared by mixing bile from an animal, preferably a bovine, with an equal volume of an 30 alcohol to produce a bile/alcohol solution; separating out the alcohol soluble fraction and isolating a solution substantially free of alcohol; removing bile pigments from the solution to obtain a colorless liquid; (d) treating the colorless liquid to substantially remove any residual alcohol; extracting the colorless liquid with ether and isolating the aqueous phase; and removing residual ether from the aqueous phase.

The composition is obtained from the bile of any animal which produces bile, preferably non-human animals.

while the composition may possess a different activity toward a specific disease if obtained from the bile of one species as opposed to another, a generally suitable source of bile is that taken from bovines, ovines and swine. In most cases, it is practical to obtain the bile of slaughtered healthy food animals, such as bovines, ovines and pigs for use in the preparation of the composition of the invention. The bile thus collected should come directly from the gall bladders of the slaughtered animals and should be substantially clear, thereby indicating that 15 the bile preparation has a low mucus content and is S S substantially free of pus or blood.

Sb In a preferred embodiment of the method, bile from 0 age bovine sources is utilized. Bovine bile is plentiful, a because, in part, relatively large quantities can be extracted from each animal. Moreover, bovines are routinely slaughtered and inspected under health-related regulations, thus such animals provide a reliable source.

6 a 4for preparing the composition of the invention.

.544 Furthermore, humans are less likely to have an allergic reaction to material of bovine origin.

4 The bile is mixed with an equal volume of an alcohol to produce a bile/alcohol solution, which is 50% al cohol.

The alcohol may be an aliphatic alcohol, preferably N methanol, ethanol, or propanol, most preferably ethanol.

*30 A solution that is substantially free of the alcohol-insoluble material may be isolated by centrifuging.

Preferably, the bile/alcohol mixture is centrifuged at 3000-5000 RPM, most preferably 4200 RPM, for at least 2 hours, at about 15-250C. The alcohol contained in the bile/alcohol-soluble fraction then may be removed by taing advantage of the different volatility of alcohol and water, using conventional methods, heating the fraction to a suitable temperature, 80-85°C, for a suitable amount of time, up to about 10 hours.

Bile pigments may be removed from the solution to obtain a colorless liquid by using activated charcoal, polyamidic microgranules, or filtration. Preferably, an activated charcoal treatment is utilised. The procedure may be repeated in order that the solution satisfies optical density and conductivity standards.

The colorless liquid is treated to remove substantially any residual alcohol, using conventional methods. Preferably the colorless liquid is filtered using S, a filter having about a 1.0-3.5 gm retention, most preferably a retention of 15 The colorless liquid is then extracted with ether and the aqueous phase is isolated. The ether used in this step is preferably dimethyl ether, ethyl ether, n-propyl ether, isopropyl ether, or n-butyl ether, most preferably ethyl ether.

Residual ether may be removed from the aqueous phase by, for example, heating the solution up to preferably up to about 40°C for about 5-15 hours, most preferably for about 10 hours.

The composition may be used without further ie.

modification simply by packaging it in vials and sterilizing. The composition also may be used in a S concentrated form. A preferred concentrated form is prepared as follows. Prior to step described S hereinabove, the colorless liquid optionally may be concentrated to about one eighth of the volume of the bile/alcohol solution by, for example, heating to a temperature of less than about 85C, preferably, to about 600-700C. After step the aqueous phase may be concentrated so that it is one tenth of the volume of the bile/ethanol solution by, for example, heating to about 80-850C.

0S In a preferred method to prepare a composition of the invention, the collected bile is mixed with an equal volume of ethyl alcohol. The bile/alcohol mixture is then centrifuged at about 4200 RPM for at least 2 1/2 hours, at about 20±20C. The supernatant liquid is decanted and checked for pH and ethanol content. Bile pigments are then removed using activated charcoal. The treated bile/ethanol solution is then monitored for optical density and conductivity. O.D. levels or conductivity levels outside acceptable specifications require that the bile/ethanol solution be given additional treatment to remove bile pigments, for example treatment again with activated carbon to achieve a reading within specification limits.

Following activated carbon treatment, the solution is filtered through a filter having a 2.5 Msm retention, the alcohol is evaporated of f by heating to less than 856C and the solution is concentrated to approximately one eighth of the original bile/ethanol solution volume. The concentrated solution is cooled to between about 20-259C.

This solution is then mixed with ethyl ether and the ether phase is discarded. Preferably, relatively small volumes of ether and strong agitation are used, such as 0. 1 -to 1 volume, preferably 0.2 to 0.5 volume. This step may be repeated once. The aqueous phase is heated to remove 25 residual -ether by heating up to 55OC for about 10 hours, and further reduced in volume to one tenth of the original bile/ethanol volume by heating to about 80-850C. This solution is then tested for appearance, biological activity, and ethanol and ether content.

The pH of the composition may be adjusted to physiological pH, i.e. 7.4-7.5, using hydrochloric acid solution and sodium hydroxide solution) and a buffered solution may be obtained using dibasic and monobasic sodium phosphate salts as buffers, using conventional methods.

*e I. 0 0~ 0 4 06 4 6* 0 kOOSO* b 6 S d The composition may be used without further modification by simply packaging it in vials and sterilizing. A preferred sterilization method is to subject the composition to three sterilization cycles by autoclaving followed by incubation.

The composition may be used in a concentrated form.

The preparation of the concentrated form is described above. The composition may also be lyophilized.

The composition and concentrated composition are clear yellowish solutions essentially free of foreign matter, containing not more than 10 ppm ethanol and not more than ppm ether. In the bioassay described in Example 4, the composition has been shown to cause non-proliferative growth at about 18 units per ml.

15 The compositions of the invention can be produced in a consistently reproducible form using the method as generally described above with demonstrated identity, 00:0 potency and purity from batch to batch. Identity and purity are determined using reverse-phase high pressure liquid chromatography. (See Example The compositions of the invention have a consistently reproducible pattern on reverse-phase HPLC, in which peaks are seen early in the 00 0.06.e exclusion fraction at about 27 and 32 minutes. Before, inbetween and after the tall peaks, there are smaller peaks that vary in intensity. The HPLC readings for three lots of 00 4) B0209 (Figure B29/3006 (Figure 6) and B15/1606 (Figure also show a very reproducible p attern. The compositions also display non-proliferative growth of about d 18 units per ml in the bioassay described in Example 4.

The compositions are also characterized by the properties hereinbef ore mentioned, for example their ability to stimulate monocytes and macrophages in vitro, etc.

.Compounds likely to be present in the present composition, considering the source, include sulfonated 14 bile acids, oxidized bile acids, other naturally occurring bile acids, and their amino acid (especially glycine and taurine) conjugates and sterols. Accordingly, it is believed that the present composition includes at least one compound having the formula 0 *e* 00 wherein the molecule may or may not be fully saturated, such that, for example, the bond between A and B, B and C, or C and D may be single or double bonds, and wherein X is H, OH, or OSO 3 H; and Y is 5*

*SS.

SO

S

COOH

OOH

I I__CH2OH

COOH

OH

CHO

COOB

CONHR; and wherein R is an amino acid residue, such as, for example, glycyl, glutamyl, or tauryl, thereby forming the glycine or taurine conjugate.

In particular, the composition of the present invention has been analyzed as to its component compounds, including organic and inorganic components. Such information was derived using standard methods of analytical chemistry, including mass spectroscopy (MS).

The results of such studies include, for example, the identification of specific bile acid compounds thought to be present, including cholic acid, glycocholic acid, deoxyglycocholic acid, ursodeoxycholic acid, cholesterol sulfate, deoxycholic acid, chenodeoxycholic acid, and taurocholic acid.

From the MS it is not distinguishable if the loss of OH and H 2 of some compounds are occurring in the MS or if the deoxy, dideoxy and unsaturated analogs of such 15 compounds are also present to begin with. These compounds may all be present as salts of ammonium, aklylammonium and inorganic cations.

The MS analysis also supports the identification in the present composition of phospholipids, sphingolipids and related agents capable of forming miscelles. Specific compounds thought to be present include: stearic acid CH 3

(CH

2 1 6

COOH,

palmitic acid CH 3

(CH

2 1 4

COOH

2oleic acid Z-9 octadecanoic acid: 25 CH3(CH 2 2CH 2

CHCH

2

(CH

2 )6COOH oxidized or hydroxylated/unsaturated short chain fatty acids: C 6

H

8 0 3 CH3CH-CHCOCH2COOH or a C 6 acid with 2 double bonds and a hydroxide) acetic acid stearic acid diglyceride palmitic acid diglyceride stearic acid, palmitic acid diglyceride stearic acid-monoglyceride-phosphocholine (a lysolecithin) stearic acid monoglyceride stearic acid triglyceride palmitic acid monoglyceride phosphocholine phosphoserine phosphosphingosine stearic acid-sphingosine sphingosine stearic acid amide stearic acid methylamide pa-laitic acid amid* lecithin sialic acid-glycerol dimer In addition, preliminary HPLC and titration evidence has been obtained which shown that shorter chain fatty acids are also present.

Phospholipid, sphingolipid, and related hydrolysis pr.oduct compounds likely to be present considering the source and the information derived from the ms and HPLC analyses include at least one compound having the formula OR, CH (C)Y 12

CH-CH

esS 5OR 2

OR'

0R 3 -X

OR'-X

0*@where 9 2

R

3 are dif ferent or the same and are H, Coe, CH=C-R X, -P or -S5(0) 20-; X is selected from the group consisting of choline, ethanol amine, malicylated etlianolamines, serine, inomitol, sugars bearing free hydroxyls, amino-sugars, sulfonated. sugars, and sialic acids; is Cj-cm aLkyl that is satur~ated or unsaturated, oxidized or hydroxylated; and R1 is an alkyl group or 0 We oxidized and/or hydroxylated analogs thereof.

S. The fatty acids and their conjugates may be present in *:25 the aforementioned aqu~eous extract as salts. The solubility of such compounds is also enhanced by other components of the mixture. Amides of the included carboxylic acids, RCONR'R 2 where R'and R 2 are the same or dif ferent and are H or alkyl, are also believed to be present.

A third class of compounds, namely, mucin and proteoglycan hydrolysis products, are also likely to be present, considering the source of the composition and the aforementioned MS analysis thereof. Sucb. compounds include hydrolysis products of mucoprotains. from bile and from the gallbladder wall, such as: chondroitln 4- and 6-oulfates, dermatan sulfate, heparin, heparin sulfate, hyaluronic acid and the hydrolysis products (monomers, dimers, oligomers and polymers) of these mucins. Chitin and other mucins may be similarly hydrolyzed, which hydrolysis products would include: N-acetyl-D-glucosamirie, N-acetyl-D-galactosamine-4 sulfate, galactose- 6-sulf ate, N-acetyl-D-glucosamine- 6-sulf ate, glucosamine- 6-sulf ate, D-glucosamine 2sulfate, D-glucosamifle 2,3 -disulfate, D-galactose-6 sulfate, glucuronic aid 2-sulfate, N-acetylneuraminic acid, sialic acid, N-acetyl chondrosine, chondroitin 4-sulfate, chondroitin 6-sulfate, D-glucosamine, Dgalactosamine, g lucuronic acid, glucose, galactose, mannose, fucose, iduronic acid, hexose, hexosamine, ester sulfate, glucuronic acid, chondrosamine, 2amino-2 -deoxy-D-ga lactose, serine, proline, threonine, alanine glycine taurifle, glutamic acid, aspartic acid, ~e :histidine, and small peptides.

similar products would be obtained by hydrolysis of mucins such as keratin sulfates, dermatan sulfates the :20 natural sugar-sugar linkages in the dimers, oligomers and polymers may be replaced by -O-Si(OH) 2 bridges between the sugar monomers or adjacent sugar chains.

In particular, specific mucin and proteoglycan hydrolysis product compounds-thought to be present include: sialic acids and their mono and diacetylated and glycolylated monomers; .'.N-acetylneuraminic acid; hexosamines; L-fucose; hexosamine-hexuronic acid (dimer) disulfate; 00 glucuronic acid or iduronic acid disulfate, monoacetylated; 0 sialic acid-glycerol (dimer); arnd 0*09* dimers, trimers, oligomers and polymers of the above monomers in acetylated and sulfated form.

go goA fourth class of compounds, namely fat-soluble 00*060 vitamins, likely to be present considering the source and the aforementioned MS analysis, include A, D, and K vitamins D1, D3, D4, K1, K2, K5, K6, K7, K-S(II), 0040 and Vitamin E acetate, for example.

In particular, specific fat-soluble vitamin compounds thought to be present include at least one of the group consisting of Vitamrin A2, Vitamin Di, Lumisterol (present f om its 'vitamin Di complex) I Vitamin E, Vitamin 1(1 oxide, and Vitamin Various miscellaneous organic comporunds are likely to be present, considering the source and the aforementioned MS analysis. such compounds include: bilirubin, and its gluconuride conjugate; biliverdin, and its gluconuride conjugate; traces of steroids; other plasma solutes, such an sugars, purines and pyrimidines;I miscellaneous dietary lipids; and glutathione and its hydrolysis products.

In particular, specific miscellaneous organic *0compou nds believed to be present in the composition include at least one of the group consisting of urea, methyl amino, dimethylamine, ethylamine, methylethylamine, diethylazine, dipropylamin., butylethylamins, ammonia, choline, taurine, glutamic acid, glycine, alanine, p-ser, p-eu, p-ea, asp thr ser sar, a-aba, cit, val, ile, leu, B-ala, G-aba, OH-lys, .:20 orn, lys, butylated liydroxy toluene (BHT) and polyethylene glycol.

Amines present in the present composition, particularly the secondary amines, may include nitrogen .00CC oxides from the air, thus forming riitroso compounds.

N-oxides-and N-carbamate byproducts may also be included.

This series of azines cited above should be extended to include all primary, secondary and tertiary alkylamines.

Certain inorganic elements have been identified and quantified (mgf 1) as follows: Tungsten 0.07 Zinc 0.666 Phosphorus 378 Cobalt 0.008 Nickel 0.2 0.022 Barium 0.032 Iron 0.022 Manganese 0.039 Chromium 0.060 Magnesium 7.46 Aluminum 0.136 Calcium 5.97 Copper 0.087 Titanium 0.01 Strontium 0.060 Sodium 9600 Potassium 483 Chloride 15400 Ammonia 218 15 Vanadium I ppm **The compositions of the invention have valuable pharmacological properties. In particular, the compositions of the invention have anti-proliferative effects, affect neoplastic growth, and affect release of tumor necrosis factor. The compositions have been shown to cause no significant toxicity and only transient adverse side effects (for example, slight fever, polydipsia, pain at injection site). They have also been found to contain no detectable components of high molecular weight matter above about 5,000 daltons) which can cause harmful immunologic reactions. The compositions may be used as agents for the prophylaxis and treatment of conditions requiring modification of the immune response, in particular infectious diseases, neoplasias, and autoimmune diseases. They may be especially useful in the treatment of various forms of neoplasia, such as leukemias, lymphomas, melanomas, adenomas, sarcomas, and carcinomas.

In particular, the composition may be useful for treating malignant melanoma, pancreatic cancer, cervico-uterine cancer, cancer of the kidney, stomach, lung, rectum, breast, bowel, gastric, liver, thyroid, neck, cervix, salivary gland, leg, tongue, lip, bile duct, pelvis, mediastinum, urethra, bronchogenic, bladder, esophagus and colon, and Kaposi's Sarcoma, which is a form of cancer associated with HIV-infected patients with Acquired Immune Deficiency Syndrome (AIDS). The composition may also be used for other anti-proliferative conditions, such as arthrosclerosis and viral infections, in particular AIDS.

It may also be used in the treatment of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Type I diabetes, myasthenia gravis, Addison's Disease, autoimmune hemolytic anaemia, Crohn's disease, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, idiopathic 15 thrombocytopenic purpura, pernicious anaemia, poststreptococcal glomerulonephritis, psoriasis, scleroderma, Sjogren's syndrome, spontaneous infertility, and pemphigus vulgaris.

*.bo The compositions of the invention may be converted 20 using customary methods into pharmaceutical agents. The pharmaceutical agents contain the composition of the invention either alone or together with other active substances. Such pharmaceutical agents can be for oral, topical, rectal, parenteral, local, inhalant, or 25 intracerebral use. They are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, and tubelets. For parenteral and intracerebral uses, those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection can be Sused, and can therefore be prepared as solutions of the compositions or as powders of the active compositions to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity which is compatible with the physiological fluids. For local us. those pr .eparations in thQ f orm of. crea-as or ointments -aor topica~l use or in thg form of pry 3 may beo considere.d; for inhalant uses. preparations in the form of sprays, for example nose sprays, may be considered. Preferably,. the composition is administered intramuscularly.

The Pharmaceutical compositio= Can be prepared by se known methods for the prepar-ation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a Mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remingtonls Ph& aeuiAl Sciences (Mack ~Publishing company, Easton, Pa., USA 18) On this basis, the pharmaceutical agents include, *&.albeit not exclusively, the composition of the invention in association with one or more Pharmaceutically acceptable vehicles or diluontg, and are contained in buffered solutions with a suitable PH and iso-osmotic with the physiological fluidS.

The compositions are indicated as therapeutic agents either alone or in conjunction with other therapeutic *0agents or other forms of treatment. For example, in the case of a malignant tumor, the present treatment may render *6:25 a tumor suitable for surgical removal whexe it was not previously operable. The compositions and agents of the S invention are intended f or administration to humans or animals.

In general, a dosage range of the composition is envisaged for administration in human medicine of from about 0.01 to 20 mg/kg, preferably from about 0.1 to 1 mg/kg, most preferably 0.1 to 1 mg/kg of body weigh~t daily may be employed. I n the case of intravenous administration, the dosage is about 0.1 to 5 mg/kg of body weight daily, and in the case of oral administration the dosage is about I to 5 mg/kg of body weighbt daily. Where the concentrated composition -is used, approximately half: the above mentioned dosages may be used. For example, for intramusculaxr administration, a dosage of a~bout 0.2 to mg/kg of body weight daily, preferably 0.275-0.75 mg/kg of body weight daily may be used.

it will be appreciated by medical practitioners that it may be necessary to deviate from the amounts mentioned and, in particular, to do so as a function of the body weight and condition of the animal to be treated, the 1o particular disease to be treated, the nature of the administration route and the therapy desired. In addition, the type of animal and its individual behaviour towards the medicine or the nature of its formulation and the time or interval at which it is administered may also indicate use 15 of amounts different from those mntioned. Thus it may suffice, in some cases, to manage with less than the abovementioned minimum amounts whilst in other cases the upper limit mentioned must be exceeded. Where major amounts ar~e it may be advisa-ble to divide these into administrations over the course of the day.

Thus, the present invention comprises a process for preparing an immunomodulator composition comprising (a) mixing bile from an animal with a water-soluble solvent to a bile/solvent solution; isolating an aqueous substantially free of solvent from the bile/solvent solution; and removing bile pigments from the substantially solvent-f ree solution to obtain a a~e colorless liquid, preferably where the water soluble solvent is an alcohol, and where the bile from the animal 30 is mixed with an equal volume of the alcohol. Preferred aspects of the aforementioned process also comprise further concentrating the col orless liquid to about one-eighth, or onetenhthe original volume of the bile/solvent '4.soe-tenh obviously, compositions produced via the above process form a preferred aspect of the invention.

The present invention also comprises a composition for use as an immunomodulator, comprising at least .one component having a molecular weight of less than about 3000 daltons, which shxows no cytotoxici~ty to human peripheral blo~od mononuclear cells, and has at least one of the following properties: is capable of stimulating monocytes and macrophages in vitro or in vivo to produce one or more cytokines; is capable of stimulating monocytes or macrophages.*to' produce tumor necrosis factor In vitro or in2 vivo; or has an anti-proliferative effect in a malignant mouse hybridoma cell line; and wheroin said component is not an endotoxin, IL-lUa, IL- TN7, IL-4, IL-6, IL-8, QM-CSI or IFN-gamina. Such compositions may be obtained fro= the bile of animals, preferably bovines, or from other sources. In a preferred Sembodiment of the composition, the composition stimulates tumor necrosis factor production In vitro or in vivo, and most preferably in humans, in the absence of exogenous

IL-

la, 1L-1$, TNF, IL-4, IL-6, IL-8, GM-CSF, and IFN-gamma, ,)Ses' The compositions of the present invention also have components -which can be characterized by column chromatography such that When said composition is dried to S obtain a solid residue, and 2 grams of said residue are **see dissolved in 20 ml of a 10% concentrated ammonium hydroxide solution in methanol, and after any insoluble material is roved, is subjected to column~ chromatography in a mehao column having dimensions of 5 Cm x 12.5 cm, and containing 102 g of 60 A flash silica gal, and operiting at a pressure of 10 pounds per square inch and a flow rate of 11 ml/min with a 10% concentrated a-mmonium hydroxide in methanol solvent solution, said component is eluted from the column in a traction taken when the total column elution is between about 180 and about 220 ml, between about 220 ml to about 260 ml, or between about 260 ml and about 300 ml.

Characterization of c=opnents may also be accomplished by ion-exchange chromatography, such that when 10 ml of said composition is subjected to anion-exchang.

chromatography in a column containing Bio-Rad

AG-I

hydroxide form resin in an amount sufficient to bind substantially all the anions present in said 10 ml of said composition, said component is eluted from the column using a step gradient of ammonium bicarbonate buffer at a buffer concentration from about 0.5 M to about 1.5 M, preferably at a buffer concentration from about 1.0 M to about 1.5 M, and most preferably at a buffer concentration of about 1.5 M.

