EP0316408A1

EP0316408A1 - Method of tumor inhibition in warm-blooded animals

Info

Publication number: EP0316408A1
Application number: EP88904876A
Authority: EP
Inventors: Loren Ralph Pickart
Original assignee: Procyte Corp
Current assignee: Procyte Corp
Priority date: 1987-05-11
Filing date: 1988-05-11
Publication date: 1989-05-24
Also published as: KR890701115A; WO1988008715A1; AU1799588A; JPH02500368A

Abstract

Sont décrits des procédés permettant d'inhiber la croissance de tumeurs chez les animaux à sang chaud. Les procédés emploient des quantités efficaces de compositions comprenant GHL-Cu (Glycidile-histidyle-lysine-Cu) ou un dérivé de celui-ci. Les compositions peuvent également comprendre de l'acide ascorbique.Methods are disclosed for inhibiting the growth of tumors in warm-blooded animals. The methods employ effective amounts of compositions comprising GHL-Cu (Glycidile-histidyl-lysine-Cu) or a derivative thereof. The compositions can also include ascorbic acid.

Description

Description
METHOD OF TUMOR INHIBITION IN WARM-BLOODED ANIMALS
Technical Field
The present invention relates to the inhibition of tumors in general, and more specifically, to the use of
GHL-Cu and derivatives thereof in the inhibition of tumors in warm-blooded animals.

Background Art
While a significant research effort has been expended in an effort to control cancerous growths, most therapies have only provided borderline effectiveness while being characterized by side effects often so severe that patients decide to avoid certain treatments altogether.

One traditional method for treating cancer is that of surgically removing the cancerous mass. However, some cancers are located in areas of the body that are relatively inaccessible to conventional surgical techniques. Further, if a surgical procedure is possible, it carries with it the risk of an increase in metastases through the spread of cancerous cells throughout the body cavity.

Another traditional method that initially had been used in concert with surgery is that of radiation therapy. However, a serious drawback to this type of treatment is the body's often discomforting reaction to the radiation.

Chemotherapy has also received relatively wide use as a cancer treatment. However, the various combinations of drugs used are often not specific to cancer cells alone, but rather destroy normal cells as well, resulting in serious side effects. In addition, some tumor cells become resistant to drugs that had previously been used to eradicate the cancer, while subpopulations of malignant cells with new antigenic characteristics may arise at any time.

More recently, while . certain drugs have been coupled to monoclonal antibodies in an effort to localize the effect of the drug, the results of these efforts have been inconsistent. In addition, recent research with monoclonal antibodies and immune modulators suggests that no single approach will suffice to treat the various types of human and animal neoplasm. The general consensus of opinion is that future tumor control will be obtained with a- therapy based upon a combination of tumor inhibitory factors which have minimal toxic side effects upon normal body functions.

Therefore, there is a need in the art for more improved, non-toxic methods for inhibiting tumors. The present invention provides such a method, while further providing other related advantages.

Disclosure of Invention
Briefly stated, the present invention discloses methods for inhibiting the growth of tumors in warm-blooded animals. In one -aspect of the present invention, the method comprises administering to the animal a therapeutically effective amount of a composition containing glycyl-L histidyl-L-lysine: copper (11).

Within a related aspect of the present invention, the method comprises administering to the animal a therapeutically effective amount of a composition containing a derivative of GHL-Cu , the derivative having the general formula:
EMI2.1
wherein R is selected from the group consisting of alkyl moieties containing from 1 to 18 carbon atoms, aryl moieties containing from 6 to 12 carbon atoms, alkoxy moieties containing from 1 to 12 carbon atoms, and aryloxy moieties containing from 6 to 12 carbon atoms.

Within preferred embodiments, GHL-Cu or a derivative thereof and copper are present in a 1:1 ratio.

The compositions described above may be admixed with a physiologically acceptable vehicle prior to administration, and further may include an effective amount of ascorbic acid.

Other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings.

Brief Description of the Drawing
Figure 1 is a photograph illustrating the effect of GHL-Cu on leg tumors.

