AU586220B2 - Creación de Inventos y Patentes con Inteligencia Artificial

AU586220B2 – Composition and method for treatment of tumors with udpg
– Google Patents

AU586220B2 – Composition and method for treatment of tumors with udpg
– Google Patents
Composition and method for treatment of tumors with udpg

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Publication number
AU586220B2

AU586220B2
AU53125/86A
AU5312586A
AU586220B2
AU 586220 B2
AU586220 B2
AU 586220B2
AU 53125/86 A
AU53125/86 A
AU 53125/86A
AU 5312586 A
AU5312586 A
AU 5312586A
AU 586220 B2
AU586220 B2
AU 586220B2
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Australia
Prior art keywords
udpg
prpp
tumor
composition
synthesis
Prior art date
1984-12-20
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AU5312586A
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M. Earl Balis
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Memorial Hospital for Cancer and Allied Diseases

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Memorial Hospital for Cancer and Allied Diseases
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1984-12-20
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1985-12-17
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1989-07-06

1985-12-17
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1986-07-22
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patent/AU5312586A/en

1989-07-06
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1989-07-06
Publication of AU586220B2
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patent/AU586220B2/en

2005-12-17
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Classifications

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES

A61K31/00—Medicinal preparations containing organic active ingredients

A61K31/70—Carbohydrates; Sugars; Derivatives thereof

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

A61P35/00—Antineoplastic agents

Abstract

UDPG shows tumor-specific inhibition of PRPP and this suggests that this action might prove to be useful in combination therapy with inhibitors of purine and pyrimidine nucleotide systhesis in various rescue regimens. High levels of PRPP in tumors were greatly affected by treatment with UDPG. UDPG can be used in treatment in conjuction with other anti-tumor drugs such as methotrexate, 6-mercaptopurine, 5-fluorouracil or 2,6-dimercaptopurine.

Description

COMPOSITION AND METHOD FOR TREATMENT OF TUMORS WITH UDPG
This application is a Continuation-in-Part of a co-pending U.S. application, serial number 683,S73 filed December 20, 1984.
This invention concerns a method for treatment of cancer using uridine-diphosphoglucose (UDPG) and UDPG in concert with other anti-tumor drugs.
BACKGROUND The UDPG composition has been known for some time. It has been in use in Italy for 20 years. Methods for its synthesis are disclosed. U.S. Patent 3,787,392 is¬ sued to Bergmeyer et al shows synthesis of UDPG from UMP (U-5′-MP). This is a chemical process using dicyclohexyl carbodiimide (DCC) to esterify UMP to yield UDPG. Ano¬ ther chemical process for UDPG by Bergmeyer et al. is shown in U.S. Patent 3,803,125 where UDPG (examples 4-6) is formed from U-5’MP amidate ester compounds.
U.S. Patent 3,725,201 to Sugimori et al. describes the process of obtaining UDPG by yeast action from U-5′- MP and glucose, (bottom Col. 2 -top Col. 3). U.S. Patent 3,717,547 to Nakayama et al. describes a bacterial fer¬ mentation process for production of UDPG form orotic acid or uracil.
5-phosphoribosyl pyrophosphate (PRPP) is a neces¬ sary metabolic intermediate in the synthesis of purine and pyrimidine nucleotides as well as other important molecules. Perturbation of the intracellular level of PRPP has been found to be relevant to the origin of certain metabolic diseases (Balis, M.E., (1976) Adv. Clin. Chem. 35.213; Kelley, W.N., (1974) A. Meister, ed. 1 :1). The inhibitory activity of several antimetabo- lites requires reaction with PRPP and therapeutic ef¬ ficacy may depend on the level of PRPP in target cells relative to normal host tissues (Higuchi, T., et al. (1976) Cancer Res. _36_-:-3779; Danks, M.K. et al.. (1979) Biochem. Pharmacol. 2_8:2733). Higuchi et al, – supra show