15 Reversed-phase (C18) HPLC can also be used for characterization of components, such that when said S. composition is lyophilized and reconstituted in 0.1% TFA in water and then subjected to reversed-phase (CI8) HPLC in a Phenomenex WP60009-C18 column, having dimensions of 250 x 4.6 m, where a first buffer of o.1% TFA in water is run through the column for about 10 minutes, then a linear gradient from 0 to 80% of a second buffer of 0.1% TPA in acetonitrile is run for about 55 minutes, followed by an 80% solution of the second buffer for about 5 minutes, and .25 an 80%-0% gradient of the second buffer for about Sminutes, and where flow rate is 1 ml/min. and the capacity of the column and buffers are not exceeded, said component is eluted from the column at a time from about 2.4 minutes to about 3.4 minutes after said reconstituted composition is applied to the column. Characterization of components of the composition can also be accomplished by an additional reversed-phase HPLC method, such that when said composition is dialyzed or dissolved in a first buffer of 0.1% TFA in water and then subjected to reversed-phase (C18) HPLC in a Bio-Rad Hi-Pore RP 318 (C18) column, having dimensions of 250 x 4.65 mm, where the first buffer is run thZrough th. column for about 10 minutes, then a lna grAdient from 0-804 Of a second buffer, of 0.14 TPA in Mc~tOnitrilo in ru for about 55 mijnutem 1 followed by an 8O* solution of the second buffer for about 5 minutes, and anl 80-0% gradient of the second buiffer for about f ive Minutes# and where the flow rate is I mi/min. and the capacitY of the column-and the buff ers are not exceeded, gaid component irn eluted from thle column at a time from about 2 minutes to about 21.4 minutes, o .r at a time from about 21.4 minutes to. about 25.6 minutes after said dialyzed composition is applied to the column.

The compositions of the present invention can also be characterized by TLC, such that when said icomposition is :subjected to thin laysr chromatogr.aphy on silica gel plates in 10% concentratedi amonium hydroxide in methanol and visualized with a ninhycirin spray, a Positive reaction with ninhydrjin oooi.rs at an Rf value fron abourt 0.80 to about 0190.

*.@The present invention also comprises a method of 20 stimulating tumor necrosis factor produ -ction in humans, comprising administering an effective amount of a composition comprising at least One Of the following' compounds:' (a)a compound Of the formula wh erQ the bonds betwO*n A-B, B-C, and C-D may be 8ingle or double bonds, and where X H, OH, or oso~x; and Y-

COON

CH 2 0O3

COOR

ON

"nCOON 0@ *eO.

S S. S

OS

S 0 *5 where R in an amino acid residue; a compound of the formula C2CH (Ole) CH2(0(O1-X) or

SOR'

Ore-X

S

S 0 0* S. S 0 5 0*

S..

S

N:

15 where iR2 and R' are H, coR 4 cH-CjH-Rs, X, P(O) (OH)0-, X in choline, ethanolaain., N-alkylated ethanolamines, serjane, inoditol, sugars bearing tree hydroxyls, aminosugars, sulfonated sugars, or sialic acids; and

R

4 is a saturated or unsaturated alkyl group having a carbon chain from about

C

1 to cm or oxidized and hydroxylated analogs thereof; and R' is an alkyl group or oxidized and hydroxylated analogs thereof 1 a mucin hydrolysis product or a proteoglycan hiydrolysis product; or.

a fat-soluble vitamin.

0@ 0

S

0*

OS..

0 0* S 0

S.

S.

*5@ *05*

SO

*0 S

SS

S S 0* 5@ S Preferably, compositions of the inventive method comprise at least one compound 5elected from the group consisting Of taurocholic acid and its BulPhated derivatives; glycocholic acid and its sulphated de-riva..

tives; sphingosine; a diacyl glycerol; lecithin; an Oligosaccharide Of less than 10 3accharide units in length, where said Oligrosaccharid. is comprised of gialic acid, fucose, hexoxamines, or dulphated hoxosamines; Vitamin A; retinolic acid derivatives; retinol derivatives; taurine; and glutaxic acid and its conjugates. The composition may also additionally comprise at least one compound selected from the group consisting of a.moni&; primary al)kyl amines; secondary alkyl amines; tertiary alkyl amines; and a carboxylic acid R'CO 2 H, wherein RI is C 1 15 C 30 alkyl that is saturated or unsaturated, and oxidized and/o r hydroxyl iz ed derivatives thereof.

The method of the invention also embraces stimulation of TNF production by administration of a composition comprising at least one compound selected from the group consisting of taurocholic acid and its sulphated derivatives; glycooholic acid and its sulphated derivatives; sphingosint; a diacyl glycerol; lecithin; an oligosaccharide of less than 10 saccharide units in length, where said oligosaccharjde is comprised of sialic' acid, fucose, heooamines, or sulphated hexosamines; Vitamin A; retinoic acid derivatives; retinol derivatives; taurine; and .glutamic acid and its conjugates.- The present invention also provides a method of treating pancreatic cancer comprising administering to a patient suffering from said cancer a therapeutically of fective amount of the compositions of the invention.

Also forming part of the present invention are compositions comprising micelles of sphingosine or sph ingosine coaplexed with a salt, or micelles of retinolic acid cr its derivaties, which have at least one of the follow~ing properties: 55 5 0S OSS S OS

S

0.5.5.

ic capable of stimulating monocytes and macrophages in vitro to produce one or more cytokinea; is capable of atimulating menocytes or macrophages to produce tuimor necrosis factor in vitro or in s vivo; or has an anti -pro lif erative effect in a malignant mouse hybridoma cell line.

The micelles may also comprise a diacyl glyceride or lecithin, and may further comprise a bile acid salt, and a source of ammoniumi or alkyl azzmoniun ions.

Finally, the. present invention also contemplates compositions comprising sphingosine, a bile acid salt and a source of ammonium or alkyl amxonium ions, a bile acid salt, sphingosine, a diacyl glycerol, a source of :15 Ammonium, or alkyl ammonium ions, and a ratinol derivative, a diacyl glyceride, lecithin, and a bile acid salt, or a diacyl glyceride, lecithin, and a mucin hydrolysis product or a protacglycan hydrolysis proftct, which has at least one of the following properties: :20 is capable of stimulating monocytes and macrophages in vitro to produce one or more cytokines; is capable of stimulating monocytes or macrophages to produce tumor necrosis factor in vitro or in e vivo; or *25 has an anti-proliferative effect in a malignant OS 0 mouse hybridoma cell line.

*0 0 0 S 0* See...

S

S.

The following non-limiting examples are illustrative of the present Invention: Prearaionof heCOM2osition 2K theA Invention Bovine bile is collected from healthy-herds at least one and one half years old which have been slaughtered for food use at a licensed and inspected abattoir. The gall bladders are collected from the slaughtered animals which have been inspected and the gall bladders are separated from the-livers and examined by a veterinarian to confirm that the gall bladders are free of parasites and evidence of infection, and thus are suitable for use as a source of bile.

Gall bladders which pass this inspection are wiped with a solution of 704 ethanol to sanitize the exterior and a syringe is inserted to remove the bile. The bile removed is vigUally examined by the veterinarian in the syringe to assure that it contains no blood or pus and is otherwise satisfactory. Bile found to be satisfactory is transferred into a graduated amber bottle containing ethyl alcohol.

The bile is a greenish fluid substantially free of blood and pus. Bile is added to each bottle to a level marked on 015 the bottle, twice the level of ethanol present to give a bile/ethanol solution. The bile/ethanol solution is a greenish fluid substantially free of foreign material in an 0*approximate 50%/50% bile/ethyl solution. It also shows positive for ethyl alcohol USP XXII Part B. These bottles 00 :20 are labelled with the date of collection which serves as the lot number. A minimum of fifty animals serve as the pool for each lot. Fragments of livers, spleen, and lymph nodes are also collected* from the animnals whose bile made up the pool. and the fragments are examined for the presence 00'.25 of parasites or other indications of disease.

The bile/alcohol mixture is then centrifuged at 4200 RPM for at least 2 1/2 hours at 20 20 C. The supernatant liquid is decanted and checked for pH and X ethanol content. The decanted liquid is then subjected to 0~3 an activated charcoal treatment. The treated bile/ethanol is then monitored for Optical Density and conductivity. O.D. levels or conductivity levels outside acceptable specifications will requ1ire that the bile ethanol solution be given additional treatment with activated carbon to achieve a reading within specification I tn:ts- S00 0 0 006 00.0 SO 0

S.

so 0 00* S it.

Following activated carbon treatment, the solution is filtered throcugh a filter (for example using filters having a 2. 5 pm retention) the alcohol is evaporated of f (for example, by heating to less than 854C) and the solution is concentrated to approximately one eighth of the original bile/ethanol solution volume. The concentrated solution is cooled to 20 25 0 C. This solution,:'is then mixed with ethyl ether and the ether phase is discarded.

This step may be repeated once. The aqueous phase is heated to remove residual ether (for example by heating-upto about 550C for about 10 hours) a-nd further reduced* in volume to one tenth of the original bile/ethanol volume by heating to about 80 85* C. The resultant composition is then tested for appearance, biological activity and ethanol 15 and ether content. The composition is a clear yellowish solution essentially free of foreign matter, and it contains not more than 10 ppm ethanol and not more than 5ppm, ether. In the bioassay described in Example 4, the non-proliferative growth is 18 Units /ml.

identity and purity are determined using reversephase high pressure liquid chromatography. Potency is assayed using the antiproliferative method as described in Example 4.

Initial batches of the composition of the invention were manufactured as a non-buffered liquid. Subsequent batches were manufactured as a buffered liquid, prepared by adjusting the pH of the composition to about 7.4 0.05, using hydrochloric acid solution and sodium hydroxide solution) as well as using dibasic and monobasic sodium phosphate salts as buffers. Bioburden reduction is conducted in a steam autoclave at 104 20C for minutes. 'The bulk solution is filled into 5 ml or 10 ml sterile bottles and capped. The filled and capped bottles are subjected to three sterilization cycles by autoclaving them at 1040C 20C for 60 minutes followed by incubation at 35 0 C for 23 I hrs. Between each cycle (autoclave plus incubation samples) are taken and tested for bioburden. Following thie last cycle, the bottles are visually inspected against a black and a white background to detect any particulates which may be present.

Following inspection, the lot is sampled and tested for conformance to specifications. Tests include identity, sterility, pyrogenicity, endotoxin, bioassay, HPLC and general safety (See Table TAML 1 FINAL PRODUCT TEST ResuIts for 1nulivida Batches BATCH BATCH R~l~ BC0227 17 14.0

S

a *0 0~e*

S

000e 0 0

S.

0S 0

S..

S

*0 S Biological Activity (18.0 Idea ty/Purity 15 Agrees with refience Safety TPam= t) Pyrogeniticy (Uamp. imcrmae sdall noc exceed 0.4 o C) Endotoxin :90.4 EU/mi Steuiiity (no growth) pH (7.40 Pass

PM

:!0.25 p-n 7.45

P-

PAN

:!Z.25

PAN

7.39

BATCH#N

BCD223 22.5

PM

pans !!0.25 pass 7.36 0e S S 0e OeOS 6 0 5* 0 0: 4**S Table 1 (Co.) FINAL PRODUCT TEST BATCH BATCH BATCH BC0226 BC0227 BCD=s Appearance Vimd (clear, light paw Pau pans yellowsh liquid wth little or no Precipitate) Appearance O.D. (passes test) 0.098 0.118 0.088 Osinlazity 5Q4 603 445 Solids (18 3mg/ml) 18 15 Amino Acids (800 10% mg/ml) 790 742 878 Ethyl Alcohol (not mom than 10 ppm) Pass PM Pan Ethyl Ether (not more than 10 ppm) Pus Pa Paw Conductivity (26 5 mMHO) 25 22 29 Physi2al Chomical and Biochemical Characteristics of the Couposition of the Invention A number of the physicochemical characteristics of the preparation (conductivity, osmolarity and total solids) are shown in Table 2. More particularly, test results for three manufactured batches of a composition prepared in accordance with Example 1 were carried out. The results shown in Table 2 demonstrate the sterility, potency, and reproducibility of the manufactured product. It is noted

S.

that the ethyl alcohol and ethyl ether are measured as inprocess tests only. A summary of the method for determination of the biological activity is provided in Example 4.

OwewOe Test Biological Activity Idetityf~urity Safety 44 0 0 04 0004 4 Ok 4 *0 0* g.e *040 op 01. 0 Pyrogenicity Endotoim Smrility pH Appearance Solids Amino Acids Osmolarity Table 2 Product Specification 18 5 unitsmi Agrees with refereace Passes test Temperature increaseshal not exceed 0.4*C 2 EU/wi No growth 7.40 .05 Cloar, light yellowish liquid with little or no precipitate 18 3 mg/mi 800 10% ug/nmJ Biological Activity

HPLC

Geeal Wiy test (mice and guinea pigs) 21 CFR part 610. 11 Pyrogen test (rabbits)

USP

Umulus Amoebocyte Lysate Test USP Steiity Tast

USP

pH test USP Visua inspection Lyophilization Trinitrobenzesne-sulfonic Acid Method Freezing point depression

USP

0 40 0 *4 006q.

4 0

S

00 .1 .4 to 15 Ethyl Alcohol Not more tha 100 ppm Direct Injection Gas Chromatography Ethyl Ether Not more thin 100 ppm Direct Injection Gas Chromatography Conductivity 26 5 mMHO Copenhagen Radiometer Model Physical and chemical properties such as conductivity, osmolarity and total solids are consistent with a composition of over 99% salt. Less than it of the solids in the composition is organic material, there are no lipids, around half are carbohydrates and the rest are amino acids. Proteins and peptides are present. SDS Gel electrophoresis conf irmed that there may be more peptides than proteins in the composition. High molecular we~ghts are not detected. This is an important feature of a peptide drug because it is not expected to be immunogenic.

HPLC and bioassay test methods for the composition of the invention were developed using the nonbuffered product.

These tests are used to characterize the product as the buffered liquid and the concentrated formula. The HPLC results described below indicate that the product is the same in all of the presentations. The bioassay shows that the activity of the concentrated composition is two and a half times greater than the original composition.

Therefore, the product used in the studies has been demonstrated as being equivalent.

R. VZRSED PHASE (Cl 5 HPLC NMALTYSIS \or THE COMPOSITION OF THE INVRTION 15 The composition of the invention has a consistently reproducible pattern on reversed phase HPLC in which peaks are seen early in the exclusion fraction and at about 27 Wes and 32 minutes. Before, in-between and after the tall S" peaks, there are smaller peaks that vary in intensity. The HPLC readings for three lots of the concentrated composition of the invention are shown in Figures 1 to 3.

RP-HPLC profiles for batches B0211 (Figure B0209 (Figure B29/3006 (Figure 6) and B15/1606 (Figure 7), also show a very reproducible pattern.

The RP-HPLC to characterize the composition of the invention was carried out as follows. Bio-Rad Hi-Pore RP 318 guard column (C 18 4.6 x 30 mm (Bio-Rad) and Bio-Rad Hi-Pore RP 318 (C 18 column, 4.6 x 250 mm was used. The samples were dialyzed in 0.1% trifluoroacetic acid (TFA S 30 Pierce) in H 2 0 (Buffer A) and applied to the column. Buffer *A was run for 10 minutes, then a linear gradient 0-80% of Buffer B TFA in 100% acetonitrile) was run for minutes. At the end of this period, 80% Buffer B was run for 5 minutes and 80-0% of Buffer B for 5 minutes. Flow rate was 1.0 ml/minute. Fractions from successive runs were collected and pooled and concentrated in a Speedvac (Model SVC 200H, Savant Instruments, Farmington, Preliminary Characterization of the Composition Peaks from HPLC were submitted for protein sequencing.

The initial sample, the major HPLC peak designated RP-HPLC -31.00 min, batch 0210 in TFA/CH 3 CN, failed to yield data when subjected to N-terminal sequence analysis. The results may be interpreted as either a quantity problem or N-terminal blockage. Quantitation of protein content of that fraction by amino acid analysis after acid hydrolysis revealed that sufficient quantity should have been subjected to sequence analysis; however, because of the composition it was thought the sample may not be a protein and thus N-terminal blockage would not be the problem. The 15 samples displayed the following composition: about 70% Glx (glutamate/glutamine) plus about 15% glycine (Gly).

s* Furthermore, analysis of the equivalent sample from RP-HPLC gave similar results: 68% Glx and 15% Gly (Table 3).

Further characterizations of unfractionated material plus several other fractions revealed the following. The starting material (32 mg/ml) yielded a sequence signal indicating a polyglutamate peptide/protein, consistent with the amino acid composition data.

Table 3 25 Mole Gbx Gly HPLC Fraction run R (see profile) Fraction Analyis I Mole SUM (07/11/92) Glx Gly Glx Gly la 393 28 44 72 lb+c 394 35 37 72 2a 395 39 36 2b 396 35 43 78 2c 397 44 30 74 Table 3 (Coat) Fraction Mote Glx Gly HPLC Fraction run RI (see profile) Anailysis MolC (07/11/92)

SUM

OGlx Gly 83 78 Analysis of several HPLC fractions (Table 3) revealed that all are very rich in Glx (Glu/Gln) (28-70 mol%) and 10 Gly (10-44 mol Each fraction, however, displayed real differences in the relative amounts of the other amino 0o°@ acids.

0 BIOLOGICAL ACTIVITY OF RACTIONl OP THB COMPOSTION The biological activity of fractions of the composition have been investigated. The biological activity of the composition is thought to be attributable Sto small molecular weight components less than 3000 daltons). This was determined through an experiment in S 20 which four fractions of the composition and unfractionated composition were tested for biological activity. The first fraction contained proteins and peptides with molecular Poo*$: weight less than 3000 daltons, while the remaining three fractions contained additional larger molecular weight proteins and polypeptides. All fractions contained **fe additional larger molecular weight proteins and S polypeptides. All fractions and unfractionated compositions demonstrated the same biological activity.

Since all fractions were as effective as the unfractionated -roduct, and since the common denominator of all fractions was the presence of the same concentration of molecules smaller than 3000 daltons, this led to the conclusion that the biological activity of the composition is due to components with molecular weights smaller than 3000 Daltons.

BammgIp 4 Effect on Malimaant Cell Lines The effect of the composition of the invention on the proliferation of cultures of four malignant cell lines (Daudi-human lymphoma cells, ME-180 human cervical carcinoma cells, T-24-human bladder carcinoma cells, and mouse hybridoma cells was measured. The composition of the invention had an antiproliferative effect on the mouse hybridoma cells. The studies suggested that the inhibitory effect of the composition in mouse hybridoma .15 cells is antiproliferative rather than cytocidal.

A bioassay based on the reproducible antiproliferative effect of the composition of the invention was designed in a mouse hybridoma cell model to facilitate characterization of the composition. The bioassay is carried out as follows. The osmolarity and pH of the composition are adjusted to match that of the cell culture medium in order to isolate the composition's biological activity from its physical and chemical properties. Serial dilutions of isotonic composition from 1:5 to 1:10,000 are prepared in .25 culture medium. Hybridoma cell samples are specifically Squantitated. Using a hemocytometer the cells in a 100 Al sample are counted. The cells are concentrated by centrifugation and then cell concentration is adjusted to 1,000 cells/ml (twice the final desired concentration) by addition of appropriate volumes of fresh media. Hybridoma Scell suspensions (1 ml) mixed with corresponding dilutions of the composition (1 ml) are incubated in 24 well plates at 37°C in a humid atmosphere with CO 2 controlled at 6%.

After 96 hours, each well is sampled at 100 Al X 3 and placed in a 96 well plate. (A blank of 100 il of medium without cells is included.) Cell density is determined using a PROMEGA CelJ.Titer 96' kit. Cell concentration is measured by reading absorbance at 595nm (650nim reference) and recorded by ELISA plate reader. For each assay, a standard curve of cell concentration is prepared. Cultured cells in the log phase of growth are sampled, counted, concentrated and resuspended in aerial dilutions. Each dilution is sampled at 10041l X 3 and placed in a 96 well plate. The standard curve is constructed by plotting cell density versus "net" OD at 595 nm after subtraction of zero cell blanks and OD at 650nm. Cell density of Iunknown samples is determined by interpolation.

To calculate biological activity, cell density as a percentage of the control (no composition) is plotted 1-9 against final composition dilution (in log and linear scale), and a curve is fitted through the points utilizing the spline curve fitting method. Then, the composition dilution that corresponds to 50% inhibition of cell proliferation is determined manually and converted directly to UNITS Of composition. By definition, one unit of composition inhibits by 50% the proliferation of 1 ml cell culture of cell line HYB 16-1, seeded at 500 cells/ml,.

after 96 hours at 370C and 6%C0 2 The average activity of the composition of the invention in the bioassay i s 18.24 1.82 Units/ml.

0 so0 0 0 0@ 0* 0

S.

S

OSSO

0.0050 RX&NRle Zf feat an azd B LVM~h0CVteu in CultUre The composition of the invention has been shown to be non-toxic to normal T and B lymphocytes in culture. The growth of human lymphocytes was examined under carefully controlled conditions in the presence and absence of the composition. Standard concentrations of lymphocytes were incubated in wells containing various concentrations of the composition. When normal T and B human lymphocytes were incubated with the composition in concentrations similar to those that are used clinically, there were no adverse effects as judged by Trypan Blue exclusion.