Figure 2 is a photograph illustrating the effect of a representative derivative of GHL-Cu on leg tumors.

Figure 3 is a photograph illustrating the effect of GHL-Cu and representative derivatives thereof on leg tumors.

Figure 4 is a graph illustrating the effect of
GHL-Cu on the growth of a mouse tumor.

Best Mode for Carrying Out the Invention
As noted above, the present invention discloses methods for inhibiting the growth of tumors in warm-blooded animals. The compositions of the present invention utilize the tripeptide glycyl-histidyl-lysine or "GHL" which has a high affinity for copper and forms glycyl-L-histidyl-Llysine:copper(II) or GHL-Cu. The affinity of GHL for copper is equivalent to that of the copper transport site on human albumin. At low concentrations, the GHL is likely to be rapidly converted into GHL-Cu in a physiological milieu. The affinity of GHL for copper avoids the problems generated through an excess of loosely-bound copper(Il), which is acutely neurotoxic in vivo, and which may cause trembling and respiratory depression.

Within the present invention, GHL-Cu or a derivative thereof may be used to inhibit the growth of tumors in animals. The derivatives of the present invention are described in detail in pending U.S. Patent Application No.

040,460 and U.S. Patent No. 4,665,054, which documents are hereby incorporated by reference. The derivatives of the present invention may be prepared by esterification, by the removal of a water molecule, or by the addition of a group (either an alcohol such as octanol, methanol, benzol alcohol or NH3) to the carboxylic acid terminus of GHL.

The overall chemical reaction in this transformation may be characterized as:
GHL-OH + R-H --- > GHL-R + H20
In practice, the reaction is most readily carried out by adding the R group to the amino acid lysine prior to the combination of lysine with the other two amino acids to
GHL. After the formation and isolation of CHL-R, the copper(II) is chelated to the molecule to form the bioactive complex.

The overall reaction to form the more lipophilic derivative of GHL-Cu may be characterized as:
(1) lysine-OH + R-H --- > lysine-R + H2O
(2) lysine-R + blocked L-histidine --- > blocked
L-histidine-L-lysine-R
(3) blocked L-histidine-L-lysine-R + blocked
glycine --- > blocked glycyl-L-histidine-L
lysine-R
(4) blocked glycyl-L-histidine-L-lysine-R --- > glycyl-L-histidine-L-lys ine-R
(5) glycyl-L-histidina-L-lysine-R + copper(II) --- > glycyl-L-histidine-L-lysine-R:copper(II)
Within preferred embodiments, the CHL or derivative thereof and copper are present in a ratio of 1:1.

In addition to the derivatives described above, other chemical modifications may be made to alter the biological activity of the derivatives of the present invention. For instance, glycine may be replaced by a variety of other small amino acids, including alanine, serine and valine. Further, the copper(II) binding affinity of the molecule may be increased by addition of an
N-terminal amino acid, such as glycine, to convert glycyl-Lhistidyl-L-lysine to glycyl-L-glycyl-L-histidyl
L-lysine. In addition, glycine may be added to a derivative as described above to create the corresponding tetrapeptide. The binding affinity for copper(II) of the imadazole group in the histidyl residue could be modified by substitution of 3-methylhistidine for histidine or by extending the lysyl side chains by adding additional carbon atoms to the chain.

As noted above, the present invention demonstrates an inhibition of tumor growth after treatment with
GHL-Cu or a derivative thereof. Within an alternative embodiment, the GHL-Cu or selected derivative may be combined with ascorbic acid. The anti-tumor action of
GHL-Cu and . ascorbic acid occurs in the absence of observable toxic side effects in mice (such as weight loss).

The transient toxicity observed after injections of GHL-Cu (or derivatives thereof) plus ascorbate can be further reduced by addition of a free amino acid, such as glycine or histidine. This raises the affinity of the copper complex and results in a slower transfer to tissues of the body.