PRPP availability to convert the anti-carcinogenic pu- rine drug 6-MP to IMP.
U.S. Patent 4,297,347 of Katsunuma shows 3*-poly- phosphate pyrimidine nucleoside activity against leuke- mia in mice. U.S. patent 4,141,972 of Nishino et al. deals with mixed 51 and 31 (2′) phosphates of .’purine neucleosides with anti-leukemic activity in mice.
Ardalan et al. in Biochem. Pharm. 3_1:1509 (1982) show an increase in PRPP levels in tumor-bearing animals given certain L-glutamine antagonist drugs-DON, azase- rine and AT-125 [6-diazo-5-oxo-L-norleucine (DON)]. Also 5-FU is reported therein as active in combination with methotrexate (P1513) and therefore DON, and AT-125 could be used instead of methotrexate in combination with 5-FU to increase the PRPP pool and direct 5-RU to nucleotides.
PRPP is formed by the interaction of ATP and ribose
5-phosphate (R-5-P) catalyzed by the enzyme PRPP synthe- tase. R-5-P is related to glucose metabolism by the pentose phosphate shunt. Alteration in glucose metabo- lism, as seen in glycogen storage disease Type I, has been found to increase the PRPP level in affected cells (Kelley, W.N. et al. (1974) Supra) . Methylene blue stimu¬ lates the oxidative pentose phosphate pathway and concur¬ rently increases PRPP availability in chick liver slices (Lipstein, B., et al. (1984) Biochim. Biophys. Acta. 521:45) . These findings seem to suggest that R-5-P con¬ centrations is not saturating for intracellular PRPP production and the availability of PRPP is directly related to purine synthesis. UDPG is known to have mutiple effects on intrahepa- tic bilirubin metabolism which in turn are related to the glycogen synthesis occurring in the liver (Casciarri, I., et al. (1977) Accademia Medica Lombarda _32^:223). The precise mechanism of these interactions is not clearly -defined, but it is believed that UDPG is

involved in the induction of various enzymes including some active in carbohydrate metabolism (Okolicsanyi, L., et al (1973) Enzyme 14:366). PRPP occupies an essential role in intermediary metabolism. It is required in the 5 purine and pyrimidine biosynthetic and recycling path¬ ways. PRPP is formed in cells by the reaction of ATP and ribose-5-phosphate by the catalytic action of PRPP syn- thetase. Perturbation of the level of PRPP in cells has been found to be related to many severe metabolic distur-
10 bances including Glycogen Storage Disease Type I ((Ba¬ lis, M.E., (1976) Adv. Clin. Chem. 1_8:213; Kelley, W.N. (1974) A. Meister, ed. 14:1).
SUMMARY The present invention determines that uridine
15 diphosphoglucose (UDPG) exerts an effect on PRPP availa¬ bility in specific cells for example tumor cells either by action on cell membranes or by incorporation per se into the cytoplasm. Therapeutic potential of such .ef¬ fects is shown.
20 Brief Description of the Drawing
Figure 1 describes UDPG transport into liver cells.
DES , CRIPTION –
Figure 1 is a plot following update of H-UDPG into liver cells at 10, 30 and 40 min.
25 Infusion of UDPG: Mice of various strains, Balb/C,
CDQ, CRF with and without tumor implants, maintained on
Purina rat chow and tap water, were infused intraperi- toneall’y as described previously, (Yip, L.C., et al.
(1984) Biochem. Pharmacol. 2_9:2888) with a phosphate-
30 buffered saline solution of UDPG at a constant rate of 0.8 mg/day for five days. Two concentrations of UDPG
! were ^±sed; 8m and 80mM. Control mice received only
;’ buffered saline solution.
After the five-day infusion the mice were killed, 35 their tissues excised and homogenized in cold PBS solu-

tion (1.5ml per gram tissue) to extract PRPP. The homo- genates were centrifuged at 48,000Xg for 10 min. Super- natants thus prepared were assayed for PRPP and enzyme activity. Pellets were resuspended in the original vo- lume of 10 mM Tris buffer (pH 7.4), 0.125M sucrose, 1 mM DTT and 2% Triton. The supernatants from the second extraction were used for the assay of membrane-bound PRPP synthetase.
Isolation of spenocytes and hepatic cells: The organs were gently teased apart with forceps over a fine tea strainer into PBS or minimum essential culture me¬ dium (MEM) containing 0.1 M potassium phosphate buffer pH7.4 with and without added effectors. The cells were incubated at 37 C for 30 min and then washed once with PBS before they were lysed in PBS by freezing and thawing three times. The supernatant solutions were as¬ sayed for PRPP.
Determination of’ PRPP concentra_tion: The PRPP was determined through the reaction of C-orotic acid with PRPP to form 14C-carboxyl orotidine-5′-phosphate in the presence of orotate phosphoribosyltransferase followed by the liberation of C0 under the catalysis by OMP decarboxylase (Tax W.J.M., et al. (1977) Clin. CHim. Acta. 73:209). The reaction mixture (100 Microliters) contained 25 microliters of 0.1M Tris-HCl (pH 7.4), 25 microliters of enzyme mix (Sigma, St. Louis, MO. contain¬ ing 2-3 units/ml in Tris buffer), 25 microliters 0.5 mM
14 C-carboxyl orotic acid (5mCi. mmole), 25 microliters of 40mM MgCl,- solution and 25 microliters of clarified tissue homogenate. The test tubes containing the reac¬ tion mixture were sealed with serum caps each of which had suspended from it a plastic well (Kontes Vineland N.J.) containing 200 microliters of protosol (New Eng¬ land Nuclear, Boston, Mass.). The tubes were incubated at 37 for 10 min before they were placed in an ice~»bath