The effect of the composition on the survival of human peripheral blood mononuclear cells (PBMN) was examined. In this experiment, PBMN were incubated for 24 and 48 hrs in plastic microwell plates with various volumes of the composition and tissue culture medium. At the end of this period, the number of surviving cells was estimated by trypan blue dye exclusion. Table 4 shows that the number of surviving cells fell at 24 and again at 48 hours; however, the number of surviving cells in the presence or absence of the composition was not different. Moreover, increasing volumes of the composition had no effect on survival (Table Thus, the composition showed no 15 cytotoxicity to human PBMN.

Table 4 Concmtratmion Viable FBMN After InwatoU 0 0

S.*

0** *00 S o@

*O

Co *o e* Patient S.Z C=ncntration (.ul/wci) 5* 0 25 ft S 50 25 100

S.

200 20 LPS (sg/well) 1 Patient

E.S

0 No. of Live PBMN per Well by Trypmn Blue (x106) 1 Zero time After 24 hra No. After 48 hrs No. viable viable 0.702 0.23(33) 0.10(14) 0.43 (61) 0.15 (21) 0.10(14) 0.23 (33) 0.15(21) 0.18(26) 0.48 (69) 0.23 (33) 1.302 0.30 (43) 0.25(36) 0.70 (54) 0.65 (50) 0.68 (52) 0.75 (58) 0.65 (50) 0.28 0.13 (18) 0.33 0.15(12) 0.38 (29) 0.23 (18) 0.20(15) Table 4 (Cont.) Concentration of Viable PBMN After Inctubation Patient S.Z Conicentration (ul/well) UPS (ag/well) 1 No. of Live PBMN per Well by Trypan Blue (x106) Z= ozimel After 24 rs No. Alta 48 hraNo. viable viable 0.60(46) 0.15 (12) 0.53 (41) 0.15 (12) *0

S.

.555 0 *0* S. S 0 *0

S.

0

*OS

*00 S *0 S 1 APProximately 1 1 106 cells plated/well in triplicate.

2 Actual nmbet of cells ounted/well1 (x106).

Example 6 cytokine -Content of Colipositiou ELISA assays for TNF-z, IL-la, IL-2, IL-4, IL-6, IL-8, 15 GM-CSF and IFN were conducted on the composition of the present invention. It was determined that the composition of the invention contained no measurable levels of cytokines (TNF, IL-i alpha, IL-1 beta, IL-4, IL-6, IL-8, GM-CSF and IFN gamma) (See Table

S*

~0 20

S

S. 0 S* 0 5 5**000

S

S

Table S ELISA DE TERMINAT1ON OF CYTOKINE IN COMPOSMTON

COMPOSITON

ThNF pg/ml 2W~ <5 iQU IL.-1/3 Pg/mi Detection Limit: 5 pg/ml Detection Limit: 4.3 pg/ml GM-CSF pg/ml 41 Table 5 (Coat.) ELISA DETERMINATION OF CYTOKINES IN COMPOSITON

COMPAOSMTON

11L-6 Pg/mI <7 Dowetion Limit: 7pg/ml IFNyp/nil Dethecdon Limit: 5 pg/mI IL-Icr pg/mi Detctioa Limit: 50 pg/mI IL-4 pg/mi 3 Detaction Limit: 3 pl/ml L-8 g/ D owetion Limit: 4.7 og/mI Exa I o 7 Physical, chemical, and biological properties, were determined for a number of batches of the composition of the invention prepared in accordance with the method as described in Example 1. In addition, the chemical :15 composition of the batches was determined and an amino acid analysis of the batches was conducted. The results are shown in Tables 6-8.

Table 6 CHEMICAL COMPOSITION Solids Batch No. mg/ml Amino Acids pg/wi B0201 B0202 B0203 B0208 B0209 B0211 B0106 B0706 B 1306 15 B2006 B2306, 15.3 15.7 15.0 7.8 8.5 5.6 32.2 .32.7 22.3 21.7 28.5 4.59 13.16 72.67 4.53 127 1.47 1.16 1.42 8.01 9.73 16.35 Sugars gc/mi 40.85 54.95 2.5.5 30 24 19.2 32.6 26.2 48 38.4 42 Lipids pgmi High M.W.

3kD PROT pg/mi ND<0.5 ND<0.S ND<0.5 ND<0.5 ND<O.5 ND<O.5 ND<0.5 ND<0.S ND<0.5 ND 0.5 ND<0.5

NA

NA

NA

ND ND ND ND .e e

C

C*

Se@@ S. C

S

C CC Ce 0

.C.

S.C..

C. S PHYSICAL, CHEMICAL AND BIOLOGICAL PROPERIES Batch No.

Conduct. Osmaor. Absorban.

pH mMHO mOsM O.D.20 nm .0 S25 -No C S o C

%A

see' B020 1 B0202 B0203 B0208 B0209 B0211 B0106 B0706 B 1306 B2006 B2306 7.37 7.35 7.3 7.00 7.31 7.35 7.57 7.57 8.02 8.56 8.01 16.9 17.3 17.7 16.1 11.2 34.9 34.3 11.6 35.6 33.9 35.1 0.98 0.777 0.67 0.453 0.594 0.287 0.341 0.387 1.147 1.024 1.054 UV, VIS Peaks 404 nm None 365 am None None None None None None None None Activity Units/Mi 10.5 21.0 8.1 6.7 17.2 23.0 17.0 21.0 19.0 Comments: 1. To batches No. B0106 and B0706 full isotonic PBS solids were added.

2. Batches B1306, B2006 B2306 were concentrated X2, no pH adjustment.

Table 7 HEMICAL COMIPOSITION Batch No.

B0213 R0201/-pH R0201 pH C0203 0-13/2109 B27/2806 B2913006 B 1511606 Solids mg/mi 31.6 52.5 55.8 36.1 12.1 17.5 28.7 26.8 Amino Acids ;&g/ml 21 1553 1530 113 149 28 26 41 Sugars ;igiml 61 216 280 42 36 37 60 45 Lipids pAgml

ND

ND

ND

ND

ND

ND

ND

75 High M.W.

>3.SkD PROT pFgmi

ND

ND

ND

ND

ND

ND

ND

ND

too.

0 0

C*

S..

C

Ge..

0 00* 0* C. C PHYSICAL, CHEMICAL AND BIOLOGICAL PROPERTIES Batch 20 r C C No.

B0213 R0201-pH R0201/+pH C0203 0-13/2109 B27/2806 B29/3006 B15/1606 Conduct Osmmar. Absobn. UV, VIS mMHO wOsM O.D.280 nm Peaks 29.5 628 0.48 none 44.5 877 1.59 271 nin 0.65 O.D.

50.0 1162 2.29 266 n 1.6 O.D.

34.8 657 0.96 none 17.0 316 0.83 none 7.60 7.90 7.73 7.71 7.67 7.84 Activity Units/il 14.5 51.5 61.5 14.5 12.4 14.0 14.0 22.0 28.8 35.0 0.49 0.55 1.04 none none none Table 8 AMINO-ACID COMPOSITION Batch B-0208 B-0209 B-0211 01106 07/06 1306 2006 2306 Aspwag~e 365 Serine Glycine 22 Histidine Arginme Threonine AIanine Proline 1092 0. Tyrosine 15 Valine 121 Metbionine Cysteine Isoleucine 2721 a 0O Leucine 0 00

S..

0 6Penylalanine Lysine 191 9 6 113 7 17 279 417 90 68 161 533 30 24 64 74 43 39 10 15 462 13 41 12 17 9 16 6 18 144 3731 938 148 949 817 205 367 107 86 232 221 45 119 5314 1335 142 1002 639 135 335 121 49 216 242 80 289 308 10371 2114 250 1423 1075 224 68 84 23 Total AA /glml

C

4.53 2.27 1.47 1.16 1.42 8.01 9.73 16.35 45 Example 8 Activation of Monocytes and Macrophages Investigations have sho-.-n that the conpositicn of the invention will activate normal monocyces to demons:race cytotoxicity towards the Chang hepatoma cell line which is used to measure monocyte toxicity and that the oncves and macrophages from cancer patients (cervical and ovarian cancer) have been stimulated by the comosition :o accack and destroy their own particular :umor cells.

More particularly, the monocytes tumoricidal function has been tested in the presence of the composicion of the invention and the basic procedure for these experiments is outlined below.

Venous blood is collected in heparinized 15 vacutainer tubes. The blood is diluted 3:1 in Hanks

**P

balanced salt solution (HBSS) layered onto lymphocyte separation medium and centrifuged to obtain a band of peripheral blood mononuclear cells (PBMN). After centrifugation, the mononuclear cell layer is recovered *0 from the interface washed twice in medium and monocytes are enumerated by latex ingestion. Monocytes are isolated by adherence in 96 well plates (for 2 hours at 370C followed by two cycles of washing). Adherent cells are estimated to be greater than 90% monocytes. Wells containing adherent 25 cells are incubated overnight in the presence of the composition 11:10 dilution) granulocyte-macrophage colony stimulating factor or PMA. Then adherent cells are washed *and incubated overnight with tumor cells. For studies using a standard cell line CR-labelled Chang hepatoma cells are used because this cell line is insensitive to natural killer cell cytotoxicity. These hepatoma targen tumor cells are added to adherent cell monolayers at an effector:target cell ratio of 20:1. This E:T ratio is used because it falls well into the plateau range on a curve prepared by varying the E:T ratio from 5:1 to 30:1.

After 24 hours H: )4 dORdp~P rC/piC 17 supernatants are collected and 51 Cr release is quantitated.

The percent specific cytotoxicity is calculated as: specific release x 100 where E CPM released from target cells in the presence of effector cells; S CPM released from target cells in the absence of effector cells; T CPM released from target cells after treatment with 2% sodium dodecyl sulfate.

Using this protocol, the composition was found to cause monocytes from healthy donors to exert cytotoxicity toward the Chang hepatoma cell line. Subsequently, whether monocytes and macrophages from a cancer patient could be stimulated by the composition to attack and destroy their own particular tumor was investigated. Using similar t protocols as described for the standard cell line (Chang 15 hepatoma cells), monocytes and/or peritoneal macrophages from cancer patients were isolated. (Peritoneal macrophages were isolated from peritoneal fluids collected at the time of laparoscopy). The composition was found to 3*r; activate peripheral monocytes and peritoneal macrophages from a patient with cervical cancer to produce cytotoxicity against the patient's own tumor cells. This effect was comparable to or better than that produced by interferon or lipopolysaccharide. Peritoneal macrophages from a patient with ovarian cancer were also found to be stimulated by the S* 25 composition to attack and destroy the ovarian tumor cells in culture.

Exale 9 Effect on TNT Studies were conducted to evaluate the effect of the composition of the invention on cytokine release from peripheral blood mononuclear cells (PBMN). ELISA assays for TNF-a, IL-la, IL-2, IL-4, IL-6, IL-8, GM-CSF and IFN were conducted.

The following methods were used in the studies described in the Example.

In the studies of TNT, whole blood was drawn from healthy subjects into heparinized Vacutainer tubes.

Peripheral blood mononuclear cells (PBMNs) were isolated by gradient centrifugation on Ficoll-Hypaque (Pharmacia). The PBMN were washed twice with phosphate buffered saline (PBS), counted and resuspended in RPMI 1640 culture medium (Gibco Labs) at a concentration of 10 6 cells/0.5 ml. These cells were cultured in 24 well, flat-bottomed tissue culture plates (Falcon, Becton, Dickinson). Of the PBMN suspension, 0.5 ml was added to each well containing Lipopolysaccharide (LPS) (from E.coli), 10 A1 fetal calf serum and the respective volumes of composition tested 300til). To neutralize the hyperosmolar effect of the composition, distilled water was added to the culture wells 0**0 15 at a volume equivalent to 10% of the volume of composition used. The total volume was then made up to 1 mi/well with S* RPMI. As control, PBS was used instead of composition.

The cells were cultured for 2, 6, 24, 48 and 72 hours at 37 C in a humidified 5% CO 2 incubator. At the end of each incubation period, the cells were harvested and cell free culture fluids were obtained by centrifugation at 9000 rpm for 10 minutes. The samples were then stored at -700C until ELISA for cytokines was carried out (within 2 weeks).

11 Protein estimation of the composition was done using 000 c* 25 the Pierce Micro BCA Protein determination technique (Smith et al., Anal. Biochem. 1985, 150:76-85). 10 Al of a sample of the composition was made up to 1 ml with distilled water. Five concentrations of Bovine Serum Albumin (0.150 ug/ml) was also made up to be used as standards. As a blank, 0.1N NaOH was used. To all these samples was added a mixture of BCA, 2% Bicinchonic Acid sodium salt) (Pierce), copper Sulfate 4% and Microreagent A .(NaC0 3 NaHCO 3 Na tartrate in 0.2N NaOH). The sample mixtures were incubated for 1 hr at 60 0 C, cooled and the resultant absorbency read at 562nm using a spectrophotometer. The amount of protein in the test sample was then compared to the plotted standard curve and the appropriate calculations made.- The protein concentration of the composition was found to be low and estimated to be 32 gg/ml.

cytokine synthesis in the supernatants were measured after stimulating human PBNN with the composition of the invention at volumes of 200 and 300 Al/well. The initial preparations of the composition show no stimulatory effect on cytokine production (Table If there was any effect there was the suggestion that cytokine production was below the constitutive level when PBM were incubated in medium alone.

.TABLE 9 Direct Effect of Canjxstioa on Cytoldnc Produdion after 24 hrs Amount of Cytoline Relased (pg/mi) Cytokine Medium Compstin P ILl 61.6± 12 59.6±:L7. 8 54.3±6.0 315 17 199± 184 218±16 188± 174 965±99

**TN'F

2 2319 151± 117 107± 120 1501± 284 11-6 928± 776 $53± 673 829± 543 2016± 41 *IL-8 126± 703 94± 503 77± 411 361 1653 OSSGM-CSF 13±4 13±7 15±11. 54 EFN-y 11±18 9±14 5±6 54±94 1-4 3.0 <3.0 <3.0 2 5 Mean of eight patient samples in duplicate 2 Mean of wse patent samples in duplicate 3n/jExperiments were performed to determine whether the composition of the invention would impa ir LPS-stimulated release. LPS was used as a positive stimulus, and the ability of the c omposition to impair LPS stimulated release of cytokines was compared for the different cytokines (Table 10). The composition clearly inhibits IL-i alpha, 49 IL-i beta and TNfF. The effects on the other cytokines IL- 6, IL-8, IYN-gamma and GM-CS? were not as marked. However, in no instance did the compositions tested augment the effect of LPS stimulated release of cytokines.

00 0@0 00 0 *0@ 0 Table Diffama betwem LPS ReLemnd Cytoine and LPS phw Cwmpoition Simulat of CylAkin Cytiae Amsyed 1 us LPS Coposiice 2 Meim Difference ug/mi IL-1a 129±135 77±2t 7 -52 IL-18 1314±723 919 460 -395 TNF 915 763 497±525 -418 EL-6 2320 1081 2320±:k1145 0 IL-8 (Dg/ml) 118±62 109 ±56 -9 :IFN-y 30±24 13 10 -17 GM-CSF 54±65 50±63 -4 'iPBMN from =i ptiaiW w=a woed in dupficata 2 COMpaition Nbac BMW0 0 000 The effect of different volumes of the compositions of the invention in inhibiting LPS stimulated release of TNF was examined. Figure 8 shows a dose-response curve of the composition inhibiting release of TNP by PBMN stimulated with LPS. Ten pl. of the composition inhibited about :and increased close to 30% inhibition at 100 141 of the composition. Another batch (B0201) of the composition :20 inhibited LPS-induced TNF production at 200, 100 and 10 M1 by 45, 21 and 12 percent, respectively.

Similarly, IL-i beta production was inhibited in a dose-dependent manner by the composition: 100, 25 and 10 Al of the composition inhibited by 16, 10 and 9 percent, respectively.

Different batches of the composition were examined f or 4A their effect on LPS-induced release of TNF. In summary, it was found that batches of the composition produced in the same way and from the same animal induced an identical ZO effect. However, changes in the method of preparation or the composition from different animal species had different effects. Batches B29/3006, B0213 (B bovine) and C0203 (goat) induced a strong release of TNP above that induced by LPS alone (Table 11).

Table 11 DoseRspom Effect on TNF Release of Strbmg S&nulatory Bathes Of Composion Difference in TNFa Ralese Betweena LPS Composition LPS Alone Batch B0213 0 0000

S

S

.OWS

*0*0 5 *0 0 B29/3006 Composition Volume (JIl) 10 100 200 10 50so 100 200 10 100 200 TNF (pg/mi) 193 j 161 858 819 2131 1742 121 102 422 78 834 811 2252 676 101 47 643 231 2650 1372 1851 980 CO203

S

0SS,

B

@6 6

S..

0S 1

S-

'V.

0 Table 12 shows the results with batches B15/1606 (concentrated preparation) and B27/2806, which were moderately stimulatory.

Table 12 Dose'Response Effect on TNF Release of Moderate Stim,111tory Batches Batch B2712806 B1511606 Difference in TNFa Release Between IYS Compociton LPS Alone Compositim TNF (pg/m) Volume (JAI) 10 -24±120 71±103 100 667±844 200 984 200 10 299±351 50 294±145 100 667±800 200 M24±446 *0 4 0

S@

0 *000 *0 S 0S 0 *0*0 S. .Table 13 shows minimally stimulatory.

that batch 013/2109 (sheep) was Table 13 Dose-Response Effect on TNF Release of Minimal Stimulatory Batch 0@ S S es 0@ *5 *5 S S S

S.

0 0S*

N

0000 S Diffmrwce in TNFx Release Between LPS LPS Aloe Batch 013/2109 Volume (al) so

TNF

(pg/mi) -9 ±73 179 ±162 178 ±373 Table 14 shows that batch R0201 (shark) was inhibitory at most concentrations for LPS-induced TNF production.

Table 14 Dose-Response Effect on TNF Release of Inhibitory Batch Difference in TNFa Raleas Betwee LPS LPS Alo1a

TNF

Batch Volume (pg/ml) R0201 50 145 256 200 -370 385 300 -400 185 Initially, the composition was shown to affect LPSinduced release of TNF from human PBMN. Thus, in the next

S.

series of experiments, the time effect of the composition on LPS-induced release of TNF was examined (Table 15). LPS stimulated release of TNF. By 2 hours the level had risen 15 to 697 pg/ml and peaked at 6 hrs at about 2006 pg/ml. At 24, 48 and 72 hrs the release of TNF progressively fell.

In fact, by 48 and 72 hrs, the TNF release was just above constitutive production levels. By contrast, Batch 0213 of the composition, which was strongly stimulatory for TNF release, showed no release of TNF above that produced by LPS alone at 2 and 6 hrs. Whereas LPS induced peak release of TNF at 6 hrs, the composition in combination with LPS induced peak release at 24 hrs at a time when the stimulatory effect of LPS had begun to fall. Unlike LPS 25 alone, composition LPS continued to stimulate TNF release at 48 and 72 hrs although the quantity of TNF released fell progressively (Table 15). Thus batch 0213 was stimulatory for TNF release. Batch B15/1606, which was only moderately Sstimulatory, inhibited LPS-induced release at 2 and 6 hrs.

At 24 hrs, B15/1606 in combination with LPS was mildly stimulatory for TNF-release. At 48 and 72 hrs, B15/1606 in combination with LPS, had a mild stimulatory effect on TNF release. Thus, batch B15/1606 had a biphasic effect; ear-y it inhibited LPS induced TNF release, and mainly at 24 hrs combination with LPS, had a mild stimulatory effect on TNF release. Thus, batch B15/1606 had a biphasic effect; early it inhibited LPS induced TNF release, and mainly at 24 hrs it caused a mild additive effect in conjunction with LPS in inducing TNF-release.

Table Time Effect on Different Batches on TNF Relase by LPS Mean Differace in TNFa (pg/ml) Releam Between LPS Composition LPS Alane by Three Different Batches Time a() TNF Released by LPS O nn/ RA21

C

C 0 0e S*

C.

S.

C

a** 0

S

15 24 48 72 697 94 2006 736 800 ±222 170 149 132 147 693 339 1949 422 2301 658 1419 447 945 367 363 189 62 ±42 1080 ±377 430 260 876 351 343 183 234 183 129 78 184 107 153 68

S

C

S

S

Batch R0201, which was inhibitory for LPS-induced release of TNF, was markedly inhibitory for LPS induced TNF 20 release at 2, 6 and 24 hrs. At 48 and 72 hrs, LPS induced minimal TNF release and batch R0201 had minimal positive or negative- affects at these times.

The direct stimulatory effect of Batch B0203 was tested at different volumes and then at different times.

25 Batch B0203 stimulated maximum TNF-release at about 100 Al (Table 16). The maximum effect was observed at 24 hrs for a volume of 200 Ll (Table 17). Thus, the composition was able to stimulate TNF-release on its own, that is in the absence of LPS and the curves for TNFrelease were similar to when the composition and LPS were combined. It appears that the comPosition by itself was less stimulatory for TN? release than the additive effect it had when combined with LPS.

Table 16 Dosi-Rasoaue of Batch B02W on Stimuitin Relew of TN? Volume (PS/Mi)

(PI)

Mam Amoiunt of TN? Relemed at 24 hr-s Nto(PBS 50 p) 123 207 223 327 10:5 ±54 189 94 0

SO

Oe

S

*5 0e *0 0 *5* 0**S 15 Table 17 Effect of Batch B0203 on Release of TN? over Tine Time 0=n) TN? Rclessedl (Pg/Mi) 0* 0 0 50 *500

S

0@ 0 S.