GHL-Cu exhibits a number of potential advantages over other copper complexes. For instance, both GHL-Cu and ascorbic acid are natural human plasma constituents. In combination, they inhibit tumor growth at concentrations that are very nontoxic to mice. Secondly, the only side effects noted were a transient (3-10 minutes) depression of breathing when very high dosages of GHL-Cu (about 0.6 ml
GHL-Cu) and ascorbic acid were administered. This was likely to have been caused by central nervous system depression through transient copper overload due to potentially loosely bound copper. As noted above, since GHL-Cu has a higher affinity for copper than other tripeptides, GHL should cause fewer side effects due to loosely bound copper.

Thirdly, even though GHL-Cu plus ascorbate had little in vivo toxicity and did not affect normal mouse weight gain, it was highly effective as a tumor inhibitor. In contrast, other copper complexes plus ascorbate, although potentially effective as tumor inhibitors, are very cytotoxic and are associated with deficient weight gain. Fourth, GHL-Cu appears to function at hormonal levels as an immune system factor. The tumor-inhibitor effect may be mediated through the immune system or, at a minimum, an additional effect of
GHL-Cu administration might be immune system stimulation.

Finally, GHL-Cu and derivatives thereof may be synthesized in nearly unlimited quantities utilizing organic synthesis procedures similar to those described herein. Therefore, the constraints of availability and cost that occur with, for instance, interferons and interleukinsr are not present.

In accordance with the present invention, one may scale-up the dosages used in mice to other warm-blooded animals (recognizing that differing metabolisms may somewhat modify the values). By way of example, Table 1 sets forth a suitable daily dosage for a warm-blooded animal weighing approximately 70 kilograms:
TABLE 1
Mouse Warm-Blooded Animal
weight = 25 g weight = 70 kg
Daily Dosage
GHL-Cu or derivative 80 mg 0.224 g Ascorbic Acid 8 ml 11.200 g
While the ascorbic acid may efficiently be given orally, the GHL-Cu or a derivative thereof may be given by intravenous or interperitoneal infusion or even injected into the tumor area itself.

In addition, GHL-Cu may be used as an oral drug through encapsulating the compound in a coating (such as hard gelatin) which would allow passage through the stomach but would release the compound in the intestine. Such methods for encapsulation are well known in the art (Baker, Richard, Controlled Release of Biologically Active Agents, John Wiley and Sons, 1986). The
GHL-Cu complex is stable between pH 4 and pH 10, and thus would persist in the intestine. As a complex, it is more stable than the free peptide against proteolytic digestion.

Further, a significant amount of GHL-Cu, or a more hydrophobic analog such as glycyl-L-histi.dyl-L-lysine octyl ester: copper(II) may pass the intestinal wall intact and pass into the blood.

The development of an orally administered, but nontoxic anti-tumor therapy as described herein permits treatment on an outpatient basis, which greatly lowers treatment costs. The therapy described herein may be especially useful after more direct tumor treatment (surgery and radiation) have reduced the main tumor masses in the body.

To summarize the examples that follow, Example 1 illustrates the synthesis of glycyl-L-histidyl-L-lysine benzyl ester:copper(II). Example 2 demonstrates the synthesis of glycyl-L-histidyl-L-lysine n-octyl ester: copper(II). Example 3 illustrates (A) the synthesis of glycyl-L-histidyl-L-lysine n-stearyl ester:copper(II), and (B) its synthesis by an alternative procedure. Based upon either procedure, one skilled in the art could substitute n-palmityl alcohol (16 carbons) for the n-stearyl alcohol (18 carbons) to yield glycyl-L-histidyl-L-lysine n-stearyl ester:copper(II). Example 4 demonstrates the effect of
GHL-Cu and ascorbate on tumors in warm-blooded animals.