and 0.25ml of 10% TCA was injected through the serum cap. Tubes were returned to a 37° water bath and incu¬ bated for 1 hour. At the end of this time the tubes were uncapped, the outsides of the wells wiped clean and the suspended wells cut. The wells were placed in scintilla¬ tion vials and 10ml of counting cocktail added. The vials were shaken vigorously and their radioactivity determined.
Enzyme assays: PRPP synthetase was assayed as des- cribed previously (Yip, L.C., et al. (1980) AM. J.
Physiol. 239:G266) . The assay is based on the coupling of the PRPP assay using C-adenine with ARPTase to form C-AMP with the synthetase reaction (in the presence of
ATP and R-5-P). Protein determination: Protein concentrations were determined by the method of Lowry et al (Lowry, O.H., et al. (1951) Biol. Chem. 193:265).
Adenine incorporation studies: Isolated balb/c mouse splenocytes and hepatocytes were incubated at 37 C in MEM with 0.1M KPB(pH 7.4) and 0.5mM l4C-adenine
(specific activity 5Ci/mole) with and without lOmM UDPG.
After 30 minutes of incubation cells were washed once with PBS before they were lysed in PBS by alternate freezing and thawing (3x). The 14C-nucleotides thus formed were assayed by chromatography on DEAE-cellulose paper and subsequent assay of the radioactivity. 3 H-UDPG incorporation studies: Isolated mouse hepatocytes were incubated at 37°C in MEM with 0.21mM 3 H-UDPG. After 30 minutes the cells were layered on top of 1.0 ml of dibenzylamine and centrifuged at 4 im¬ mediately to separate the cells from the incubation medium. The cells were lysed in 0.2 ml of KPB (pH 7.4) by vigorous vortexing. The solution was clarified by centrifugation at 30,000 xg for 15 minutes, and 25 microliters of 10% TCA were added to 0.1 -ml–o-f superna-

tant solution. The initial pellet (containing cell mem¬ branes) was solubilized in 2% Triton in lOmM Tris (pH 7.4) buffer and deproteinized. Solubilized membrane and cytoplasm preparations were anlysed for H-containing components by HPLC fractionation through a PXS-102SAX column with a linear gradient from 0.007M KH^PO, to 0.25M KH2P04, 0.5M KC1 (pH4.5). The flow rate was 0.8 ml/ minute and fractions were 0.4ml each. The uv absorb- tion and the radioactivity profiles were compared to values of known standards. The radioactivities of the fractions were determined by liquid scintillation count¬ ing.
The following examples are for illustrative pur¬ poses only and are not meant to limit the invention to the specific examples shown.
EXAMPLE I Changes in PRPP levels and PRPP synthetase of Balb/c mouse liver and spleen upon UDPG treatment: Table I shows that treatment with UDPG increased amount of PRPP found in the liver of Balb/c mice. Lower UDPG concentration, introduced by injection of 0.5 ml of 2mg/ml of UDPG per day caused only a 137= increase of liver PRPP. At slightly higher dose level produced by continuous infusion a 3.3. fold increase in PRPP level was observed. Contrary to expectatation, a 10 fold in¬ crease in UDPG concentration gave only a 197° increase in PRPP level. The changes in liver PRPP synthetase acti¬ vity did not correspond well with that observed with its reaction product. The 30-37% increase of the enzyme activity.- at » low dosage UDPG treatment was diminished when the higher* concentration of UDPG was used. Mouse spleen was shown ‘ to be less sensitive to the UDPG effect. No change, in either PRPP or synthetase. level was seen at low UDPG .concentration. A slight (about 30%,) increase in–PRPP and synthetase. in the cytosol-was noted
SUBSTITUTE SHEET