S.

*0S*S*

S

47 154 106 73 20 13 _t 8 6 2 1 Mediuan 425 In summary, the experimental results indicate that' some batches of the composition are able to inhibit TNF release when human PBMN are stimulated with LPS. Other batches have biphasic effects suggesting that they partially inhibit LPS-induced TNF release and have, as a late effect, the ability to induce a mild release of TNF.

A third preparation had no inhibitory effect on LPS-induced release of TNF; but at a different time point than LPS, the preparation was able to stimulate human PBMN to release TNF. In conclusion, the composition of the invention can modulate TNF production, an important mediator of antitumor responses. A summary of the data is shown in Figure 9 and in Table 18.

Ta 18 TNF Bimuay Reuts RP-HPLC Normal or Batch No. Peaks I Min Source Concenaed Buffer TNF Release B0213 27,32 Bovine Normal Yes t t C0203 27,32 Ciprine Normal Yes f t 013/2109 27.32. 21:50, Ovine Concentrated Yes 4 I t R0201 21-25. 28, 29, Shark Normal Yes 4 1 29.5,27,32 20 B29/3006 27,32 Bovine Normal Yes t t 0 B27/2806 427, 432 Bovine Normal Yes t B 15/1606 27, 32, 22, 28 Bovine Concentrated Yes t :Yes 4 ao ~ElaNple This Example demonstrates, in summary, the following: The composition has TNF-a releasing activity and the TNF-a releasing activity is not related to any contamination with endotoxin. Priming of macrophages enhances the ability of the composition to stimulate release of TNF-a. The hyperosmolarity of the composition is not responsible for TNF-a releasing activity. The TNF-a releasing activity of the composition can be separated, in part, from other

S

0 Oe 0* 0< constituents. The TNF-a releasing activity of the composition does not bind or binds poorly to c 18

RP-HPLC.

Most of compositions activity elutes early from RP-HPLC.

Less than 20% of the activity of the composition is recoverable from the fractions that are retained on the RP-HPLC and elute later. The TNF-a releasing activity is precipitated by 80% acetonitrile, a high content of organic buffer. The precipitated material when reconstituted in aqueous buffer and analyzed on RP-HPLC shows great similarity to the excluded peak on RP-HPLC of the composition. It is possible to separate and concentrate the active component of the composition in a fraction that constitutes about 30% of the original material.

A. Polyayzin and TNT-alpha Release 15 To eliminate any possibility of an endotoxin effect of the composition experiments were performed with Polymyxin added to the reactants. Polymyxin inhibits the action of endotoxin on leukocytes. Table 19 shows that polymyxin completely inhibits the LPS-induced release of TNF-a. In 20 the absence of polymyxin, LPS induces 517 pg/ml of TNF-a, whereas in the presence of Polymyxin 11 pg/ml of TNF-a is released. The composition, on the other hand, releases 1591 pg/ml of TNF-a in the presence of Polymyxin. In the absence of Polymyxin, LPS and the composition show more than just an additive effect of the stimulators, suggesting that the composition acts with greater intensity when macrophages are primed.

Table 19 Effect of Polymyxin on TNF Relase by LPS Comiposition TN'F Released (pg/mi) Total -LPS Sample Tested Additive PolymyXin None Polymyxin None Composition (#B0213) 11 7 517.:+ 118 1591 413 5256 2585 1581 4738 Notes: 1. Total TNF Released is corrected for TNF release by 1640 Medium.

Polymyxin concentration: 50,000 unitslinl.

3. Cmpostio volme:200 pl.

With polymyxifl, 8 patients twted. With no additive, 3 patients tested.

5. LPS concentration: 50ng/10 A.

B. TNl7-Releasizig Activity of Reversed-PbAsed High Pressure Licruid Chrostatoarsay (RP-HPLCI Practions Figure 10 shows the Cis RP-HPLC profile of the composition, Batch 0213 and the 5 separate fractions that were tested for TNF-releasing activity.

Initially, the effect of different fractions were examined in the presence of Polymyxin. Table 20 shows that the early eluting fraction (2:05 to 21:20 minutes) from RP- HPLC had most of the detectable TNF-releasing activity.

*4 25 However, this activity was only about 50% of the starting composition. The right hand column of Table 20 shows the* osmolarity of the samples. Batch B0213 was 369 and high.

Fractions from 21 minutes and later had normal osmolarity, whereas the first fraction which was active was even more hyperosmolar than the starting material, indicating that much of the salt in the compo sition also eluted early.

Therefore, whether the hyperosmolarity of the samples was inhibiting or enhancing TNF-a release was investigated as set out below.

Table Separaton and Tstig of Differet HBpLC Fractions of Ccmnposition in &he Presac of polyinyxin Sample Tested 1640 RPMI Medium LPS (50 ng1gla) Composition #=013 TNF Released (pg/mi) Total -LPS 0 40±25 0 1=22448 1192 OS0sx11xity (mOsm) 287 369

S

a a 0* b 455 5* 0a

S.

a sea 15

S

5- S Fractions (minutes) 2:05-21:20 21:20-25:32 25:32-31:55 31:55-34:58 34:58-46:55 554 ±394 7±7 0 4±4 32 ±28 Note: a.

S

C.

40 Sb a.

55 1 S. C S S.

Total TNF -Released is coffrced for TNF release by RLPMI Medium.

Fractions are reconstituted in PBS.

Volume of composition and fractions: 200 A'l.

Polymyxin concerionac: 50,000 units/nil.

Number of p~atients tested: Osmoimity not massured. for LPS.

a

S

i

S

S

4 .5eSf 5

'I'

Table 21 shows additional testing on two patients with composition fractions 1 (2:05-21:20) and 2 (21:20-25.32).- Once more, most of the activity was recovered in fraction 1, bu t there was some activity in fraction 2. However, fraction 1 and 2 had only about 50% of the starting activity of the composition.

Table 21 Repeat Evaluation of Composition NPLC Fractions 2:05-21:20 and 21t20- 25:32 minutes for release of =r in the Presence of Polynyxin.

TNT Released (pg/m1) Sample Tested 1640 RPMI Medium LPS (50 ng/10 4l) composition #B0213 Fractions (minutes): Total -LPS 31 31 2013 1 726 526 1 126 132 108 1983 2:05-21:20 21:20-25:32 ¢,ee 0* (0 *afJ 15 Note: 1.

2.

3.

4.

5.

Total TNF Released is corrected for release by RPMI Medium.

Fractions are reconstituted in PBS.

Volume of composition and fractions: 200 IA.

Polymyxin concentration: 50.000 units/ml.

Number of patients tested: 2 To determine whether there was any nonspecific activity in the fractions, a blank run, was made and the same fractions were collected, concentrated and tested.

The blank fractions produced virtually no TNF release.

This result indicated that fractions-from RP-HPLC could be used to test for TNF-a releasing activity without concern that the column or buffers contributed to TNF-a releasing activity.

Next the fractions of the composition were tested in the absence of Polymyxin in order to have the priming effect of LPS. Table 22 shows that Batch B0213 induced a marked release of TNF-a. There were 5 fractions of the composition from the RP-HPLC, with the elution times as indicated. Once again, fraction 1 had the most TNF-a releasing activity. However, with the priming effect of LPS, fractions 2 through 4 had some TNF-a releasing activity. Fraction 2 (21:20 25:32) had about 25% of the activity of fraction 1, and double the activity of the later fractions.

Table 2-2 Separaton and Testing of HPLC Fria*iozu of Compositon in the Abm=~ of Polywyxin.

TNF Released (pglml) O1mIarity Sample Tested Total -LPS (mosm) 1640 RPMI Medium 0 -299 U'S (50og110 Al) 219 0 305 Comnposition#110213 1575 ±470 1356 376 Fractions (minutes): 2:05-21:20 656 ±206 436 321 .:21:20-25:32 345 8 126 309 25:32-31:55 287 ±70 68 305 .:31:55-34:58 262 ±50 43 304, 34:5"4:55 237 ±59 18 376 15 1. Total TNF Released is corrected for releas by RPMI Medium.

2. Fraction Reconstitution: 2:05-21:20 in water, 21:20 to 48:55 in PBS.

3. Volume of compositic and fractions: 200 id.

4. Number of patients tested for TNF relem OsmoLarities are averages for 2 of 5 patiets; standard effors very small, therefore, not reported.

Whereas Table 22 shows the results of using 200 A.l volumes, Table 23 shows the results of testing an additional three patients with 100 Al& of the composition and 100 Al of its RP-HPLC fractions in the absence of .:Polymyxin. Although the release by the composition is 25 lower than with 200 gl of the composition (Table 22), the results are similar. Fraction 1 contains most of the activity with some. activity in the later fractions 2 and 3.

Sepatio an TesiM f EPLCFractions of Composition in the Absence of Polymyxin.

TN'F Released (pg/mi) Osmolaicy Sample Tested Total -LPS (MOSM) 1640 RPMI Medium 0 303 LPS (S0ng/1O pl) 195 ±72 0 302 Composition #BG213 692 ±266 497 347 Fractions (minutes): 2:05-21:20 575 2 379 310 21:20-25:32 226 ±65 31 337 25:32-31:55 210 71 14 305 31:55-34:58 192 ±40 0 313 3.4:58-46:55 182 ±73 0 344 1. Total TNF Rekased is corrected for release by RPMI Medium.

2. Fraction Reconstituion: 2:05-21:20 in water. 21:20 to 48:55 in PBS.

3. Volume of composition and fractons: 200 ;d.

4. Number of patients tested for TNF release: 3.

Osmolarities amn averages of patients tested; stadard errors very small. therefore, not reported.

15 C. Uf foot of Oamolarity of TWl Release by the Coap~gsition 00 15 The composition of the invention is hyperosmolar. The effect of the hyperosmolarity of the composition on TNF-a releasing activity was studied. It was f ound that the composition, when adjusted for osmolality even to the point of being hypoosmolar, continued to release TNF-a.

00 20 D. Physicochemical Separation of the Composition by Precipitation with High Content of organic Solvent Since most of the TNF-releasing activity of the composition did not bind to the RP-HPLC as evidenced by its quick elution, it was decided to use a column that acts on the inverse principle of reversed-phase chromatography separation where the sample is in a high content of organic Nsolvent and permits hydrophilic interaction. This separation technique is used for *small polar substances.

£However, when the composition was brought to acetonitrile a precipitate formed. Thus, some of the contents of the composition in a high organic solvent buffer precipitated. The precipitate and the soluble fraction were separated. Both the precipitate and soluble fraction were taken to dryness by lyophilization. The precipitate and soluble fraction were reconstituted in aqueous solutions and both analyzed by RP-HPLC and tested for TNF-a releasing activity. Table 24 shows that most of the TNF-a releasing activity was contained in the precipitated material.

Table 24 TNF-Releasing Activity of Fractions of Composition Prepared by Precipitation in Acetoatitrle.

TNF Reased (pg/ml) Sample Tested IX 199 Medium

LPS

0 0 s Composition #B0213 oo0o 15Supenatant e 0e* o Precipitate 0 0 0 Volume/ Concentration 50 ng/10 l 100 Al 200 A] 100 Ad 200 HI 100 A1 2 00 pl Total 161 50 471 ±304 505 210 192 63 221 69 626 ±212 1299± 565 0 310 344 31 60 465 1138 Osmolarity (mOSm) 306 301 307 318 309 310 307 346 Note: 'e 20 oo@S e 0

C

25 *4b *0**00 0<3 1. Total TNF Released is corrected for IX 199 Medium.

2. Reconstitution: supernamtant in PBS, precipitate in double distilled water.

3. Precipitate is in (hypotonic) 70% IX 199 Medium.

4. Number of patients tested: 5. Osmolarities are averages of 4 of 5 patients tested; standard errors very small, therefore, not reported.

RP-HPLC analysis of both the precipitated (Figure 11) and soluble fractions (Figure 12) of the composition shows that the precipitate is principally the material contained in fraction 1 of the RP-HPLC of the composition (Figure 10), and the soluble material contains the other fractions of the RP-HPLC of the composition (Figure 10). Thus in two different ways, it was shown that the composition's activity is contained in the fraction that is minimally retained by RP-HPLC. In fact, the precipitate had equal activity to unprecipitated composition when tested at 100 and 200 4l.

Only the 200 4I precipitate had above normal osmolarity. Consequently, the precipitate and soluble fractions (supernatant) of the composition were separated by RP-HPLC (Figures 11 and 12) and divided into two fractions (see profiles of RP-HPLC). Fraction 1 (2:00- 21:10 min) was equivalent to the same fraction 1 of the composition separated by RP-HPLC and fraction 2 was equivalent to fractions 2 through 5 of the composition separated by RP-HPLC. The isolates were then tested for their TNF-a releasing activity. Table 26 shows that for two patients neither the precipitate or supernatant after RP-HPLC separation had any TNF-releasing activity.

However, the results for the initial two patients (Table suggested the possibility that the precipitate may not have been tested at the ideal volume. Consequently, the fractions were retested on two additional patients and at one lower concentration. Table 26 shows that at 50 and 100 20 41, fraction 1 of the precipitate had the most activity.

At 50 4i it released twice as much TNF-a as did 50 ng LPS.

However, minor releasing activity was also found in RP-HPLC fraction 2 of the precipitate as well as minor activity was found on fractions 1 and 2 of RP-HPLC of the supernatant.

0veS *o o 0 *0

S

S SO S Se

S

N

eSSeOS

C

Table TNF-Rdcailn Activity Of HPLC SePvraed Fraclions of 80% Acetonitrile Precipitated Comnpiodn.

TNF Released (pg/mi) Sample Tesed 199 Medium Used Volume/Conc.

Total Medium

LPS

(#M013) Ix IX-70%*IX 50 ng/10 yJ 194±: 93 -LS Osmolf'isy LS (3105m) 294 0 294 661 281 732 29e' 0 269 C. C C C

C.

*0S@

C

e.g.

C. C C C

C.

C.

C

C..

C C C. Supernutant 2:15-21:28 21:48-46:13 Precipitate: 1:60-21: 10 21:10-46:20 70% 1X 70% tX too At 2 0 0 Al 10ooAl 200 At 100 Al 2 0 0 IL 2 0 0 At 2 0 0 At 855 ±88 926 ±163 92 ±75 25±25 61 56 2±2 0 62 61 0 0 297 0 305 0 354 Ix 70% 1X

IX

Note:

C*

C C

C.

*0*S C C. S C. C C SC CSS

C

e Total TNF releaed is corrected for TNF release by 199 Medium IX.

2. Reconstitution: first fractions in double distilled water, second fr-actions in PBS.

3. Number of patzits tesWe: 2.

4. Osmolarities are averages of patients tested: standard errors very small, therefore not reported.

5. Averages of valus inIX and 70% IX 199Mediun.

Table 26 TNF Reie with furthe Titratoa of Preipitate and Supernatant from 80% Aceonhile Frationd.

TNF Released (pg/ml) Osmority kample Teted Volume/Conc. Total -LPS 1X199 Medium

PS

#B0213 50 ng/l10 A 100 l6 0 136 274 317 320 3*17 10

S

C*

0 0Se

S.

0

S

Supernnt 2:15-21:28 21:48-46:13 Precipitate 2:00-21:10 21:10-46:20 Soo 1.00 A Sop' 100 At too At 2001,1 204± 148 198 1299

S

S

S

S

Oe S

S

OS

Note: 1. Total TNF released is corrected for TNT releae by 199 Medium IX.

2. Reconstitution: first fractions in double distilled water, second fractions in PBS.

3. Numer of patients tested: 2.

4. Osmolarities are averages of patients tested standard errors very small, therefore, not reported.

5. All samples in IX 199 Medium.

E, TNP-a Releasing-bV Different )fedjA It was observed that the seitch from RPMI 1640 to Medium 199 resulted in a lower TNF-a release was evaluated in Medium 199 and RPMI 1640 (Table 27). The results show that LPD from 10 to 200 rig is much more effective in releasing TNF-a in RPMI 1640 Medium than in Medium 199.

Presumably the composition also gives greater release in RPMI 1640 Medium. Thus, cultural conditions can influence the degree of TNF-a release.

S

5 0 *5

S

0 0550 5* S 0 *0 0* *5* 15

SO

S

Table 27 Evalution of TNF Release in Difffttnt Media IX 199 Medium 1640 RPMI Medium TNF Releasd Osmolarity TNF Released Osmolarity (pg/mi) (MOSM) (pg/wi) (mOsm) 23 296 47 Sample Tested Medium

LPS:

10 ngII0 Al 50 ng/l0 yd 100 ng/IO 0 200 ngI10 Ad 1000 rig/b 11 .5

S

S.

SS *4 *5 0 *5 S S *5 SO S SO

S

Note: 1. Number of patients tested: 1.

F. Osmolarity of the Coumosition Table 28 shows the osmolarities of different batches of the composition. B0213 is moderately high at 675 mosm.

B0222 shown to have TNF-releasing activity even better than B0213 is less hyperosmolar, 581 mosm. The fractions B0226, BC11-06 and BC11-09 range from 540 to 603 mOsm.

Table 28 OSMOLARITIES OF WHOLE BATCHES Batf-h #P 8OIAity (MORn) B0222 pr"-H 411 B02=2 pH adjusted 581 B0216 pH adjusted87 B0219 pH adjusted 886 Noccoocaitaed:.

30221 pre-pH 652 30221 pH adjusted 533 30213 pH adjusted 675 B0225 pH adjusted 590 B 0226 pH adjusted 5.40 is E5 1 C 11-06 RC 11I-09 pH adjusted 603 A. Tuor Necrosis Factor (TN7) Rleauing Activity of a Compouition of the Invention, S 20 1. Acetonitrile Precipitate anid Suvernatant of the composition As, shown in the prior Example, 80% acetonitrile precipitated material form a composition of the invention.

The preci pitated material and the unprecipitated (hence forth called Supernatant) composition were tested further to determine where the TNF-releasing activity resided.

Table 29 shows that the TNF-releasing component of the composition is precipitated by 804 acetonitrile, an organic solvent. Whereas the whole composition at 0.04 al released about 15 pg/ml of TNF, 0.05 ml of precipitated Batches of the composition released 58 (B0222), 0 (B0221), and 17 (B0213) pg/ml, suggesting recovery of the TNF-releasing 69 component by 80% acetonitrile precipitation. The precipitated composition was reconstituted in the same volume of liquid from which it has been precipitated.

Thus, 0.1 ml of precipitate comes from 0.1 ml of whole composition and ecuals 0.1 ml whole composition.

Table 29 TNF Releasing Activity of Precipitates of Composition of the Invention Prepared in 80% Acetonitrile TNF Released (pg/ml) Total -LPS 0@ 0 3 0@00 S. S Sample Tested

LPS

Batch: B0222 Whole Precipitate Batch: B0221 Whole Precipitate Batch: B0213 Whole Precipitate 199 Medium Used

IX

80% IX 70% 1X 70% 1X 70% IX 70% 1X 90% IX 70% 1X 70% IX 1X 1X 90% IX 70% IX 70% 1X 70% 1X 70% IX Quantity in Wells 50 ng 40 iul 200 ul 100 ul 50 il 25 u 40 l 200 ai 100 al 50 l 25 ,ul 40 gl 200 ul 100 ,l 50 ji 25 gli 322 115 337 107 1091 137 620 186 380 132 312 137 282 75 981 205 526 169 308 104 318 185 383 72 1143 172 687 186 339 133 300 144 0 15 769 298 58 0 0 660 205 0 0 61 821 365 17 0 Osmolaricy (mOsm) 308 312 351.

317 297 294 306 348 314 298 292 312 366 326 305 298 0S 0 0* 0000

S.

00 Note: 00 0 OSr @00 0 0.003 1. Number of patients tested: 2. Total TNF released is corrected for TNF release by 1X 199 Medium 3. Osmolarities not corrected for 1X 199 Medium (311 mOsm) 4. Average of osmolarities given; standard errors not reported as values are very low.

Precipitate reconstituted in double distilled water.

6. LPS volume added to wells was 10 ul.

7. Wells contained a total volume of 1000 ptl.

8. Sample volumes are equivalent.

H:\lnn\ y\ 5 744-94.cto c L7/L..94 It is of interest to note that in earlier studies generally between 0.1 and 0.2 ml of the composition was used to stimulate TNF-release. The precipitates of B0222, B0221 and B0213, reconstituted to 0.1 and 0.2 ml released between 205 and 821 pg/ml of TNF. At 0.1 ml of precipitate, batches B0222, B0221 and B0213 released between 205 and 365 pg/ml of TNF, a very similar quantity of TNF, indicating the consistency of the TNF-releasing activity of the three different batches.

On a separate group of donor leukocytes, the supernatants remaining after 80% acetonitrile precipitation of the composition were tested. Whereas 0.04 ml of whole batches of the composition (B0222 and B0213) released 175 and 233 pg/ml of TNF, 0.05 ml of the supernatant released 15 42 and 41 pg/ml about 33% of the activity of the whole composition. Blanks prepared in the same fashion with acetonitrile had no TNF-releasing activity. Thus, TNFreleasing activity in the precipitate was not the result of some residual substances in the buffers used to induce the precipitate.

Whereas in the aforedescribed studies the precipitate and supernatant fractions were tested on leukocytes from different donors, another study was conducted in which the two fractions were tested on leukocytes from the same S 25 donors. For the two tested batches, the whole composition at 0.04 ml released between 0 and 41 pg/ml of TNF. In comparison, the precipitate of the same batches of *composition at 0.05 ml released 141 and 749 pg/ml of TNF, whereas 0.05 ml of the supernatant fraction released between 6 and 57 pg/ml of TNF. Thus the precipitate contained much of the TNF releasing activity. The supernatant fraction still had some TNF-releasing activity but much less than the precipitate and no more than the whole composition.