Example 5 demonstrates the effect of a representative derivative of GHL-Cu and ascorbate on tumors in warmblooded animals. Example 6 also demonstrates the effect of representative derivatives of GHL-Cu and ascorbate on tumors in warm-blooded animals. Example 7 demonstrates the effect of GHL-Cu and representative derivatives thereof on tumors in warm-blooded animals.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES
Preparation of GHL-Cu for Use in Animals
CHL was purified by dissolving in glass distilled water (50 mg/ml), then centrifuging at 20,000 x g for 1 hour at 30C. This removes poorly water-soluble material remaining from the synthetic procedure. The supernatent is lyophilized, then passed through a Sephadex G-10 column at 30C in a solvent of 0.5t acetic acid. The main peak that elutes behind the solvent front (monitored by absorption at 254 nanometers) is lyophilized to dryness. GHL-Cu was prepared by combining purified GHL with equimolar amounts of cupric acetate and sodium hydroxide, then precipitated by use of ethanol addition and low temperature by published methods (Perkins et al., Inorg. Chim. Acta 67: 93-99, 1984).

Sources of chemical. Chemicals and peptide intermediates utilized in the following examples may be purchased from the following suppliers: Sigma Chemical Co.

(St. Louisr MO); Peninsula Laboratories (San Carlos, CA); Aldridge. Chemical Co. (Milwaukee, WI); Vega Biochemicals (Tucson, AZ); Pierce Chemical Co. (Rockford, IL); Research
Biochemicals (Cleveland, OH); Van Waters and Rogers (South
San Francisco, CA); Bachem, Inc. (Torrance, CA).

EXAMPLE 1
Synthesis of glycyl-L-histidyl-L-lysine benzyl ester: copper(II)
Ne-benzyloxycarbonyl-L-lysine benzyl ester was dissolved in 1:1 hexane-ethyl acetate and coupled to Na-t-butyloxycarbonyl-Nlm-benzyloxycarbonyl-L-histidine using dicyclohexylcarbodiimide as a coupling agent. Sodium bicarbonate (10%) was added and the product extracted into the organic layer. The product, Na-t-butyloxycarbony1-Nim- benzylOxyCarbonyl-L-histidyl-Ne-benzyloxycarbonyl-L-lysine benzyl ester, was crystallized from solution. The
N-terminal group of the blocked dipeptide was removed by stirring in 50% trifluoroacetic acid in dichloromethane for 30 minutes, then vacuum evaporated.

The product, Nim- benzylOxyCarbonyl-L-histidyl-Ne-benzoylcarbonyl-L-lysine benzyl ester, was coupled to benzyloxycarbonyl-glycine with dicyclohexylcarbodiimide as a coupling agent. Blocking groups were removed by catalytic hydrogenation using 10% palladium on carbon in glacial acetic acid. After lyophilization, the product, glycyl-L-histidyl-L-lysine benzyl ester, was dissolved in water and purified by ionexchange chromatography on Dowex 50 X-4 cation-exchange resin and elution with 0.1 M ammonium hydroxide, the eluate being immediately neutralized with acetic acid. A further passage through an anion-exchange column BioRex 63 at neutral pH removed breakdown products with free carboxylic acid groups.

The glycyl-L-histidyl-L-lysine benzyl ester was dissolved in water with equimolar copper acetate added.

The pH was raised to neutrality with sodium hydroxide. The solution was centrifuged at 20,000 x g for 1 hour at 30C to remove poorly water-soluble material. The supernatant was lyophilized to obtain glycyl-L-histidyl-L-lysine benzyl ester:copper(II).

EXAMPLE 2
Synthesis of glycyl-L-histidyl-L-lysine n-octyl ester: copper(II)
A mixture of Ne-benzyloxycarbonyl-L-lysine, n-octanol, benzene, and p-toluenesulfonic acid monohydrate was refluxed overnight using a Dean-Stark trap to remove water. After cooling, dry ethyl ether was added. The solution was then allowed to precipitate at OOC overnight.

A portion of the precipitated solid was added to 50 ml potassium carbonate solution and 50 ml dichloromethane.

After extraction, the layers were separated and the organic phase washed with water and brine, then dried with anhydrous magnesium sulfate. Filtration, evaporation and purification by flash column chromatography gave n-octyl
Ne-benzyloxycarbonyl-L-lysinate. The product was dissolved in tetrahydrofuran and mixed with Na-t-butyloxycarbonyl-L- Nlm-benzyloxyzarbonyl-L-histidine, isobutyl chloroformate and N-methylmorpholine. After evaporation, water and ethyl acetate were added. The product was extracted into the organic phase, which was dried with anhydrous magnesium sulfate. Filtration, evaporation and purification by flash column chromatography gave n-octyl Na-t-butyloxycarbonyl-
Nim-benzylOxycarbonyl-L-histidyl-Ne-benzyloxyCarbonyl-L- lysinate.