when 80 mM was infused. UDPG was found to have no effect on PRPP synthetase ; activity when it was added to the reaction mixture at concentrations up to lOmM.
EXAMPLE II
Effects of UDPG and G-6-P on spenocyte PRPP level: Isolated splenocytes incubated in media containing vari¬ ous amounts of UDPG or G-6-P were observed to have an increased intracellular PRPP level (Table II). However, the effect of G-6-P was not evident below a concentra¬ tion of 5.5mM, while UDPG effect was essentially maximal at 0.7mM. The effect of G-6-P increased with concentra¬ tion up till 22 mM while at the highest concentration of UDPG used (l4.4mM) the effect of UDPG appeared to begin to decrease.
EXAMPLE III
Effects of UDPG on tumor-bearing mice: Mammary tumorbearing DCR mice and CRF mice with transplanted colon tumors were infused with UDPG for 5 days . At the end of the treatment the tumors were excised and their weight and the level of PRPP and PRPP synthetase were determined. In one set of experiments the average size of the mammary tumors was 50% of the controls. The decrease although not statistically significant was de¬ termined after only five days of treatment. Nevertheless the effect is suggestive of possible specificity as is the decrease in tumor level of PRPP compared to the essentially unchanged levels in the host mice (Table III). In a similar experiment (Table IV) PRPP levels of two other proliferating tissues, bone marrow and intes¬ tinal» mucosa, .were also assayed. Although there was decrease in vcol»onic and bone marrow cells it was much less than that • observed in the tumors. The low values seen in the small bowel may be related to the very high phosphatase level found in this tissue.

EXAMPLE IV
Studies on the incorporation in vitro of C- adenine into splenocytes: Incorporation of adenine into isolated splenocytes was stimulated by the presence of lOmM UDPG in the incubation medium (Table 5). This result is consistent with the observation (Table II) that UDPG increased the level of PRPP. The PRPP partici¬ pates in the retention of purine bases by phosphoribosy- lation of the aglycone (Wohlhueter, R.M., et al. (1982) J.B.C. 257:2691).
EXAMPLE V
Studies on the incorporation in vitro of UDPG into hepatocytes: A study of the transport of triti-ated UDPG into liver cells was done at three time periods; 10., 30 and 40 minutes. The cells were prepared as single cell suspensions under conditions that left them viable in terms of several biochemical parameters and vital dye exclusion. The enclosed plot (fig.l) shows the fraction of the material in each of the three compounds that were derived from the glucose moiety of the UDPG.
As can be seen there is an uptake of intact UDPG which is maximal at thirty minutes. By forty minutes the cells have begun to die in this medium and the UDPG has broken down. At thirty minutes about 1/6 of the label is as UDPG. Thus it is quite apparent that a major amount of the UDPG enters the cells intact. The data further show that the initial product is glucose phosphate which in time is dephosphorylated to glucose.
It is obvious that the UDPG does not break down before entering the cell both from its presence intact, but also -from the fact that the breakdown products do not cause the same metabolic response that UDPG does.
Other preliminary studies show the presence of some intact UDPG. in the cell membranes. This suggests that

there may be an active transport mechanism responsible for the entry of UDPG into the hepatic cell.
The results presented here indicate that the intra- cellular level of PRPP in animal tissues is greatly affected by the extracellular presence of UDPG. The – resulting alteration of PRPP level by UDPG is not linearly dose-related. When higher concentrations of UDPG were used Ln vivo a maximal value was reached and concentrations beyond this had a negative effect. The changes in PRPP that were induced by UDPG were also tissue-specific, mouse liver was more sensitive than was the spleen. This latter specificity may well be related to the therapeutically beneficial effects of UDPG.
Our data on the permeability of cell membranes to UDPG, indicate that a significant amount (about 20%) of the UDPG, even though it is a highly polar compound, does readily pass through the membrane unchanged. A large fraction of the UDPG added to incubation media, however is found in the cell as glucose phosphate, indicating cleavage by membrane-bound UDPG phosphatases after penetration of the cell. Eventually all the UDPG that entered the cells was degraded to glucose phosphate and glucose. The in vitro studies show that G-6-P is no ‘ the active form and several tests show that uridine does not lead to changes like those seen with UDPG (Bossa, R., et al. (1975) Biochem. 3_:5315 Pinelli, A., et al. (1976) Biochem. Pharm. 2^:623; Pinelli, A. et aϊ.- (1981) IN: Alcuni Aspetti Fisiopatolieis Diagnostici e Tera- peutici in Epatologia. Pacini Ced. P. 121-128). Thus,’ the most likely mechanism of action is through- -the membrane. The effects of UDPG on cell metabolism .do-not – appear to be limited to enzyme induction. Others have reported that UDPG can have effects under, conditions that bar enzyme induction (Chiesara, E., et al. (1980) Rivista di Farmacologia e Terapia XI, 103-111).