2. Reversed Phase-HPLC Separated Fractions of the Composition of the Invention As the precipitate of the composition was shown to contain much of the TNF-releasing activity, the profile of the precipitate was examined by C 18 RP-HPLC. Figures 18, 19 and 20 show the RP-HPLC profiles of whole composition, the precipitate and the supernatant, respectively. The precipitate's profile shows principally the early eluting peak of whole composition (Figure 19), whereas the supernatant's profile (Fig. 20) is similar to the whole composition except that the early peak is less intense.

In the next series of experiments, the activity of the precipitate and supernatants were evaluated after their separation by C 18 RP-HPLC. The precipitate and supernatant 15 were collected as 2 pools from RP-HPLC: 2 to 21 min and 21 to 46 min. Earlier studies of fractions of whole composition separated by RP-HPLC indicated that TNF- S-releasing activity eluted principally in the 2 to 20 min fraction. Table 30 shows the results of testing whole 20 composition and RP-HPLC fractions of the precipitate and supernatant. In this particular experiment, only 10 M1 of the whole composition was used and caused no TNF-release.

The precipitate pool from 2 to 21 min released TNF, whereas the 21 to 46 min pool did not (Table 30). The supernatant pool from 2 to 21 min also released TNF activity, whereas the 21 to 46 min pool released minimal quantities of TNF (Table 30). Thus for both the precipitate and supernatant, "the TNF-releasing activity resides principally in the early eluting fraction from C 18

RP-HPLC.

72 Table TNF Releasing Activity of HPLC Fractions of Precipitate and Supernatants of Composition of the Invention Prepared in Acetonitrile (2x wash) TNF Released (c ml) Sample 199 Medium Quantity Total -LPS Osmolaric Tested Used in Well (mOsm) LPS 1X 50 ng 99 43 0 07 Batch: B0222 Whole 30% 1X 10 ul 83 33 0 29 HPLC Precioitate: 2:00-21:36 70% 1X 100 ul 153 43 54 305 lX 50 ul 50 14 0 289 21:36-46:28 IX 100 ul 97 29 0 306 1X 50 il 103 43 3 306 HPLC Supernatant: 2:00-21:35 1X 100 gi 192 55 92 330 x 50 .l 183 72 83 320 21:35-46:30 IX 100 ul 65 21 0 311 1 X 50 i1 142 40 43 309 Batch: B0213 Whole 90% 1I 10 g1 93 36 0 296 HPLC Precipitate 2:00-21:12 70% 1X 100 p. 165 52 66 310 70% 1X 50 ul 48 15 0 287 S* 21:12-46:12 iX 100 .l 88 31 0 307 1X 50 l 101 38 1 307 HPLC Supernatant 2:00-21:15 IX 100 .l 193 76 93 317 I 1X 50 p. 126 43 26 306 21:15-46:20 IX 100 p1 59 24 0 309 1X 50 pl 125 32 26 312 e Note: 1. Number of patients tested: 2. Total TNF released is corrected for TNF release by IX *199 Medium.

3. Osmolarities not corrected for 1X 199 Medium (309 mOsm); standard errors are very low and, therefore, not reported.

4. Reconstitution: precipitates in Type 1 water, supernatants in PBS buffer 5. LPS volume added to wells was 10 Il.

6. Wells contained a total volume of 1000 il.

H: \<inn.i\K.dCpP.et/ypes\64.9-94 .doc 17 L2/ 98 73 The results suggest that the TNF-releasiig substance in the precipitate and superniatant are likely closely related molecules, if not identical, with the only difference, if any, perhaps being the degree of solubility in 80% acetonitrile.

In another experiment the TNF-releasing activity of the two RP-HPLC pools of the precipitate were examined for three batches of the composition. Table 31 shows again that for three different batches (B0222, B0221, and B02 13), the TNF-releasing activity is principally in the pool 2 to 21 min. Thus different batches are consistent.

74 Table 31 TNF Releasing Activity of HPLC Precipitate Fractions of Composition of the Invention Prepared in 80% Acetonitrile Samcl: 199 Medium Quar:i -a Osmclar Tes Used In il (mOs=) LPS 1x 50 nc 0 299 LPS k1) 1X 50 3 O6 Batch: B0222 Whole lX 40 ul 73 27 5 329 ul 113 1A 38 303 HPLC Precioitate: 2:00-21:25 70% 1X 200 U 20 0 354 100 ul iS 74 307 21:25-46:20 1X 200 u 57 39 0 313 100 ul 73 1 0 310 Batch: B0221 Whole 90% 1ix 40 p1 526 490 315 10 p1 52 31 0 290 HPLC ?recipitate: 2:00-21:45 70% 1X 200 p1 20 7 0 365 min a a 100 pl 170 6 90 329 21:45-46:50 1X 200 u 45 5 0 336 o* min 100 p1 73 i 15 0 311 Batch: B0213 Whole 90% 1X 40 p1 620 123 .484 315 ul 30 17 0 290 HPLC Precipitate 2:00-21:30 70% 1X 200 g1 6 4 0 393 100 p1 153 45 72 314 21:30-46:30 1X 200 .ul 182 96 47 312 min 100 U1 78 20 0 310 Note: 1. 7or 100 10 ul samples; 3 pazients tested for these samples 2. For 200 40 p1 samples: 4 patients tesced for these samples \i 3. Total TNF released is correczed for TNF release by 199 Medium 1X.

4. 199 Medium 1X@100 10 pl 306 mosm, 200 S 40 ul 305 mrr.0sm.

S 10 5. Osmolarities are averages and noc corrected for 199 Medium 1X; scandard errors are not reoor:ed as values are very low.

6. IPS volume added to wells was 10 ul.

7. Wells contained a total volune of 1000 ul.

8. Sample volumes are equivalen-.

9. Reconstitution: first fractifcns in double discilled water, second in PBS buffer.

H: :annal ep\Retypes\l?5419-94.d-,oc 17:12/91 In a further experiment, the effect of washing the precipitate with 80% acetonitrile was examined. The point of the experiment was to prove that the TNF releasing activity was not being simply trapped. Whole composition, 0.04 ml, released 325 pg/ml of TNF. The precipitate pool from 2 to 24 min at 0.1 ml released 324 pg/ml of TNF and at 0.05 ml, 3 pg/ml. The pool from 24 mrin to 46 min released no TNF. Likewise the supernatant pool from 2 to 24 min released TNF at 0.1 and 0.05 ml, whereas the pool from 24 to 46 min had some, but considerably less, TNF-releasing activity on RP-HPLC.

To be certain that the handling of RP-HPLC isolates of the composition were not responsible for the presence of TNF-releasing activity, samples were prepared in the same fashion but without the composition. RP-HPLC eQg.

profiles of PBS and H20 blanks, their precipitates and supernatants were essentially free of any peaks.

*,*Using mononuclear cells from identical donors, the *6 ,samples were tested on leukocytes from the same donors.

Whole composition and the precipitate eluting from 2 to 24 min released TNF, whereas PBS, H 2 0 or their precipitates separated on RP-HPLC had no TNF releasing activity in the pool from 2 to 23 min. Thus, the precipitate eluting from 2 to 24 min causes specific TNF-release.

24 The precipitate pool of the composition eluting from *24 to 46 min released no TNF. The controls of water and PBS showed release of 114 and 40 pg/ml, respectively.

Thus there was no specific release of TNF from the precipitate pool 24 to 46 min.

*0,e00 The supernatant fraction pool 2 to 24 min and 24 to 46 min released 82 and 68 pg/ml of TNF, respectively.

The water and PBS blank pools from RP-HPLC released some TNF activity. The water pool 2 to 25 min and 25 to 46 released 149 and 216 pg/ml of TNF e e: a..7c z~?8S pCC13 released 0 and !26 restec-z v Thus, nzearcioitated and suer-':,:-r-'cn had -IVT-releasirng activ4iy. .P-H-PC seaat:cn c- activitv showed that both eluted ear-- Rp-.:LC, suggesting-: that the actiJVe COm=CnentS are oh vs cally. Very siilJ-ar ji1 not identical.

Tal 2 ::iesasi-zy result of fr-e testing, for SO13 acatonit.-ile precipitates and io su-ernatants aftrRP-HLC separation, minus theacVt in similarly prepared blank samples.

Table 32 .Releasing Activity of Comosition of;: tne Invention Less Release byv Reconstitution Solution-s 199 mo4ix Quantity ACZ2Wa Rel-se (plimA) 0s~clnrry.

5 Salo Tested. Used in Weil TNFA GM-CSF U..-13 (C06) Batch: B0222 Wbole 80% Ix 40,gl 178 136 142 -110 1-PLC Prcipit= 2:20-24:10 70i X 100 Al 75 13 is 314 020 24:10-46:20 70% IX too i 0 16 0 292 0 0 HLC Supecratant (min): 2:00-23:55 Ix 100 ud 82 86 0 3 -,6 Z 3:55-46:20 Ix I00 A]l 0 45 29 316 o: Note: 1. Damz da'ived from SuImtable Table 2A.4.

Reconstitution: -1st proi=zt ftr~o in Type I ww~er, aLl otbe &-actons in ?BS.

3. Whole amounts sr-ur~tne and tbfcr-- not corITc.

Whole composition releases TNF, GM~-CSF and -Bf vi~or from mononuclear cells at 24 The acetcnitrile prec izitate contains the same releasin activity and most elutes in the early fraction frc= P=h?-LC. The suzernatant fraction retains releasin.g activity and most elutes in the early fraction from RP- HPLC. The supernatant fraction retains releasing activity, but it also elutes in the early eluting RP-HPLC fraction for TNF and GM-CSF. The results suggest that likely the same component releases the three cytokines.

For GM-CSF and IL-16, the fact that some releasing activity elutes in the late fraction from RP-HPLC suggest that there may be another substance in the composition that can act on monocytes to release GM-CSF and IL-1$.

Physiaooheical Analysis SD8 Gel ElectroDhoresis Having identified that TNF, IL-lI and GM-CSF releasing activity can be precipitated, in part, by acetonitrile and that much of the releasing activity oo..

15 elutes early from C 1 8 RP-HPLC, the physicochemical properties of the precipitate traction have been studied and compared to the whole composition and supernatant fraction of the composition.

S

Figure 21 shovs an SDS gel electrophoresis of whole composition and precipitates and supernatants of the composition. In all three instances, the composition runs near the SDS front, indicating a low molecular weight. The smallest standard used was 14,400 daltons.

o

S

Xolecular sieve HRPLC S 25 The molecular size of the composition was also examined by determining its time of elution from a Smolecular sieve HPLC column. The elution times of whole composition, precipitate and supernatant compared to standards. All three eluted later than insulin, which 30 eluted at 24.5 min. Once again, physicochemical analysis j4 indicates a mol. wt. less than 2,400 daltons.

Hydrophilic (PolvhTdrollethvl) gpLC The TNF-releasing component elutes early. Thus a column with the opposite effect was chosen, a hydrophilic column in the presence of organic solvents. The ideal eluting conditions for the polyhydroxyethyl column is acetonitrile. However, as indicated in the prior Example, some of the substances in the preparation precipitated at this concentration. Consequently, the composition was analyzed at a low concentration of acetonitrile where the column functions mostly as a molecular sieve column. Figures 22 and 23 show the profile of whole supernatant and precipitate. The front sheet summarizes the elution time for the different peaks. The elution times indicate the active componet of 15 the composition has a low molecular weight.

Amino caid Analysis and Bequenainq of the Ime~initated Caemunna 0

S*

0 S 0 *0 0 0 0@ 5

S..

S*

S 00 0 *0 S S

S.

000@ S 0* S. S

S

0* 0@O 0.@

S

N:

of the Preginitated Caannnam* Two samples were submitted for protein analysis by amino acid compositional analysis before and after acid hydrolysis: the acetonitrile precipitate and 2-20 min RP- HPLC eluted pool of the precipitate. The two samples were very similar by comparison of amino acid content before and post acid hydrolysis. There are, however, significant differences between the amino acid 25 composition (post acid hydrolysis), and the free amino acid content which suggests that peptide bonds were hydrolyzed. The composition of the samples were peculiar in that they were very rich in glycine plus glutamate/glutamine.

From the foregoing, it may speculated that at least one of the active components is proteineous. Analysis reveals potentially significant quantities of unidentified ninhydrin positive (most likely amino acid compounds, but other compounds may yield a response) components that appear to be stable to acid hydrolysis.

The principal amino acids per 1000 residues in the sample are Asx (asparagine) 143, Thr (Threonine) 31, Glx (glutamate) 381, Gly (glycine) 187, and Ala (alanine) 170.

A comparison was made between free and released (acid hydrolysis) amino acids. This shows the following amino acids per 1000 residues: Asx (asparagine) 51, Threonine 8.6, Serine 18, Glx (glutamine) 375, Pro (proline) 17, Glycine-(Glyj 429 and Alanine 86.

The ratio of Free/1000 is as follows: Released/1000

S

U

B

B.

0 0 *0e 0 r.

0* *0@ Asx Thr Ser Glx 4.09 2.03 1.65 1.18 Gly 0.37 Ala 1.69 There are 5 unidentified (ninhydrin positive) 20 components. Highly speculative assignments are cysteic acid, glucosaminic acid and sarcosine. Also there may be methionine sulfoxide and methionine sulfone. The major unidentified ninhydrin component appears to be free components in the sample.

330 Release of IL-1 and IL-8 Table 33 shows that the composition of the invention stimulates human mononuclear cells in culture to release and the 80% acetonitrile precipitate of the composition releases more than the remaining supernatant.

Whereas the whole composition does not stimulate IL-8 release, fractionated composition seems to release some IL-8. The results shown in Table 33 are minus the release of IL-:.O and IL-8 with mock samples.

Table 33 Releasing Activity of Composition of the Invention less Release by Reconstitution Solutions 199 Mdia .Qualuity Actua Relased Oszn,1arity Sample Tested Used in Well IL-I(pg/rnI) RL-g (ng/ml) (mourn) Batch: B0222 Whole 80% IX 40 Al 171 0 304 Precipitate 70%1IX 100 Al 160 71 292 Superna~t 90% IX 100 Al 17 87 335 Note: 1. Number of pazi±s tested: 5. buf.

2. Rconastnton: precipitate in Type IwW p~tti B ufr 3. Sa les are comed fbr release by IX 199 Medium sad LPS: 154. 1065 pg/ml. and 68. 294 ng/I.reseciveyfbrIL16and L8 Oa~1nariie for md;- and LPS am~ 311 and 309, respeclively.

5 5. LPS voll-add o 6. Total vohzme of wells: 10OO;i1.

Sample volumes amc equivalent.

Physicochemical Cbaracteriatics The composition and its precipitate and supernatant were separated by ion-exchange HPLC. Both by AX300 (anion exchange) chromatography and by CMX 300 (cation exchange) chromatography, there was no significant separation of components. Hydrophobic reverse phase chromatography did not separate the peaks.

cairilla.. electrophoreus The precipitate was analyzed by capillary electrophoresis. At high pH, a W absorbing peak was observed at 190 rim but completely disappeared at 200 rim.

There were no significant peaks at 214 rim Uv absorption.

Free amino acid are not visualized unless they are derivatized. It is thought that the W peak at 190 nm is likely a salt.

rExamale 1 3 The composition was evaluated for stimulatory activity in the following 3 indicator systems: 1) Stimulation of lymphocyte DNA synthesis; 2) Induction of lymphocyte-mediated cytotoxic function; and 3) Induction of monocyte/macrophage-mediated cytotoxic function.

These tests were chosen for the screen because they measure immunological functions which have been shown to be associated with different clinical parameters in patients with malignant disease. These indicators of immune function also can be modulated in cancer patients 15 who are treated with different biological response modifying agents such as interferon or interleukin-2.

The results of the initial screening procedure are presented below.

1) Stimulation of Lvmphocvte DNA synthesis: comparison with an optimal stimulating concentration of phvtohemaaqlutinin (PHA) SStimulant Counts per Minute Medium 374 PHA 125,817 Composition (#222) 1,116 Composition (1:10) 1,021 Composition (1:50) 649 f Results: Unlike the prototypic mitogen, PHA, the Composition does not stimulate lymphocytes to undergo blastogenesis and cell division.

2) Stimulation of Lymphocyte-mediated Ctotoxic comparison with an oDtimal stimulating concentration of Interleukin-2 (IL-2) Stimulant Lytic Units Medium 30.8 IL-2 472.5 Composition (neat) 48.1 Composition (1:10) 33.3 Composition (1:50) 44.8 Results: Unlike the prototypic stimulator of lymphocyte cytotoxic function, Interleukin-2, the composition does not elicit lymphocyte cytotoxicity.

3) Stimulation of Monocvte-Mediated Cvtotoxic Function by the Composition: Comoarison with Gamma Interferon Endotoxin (y-IFN LPS e o *o 0 a Stimulant (E/T-20/1) Medium IFN LPS Composition (neat) Composition (1:10) Composition (1:50) Cvtotoxicitv 4.3 24.4 19.7 20.0 11.5 ft f

S

9 e 2 5 Results: The composition is capable of stimulating peripheral blood monocytes to express tumoricidal function in a dose dependent manner. The magnitude of stimulation is comparable to that elicited by the prototypic macrophage activator combination of yIFN LPS. It is important to recognize that the action of the composition in these in vitro assays did not require the addition of endotoxin as in the case with any other macrophage activators. If the composition is free of endotoxin contamination, its biological activity in this assay of macrophage activation would be considered biologically significant.

Xonocvte/MacroDhae Studies vith the Composition Because the screening procedures demonstrated that the composition does not stimulate lymphocyte functions but can stimulate monocyte functions, subsequent studies were aimed at further characterization of the monocyte/macrophage stimulatory activities of this compound. A number of comparative studies aimed at determining the dose response characteristics of the composition in stimulating monocyte/macrophage tumoricidal function, were performed as well as testing different batches of the compound. The main emphasis of the studies was to test the capacity of the composition to simulate tumoricidal function in monocytes and macrophages from different anatomical sites of cancer patients. The central hypothesis guiding these studies is that the therapeutic efficacy of any biological stimulator will depend, in large part, on its ability to elicit tumoricidal function in environments which contain malignant disease. That could come about by direct stimulation of resident immune cells in tumor microenvironments. Alternatively, this could come about S* by stimulation of circulating immune cells if those cells 25 were then able to home on sites of malignant disease and to function in that environment. For these investigations, the following were relied upon: 1) peripheral blood monocytes from cancer patients and N* control subjects; 2) alveolar macrophages from lung 30 cancer patients and control patients with non-malignant lung diseases; and 3) Peritoneal macrophages from patients with gynecological malignancies.

1. Dose Response and Different Batch Studies with the Composition These studies relied on peripheral blood monocytes to test the stimulatory activities of different doses and different batches of the composition. Three batches of the composition were provided for testing. These were designated as batch #s 216, 219 and 222. Each batch of the composition was tested without dilution (neat), a 1:10 dilution and a 1:50 dilution of material. The results are depicted graphically in Figure 24.

Results: Batch #222 and 216 stimulate monocyte tumoricidal function, Batch #219 did not. It appeared that #222 was superior to 216 in these preliminary investigations. Batch #222 appears to stimulate equivalent levels of tumoricidal function at the undiluted (neat) and 1:10 dilution concentration with 15 less, but still detectable activity at the 1:50 dilution.

999@ Batch #216 gave the greatest stimulation of tumoricidal function at the undiluted (neat) concentration, with less activity at the 1:10 dilution and no detectable activity "at the 1:50 dilution. As stated above, Batch #219 did 20 not elicit detectable monocyte tumoricidal function at any concentration tested.

2. Tumoricidal Function in Peripheral Blood Monocytes Tests have been performed on 4 peripheral blood monocyte samples from control subjects. These tests utilized an optimal stimulating concentration of the composition (1:10 dilution of batch #222) and an optimal stimulating concentration of 7-IFN LPS. The target cells in these studies were a cultured, N-insensitive cell line, the Chang Hepatoma.

Cvtotoxicity Medium 5.4 1 y-IFN LPS 18.6 4 composition 22.3 6 A test was also performed on 1 monocyte sample from a patient with cervical cancer. This test was important because the patient's own tumor cells were available to be used as target cells in the assay. As before, this test utilized an optimal stimulating concentration of the composition (1:10 dilution of batch #222) and an optimal stimulating concentration of 7-IFN LPS. Also, the effector/target cell ratio was reduced to 15/1 to conserve patient tumor cells.

Stimulant Cvtotoxicitv (E/T=20/11 Medium 7-IFN LPS 14.4 Composition 20.9 15 Results: In the peripheral blood monocytes from control subjects, the composition stimulated monocyte tumoricidal function against the Chang Hepatoma at a level equal to or greater than the level elicited by an optimal stimulating concentration of yIFN LPS. In the peripheral blood monocytes from a patient with cervical cancer, the composition stimulated tumoricidal function against the patient's own tumor cells at a level which exceeded that elicited by 7-IFN LPS by a 0 0 3. Tumoricidal Function in Peritoneal Macrophaaes from 25 Patients with Gvnecoloaical Malignancies These tests were performed on peritoneal macrophage samples isolated from lavage fluids of 1 patient with cervical cancer and 1 patient with Ovarian Cancer. These tests were performed with the patient's own tumor cells as target cells in the assay. As before, an optimal stimulating concentration of the composition (1:10 dilution of batch #222) and an optimal stimulating concentration of y-IFN LPS were compared. Also, the effector/target cell ratio was reduced to 15/1 to conserve patient tumor cells.

til -antCervical Cancer Ovarian Cancer Medium 8.2 0.6 IFN LPS 29.8 4.1 Composition 13.2 8.9 (1:10) Results: These test results highlighted the fact that the local tumor environment may be a determinant of the response of immune cells to immunological activators.