The product was dissolved in 50% trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated, forming n-octyl Nim-benzyloxycarbonyl-l-histidyl-Ne- benzyloxycarbonyl-L-lysinate. This was dissolved in tetrahydrofuran, and isobutyl chloroformate, N-methylmorpholine and benzyloxycarbonylglycine were added to form n-octyl benzylOxyCarbonylglycyl-Nim-benzyloxycarbonyl-L-histidyl-Ne- benzyloxycarbonyl-L-lysinate. This was dissolved in glacial acetic acid and hydrogenated overnight.

The resultant n-octyl ester of glycyl-L-histidyl
L-lysine was converted to the copper complex by the addition of an equimolar quantity of copper diacetate. The pH was raised to neutrality with sodium hydroxide. The solution was centrifuged at 20,000 x g for 1 hour at 30C to remove poorly water-soluble material. The supernatant was lyophilized to obtain glycyl-L-histidyl-L-lysine n-octyl ester: copper(II).

EXAMPLE 3
A. Synthesis of glycyl-L-histidyl-L-lysine n-stearyl
ester: copper(II)
A mixture of Ne-benzyloxycarbonyl-L-lysine, n-stearyl alcohol, benzene, and p-toluenesulfonic acid monohydrate was refluxed overnight using a Dean-Stark trap to remove water. After cooling, dry propyl ether was added to increase the total volume sixfold. The product was allowed to precipitate at OOC overnight and filtered. A portion of the filtrate was added to 50 ml potassium carbonate and 50 ml dichloromethane. After extraction, the layers were separated, and the organic phase was washed with water and brine, then dried with anhydrous magnesium sulfate. Filtration, evaporation and purification by flash column chromatography gave n-stearyl Ne-benzyloxycarbonyl-
L-lysinate.

The product was dissolved in tetrahydrofuran and mixed with Na-t-butyloxycarbonyl-Nim-benzyloxycarbonyl-
L-histidine and isobutyl chloroformate and N-methylmorpholine. After evaporation, water and propyl acetate were added and the product was extracted into the organic phase, then dried with anhydrous magnesium sulfate. Filtration, evaporation and purification by flash column chromatography gave n-stearyl Na-t-butyloxycarbonyl-Nim-benzyloxycarbonyl- L-histidyl-Ne-benzyloxycarbonyl-L-lysinate.

The product was dissolved in 50% trifluoroacetic acid in dichloromethane for 30 minutes, than evaporated, forming n-stearyl Nim-benzyloxycarbonyl-L-histidyl-Ne- benzyloxycarbonyl-L-lysinate, which was dissolved in tetrahydrofuran, isobutyl chloroformate, N-methylmorpholine and benzyloxycarbonylglycine to form n-stearyl benzyloxy carbonylglyCyl-Nim-benzyloxycarbonyl-L-histidyl-Ne-benzyl- oxycarbonyl-L-lysinate. The product was dissolved in 50% trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated, forming n-stearyl ester glycyl-L-histidyl
L-lysine.

The resultant molecule, glycyl-L-histidyl-Llysine n-stearyl ester, was converted to the copper complex by the addition of an equimolar quantity of copper diacetate. The pH was raised to neutrality with sodium hydroxide to obtain a product useful for animal studies.

By substituting n-palmityl alcohol for the n-stearyl alcohol, glycyl-L-histidyl-L-lysine n-palmityl ester may be similarly synthesized.