Since PRPP is involved in many metabolic pathways, changes in intracellular PRPP levels are often not accom¬ panied by obvious changes in the synthetase. PRPP synthe¬ tase has been shown to in many respects to be a self regulating enzyme (Becker, M.A. , et al. (1977) J. Biol. Chem. 252:3911) . Its activity can be controlled by fur¬ ther aggregation or dissociation of molecular aggregates (Becker, M.A. , et al. (1977) J. Biol. Chem. 252:3911, Yip, L.C., et al. (1978) Biochem. 17:3286). This pheno- menon may explain some of the observations reported here and also suggests that the effect of UDPG or PRPP levels is most likely not due to the induction of additional synthesis of the enzyme, but to alteration of the confor¬ mation of the enzyme and the expression of this in a different catalytic function of the synthetase. Under the assay conditions employed it is quite possible that these differences are masked. This line of reasoning is consistent with .the rapid increase in PRPP seen in spenocytes following incubation in medium containing UDPG.
Glucose-6-phosphate ~ can easily be converted intra- cellularly into ribose-5-phosphate and thus activate the production of PRPP. However,- the different effects that we have observed between the G 6-P and UDPG incubation indicates that the UDPG action is not due solely to its increase in intracellular glucose concentration.
Contrary to^ the effect of UDPG on normal cells, UDPG was seen to» decrease PRPP concentration in tumor cells. Since with tumor _cells only one very high UDPG concentration was studied it is difficult to assert that the reverse effect is due to..the specific sensitivity of the tumor, as was notecl between liver and spleen, or due to the nature of the tumor cell membrane. Nevertheless other rapidly proliferating tissues were also affected albeit less so. Others have reported diferent changes in

_u_
PRPP pool sizes in tumors and gut (Ardalan, B., et al. (1982) Biochem. Pharmacol. _31:1509). These observations plus the fact that the weights of the mammary tumor were actually decreased after only five days of exposure to UDPG suggests UDPG as a therapeutic agent especially in combination with inhibitors of the PRPP utilizing steps in purine and pyrimidine synthesis.
Thus the present invention contemplates the use of UDPG alone, ore preferably in combination with known anticancer drugs, preferably of the class of compounds which deplete the purine and pyrimidine pool, to treat tumors in mammals. The types of tumors to be treated, especially in humans, would include tumors of the lung, colon, rectum, breast, prostate, ovaries, head and neck, bone, pancreas, liver, kidney, stomach, bladder and gene- talia, and melanomas, soft tissue sarcomas, lymphomas and leukemias.
The UDPG could be administered alone or in combina¬ tion with other anti-cancer drugs, in single or multiple dosages, by the parenteral (I.V. or I.M.) route. In normal formulations the UDPG when administered orally will tend to break down in the stomach. Special formula¬ tions might be developed which would prevent such UDPG decomposition, and in that event such UDPG containing special formulations could be administered by the oral route.
The UDPG will be administered to the patient in the amount of 1 to about 300 mg per kg of body weight per day, preferably about 50 to 200 mg per kg per day, and most preferably about 100 mg per kg per day. The higher dosage levels of UDPG, such as in the range of 100 to 300 mg per kg per day, would normally be given by continuous infusion. The inhibition of PRPP synthesis per se is believed to be toxic to tumors, so that the –

UDPG could be administered alone to inhibit tumors. However, it is most preferred that the UDPG be admini¬ stered together with an anti-tumor compound which is an inhibitor of purine synthesis, pyrimidine synthesis, or glutamine metabolism. These other anti-tumor drugs can be administered in combination with the UDPG, or in a sequential fashion with the UDPG.
The purine synthesis inhibitors can be antifolates in general, and especially methotrexate. The metho- trexate or other antifolate would be administered in conventional amounts, using conventional methods of ad¬ ministration, such as, for instance, an IV administra¬ tion once a week of from 4 to 400 mg/kg per single weekly dose, or in corresponding lesser daily maounts. Normally the dose would be monitored by following the serum methotrexate level. It is known to use a rescue technique when methotrexate is administered in the treat¬ ment of tumors, and it is contemplated that such a rescue technique would be used if UDPG and methotrexate were administered together. The rescue agent is pre¬ ferably thymidine which could be administered, for in¬ stance, in an amount of 1 gm per sq. meter of patient body surface per 24 hour period, plus serine in an amount of 100 to 300 mg/day in divided doses, and a purine (such as for instance, hypozanthine in am amount of about 300 and 1500 mg per day in divided doses, and allopurinol at 200 to 400 mg/day in divided doses), or other known rescue agent could be used.
It is known that inhibitors of glutamine metabolism also block purine synthesis, as glutamine is a precursor to purine. Glutamine metabolism is inhibited by azase- rine, 5-diazo-5-oxo-L-norleucine (DON) , and other com¬ pounds and it is contemplated that these glutamine syn¬ thesis inhibitors could also be administered with UDPG, with t-he– -gliitam-ύae synthesis inhibitor being admini-