In this case of cervical cancer, there was no pathological evidence of malignant disease within the peritoneal cavity and the development of tumoricidal function against the autologous tumor was better with y- 15 IFN LPS than the composition. In the patient with ovarian cancer, there was significant tumor in the peritoneal cavity. The response against the patient's own tumor to 7IFN LPS was minimal at best, whereas the response to the composition was greater.

@0 B 0 0

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0.eg 4 0* S 0 0 *0

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0 *eg *000

S

@9 4. Tunoricidal Function in Alveolar MacronhaQes from Lung Cancer Patients and Control Sublects "These tests were performed on alveolar macrophage ~samples isolated from broncholveolar lavage fluids of a patient with non-small cell lung cancer and 3 patients 25 with non-malignant diseases of the lung. These tests utilized an optimal stimulating concentration of the composition (1:10 dilution of batch #222) and an optimal stimulating concentration of 7-IFN+LPS. The target cells in these studies were the Chang Hepatoma cells and the effector/ target cell ratio was 20/1.

Stimulant Cancer Patients Control.

Medium 2.6 2 19.5 4 y-IFN LPS 10.9 13 1.2 Composition 5.2 2 18.6 8 Results: Alveolar macrophages from lung cancer patients are impaired in their development of tumoricidal function in response to conventional macrophage activators such as yIFN LPS. These results are consistent with this observation; they show that the tumoricidal function of alveolar macrophages from lung cancer patients is greatly reduced compared to control subjects. They also show that the composition does not activate tumoricidal function in either the alveolar macrophages of lung cancer patients or the alveolar macrophages of control subjects with non-malignant lung diseases.

These preliminary in vitro tests with the composition demonstrate that it is a macrophage 15 activator. The material provided was able to elicit tumoricidal activity in a standard cytotoxicity assay against both an NKinsensitive cell line and against freshly dissociated human tumor cells. The activity elicited was also found to be concentration dependent in 20 these tests. The capacity of the composition to active macrophage tumoricidal function in vitro is comparable to that of the best macrophage activating combination presently available, namely, yIFN endotoxin. As stated above, the capacity of the composition to elicit this 25 level of tumoricidal function in the absence of endotoxin would be considered important biologically if the material is free of endotoxin contamination.

As has been found for other macrophage activators, the activity of the composition in stimulating macrophage tumoricidal function varies with the source of the macrophages. It appears that the composition is an excellent activator of peripheral blood monocytes being equivalent to yIFN LPS with normal donors and possibly superior to yIFN LPS with cancer patient donors.

Malignant disease has a significant impact on the development of monocyte tumoricidal function depending on the activator used (Braun et al, 1991). One determinant of the biological activity of different macrophage activators in cancer patients monocytes is the sensitivity of the activator to arachidonic acid metabolism and the secretion, by the cell of prostaglandins. From these initial studies with the composition, it appears that activity elicited with the compound is not sensitive to the inhibitory effects of prostaglandins. If prostaglandin insensitivity can be proven definitively for cancer patient monocytes stimulated with the composition, this would be considered important therapeutically since the effectiveness of many other biological activators is limited by prostaglandins.

Preliminary studies with 2 specimens indicate that the 15 composition may have good activity in peritoneal macrophages, particularly when malignant disease is °0 present in the peritoneal cavity.

These preliminary results also illustrate what has been found when comparing the capacity of different 0 activators to stimulate tumoricidal function in peritoneal macrophages of patients with different gynecological malignancies. In those studies, it was found that the presence of malignant disease within the peritoneal cavity influences the responsiveness of the 25 peritoneal macrophages to specific activators. In patients with cervical cancer, malignant disease is not *present in the peritoneal cavity in general, and thus, the response of the resident macrophages to 7IFN LPS is normal. When disease is present in the cavity, however, as in the case with ovarian cancer, the response to ylFN LPS is suppressed. This is related, in part, to changes in the arachidonic acid metabolism of the peritoneal macrophages when malignant disease is present (Braun et al, 1993). The fact that the composition apparently can activate tumoricidal function in peritoneal macrophages from ovarian cancer patients against the patient's own tumor cells may reflect, once again, a mechanism for activation which is independent of the arachidonic acid metabolic pathway.

On the other hand, the composition clearly does not activate alveolar macrophages to become tumoricidal whether malignant disease is present in the lung or not.

Alveolar macrophages from lung cancer patients have been found to be inhibited significantly in their development of tumoricidal function when compared to either peripheral blood monocytes from the same patients or to control alveolar macrophages from patients with nonmalignant lung diseases (Siziopikou et al., 1991). Thus, the lack of activity of the composition in this case is not surprising.

15 xmrle 14 The development of tumoricidal function in response to the composition of the invention and other macrophage activators was investigated in peripheral blood monocytes and peritoneal macrophages from patients with gynecological diseases. More particularly, the patient population consisted of 7 patients, 3 with benign disease and 4 with malignant disease (2 ovarian cancers, 1 endometrial cancer, and 1 cervical cancer). Samples were removed from patients at the time of surgical procedure.

Preparations containing peripheral blood monocytes were S: isolated from blood samples using the procedure set out in Braun et al. Cancer Immunol. Immunother 32:55-61, 1990 and preparations containing peritoneal macrophages were isolated as set out in Braun et al., Cancer Research the composition of the invention (1:10 dilution of stock batch 222) and other activators namely gamma interferon (100 U/ml), interleukin-12 (500 U/ml), and monocyte-CSF (500 U/ml) was assessed using the monocyte cytotoxicity assay described in Braun et al. Cancer Immunol.

Immunother 32:55-61, 1990.

The results as shown in Table 34, demonstrate that the composition of the invention stimulates tumoricidal function in both the peripheral blood monocytes and the paritoneal macrophages from patients with malignant and non-malignant gynecological diseases. The tumor cytotoxicity elicited by the composition of the invention is equal to or greater than that elicited by the other biological stimulators which ware tested.

TABLE 34 The Development of Twnriaddal Functioa in Response to the Conipaistion of the invention So=nd odu' Macraphage Activators Wn Pipheral Blood Mcumocytes and Peitonmi Maopbages 0:90 fromi Patients with Gyfleokgical Qa( bmign dnse, 4 malignant dsse) Tumor Cyuooxicty(+/- S.E.) Ac~Agr t Monoctwrrtaw Cell ratio 15/1 Acti* Periphma Peritonea' Medium 8.6 :t 3 3.1 ±1 Gamma Interfron 18.3±2 9.5±1 Intedeukin-12 26.0±4 8.5 2 **Monoqyte-CSF 16.0 ±2 7.0±2 Composition of the 23.0±6 12.5 2 Invention Rzazvle The effect of indomethacin, a prostaglandin funtein inhrespose on the comepit of the rivion synteisn inhibitore to the developmein of theoicvion and thermacrophage activators in peripheral blood monocytes from cancer patients wa s also investigated.

Samples from the Patients with malignant disease in Example 14 were tested using the assay system as described in Example 14 with the exception that indomethacin (up to 5ng/ml) was simultaneously added with the composition of the invention, ±4nterleuin-12 (500 u/ml) ,and monocyte-CSF (500 u/ml).

The results as shown in Table 35 indicate that indomethacin augments cytotoxicity in response to iFa, Gm-CSF and ?I-CSF. Thus,. the development of tumoricidal function in response to IFN-y, GM-CSF, and M-CSF was regulated by an indomethacin-sensitive function. in contrast, the development of tumoridical function in response to Phorbol Ester (P1NA) IL-12 and the composition of the invention was not regulated by an indomethacinsensitive function i.e. indomethacin did not augment cytotoxicity in response to the composition of the invention, IL-12 and PNA.

15 TABLE- nTfed dE~. hoinh a Nintoafi bdtw m d Dwdsbm of Tmwkd Fcwdm in Rwp. to do C~pad o dw Wb. Iu d etar Macr"ph Aaivaau ihma She" Md Moma. frm *M 02600 iActivation Conditio#nsmcy oict OIFN-y 23 11.9±9 *ITh..r Indomethacin 25.2 ±17 0GM-CSF 10 7.8±6 OGM-CSF lndomdvcin 17.9 6 27.3 ±14 *PMA+ Indomedacin 22.0±17 IL-: 12 3 24.7 IL-12 Indomethcin 25.6 ±6 *6M-CSF 3 14.1±3 M-CSF Indowcthacin 19.0 ±3 ComPosition 4 18.7±6 Composition 16.4 ±6 Indoeicn The effect of prostaglandin E 2 on the development of tumoricidal function in response to the composition of the invention in the presence of indomethacin was investigated. The subject population- consisted of one normal and eight patients (one patient with a pancreatic tumor, two patients with head and neck tumors, one with endometriosis, and four with HIV) Preparations containing peripheral blood monocytes were isolated from blood samples from the patients using the procedure set out in Braun et al. Cancer Immunol. Immunother 32:55-61, 1990. -Tumor cell crytotoxicity in response to the composition of the invention (1:10 dilution of stock batch 222) and indomethacin (up to 5-g/ml), with or rose 15 without PGE 2 (10 8 M) was assessed using the monocyte C cytotoxicity assay described in Braun et al., Cancer Immunol. Immunother 32:55-61, 1990.

0 The results in Table 36 show that 36 pathophys io logical levels of PGE 2 (10 8 M) tailed to suppress the level of tumoricidal function-which developed in response to the composition of the invention. This is in contrast to the capacity of PGE 2 to suppress tumoricidal 0function in monocytes stimulated with 7-interferon (Braun see 400et al, Cancer Research 53:3362, 1993).

00.

TABLE 36 44* The Erhci d ?rotagw~ Ex so d DevAmet of TmaciiM Fmco in Rapi to the Campitim@ othe S .Invmb in the Pru= of hwadhcl 4. Tint Cy k~aziiy at M..ocyt(rinr Coa ra6iu 1511 0 i CGOVmpon Ccoaodonk l~adwtsrn Ladom.aacin 3NOA19 1.0 27 PuWMtcr~ 15 14 2 1H4SCC 9 12 HNSCC 11 3 12 En m ioaoj 37 3-1 f.d.

HIV 6 7 8 HEIV 15 12 19 MV 21 16 lIV 23 mnd.

TiP51 7.

The development of tumoridical function against autologous tumor cells in monocytes stimulated with the composition of the invention was investigated.

Preparations containing peripheral blood monocytes were isolated from blood samples from 6 patients (three ovarian cancers, one endometrial cancer, one cervical g. cancer and one ENT cancer) using the procedure set out in Braun et al., 1990. Tumor cell cytotoxicity in response to the composition of the invention (1:10 dilution of 15 stock batch 222) and indomethacin (up to 5-g/ml), with or *without PGE(10 8 N) was assessed using the monocyte ocytotoxicity assay described in Braun et al., 1990, with 4 the exception that the patient's tumor cells were used in place of the Chang hepatoma cells. The patient's tumor cells were treated with collagenase and DNase, single cell preparations were prepared, and the cells were

CS

labelled as described in Braun et al. 1990.

The results shown in Table 37 demonstrate that the composition of the invention is capable of activating the 25 patient's own monocytes to kill the patient's tumor. The composition of the inventon is a- least as effective as

C

the standard biological activators which are currently 0 being used.

Table 37 The Development of Tumoridical Function against Antolwp ~s Thamor Calls in Wonocytes stimlated with the Composition of the invention Diagnosis Cultu~re Conditions %Tumor Cytotoxic ity (3/T 15/1) Orvarian Cancer ovarian Cancer Medium Coatpos it ion Mdium y-Interferan LPS composition ovarian Cancer Mdium es S 0 0 0@ *000 S S *5 *0 0 0*S S. S 5 0 *5 0 y-Irteron LIPS Composition Endomtrial Cancer. Medium y-Interferon +4 LPS Composition Cervical Cancer Medium y-Interferon LPS Composit ion 0* 0O S 0* 0OS 5@ *5 S

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0 ENT Cancer Medium *-Interferon UPS 12 Composition The experimental results in Examples 14 to 17 indicate that the composition of the invention is capable of activating monocytes. to express tumoricidal function, and it is at least as effective-as other activators currently being used in the clinical setting; it works in the blood with peritoneal macrophages; and, it appears to not be subject to the inhibitory effects of prostaglandins, which is one of the principle forms of immunosuppression in patients. The experimental data also supports the utility of the composition in the treatment of peritoneal and gynecological malignancies.

ExaMple 18 Early Toxicity Studies Toxicity studies were conducted on a variety of animal species. The studies are summarized in Table 38.

All animals were assessed on the basis of daily clinical observation while receiving the injections on days 14, 21 and 30 thereafter. No adverse effects were noted throughout the period that injections were administered 15 or during the follow-up period (one month for all species except the dogs which were followed for 4 months).

Table 38 Summary of Early Toxidty Studie S* S S 0 S0 0 0*S0 S@ S 5 5 0*

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6050 0 S. 0 50 0 0 0 *0 000. .0 0 20 White Mice Male Wistar Rats Golden Hamsters Guinea Pigs Rabbits Cats Dogs I00lf 100 100 60 60 15 10 12 Doa 0.2 ml i.m. at three day intervals four times 2.0 ml i.m at three day intervals four times 1.5 ml i.m. at four day intervals four times 3.0 ml at three day intervals four times 5.0 ml i.m. at three day intervals four times 3.0 ml i.m. at three day intervals six times 2 ml/kg i.m. given once observed for four months' Hematologic data collected every third day for the first 30 days and once monthly thereafter.

A toxicity study was conducted to determine the effect of a single large intramuscular dose of the composition. Thirteen rats received a single intramuscular dose of 5 ml/kg of the composition. Three rats were observed for 7 days. Ten rats were observed for 14 days followed by euthanasia and necropsy. No symptoms of toxicity were observed in either group and no gross pathologic findings were observed in the animals that were necropsied. Based on these observations the LDso for intramuscular administration of the composition in rats was determined to be greater than 5 ml/kg. Table 39 summarizes these results.

Table 39 dF.dman of LDs in Spragu-Dawley Rats 15 Rotma of Animal Quantity Admin Dose Unit/kg* o Sprague 3 male im. 5 ml/kg 52.5 Dawley Rats 10 i.m. 5 mYkg 52.5 >5 ml/kg male/ feade) *tUnits calculated on the expectd range of biortivity of Lot B80201 measured at 10.5 units/ml.

Toxicity Trial In Doas 25 In a study conducted by the Ontario Veterinary College, the composition was administered to two mixed breed dogs. The protocol is summarized in Table 40. In each case one dose was given in the right leg and the second dose 7 days later was given in the left rear leg.

Both dogs were observed for 14 days after the first injection. Appetite, activity, temperature, pulse rate, respiratory rate were monitored twice daily throughout the study. Routine u-rinalyses, hematology and serum chemistry profiles were Performed, pretreatment and 24 hours, 72 hours, 7 days and 14 days after the first injection. Neither animal showed signs of pain associated with either injection. There was no evidence of anaphylaxis associated with the second injection. No abnormalities or changes in physical or laboratory parameters were observed that could be attributed to the drug. The drug appeared to be well tolerated by healthy dogs.

Table Toxcity Study in Dogs 0O Ani-I Mixed Breed Female Mixed Breed Age and Wei&h Adult 5 kg 6 months 13 kg Dose I Units Calculated* 5.5 ml i.m.

28.6-50.6 Units 12.5 ml i.in.

65.0-115.0 Units Dowe 2 Units Calacuated* 0.6 ml i.m.

3.1-5.5 Units 1.3 ml iLm.

6. 8-12.0 Units Doe Interval 7 days 7 days Treatment Of ARIMAle with Maliamant Neoplasmas The composition of the invention was used clinically in a veterinary hospital for the treatment of various malignant tumors in companion animals. E leven cats and ten dogs with advanced neoplastic disease that was not :responding to conventional therapy were treated with the composition given intramuscularly in weekly doses. Table 41 summarizes the individual clinical cases in this study. The number of injections ranged from 2 to 69, with volumes up to 7.5 ml given into a single intramuscular site. Protocols of weekly injections allowed for examinations and careful monitoring of the individual cases with diagnostic tests determined individually for each case. The clinician noted that there was no local irritation nor severe allergic reactions, including anaphylaxis. The clinician and the owners of the animals did not observe any systemic adverse reactions. The investigators noted some clinical, improvements consisting of minor reductions, improved appetite and activity levels, significant weight gain in few animals and a decrease in pain and/or discomfort.

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if, 0 4 0 0* 0 0 0 0 8 0 0 @0 0 S @0 4 0 0 dIe 000 0. S 0.e S 8 0 S 0* 00 00 I C S S 0 I* 0 0 lOt 0 I 0 S 0 S *0 eel 0 000 Table 41 Table 5-5 Summary of Results from Treatment of Animals with Malignant Neoplasmis NO. Narne-Ae Species-Sex Diagnoss 01 Bandit 13 Canine-NI/n Orinasal Fibrosarcoma 02 Bob 5 Feline-NI/n Focal Osseous Motaplasia with Ositeosarcomatous development, Spindle Cell Sarcoma, Feline Fibrosarcoma, Squamous, Recurrent Spindile Ccli Sarcoma invasive (necrupsy diagnosis) From-To 01.3 1.87- 05.19.87 04.02.87- 08.10.87 11.68.88- 02.21.90 Injections Surperies Minor Partial Response Progressive Disease Euthanasia First recurrence 16 months all complete response Response with subsequent treatment evolving to Progresstve Disease (tumor became Rapidly invasive) Euthanasia Complete R"Wpnse.

Curesy Asymuptoinatic (4 months) Stable (No Change), Limb amputation.

No recurrence Initial Major partial Response. Subsequent R ekmlts 01 1) 7 Canine-MMn Oral Amelanotic Melanoma, Benign Papilloma Recurrent Round Cell Sarcoma 04 Mimi 7 Canine-F/s Invasive Fibrusarcuina recurrent 03 .02.02.03.87 08.24.87 12.14.87-04.05.88 04.Q4.89-08.01 .89 11.09.89-11.30.89 241290 25029189 02.27.87-08.10.87 Goliath 17 Feline-NI/n Malignant Melanoma 04.02.87-09.14.87 Fibrosarcoma '1mbS.* Mo Ea 0S 00 0 0 *00 054 0 0 0 0 00 0 00 0. 0 4 0 0 0 0 0 a *o 0 009 0 00 .50 0 000 Table 41 (Cont.) Table 5-5 Summary of Results from Treatment of Animals with Malignant Neoplasms No. Name-Agze Species-Sex Diagznosis From-To lniections Surgerde Remuits 11.30.87-07.25.88 06 Diablo 15 Feline-M/n Malignant 05.28.89-06.29.87 Squamnous Cell carcinoma 07 Ofivcr 10 Canine-M/n Malignant Round 02.24.89-05.30.89 Cell Sarcoma 08 Kani 7 Canine-M/n Mucinous Intestinal 06.01.87-09.14.87 Carcinoma Metastatic 09 Puppy 12 Feline-M/n Ceniminoua 07.22.88-10.04.88 Gland Adenocaz-cinoma Minor Partial Resaponse.

Progressive Disease Euthanasia.

Initial Minor Response Then Progressive Disease Euthanasia Complete Response.

Asymptomatio 1 year.

Minor Partial Res-ponse.

Then Progressive Disease.

Euthanasia Minor Partial Response.

Then Progressive Disease.

Died 10.10.87 Minor Partial Response.

Then Progressive Disease.

Euthanasia 03.26.87 Minor Partial Response.

Then Progressive Disease.

Died 12.21.87 Grandpa 16 FeLine-M/n Anaplastic Neoplasm High Gra, Malignancy *01.17.89-03.20.89 I11.02.87-12.21.87 I1I Sam 7 Fefine-F/s Mediastina] Lymphoma

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S* S S 0 5* Table 41 (Cont.) Table 5-5 Summary of Results from Treatment of Animals with Malignant Neoplasms ~Z zu-Aec Species-Sex D iagnosis From-To Inaections Surkeries -3 Feline-M/n Acute Feline Leukemia 04. 13 .87-05. 11.87 i3 Nlidnight 8 Feline-M/n 14 Sturmy r 10 Canine-M/n Feline Leukemnia 11.24.87-01.01.88 03.02.87-07.04.87 Asnelanofic Melanoma Transient Minor Partial Response. Then Progressive Disease. Died 05.05.87 Prog-essve Diweas Euthuanas 01.07.88 Complete Response, Recurrence After Nine Months, Progressive Diseas Partia Response<?) Progressive Disease Complete Response, Recurrence After T1hree Months Stable Minor Partial Response, No Change After 26 Months Stable Minor Partal Response Is Penny 10 Canine-F Malignant Melanoma 16 Muky 5 Feline-F Amaplastic Carcinoma 03.02.87-07.08.87 02.09.87-06.08.87 03 .02.87-08.04.87 04.02.87-08.31.87 17 George 10 FeLine-M 18 Simon 13 Canine-M Malignant Melanoma Benign prostatic Ityperplasia 6 15 10

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4'' a *0e a 0 L I p 0 C, 0 4 oh 0 4 *99 U a. 0 00 4) 0 06.0 0 *16 Table 41 (Cont.) Table 5-5 Summary of Results from Treatment ot Animals with Malignant Neoplasms N o. Naii-Age Spccics-Sex DiAemoi~ Ftom -Tg 1flieci1 Surizerce R etst 19 Tequila 14 Canine-F/a Malignant Inltetinll Adenocarcinoma 11.24.89-08.09.90 Sheba 12 Canine-F/s Invasive Outcosarcoma Skutt 12.06.89-06.28.90 02.07.89-05.06.89 Minor Partial Re~pnse Euthanasia 08.09.90.