B. Alternative synthesis of glycvl-L-histidyl-L-lvsine
n-stearyl ester: copper (11)
N( )-benzyloxyarbonyl-L-lysine n-stearyl alcohol, p-toluenesulfonic acid monohydrate, and benzene are refluxed together using a Dean-Stark trap to azeotropically remove the evolved water. After cooling to room temperature and then adding dry ethyl ether, n-stearyl Nt )-benzyloxyzarbonyl-L-lysinate p-toluenesulfonate salt is collected by filtration, treated with- 2 M aqueous potassium bicarbonate solution, and extracted into dichloromethane. Evaporation gives the free amine, which is redissolved in dry tetrahydrofuran (THF) and added to a stirring solution of N( )-t-butyloxycarbonyl-N(im)-benzyloxycarbonyl-L-histidine, N-methylmorpholine, and isobutyl chloroformate in dry THF at -150C.

The resulting fully protected dipeptide ester is treated with ' 1/1 trifluoroacetic acid/dichloromethane at room temperature, neutralized with saturated aqueous sodium bicarbonate solution, and extracted into ethyl acetate. Evaporation gives the partially deblocked dipeptide, which is redissolved in dry
THF and added to a stirring solution of benzyloxycarbonylglycine, N-methylm6rpholine and isobutyl chloroformate in dry THF at -150C. The formed, fully protected tripeptide ester is totally deblocked by treatment with hydrogen gas in glacial acetic acid at room temperature in the presence of Pd-C catalyst. Filtration, evaporation and purification on a microcrystalline cellulose column followed by lyophili zation give the desired tripeptide ester as its triacetate salt.

As described below, GHL-Cu and ascorbic acid were tested on the growth of a solid sarcoma-180 in mice. The daily administration of 0.2 ml of 10-3 M GHL-Cu and 0.2 ml of 4% ascorbic acid (starting one day after tumor cell injection and continuing for 5 or 12 days) produced a marked inhibiting of tumor growth in three separate experiments.. Sarcoma-180 cells were injected into mice in the muscle mass of a rear leg. The sarcoma grew as a solid tumor. After 14 to 17 days, the total weight of a tumorbearing leg was compared to a nontumorous leg to determine tumor weight. The details of this method are given below.

The sarcoma-180 tumor line is carried as an ascites tumor. For testing, ascites fluid containing the tumor cells is removed from a mouse, then diluted in phosphate-buffered saline to obtain a concentration of 107 cells per milliliter.

The tumor is implanted into a rear leg of a
Swiss-Webster mouse weighing 15 to 20 grams by injecting 106 cells contained in 0.1 milliliter into the muscle mass.

The tumor then grows as a solid mass for about three weeks.

To assay tumor mass at the end of the experiment, the tumor-containing leg is excised and the skin removed. the legs are photographed and weighed. Tumor weight is taken as the average weight of tumor-bearing legs minus the weight of legs in control animals.

Treatment will be started three days after tumor implantation. (Treatment started after one day is too readily inhibited.) Mice are injected intraperitoneally with 0.2 milliliters of 10-3 M GHL-Cu in saline (pH 7.4) plus 0.2 milliliters of 4% ascorbic acid (pH 7.4). These are injected as separate solutions. Mice are treated daily for the desired treatment length. Control mice receive saline injections.

Tumor growth is determined after completion of treatment. This is minimally at about 16 to 20 days after the start of the experiment, depending on how well the control tumors are growing. There was no significant effect of this treatment on the rate of normal weight gain of the young mice.

EXAMPLE 4
Mice were injected with 107 cells, then treated for 5 to 12 days. This procedure produced an 81% to 89% reduction in average tumor size, as shown in Figure 1. In some mice, large tumors appeared to be regressing in size toward the end of the experiment.

Mice with Average Percent reduction
Test Tumors Tumor Size in Tumor Size
Control
tumors 6/6 7.4 + 1.4
GHL-Cu +
ascorbate
(12 days) 5/5 0.7 + 0.2 -89%
GHL-Cu +
ascorbate
(5 days) 4/4 1.5 + 0.4 -81%
EXAMPLE 5
Mice were injected with 106 cells, then treated for 12 days. In addition to GHL-Cu, several analogs were tested, including Gly-L-His-L-Lys-octyl ester: Cu(II) or
GHL-octyl-Cu (Figure 2).