stered in conventional methods of administration and in conventional amounts, such as, for instance, 5 to 15 mg/kg/day, for 2 to 10 days for DON, either alone or sequentially with the UDPG. It would normally be prefer- red to use a rescue technique with glutamine synthesis inhibitors, and the rescue -would be with the purine rescue agents described hereinabove.
Inhibitors of pyrmidine synthesis include inhibi¬ tors of aspartate transcarbamylase such as N(phos- phonacetyD-Laspartate (PALA), orotidylate decarboxylase such as pyrazafurin, dihydroorotase if reasonably potent compounds become available and general depletors of available cellular pyrimidine pools such as galactosa- mine. The pyrimidine synthesis inhibitors are usually used in conjunction with a rescue technique, analogous to that for purine synthesis, with uracil or cytosine being the rescue compound. The rescue compound and the pyrimidine synthesis inhibitor are administered by con¬ ventional methods of administration and in conventional amounts. For instance, the PALA pyrimidine synthesis inhibitor can be administered in amounts of 0.25 to 5
2 g/m /day by injection, and pyrazafurin can be admini-
2 stered by I.V. in a single dose of 1-300 mg/m , or by infusion at a dose of say 250 mg/day for 1-6 days, and the cytosine rescue compound in amounts of 5 to 100 mg/kg/day by infusion, and for uracil approximately 3 to
50 mg/kg/day by infusion.
As will be apparent from the above, the presence of
UDPG in the body of a patient having a tumor decreases the PRPP levels in the tumor cells. At the same time, the presence of UDPG maintains or increases the PRPP levels in normal living cells, so that the use of UDPG assists in the specificity of treatment of the tumor cells as opposed to the normal living cells. The average size of the tumor is decreased’ after prolonged treatment

with high dosages of the drug UDPG. The reduction of the mass of the tumor in test animals after treatment with UDPG suggests that the life-span of the treated animals be prolonged, which in turn suggests that the life of human tumor patients could be correspondingly prolonged. The dual action of UDPG, which decreases the PRPP level in tumor cells while increasing or at least main¬ taining the PRPP levels in non-tumor, normal cells, even if these normal cells are damaged, is of considerable therapeutic relevance. The dualistic behavior of UDPG may favor the susceptibility of the tumor cells to the action of the antimetabolites and/or other antineoplas- tic agents, while at the same time exerting a protective affect on the normal cells of the tumor patient. The selective decrease of PRPP in the tumor cells, incuded by the drug UDPG, makes these tumor cells more acces¬ sible to ‘anti-tumor agents, while at the same time protecting the other cells of the mammal from the action of the same anti-tumoral agents. As will be clear from the therapeutic viewpoint, an immediate and approximate result of the above could be not only better efficacy of the antieoplastic treatment, but also a reduction of the cumulative dosage level of co-administered anti-tumor agents. It is therefore be- lieved that the relevant discoveries of the present invention include the following: a. Direct anti-tumor effect of UDPG when admini¬ stered at various dosages. b. More efficacy of known anti-tumor agents when co- administered with UDPG. c. Reduction of the side effects of the known anti- tumor agents when administered with UDPG. d. Better tolerability and drastic decrease of the toxic effects of known anti-tumor agents when co- administered with UDPG.

The prior use of UDPG acknowledged above has been in the treatment of chronic and acute hepatitis, the hyperbilirubinemias and as adjuvant in the hepatic de- toxication processes. The administration has been by the parenteral route (I.V. or I.M.) and the daily dosages has varied from 50 to 3-500 mg. Thus it will be noted that the prior use of UDPG has been at a generally lower daily dosage level than contemplated for the treatment of tumors described herein, as it is generally preferred to administer UDPG in an amount of at least 2000 mg per day in the treatment of tumors. Also, of course, in the prior use of UDPG to treat liver diseases, inhibitors of purine synthesis, pyrimidine synthesis or glutamine mtabolism would not be administered. Furthermore, the prior use of UDPG in the treatment of liver diseases has not involved administration by the oral route. Thus in addition to the method of treatment, the present inven¬ tion generally contemplates significantly different phar¬ maceutical compositions to be used for the treatment of tumors.
Based on the test data obtained to date, which data is reported above, it is believed that the best mode of the present invention would be the use of UDPG in an amount of about 100 mg per kg per day, combined with the administration of methotrexate, followed by a purine synthesis rescue technique using thymidine, serine and a purine.