Initial Minor Partial Response. Then Progressive Dibcabe Euthanasia Limb Amputation.

Complete response. No recurrence or meastases I yr.

21 Mesha 14 Feline-F/s Osicosarcoma 103 The clinical results are summarized as follows: Six animals (3/10 canines and 3/11 felines) experienced complete response. One animal (1/11 felines) had initial major partial response. Eleven animals (5/10 canines and 6/11 feline) experienced minor partial response. One animal (1/10 canines) remained stable and one animal (1/11 felines) did not respond. Table 42 provides definitions of each treatment. The clinical experience in animals suggested a potential role for the composition in the treatment of malignant neoplasms.

Table 42 •ee e@ *e Ragonse, Complete Response Partial Response Major Minor Stable Disease Defintion of Tremsent Respomses Dofiitio Disappearance of all clinical evidence of active tmnors. The patient mat be free of all known disease as determined by two observations not less than four weeks spart.

Where there is a greater than 50% xreduction in the sum of the product of the perpendicular dimesion of all measurable tumor with no new lesions appearing elsewhere.

Where there is a 25-50% shrinkage in the sum of the products of the perpendicular diameter of all measurable tumors; or subjective responses such as improvement in performance status, appetite and feeling of well being; or tumor necrosis or lysis as seen on ultrasound, x-rays, or changes in consistency and character of the tumors suggesting a decrease in adhesions and an increase in tumor mobility.

Les than 25 increase or decrease in the size of one or more measurable lesions without tumoral lysis, or appearance of new lesions.

tt Progressive Disease Increase of 25% in the size of one or more measuratble lesions without umnoral lysis, or appearance of new lesions.

Clinical Trials Prexli 1inar9 Prelimina-rY Patients with untreatable tumors were treated with 0.11 ml per kilogram of the composition of the invention as prepared in accordance with the methods set out in Example i. The composition was given intramuscularly every three to five days. Of the 58 patients treated there was absolutely no significant toxicity. In the 37 evaluable patients three patients had minor responses i.e. tumor shrinkage of between 25 and 50 percent, and five patients had stable disease of at least eight weeks in duration.

The most interesting results were in the pancreatic patients who seemed to have the most encouraging results. Of the seven patients one patient had the disease stabilization for a full 11 months. And a second patient with extremely advanced disease had disease stabilization for four months. It was based on these results that carcinoma of the pancreas was selected for a basic study of the composition of the invention. The objective of the study was to determine the safety and efficacy of the composition 4"7 in this group of patients.

.I The treatment consisted of the composition of the 20 invention 0.11 ml per kilogram with a minimum dose of 7.5 ml given as a single deep intramuscular injection to the gluteus maximus. Patients received the treatment three times weekly during the first week and then twice a week until disease progression. In all, 22 patients were enrolled in this study and all were evaluable for toxicity.

Only 17 patients were evaluable for efficacy. With a total of 570 injections there was no toxicity of any kind reported in either local or systemic.

There were also no objective responses. Six opatients however had stable disease for three months or longer but the rest of the patients progressed within the first three months. The median survival was eight months from the time of diagnosis. There 35 was a median survival of four months from the first injection. There were three patients who had stable disease for longer than six months. One patient, a 105 year old man relapsed with liver metastasis 18 months after a liberal procedure. He remained absolutely stable on the composition for eight months before progressing. A 71 year old woman with unresectable disease remained stable for at least ten months and continued to work full time. A 64 year old woman who relapsed regionally four months after the procedure was stable for at least eight months.

A fourth patient who had inoperable carcinoma of the pancreas and could not be enrolled in the study because tissue could not be obtained for a pathologic diagnosis, was stable for almost a year although his tumor progressed despite higher doses of the composition. In summary, the composition has no site of toxic activity against pancreatic cancer at this dosage schedule.* There was a suggestion of temporary anti-proliferative activity in a minority of cases with this disease using the composition.

Eape2 pancreatic cancer clinical studies A Phase II trial with the composition of the invention was begun for patients with-measurable, go biopsy-proven pancreatic cancer. The composition 25 was administered as a 7.5 ml (0.11 ml/kg) intramuscular injection 3 times weekly for 1 week then twice weekly until disease progression.

Details of the study are set out below.

0.000:d Treatment consisted of the composition prepared as in Example 1, 0.11 mlfkg (minimum dose 7.5 ml) administered with a single deep intramuscular injection to the gluteus maximus, alternating buttocks with each dose. Patients received 3 injections during the first week followed by twiceweekly injections until t-rinor progression.

00 0 GO 0 .0 *0 0 0*S S-04e% Response was defined using standard criteria.

Miller et al., Cancer 1981; 47:207-214). A complete response (CR) was defined as complete disappearance of all evidence of disease for at least 4 weeks. A partial response (PR) was defined as a reduction in the product of the two largest perpendicular diameters of the largest measurable lesion, with no new lesions or progression of any lesion, for at least 4 weeks. Progressive disease was defined as a 25%, or more increase in the size of one or more measurable lesions or the appearance of new lesions. Disease not meeting criteria for response or progressive disease was termed stable disease.

Resulto A total of 22 patients were enrolled in the study, but five patients were considered inevaluable for efficacy. There were no complete or partial responses. Three patients had disease progression within the first month. Six patients had disease stabilization for more than 3 months 3.5, 8, 12+, Median survival for the entire group was 8 months from the date of diagnosis and 5 months from the start of treatment. One patient with biopsy-proven liver metastases and a CEA of 37 ng/ml (normal <3 ng/ml), had absolute stabilization of the liver metastases and CEA for a months. One had stable disease for 5 months. one patient had disease relapse in her pancreatic bed 4 months after a Whipple procedure and was been stable on the composition for at least one year, with the exception of a slowing rising CE.A. A third patient had a percutaneous stent inserted and continued to work full-time for at least 14 months with no evidence of tumor progression.

107 All 22 patients were evaluable fortoxicity, having received a total of over 500 injections.

None developed any clinical or laboratory evidence of drug-related toxicity. There was no detrimental effect on Quality of Life which generally parallelled disease activity. No significant changes in total white blood cell counts, absolute lymphocyte counts on serum immunoglobulins were seen.

Survival curves representing the survival times from diagnosis and from treatment initiation are presented in Figures 13 and 14, respectively. For comparison, an historical survival curve for Gudjonsson (1987) has been superimposed in Figure 13. Another example of a comparable historical survival curve may be found in Bakkevold, Petterson, •Arnesjo and Espenhaug (1990).

The results of the survival analyses are summarized in Table 43. The mean survival time from diagnosis was 281 days (Figure 13). The median survival was 182 days (approximately 5 months). For comparison, Gudjonsson (1987) reported the mean survival of his 188 surgical patients as 208 days with a medium survival of 120 days. The mean survival time from treatment start was 166 days (Figure 14). The median survival was 133 days (approximately 4 months and 1 week).

o••e 0 0 0• Table 43 Protocol CO2-104 Survival Estimates Mean Survival Standar Medin Survival Parieat Popultion (days) Deviation Survival (days) From Protoco1 C02-104 231 203 132 diagnosis patients Protocol C02-104 3C4 157 219 evuhiable patients From Protocol C02-104 166 135 133 treatmnt patents Protocol C02-104 220 132 146 evaluable patients Survival times were also estimated among a subset 10 of evaluable patients who had each received at least 13 injections. Fourteen of the 22 patient were evaluable. Among these patients (Table 43), the median survival from diagnosis was 219 days (approximately 7 months and 1 week). The median 969:0 15 survival from treatment start was 146 days (approximately 5 months).

Example 21 Clinical Trials re Maliqnant Melanoma Advanced malignant melanoma was defined to include 20 all stage III or IV patients and all loco-regional or distant relapses occurring after primary treatment. The standard treatment by which all other treatments are judged is DTIC (dacarbazine) which has a reported response rate of about The median response is 3-6 months, and carries with it severe nausea and vomiting, and a potentially lethal side effect of acute liver necrosis by thrombosis of the hepatic veins. This treatment fails to show any definitive survival advantages.

This study was -Dnducted to determine the safety and efficacy of the composition of the invention and go S

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C C 5* 0 5* S 5 0

C.

0 505050 0

S

0w*OS@ to determine its effect on survival and on quality of life, when used in patients with advanced malignant melanoma. The study, was a noncomparative, multicenter trial.

An initial dosing schedule of 7.5 ml injections of the composition of the invention intramuscularly 3 times per week was used. After no organ or marrow toxicity was observed, the loading schedule was increased to daily injections for 15 days, followed by maintenance of 3 injections per week.

subsequently the loading dose was increased to days. Duration of treatment was 36 weeks and then reduced to 16 weeks, after which patients were given.

the option of entering a continuation protocol.

Thirty-three patients with advanced melanoma were, included in the study population (17 females and 16 males), ranging in age from 17 to 85 years of age.

64% had been previously treated and 36% were untreated. Of the 33 patients included in the study population, twenty five were evaluable. The Karnof sky Performance Status (baseline) was in the range of. 40-100.%, median 80%. Eleven patients were alive at the end of the study period and five of these were under treatment.

A minor partial response was observed in 16/33 patients One patient had a reduction of 33% in the lungs, six patients had pain reductions and eight patients gained more than 1000 grams in weight for more than a month (Range 1000 2600 grams). A stable condition was observed in 19/33 patients (Range 60 1 70 days, median 77 days).

Figures 15, 16 and 17 show the survival of patients treated with the composition of the invention compared to historical controls, measured as survival from diagnosis of metastases/recurrence in days. The solid line represents the survival curve for patients treated with the composit- on of 00 0 00 00 0000 .0 0000 00 0 0 0 00 00 0 *00 0000 00 00 0 the invention and the broken line represents the historical survival curve (Balch, C. X. et al., Cutaneous Melanoma, 2nd. ed. 1992, Chaoters. 14 and e pp. 165-187 499-508, Lippincott Co., Philadelphia, Penn.) The survival of all patients treated with the composition of the invention, including patients with one to over three tumor sites, is shown in Figure 15. Survival of patients with two tumor sites and with three or more tumor sites is shown in Figures 16 and 17 respectively.

The group of all patients treated with the composition of the invention had a 39% survival (Kaplan-Meier estimation) at one year. The survival' rate at one year for all advanced malignant melanoma (AIO() patients is approximately 11% in historical controls (matched by number of tumor sites) The group had a median aurvival of 315 days compared to the historical median of 89 days.

With two tumor sites the one year survival was 49% in the patients treated with the composition of the invention, as compared with 13% in historical controls. This group had a median survival of 360 days compared to the historical median of 120 days.

With three or more tumor sites the one year survival was 31% in the patients treated with the composition of the invention, as compared with 0% in historical controls. The group writh three or more tumors had a median survival of 205 days compared to the historical median of 60 days.

Quality of life was assessed by weight gain, performance status (Karnof sky), Quality of Life Index (Spitzer) and pain scale (Linear knalogue) Weight gain over time is shown in Table 39.

000 00 0 0 06 *000*9 0 000.

d Table 44 Numbr of ffvaluable 1st 2ad month Month 3rd 4th 5th 6th menth mnnth momth nmoth 11/25 12/25 44% 48% 4/25 4/25 1/25 1/25 16% 16% 4% 4% Percait R&nge 100- 200- 2400 6000 100- 100- 1000 2000 Averag (gr0 900 1480 525 775 100 2000 *0 0O 0*

S

*4* S. S S 0*

B

5*5 06O*

S.

a. 0 By 0 B

B*

OOBS

S

#4 0 0* S

B.

is 0 60.40.

S

The Karnofsky and Spitzer scales are both subjective and were found to approximately agree in each individual. Fifteen patients reported no change in these parameters. Four patients showed fluctuations which later returned to previous levels. One patient had a decrease (from 40-20%).

The results of pain evaluation showed that in six patients by week 4 the pain dropped from 5 (worst possible) to 2 (moderate) or 0 (no pain). One patient had a drop in pain from 3 to 0. One patient with hepatic metastasis had pain reduction to 0 and stabilisation for 11 months. Nine patients who entered the study with 0 pain maintained that level throughout the study. Five patients had a moderate (2 unit) increase in pain. Three patients had transient pain increases (1 to 2 units).during the second or third month.

Out of 1734 injections administered to 33 patients, 21 patients had no adverse drug reactions.

Fourteen adverse drug reactions were reported in 12 patients. The adverse drug reactions usually occurred at weeks 4 or 8 and were mild to transient, and most frequently were a low grade fever.

4 4

S

A

112 The difference in survival between the historical groups and the protocol groups treated with the composition of the invention suggests a survival benefit for patients treated with the composition of the invention. The cancer seemed to stabilize in 19 patients. All patients treated for AMM were included in the survival data. Also included were 21 previously treated patients (many clinical trials require untreated patients, because of the poor prognosis of failed previous treatments). The tumor burden was high (82% had more than one metastatic site).

The survival and quality of life data suggest that most patients received some benefit from the treatment. Eleven patients were still alive at the *end of testudy period and of those 11, 5 continued treatment.

zzample 22 PATHOLOGY PROTOCOL XALIGNANT MN NA 20 The following is a report of a 73 year old female with progressive malignant melanoma of the hard palate and gums. Figure 25 shows two views of malignant melanoma as seen under the microscope. In .*~Figure 25a, looking from top to bottom, one can see the epithelial layer with accompanying keratin, beneath which the malignant cells start to became more apparent. These melanoma cells can be seen to be rounded or oval, with an abundant eosinophilic cytoplasm, and pleomorphic hyperchromatic nuclei.

These cells have substituted the normal submucosal tissue. The blood vessels which are seen appear normal, and there is a paucity of any kind of inf lammnatory/ immune response as would be represented by the presence of leukocytes (polymorphonuclear and 35 mononuclear cells). This is an example of tumor tissue which is thriving, i.e. the tu~moral architecture is intact.

In Figure 25(b) a tumor tissue sample is shown from the same patient, who had been treated with the composition for two months. Starting from top to b~ottom, one can see that the continuity of the epithelium has been disrupted by a necrotic process.

This necrosis, while common in the center of any tumor that has reached a critical mass, is rarely seen on the periphery, especially in malignant melanoma, and is a sign that the host's immune response is mounting an attack against the tumor.

Throughout the photo are a massive number of cells different from the original tumor cells. These are is1 the immune cells-neutrophils, lymphocytes, macrophages- which have orchestrated the disruption of the typical tumoral architecture. The blood vessel walls have become densely infiltrated with a large number of host immune cells (arrow). This cellular infiltrate subsequently will cause the destruction of the blood vessel, which in turn prevents the tumor from receiving its supply of nutrients and oxygen (ischemic necrosis). This immune response which contributed to the tumoral 25 disruption seen in this patient's tissue slide is consistent with reported changes known to be brought S about by TN? (tumor necrosis factor) and with the results of the work described in the previous a..examples.

The immune response demonstrated in the after treatment with the composition slide (Figure strongly links the in vitro TNF immune modulation by the composition with known in vivo anti-tumoral TNF effects.

3 5 From the foregoing, it will be appreciated that, although specific embodiments of the invention have b~eni described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Accordingly, the invention is not limited except as by the appended claims.

Eape2 Isolation of Active Fractions A 300 ml sample of the composition was evaporated to dryness on a rotovap in which the temperature of the bath did not exceed 400C. In order to ensure that the solution remained basic during the evaporation, 5 drops of a concentrated ammonium hydroxide solution was added every half hour to the sO composition until the evaporation was complete. The resulting residue had a weight of 11.6g.

Os 15 20 ml of a 10% concentrated ammonium hydroxide in methanol solution was then added to 2 g of the above so:, residue. The insoluble material was filtered off and the filtrate was chromatographed through 101.93g of 60JL flash silica gel in a column with dimensions of 5cm x 12.5cm. The solvent system used was concentrated ammonium hydroxide in methanol solution. The column was run at a pressure of p.s.i. and a flow rate of 11 ml/min. After 100 ml of solvent had passed through the column, twelve 25 ml. fractions were collected. The collection of S. these fractions correlated to the appearance of an off-white band that was quickly moving down the column.

Thin layer chromatography (TLC) of these fractions 5 30 was run on silica gel plates in a 10% concentrated ammonium hydroxide solution in methanol and visualized with a ninhydrmn spray. Fractions having similar TLC profiles were combined, resulting in the following fraction combinations, which were dried on a rotovap.

115 Volume Through Column to Obtain Tractions Fraction Yield (g) 1-4 100-180 0 5-6 180-220 0.1175 7-8 220-260 0.1969 9-10 260-300 0.0151 11-12 300-340 0.0053 Fractions 5-6, 7-8 and 9-10 had a positive reaction with ninhydrin at an Rf value of 0.81.

xample 24 10 Fractions 5-6 and 9-10 from Example 23 were tested in vitro, for anti-proliferative effect (in accordance with the procedure of Example 4) and TNF stimulation (in accordance with Example The results are shown below: 15 FRACTION ASSAY ACTIVITY 5-6 Anti-Proliferative Effect 11.57 unmit/mg 5-6 TNF Stimulation LPS 50 pg/mg 9-10 Anti-Proliferative Effect 2.6 unitamg 9-10 TNF Stimulation LPS 1814 pg/mg Thus, fraction 5-6 was an active antiproliferative, and fraction 9-10 was an extremely active TNF stimulator, and a moderately active antiproliferative.

Example 2 0 25 Samples of Fraction 5-6 was analyzed by Electron Impact Mass Spectroscopy (El MS) and Electrospray Mass Spectroscopy to identify specific compounds likely to be present in the fraction. The Electrospray MS was performed on a Perkin-Elmer Sciex API-III spectrometer, using 5% acetic acid in water as the solute. In some instances, methanol was added to aid dissolution. The El MS using a 116 direct insertion probe was performed on a VG Analytical model ZAB-SE spectrometer using glycerol as a matrix, and using a DCI probe on a Kratos Analytical Profile Mass Spectrometer.

A review of the resultant spectra indicated that the following compounds were likely present in Fraction 5-6: phosphocholine, taurocholic acid, choline-stearic acid diglyceride, stearic acid, stearic acid diglyceride, palmitic acid-stearic acid diglyceride, and a sphingosine-oleic acid conjugate.

gxamp* 26 100 ml of the composition was acidified with 4 ml of a 1N HC1 solution such that the pH of the oo. 1composition was equal to 3. The composition was 15 then extracted with three 100 ml. portions of HPLC grade dichloromethane. The dichloromethane fractions were then combined and dried over a small amount of anhydrous sodium sulfate. The dichloromethane solution was then filtered through paper into a round bottom flask and evaporated to dryness on a rotovap to yield 0.0049g of a brown film.

0.0017g of this film was dissolved in a 214 ppm

NH

3 *H20 solution at pH 7, and was screened for anti- 25 proliferation activity as is set forth in Example 4.

This screen revealed that the solution was an active anti-proliferative, with an activity of 14 Units/mg.

zxamle 27 Example 23 was repeated on a larger scale, as follows. 10 ml of a concentrated ammonium hydroxide solution was added to 900 ml of the composition and the resulting solution evaporated to dryness on a rotovap in which the temperature of the bath did not exceed 40*C. In order to ensure that the solution remained basic during the evaporation, 5 drops of a 117 concentrated ammonium hydroxide solution was added every half hour to the composition until the evaporation was complete, leaving a residue.

150 ml of a 10% concentrated ammonium hydroxide in methanol solution was then added to the total residue. The solution was sonicated for 15 min. and the insoluble material was filtered off. The filtrate was chromatographed through 1695g of flash silica gel in a column with dimensions of .0 x 12cm. The solvent system used was concentrated ammonium hydroxide in methanol solution. The column was run at a pressure of 6 p.s.i. and a flow rate of 30 ml./min. The results of the column are summarized in the table below.

em C C em .me.

C. C C 0 em

S.

m

C..

me C S

C

Fraction I Volue of each fraction (mL) 550 450 400 150 C C e0CC C 0 0 6-7 8-13 14 15-35 36-40 Obsrvadons colord" colodu colodes colodres colories colorless tan colored solution begins to elute tan colored solution colorless O 30 TLC was run on silica gel plates in a concentration ammonium hydroxide solution and visualized with a ninhydrin spray. Fractions having similar TLC profiles were combined, resulting in the following fraction combinations, which were dried on a rotovap.

118 Fraction Volume Through Yied Coments Co,,inn to Obtain Fraction 3 10C00-1400 0.0504 white podery solid 1400-1650 0.0855 white powdery solid 6-8 1650.1850 0.1555 white powdery solid 9-12 1850-2050 0.3014 white pow ery solid 13-14 2050-2150 0.3595 white powdery solid 15-16 2150-2250 0.6914 slight brown color solid is tacky 17-18 2250-2350 1.024 tan color- solid is clupy *0 S19 2350-2400 0.3432 tan color solid is 0.e* S* clumpy 10 20-23 2400-2600 1.1531 brown color solid is clumpy 24-30 2600-2950 0.8517 brown color solid is clumpy 31-34 2950-3150 0.0813 brown oil All fraction combinations from 15-16 through Fraction 31-34 had a positive reaction with 0o 15 ninhydrin at an Rf value of 0.87, a value very similar to the Rf value for the active fractions of *0 0 Example 23. Fractions 24-30 and 31-34 had an additional positive reaction with ninhydrin at an R value of 0.85.