This experiment was similar to Example 4, except care was taken to avoid absorption of the copper complexes on the membranes used for cold sterilization. To avoid this, concentrated solutions of GHL-Cu (20 mg/ml) were sterilized, then diluted to the desired concentration.

Mice with Average Percent reduction
Test Tumors Tumor Size in Tumor Size
Control
tumors 7/7 2.0
GHL-Cu +
ascorbate 2/6 0.23 -89
GHL-octyl-Cu +
ascorbate 2/4 0.31 -85%
This experiment indicated that GHL-Cu is superior to other known compositions, especially in terms of toxicity. For instance, in terms of reducing normal weight gain, PCPH-Cu was very toxic to the mice, while
GH-cadvarine was slightly toxic. (Final average weights: control mice, 25.2 grams; GHL-CU-treated mice, 24.7 grams;
GHL-octyl-Cu-treated mice, 27.2 grams; GH-cadvarine-treated mice, 22.7 grams; PCPH-Cu-treated mice, 17.1 grams.)
GHL-octyl-Cu was not toxic and appeared to be equivalent to
GHL-Cu at tumor inhibition.

EXAMPLE 6
Mice were injected with 106 cells, then treated for 12 days. Ternary complexes in which a third ligand (the amino acid, histidine) was added to raise the stability of the complex were also tested.

Mice With Average Percent reduction
Test Tumors Tumor Size in Tumor Size
Control
tumors 14/14 1.42 + 0.19
for 7 tumors
GHL-Cu +
ascorbate 0/13 None 100%
GHL-Cu-His +
ascorbate 0/7 None 100%
GHL-Cu-His
(0.5 ml) +
ascorbate 0/7 None 100%
In this experiment, all of the control mice developed tumors, but none of the treated mice (Figure 4).

At the termination of this experiment, seven of the tumor-bearing mice were used to obtain average tumor weight.

In the remaining seven control mice (with tumors about 1.4 gram), treatment with GHL-Cu and ascorbate was started, and continued for 12 days. After an additional 7 days, mice were sacrificed and tumors examined. At this time, four mice were apparently tumor-free, one had a possible tumor, and two had tumors of about 0.3 gram. Thus, there was regression of even well-established tumors by these compounds.

EXAMPLE 7
This example demonstrates the effect of GHL-Cu and derivatives thereof on the growth of tumors in mice, absent a combination with ascorbate. Briefly, mouse sarcoma M5076 was induced by injection of 106 tumorous cells into the leg muscle of mice. After day one, treatment with GHL-Cu or a derivative thereof was initiated.

By day 11, the treatment had consistently reduced the size of the tumors by 45%.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been 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.

Claims

1. A composition comprising glycyl-L-histidyl-Llysine: copper(II), for use in inhibiting the growth of tumors in warm-blooded animals.

2. A composition comprising a derivative of GHL-Cu having the general formula: EMI18.1 wherein R is selected from the group consisting of an NH 2 moiety, alkyl moieties containing from 1 to 18 carbon atoms, aryl moieties containing from 6 to 12 carbon atoms, alkoxy moieties containing from 1 to 12 carbon atoms, and aryloxy moieties containing from :6 to 12 carbon atoms, and ascorbic acid, for use -in inhibiting the growth of tumors in warm-blooded animals.

3. A composition comprising a derivative of GHL-Cu having the general formula: EMI18.2 wherein X is glycyl-L-alanyl, glycyl-L-seryl, or glycyl-L valyl, and wherein R is selected from the group consisting of an NH2 moiety, alkyl moieties containing from 1 to 18 carbon atoms, aryl moieties containing from 6 to 12 carbon atoms, alkoxy moieties containing from 1 to 12 carbon atoms, and aryloxy moieties containing from 6 to 12 carbon atoms, and ascorbic acid, for use in inhibiting the growth of tumors in warm-blooded animals.

4. The composition of any of claims 1-3 including a physiologically acceptable carrier or diluent.

-5. The composition of claim 1 wherein said composition includes ascorbic acid.