TABLE I

CHANGES IN PRPP LEVEL AND PRPP SYNTHETASE AFTER UDPG
TREATMENT
LIVER SPLEEN
, 5′ PRPP PS PRPP PS ow
Inject lmg/day 206+58 233+20 4.7+18 6.5+3.5
• Infuse
237+95 772+54 4.6+1.6 6.0+1.6 242+60 213+32 7.7+0.5 7.4+3.7
276+12 328+43 4.5+0.4 4.4+0.6 228+32 302+27 7.6+0.8 9.4+1.4

Balb/c mice were used .C = control, T treated animals. Control mice received injections or infusions of PBS.

TABLE II
EFFECTS OF UDPG AND B-6-P ON PRPPP LEVELS OF ISOLATED SPLENOCYTES
Concentration of drugs (mM) PRPP levels pmoles/mg protein G-6-P 0 981
1.1 969
5 .5 1418
11 1510
22 1998 UDPG 0 981
0.7 1825
3 .6 1937
7 .2 2135
14.4 1508 Cells were incubated in vitro in MEM containing indicated compound. Assays were performed on soluble fraction of homo- genates.
UBSTITUTE SHEET

TABLE III
EFFECTS OF UDPG ON TUMOR-BEARING MICE
LIVER SPLEEN TUMOR
Cntr Tr Cntr Tr Cntr Tr MAMMARY CARCINOMA IN CDg MICE
PRPP nmoles per 153.08 119.58 100.37 127.16 1322.57 319.85 g protein
SYNTHETASE I.U. in 4.32 3.81 1.34 1.17 2.25 1.56 lysate
SYNTHETASE
I.U. in 1.54 1.19 2.15 2.12 1.56 1.22 membrane
COLON TUMOR IN CRF MICE
PRPP nmoles per 213.75 218.16 176.7 197.92 1116.83 625.56 g protein
SYNTHETASE I.U. in 4.8 4.58 8.43 6.72 6.76 5.77 lysate
SYNTHETASE
I.U. in 1.32 1.37 4.60 4.18 2.25 2.51 membrane Tr = UDPG infused; Cntr = Saline infused controls

TABLE IV
EFFECT OF UDPG ON PRPP LEVELS OF TISSUES
OF TUMOR-BEARING MICE
TISSUE CONTROL TREATED
TUMOR 1704 385
COLON 230 112
JEJUNUM 20 49
BONE MARROW 131 82
Mice were CDQ carrying second generation mammary tumors

TABLE V
EFFECT OF UDPG ON THE INCORPORATION
OF 1 C-AD
INTO SPLEEN AND LIVER CELLS
14C-AMP FORMED pmoles/mg protein/minute
Hepatocytes in PBS 93
Hepatocytes in lOmM UDPG 139
Splenocytes in PBS 76 Splenocytes in lOmM UDPG 204

Claims (27)

What is Claimed:

1. Method of affecting the intracellular PRPP pool size in human cells which comprises exposing normal or tumor cells to pharmacologically active levels of UDPG wherein the PRPP levels in normal cells are increased and the PRPP levels in tumor cells are decreased.

2. Method of treating cancer in mammals which comprises exposing mammals with cancer to pharmacologically ef¬ fective doses of UDPG.

3. Method of Claim 2 wherein the treatment with UDPG is combined with pharmacologically effective doses of anticancer drugs.

4. Method of Claim 3 wherein the anti-cancer drugs are selected from the group consisting of purine and pyrimidine analogs and mixtures thereof.

5. Method of Claim 4 wherein the analogs are selected from the group consisting of 5-fluorouracil, 6-mercap- topurine, 2,6-diaminopurine and mixtures thereof.