S

zaavle 28 S Fractions 4-5, 15-16 and 17-18 were tested in *vitro for anti-proliferative effect (in accordance with Example 4) and TNF stimulation (in accordance with Example The results are shown below: Fraction Assay Activity Anti-proliferative 4.7 units/mg Effect TNF Stimulation 119 15-16 Anti-proliferative 4.5 units/mg Effect 15-16 TNF Stimulation 17-18 Anti-proliferative 3.9 units/mg Effect 17-18 TNF Stimulation Thus, fractions 4-5, 15-16, and 17-18 showed antiproliferative activity, but no TNF stimulation activity. Elemental analysis of the above fractions showed them to be high in NH 4 CI, which inhibits TNF production.

0O1*2 see: Sample of fractions 15-16 and 24-30 was dialyzed and then was analyzed by mass spectroscopy, using the methods described in Example 25. Undialyzed samples from fractions 17-18 and 24-30 were also analyzed. A review of the resultant spectra indicated that the following compounds were likely present: glycocholic acid, a trihexosamine trimer, and taurocholic acid (Fraction 15-16); stearic acid, ["and a hexosamine dimer; and glycocholic acid 0OO• 20 (Fraction 24-30).

XAM21 e* The composition was dialyzed in separate dialysis tubing as follows: 100 ml of the composition was placed inside a Spectra/PorO CE membrane tubing which had a molecular weight cut off of 100. The ends of the tubing were sealed with clips and the tubing was placed into a stirred bath of 10 L of distilled water. The dialysis was monitored daily by removing 1 ml. of solution from the dialysis tubing and adding 3-4 drops of a 1/10 N silver nitrate solution. The presence of chloride indicated that the dialysis was not complete. If the dialysis was 120 0 C C 0@ ewe.

C

*0@ 0* *5

S.

not complete the bath was replaced with fresh distilled water. Dialysis completion occurred after 3-4 days. After dialysis was complete, the dialyzed material was dried on a rotovap to yield an average of 0.3 mg of solid per ml of original volume.

A sample of the solid material was then dissolved in HPLC grade water, and TLC was run on silica gel plates in a 10% concentrated ammonium hydroxide solution in methanol, and visualized with a ninhydrin spray. A positive reaction with ninhydrin was obtained at an Rf value of 0.83.

xamale 31 A sample of the solid material from Example 30 was also analyzed by mass spectroscopy, using the methods described in Example 25. A review of the resultant spectra indicated that the following compounds were likely present: a sphingosine-oleic acid conjugate, diacetyl sialic acid, a fucosehexosamine dimer, deoxyglycocholic acid, taurocholic acid, a sialic acid-fucose dimer, and a di(fucose)hexosamine trimer.

Example 32 Previous results indicated that the active components of the composition (at least according to a TNFa release assay) were present in the unbound fractions (void volume) after reversed-phase high performance liquid chromatography (RP-HPLC) on a C18 ABondapack column. As well, most of the mass of the composition extract, which was loaded onto a C18 IBondapack column, eluted in the void volume.

These results suggested the active components of the composition are, very likely, very polar or even ionic molecules.

To further examine the above results, purification of the active components by ion-exchange chronatography was performed. Negatively charged

C

S.

C

S.C See

C

25 active components (asseased by its antiproliferation affect on tumor cells and not on normal cells, and as wall by its TN?a releaseinducing activity) if present, would thus become bond to an anion exchange resin.

Experimental Tprncedurt ml of total xtctwas loaded onto an aflion-exchange cb-romatography column (Ejo-Rad AGhydroxide form, totAl resin wet volume was 10 ml' lo ~(column dimensions 1.5 am X 6.0 cm), equilibrated :with Millipore deionized water). The volume of go.::resin used was calculated to be sufficient for the 0000 binding of all the anions present In the extract.

*0 0 The unbound fraction was collected and reloaded onto 15 the column in order to maximize the binding to the resin. The unbound fraction from this second 5 passage was collected and saved. Any unbound material remaining on tho column's void volume was removed by washing with deionized water (2X20 ml).

Bound molecules were eluted with a step gradient of **Sammonium bicarbonate (NH4HCo 3 (20 Ml/StOP).

elution steps were 0 M 0@ S0.1

K

0.2 M 0.3 M 0.4 X 0.5 K .0.6

M

M

1.5 M toa=ple 3 3 iA Samples from all the fractions of Example 32 were analyzed for anti--proliferation activity and TNF timlaton ctiity inaccordance with the poceuresof xamles an 9,respectively.

The results are shown below: 122 Anti-prolifertive TNFa release- Sample activity inducing activity-LPS (U/mg) (pg/ml) 0 M 0 0.1 M 0 -79 0.2 M 0 -76 0.3M 0 0.4 M M 555.6 0.6 M 0 107 M 0 105 S; 10 1.5 M 0 189 The results from the activity assays show that "*9e .TNF production stimulation was found in the 0.6 M, *1.0 M, 1.5 M fractions. Anti-proliferative activity was found in fractions 0.4 M (minor activity) and 15 0.5 M (major active fraction).

Example 34 Thin layer chromatography analysis of the active fractions revealed a mixture of several components. A sample of the 1.0 M fraction from 20 Example 32 was analyzed by mass spectroscopy in accordance with Example 25. A review of the spectra !generated suggested that the following compounds may be present: a sialic acid-glycerol dimer, cholesterol sulfate, and taurocholic acid.

Example *Reversed Phase (C18) HPLC analysis was performed on a sample of the composition in accordance with the procedure of Example 2, except that a Phenomenex WP60009-CB18 column, 250 x 4.6 mm, was used, the sample was lyophilized and then reconstituted in 0.1% trifluoroacetic acid (TFA) in water, and 150 i1 of the reconstituted sample was applied to the column.

Various fractions of eluent were collected, including a fraction which eluted at approximately 2.40-3.40 minutes after the reconstituted sample was applied to the column.

Example 36 A sample of the fraction eluting at approximately 2.40-3.40 minutes from Example 35 was analyzed three times for TNF stimulation activity in accordance with the procedure of Example 9. The following results were obtained.

Asay I TNF Stimulation LPS 1 259 pg/ml 107 pg/ml 2 311 pg/ml ±14 pg/ml 3 572 pg/ml 176 pg/mi o*

*O

0: 3 15 Zxanple 37 A sample of the fraction eluting at approximately 2.40-3.40 minutes from Example 35 was subjected to tandem column reversed-phase (C18) HPLC as follows. The column from Example 35 was used in 20 tandem with a Phenomenex prime-sphere HC-C18 column, 250 x 4.6 mm. The sample was lyophilized, reconstituted in 0.1% TFA in water (Buffer A) and 150 Al of reconstituted sample was applied to the column. Buffer A was run for twenty minutes, then a 25 linear gradient of 0-80% of 0.1% TFA in acetonitrile (Buffer B) was run for 35 minutes. At the end of this period, 80-0% Buffer B was run for 5 minutes.

Flow rate was 0.9 ml/min. Six eluent fractions were Scollected, at the following approximate times from injection: 124 Fracalon TIme Omm..) 1 5.6-4.25 2 6.25-6.6 3 6.6-7.1 4 7.1-3.2 8.M-.6 6 14.7-16 A sample of Fraction 1 and a sample of Fraction 2 from Ex~ample 37 were lyophili3,d and reconstituted in 214 pa KH3 1

*H

2 0. These reconstituted samples were'then analyzed for antiproliferative effect in accordance with the *procedure of Example 4. The following results were is obtained: e* j .a-miatv .fe 1 Un.wLini 2 0 00 Ml*4 Samples from each of the six fractions of Example 37 were analyzed by mass spectroscopy in accordance with Example 25. A review of the resultant spectra for the six fractions indicated that the following compounds were likely present: taurocholic acid, a sialic acid-glycerol dimer, NaCi, trimathylamine, methylethylanine, and propylamine.

Anti-proliferative effect, according to the method of Example 4, was measured for the following three samples: 10-11 mg taurocholic acid in 2 m. of H1 2 0; 2:4 PPM NIT 3

.H

2 0; and 0.7 mg of taurocholic acid ill 4.0 ml1 of 214 ppm NXU,*H 2 o.

Neither sample nor sample had any detectable 125 anti-prolifarativo effect. sample however, had an anti-proliferative effect of 14 units/mg.

This result indicates that bile acids, such as taurocholic acid, in combination with ammonium ions, exhibits anti-proliferative activity at concentrations below that which the components, tested individually, show no activity. Thus, the combination of these two components, apparently synergistically, affects anti-proliferative activity.

The entire disclosure in the complete •specification of our Australian Patent Application No. 76489/94 is by this cross-reference incorporated into the present specification.

S

S O *2o eg S O *ooooo *ooo.

Claims (44)

1. A medicament derived from a water-soluble or water-miscible extract of animal bile that has been treated to remove all molecules of molecular weight over 3,000 daltons, bile pigments, and fatty organic materials, wherein greater that 90% of solid material is inorganic salts, and said medicament exhibits the following properties: a) is capable of stimulating monocytes and/or macrophages in vitro; b) is capable of modulating tumor necrosis factor production; S* c) contains no measurable level of IL-1u, 1 IL-1P; TNF;, IL-6, IL-4, GM-CSF or IFN-y; d) shows no cytotoxicity against human peripheral blood mononuclear cells; and e) is not an endotoxin.

2. A medicament according to Claim 1, wherein said animals are bovine. 20

3. A medicament according to any one of Claims 1 or Claim 2, wherein the medicament stimulates the production and/or release of tumor necrosis factor from human peripheral blood mononuclear cells.

4. A process for preparing the medicament of any one 25 of Claims 1 to 3, comprising the steps of: a) mixing bile from an animal with an equal I. volume of an alcohol to produce a bile/alcohol solution; b) separating out the alcohol soluble fraction and isolating a solution substantially free of alcohol; c) removing bile pigments from the solution to obtain a colorless liquid; d) treating the colorless liquid to substantially remove any residual alcohol; e) extracting the colorless liquid with ether and isolating the aqueous phase; and f) removing residual ether from the aqueous phase. H:\anna\Kee\Retypes\7$5439-94 DIV.doc 21112/98 127 0* q@00 *0 S S. S* C

*5 S A process according to Claim 4, wherein prior to step the colorless liquid is concentrated to about one eight of the volume of the bile/alcohol solution and after step the aqueous phase is concentrated so that it is one tenth of the volume of the bile/ethanol solution.

6. A process for preparing an immunomodulating medicament from the bile of animals, comprising the steps of: a) mixing bile from an animal with a water soluble solvent to produce a bile/solvent solution; b) isolating an aqueous solution substantially free of solvent from the bile/solvent solution; c) removing bile pigments from the solution to obtain a colorless liquid; 15 d) treating the colorless liquid to substantially remove any residual alcohol; e) extracting the colorless liquid with ether and isolating the aqueous phase; and f) removing residual ether from the aqueous phase.

7. A process according to Claim 6, wherein the water soluble solvent is an alcohol.

8. A process according to Claim 7, wherein the bile from an animal is mixed with an equal volume of an alcohol 25

9. The process according to any one of Claims 6 to 8, further comprising the step of concentrating the colorless liquid to about one-eighth the original volume of the bile/solvent solution.

The process according to any one of Claims 6 to 8, further comprising the step of concentrating the colorless liquid to about one-tenth the original volume of the bile/solvent solution.

11. A water-soluble or water-miscible medicament extracted from the bile of animals, comprising at least one component having a molecular weight of less than about 3000 Daltons, wherein said component shows no cytotoxicity to human peripheral blood mononuclear cells, is not an H:\anna\Keep\Retypes\7649-94 DIV.doc 21/12/98 128 endotoxin, IL-la, IL-13, TNF, IL-4, IL-6, IL-8, GM-CSF or IFN-y, and has at least one of the following properties: a) is capable of stimulating monocytes and/or macrophages in vitro to produce and/or release one or more cytokines; or b) is capable of stimulating monocytes and/or macrophages to produce and/or release tumor necrosis factor in vitro or in vivo.

12. A medicament according to Claim 11, wherein said animal is bovine.

13. The medicament according to Claim 11 or Claim 12, wherein the medicament stimulates tumor necrosis factor production and/or release in vitro or in vivo in the absence of IL-la, IL-13, TNF, IL-4, IL-6, IL-8, GM-CSF or 15 IFN-y.

14. The medicament according to Claim 13, wherein the medicament stimulates tumor necrosis factor production and/or release in humans.

The medicament according to any one of Claims 11 20 to 14, wherein when said medicament is dried to obtain a solid residue, and 2 grams of said residue are dissolved in 20 ml of a 10% concentrated ammonium hydroxide solution in methanol, and after any insoluble material is removed, is subjected to column chromatography in a methanol column 25 having dimensions of 5 cm x 12.5 cm, and containing 102 g of 60 A flash silica gel, and operating at a pressure of pounds per square inch and a flow rate of 11 ml/min with a concentrated ammonium hydroxide in methanol solvent solution, said component is eluted from the column in a fraction taken when the total column elution is between about 180 and about 220 ml, between about 220 ml to about 260 ml, or between about 260 ml and about 300 ml.

16. The medicament according to Claim 11, wherein when 10 ml of said medicament is subjected to anion- exchange chromatography in a column containing Bio-Rad AG-1 hydroxide form resin in an amount sufficient to bind substantially all the anions present in said 10 ml of said H:\ann\Keep\RetYPsy76489-94 DIV.doc 21/12/98 129 medicament, said component is eluted from the column using a step gradient of ammonium bicarbonate buffer at a buffer concentration from about 0.5 M to about 1.5 M.

17. The medicament according to Claim 16, wherein said component elutes from the column at'a buffer concentration of from about 1.0 M to about 1.5 M.

18. The medicament according to Claim 17, wherein said component elutes from the column at a buffer concentration of about 1.5 M.

19. The medicament according to Claim 11, wherein when said medicament is lyophilized and reconstituted in 0.1% TFA in water and then subjected to reversed-phased (C18) HPLC in a Phenomenex WP60009-C18 column, having dimensions of 250 x 4.6 mm, where a first buffer of 0.1% 15 TFA in water is run through the column for about 10 minutes, then a linear gradient from 0 to 80% of a secondbuffer of 0.1% TFA in acetonitrile is run for about minutes, followed by an 80% solution of the second buffer for about 5 minutes, and an 80%-0% gradient of the second buffer for about 5 minutes, and where flow rate is 1 ml/min. and the capacity of the column and buffers are not exceeded, said component is eluted from the column at a time from about 2.4 minutes to about 3.4 minutes after said reconstituted medicament is applied to the column. 25

20. The medicament according to Claim 11, wherein when said medicament is dialyzed in a first buffer of 0.1% TFA in water and then subjected to reversed-phase (C18) HPLC in a Bio-Rad Hi-Pore RP 318 (C18) column, having dimensions of 250 x 4.6 mm, where the first buffer is run through the column for about 10 minutes, then a linear gradient from 0-80% of a second buffer of 0.1% TFA in acetonitrile is run for about 55 minutes, followed by an solution of the second buffer for about 5 minutes, and an 80-0% gradient of the second buffer for about 5 minutes, and where the flow rate is 1 ml/min. and the capacity of the column and the buffers are not exceeded, said component is eluted from the column at a time from about 2 minutes to H:\anna\Keep\RetypeS\765489-94 DI'.doc 21/12/98 130 about 21.4 minutes, or at a time from about 21.4 minutes to about 25.6 minutes after said dialyzed medicament is applied to the column.

21. The medicament according to Claim 11, wherein when said medicament is subjected to thin layer chromatography on silica gel plates in 10% concentrated ammonium hydroxide in methanol and visualized with a ninhydrin spray, a positive reaction with ninhydrin occurs at a Rf value from about 0.80 to about 0.90.

22. A medicament derived from animal bile, comprising micelles of sphingosine or sphingosine complexed with a salt, which has at least one of the following properties: is capable of stimulating monocytes and/or macrophages in vitro to produce and/or release one or more cytokines; or is capable of stimulating monocytes and/or macrophages to produce and/or release tumor necrosis factor in vitro or in vivo.

23. The medicament according to Claim 22, further comprising a bile acid salt, and a source of ammonium or alkyl ammonium ions.

24. A medicament derived from animal bile, comprising sphingosine, a bile acid salt and a source of ammonium or alkyl ammonium ions, which has at least one of the 25 following properties: is capable of stimulating monocytes and/or S" macrophages in vitro to produce and/or release one or more cytokines; or is capable of stimulating monocytes and/or macrophages to produce and/or release tumor necrosis factor in vitro or in vivo.

A medicament derived from animal bile, comprising a bile acid salt, sphingosine, a diacyl glycerol, a source of ammonium or alkyl ammonium ions, and a retinolic acid derivative, which has at least one of the following properties: \\melb-fi es\home$\WendyS\Keep\species\9724298 Lorus.doc 8/02/01 131 is capable of stimulating monocytes and/or macrophages in vitro to produce and/or release one or more cytokines; or is capable of stimulating monocytes and/or macrophages to produce and/or release tumor necrosis factor in vitro or in vivo.

26. A medicament derived from animal bile, comprising a diacyl glyceride, lecithin, and a bile acid salt, which has at least one of the following properties: is capable of stimulating monocytes and/or macrophages in vitro to produce and/or release one or more cytokines; or is capable of stimulating monocytes and/or macrophages to produce and/or release tumor necrosis factor in vitro or in vivo.

27. A medicament derived from animal bile, comprising a diacyl glyceride, lecithin, and a mucin hydrolysis product or a proteoglycan hydrolysis product, which has at least one of the following properties: is capable of stimulating monocytes and/or macrophages in vitro to produce and/or release one or more cytokines; is capable of stimulating monocytes and/or macrophages to produce and/or tumor necrosis factor in vitro or in vivo.

28. A medicament derived from a water soluble or water miscible extract of animal bile that has been treated to remove all molecules over 3,000 daltons, wherein said medicament comprises organic and inorganic compounds, said inorganic compounds being selected from the group consisting of tungsten, zinc, phosphorus, nickel, barium, manganese, chromium, magnesium, calcium, copper, strontium, sodium, potassium, chloride and ammonia, said organic compounds being selected from the group comprising acetic acid, choline, phosphocholine, taurine and urea, wherein said medicament exhibits at least one of the following properties: \\melbfiles\home$\WendyS\Keep\species\97242-98 Lorus.doc 8/02/01 132 a) is capable of stimulating monocytes and/or macrophages in vitro; b) is capable of modulating tumor necrosis factor production; c) contains no measurable level of IL-la; TNF; IL-6, IL-4, GM-CSF or IFN-y, d) shows no cytotoxicity against human peripheral blood mononuclear cells; and e) is not an endotoxin.

29. The medicament of Claim 27, wherein said inorganic compounds constitute about 95-99% of all compounds in said medicament.

The medicament of Claim 29, wherein said organic compounds constitute about 1-5% of all compounds in said medicament.

31. A composition comprising as active agent a medicament according to any one of Claims 1.to 4 or 11 to 33, together with a pharmaceutically-acceptable carrier or diluent. 20

32. A method of modulating the immune response in a subject in need of such treatment, comprising the step of administering an effective amount of a medicament according to any one of Claims 1 to 4 or 11 to 30 to said subject.

33. A method of prophylaxis and/or treatment of a 25 disease or condition associated with altered immune .response, comprising the step of administering an effective amount of a medicament according to any one of Claims 1 to 4 or 11 to 30 to a subject in need of such treatment.

34. A method according to Claim 32 or Claim 33, in which the composition is administered to the subject in an amount effective to modulate the subject's immune system.

A method according to any one of Claims 32. to 34 in which the subject is a human.

36. A method of treating pancreatic cancer, comprising the step of administering to a patient suffering from said cancer a therapeutically effective amount of the Smedicament of any one of Claims 1 to 4 or 11 to \\melb_files\home$\WendyS\Keep\species\97242-98 Lorus.doc 8/02/01 133

37. Use of a medicament according to any one of Claims 1 to 4 or 11 to 30 in the manufacture of a medicament for modulating the immune response.

38. Use of a medicament according to any one of Claims 1 to 4 or 11 to 30 in the manufacture of a medicament for the prophylaxis and/or treatment of a disease associated with altered immune response.

39. Use of a medicament according to any one of Claims 1 to 4 or 11 to 30 in the manufacture of a medicament for the treatment of infectious diseases or neoplasias.

Use of a composition according to any one of Claims 1 to 4 or 11 to 30 in the manufacture of a medicament for the treatment of pancreatic cancer.

41. A method of treatment of an infectious disease or a neoplasia, comprising the step of administering an effective amount of a medicament according to any one of Claims 1 to 4 or 11 to 30 to a subject in need of such treatment. 20

42. A medicament according to Claims 1, 11 or 22 to 27, substantially as herein described with reference to the examples and drawings. S*

43. A process according to Claim 4 or Claim 6, substantially as herein described with reference to the 25 examples and drawings.

44. A method according to Claim 30 or a use according to Claim 41, substantially as herein described with S...references to the examples and drawings. DATED this 9 t h day of February 2001 LORUS THERAPEUTICS INC. By Their Patent Attorneys: GRIFFITH HACK Fellows Institute of Patent 0 Attorneys of Australia \\melbfiles\home$\WendyS\Keep\species\97242-98 Lorus.doc 8/02/01 nI-,