6. Method of Claim 2 wherein the UDPG is active in liver and spleen cells.

7. Method of Claim 3 wherein the drug plus UDPG is .. active in liver and spleen cells.

8. Method of treating a tumor in a patient in need of such treatment, said method comprising administering to said patient’ a anti-tumor effective amount of UDPG.
TITUTE SHEET

9. Method of Claim 8, wherein the tumor is selected from the group consisting of tumors of the lung, colon, rectum, breast, prostate, ovaries, head and neck, bone, pancreas, liver, kidney, stomach, bladder, and genetalia, and melanomas, soft tissue carcomas, lym- phomas and leukemias.

10. Method of Claim 9, wherein said effective amount is about 3 to about 300 mg/kg of body weight of said pa¬ tient/day.

11. Method of Claim 10, wherein said amount is 50 to 200 mg/kg/day.

12. Method of Claim 11, wherein said amount is about 100 mg/kg/day.

13. Method of Claim 12, wherein the UDPG is administered parenterally.

14. Method of Claim 13, wherein the UDPG is administered by continuous infusion.

15. Method of Claim 8, wherein the method also includes administering to said patient an effective amount of at least one member selected from the group consis¬ ting of inhibitors of purine synthesis, inhibitors of pyrimidine synthesis, and inhibitors of glutamine metabolism. . v
I

16. Method of Claim 15, wherein said member i’s an inhibi¬ tor of purine synthesis.

17. Method of Claim 16, wherein said inhibitor is metho¬ trexate. .

18. Method of Claim 15, wherein said member is an inhibi¬ tor of pyrimidine synthesis.

19. Method of Claim 15, wherein said member in an inhibi¬ tor of glutamine metabolism.

20. Composition for the treatment of tumors comprising an anti-tumor-effective amount of UDPG and a pharma¬ ceutically acceptable carrier therefor, said composi¬ tion in a form suitable for parental administration in an amount of at least 2000 mg per day of said UDPG.

21. Composition for the treatment of tumors, said com¬ position comprising UDPG in an amount sufficient to reduce the level of 5-phosphoribosyl pyrophosphate in the tumor cells and an anti-tumor-effective amount of at least one member selected from the group consisting of inhibitors of purine synthesis, inhibitors of glutamine synthesis and inhibitors of pyrimidine synthesis.

22. Composition of Claim 21, wherein the UDPG is in a form to be administered parenterally.

23. Composition of Claim 22, wherein the UDPG is in a form to be administered by continuous infusion.

24. Composition of Claim ’21, wherein said member is an inhibitor of purine synthesis.

25. Composition of Claim 24, wherein said inhibitor is methotrexate.

26. Composition of Claim 21, wherein said member is an inhibitor of pyrimidine synthesis.

27. Composition of Claim 21, wherein said member is an inhibitor of glutamine metabolism.

AU53125/86A
1984-12-20
1985-12-17
Composition and method for treatment of tumors with udpg

Ceased

AU586220B2
(en)

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1984-12-20

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Udpg as a rescue agent in cancer therapy after the¹administration of antipyrimidine or related anti-tumor¹agents with or without bau

AU4100193A
(en)

1991-12-16
1993-08-03
Boehringer Mannheim Italia S.P.A.
Use of udpg as cancer therapy rescue agent

US6472378B2
(en)

1998-08-31
2002-10-29
Pro-Neuron, Inc.
Compositions and methods for treatment of mitochondrial diseases

US6683060B2
(en)

2002-02-25
2004-01-27
Advanced Gene Technology Corp.
Matrix metalloproteinase and tumor necrosis factor inhibitors

WO2007057440A2
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2005-11-17
2007-05-24
Innate Pharma
Improved methods of using phosphoantigen for the treatment of cancer

WO2008059052A1
(en)

2006-11-17
2008-05-22
Innate Pharma
Improved methods of using phosphoantigen for the treatment of cancer

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(en)

2019-06-25
2023-08-18
中国科学院分子细胞科学卓越创新中心
Use of UDP-Glc in lung cancer metastasis assessment

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2019-12-20
2021-12-21
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Boehringer Mannheim Gmbh, 6800 Mannheim

Process for the production of nucleoside diphosphates or their esters

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1973-04-03
Marukin Shoyu Kk
Manufacture of uridine-5-diphosphoglucuronic acid

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1994-07-28

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1990-01-08

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1986-08-08

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1994-06-22

JPH0660103B2
(en)

1994-08-10

NZ214616A
(en)

1989-04-26

DK379886A
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1986-08-08

JPS62501704A
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1987-07-09

AU5312586A
(en)

1986-07-22

EP0205586A1
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1986-12-30

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1986-05-22

WO1986003678A1
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1986-07-03

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1994-09-29

ATE107509T1
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1994-07-15

ZA859740B
(en)

1986-07-30

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