Use of Phlorizin Or Its Derivatives in Combination with a Chemotherapeutic Agent for the Treatment of Cancer

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~" ' MM II II II Ml I Ml I Ml II II II I II J European Patent Office CO Publication number: 0 172 721 B1 Office europeen„ des brevets

@ Date of filing: 13.08.85

The file contains technical information submitted after the application was filed and not included in this specification

® Priority: 13.08.84 US 640170 © Proprietor: LeVeen, Harry H. 321 Confederate Circle @ Date of publication of application: Charleston, SC 29407(US) 26.02.86 Bulletin 86/09 Proprietor: Leveen, Eric G. © Publication of the grant of the patent: 321 Confederate Circle 03.03.93 Bulletin 93/09 Charleston South Carolina 29407(US)

© Designated Contracting States: Proprietor: LeVeen, Robert F. DE FR GB IT 312 Lombard Street Philadelphia Pennsylvania 19147(US) © References cited: US-A- 3 523 937 @ Inventor: LeVeen, Harry H. 321 Confederate Circle CHEMICAL ABSTRACTS, vol. 67, no. 25, De- Charleston, SC 29407(US) cember 18, 1967, page 10834, ref. no. Inventor: Leveen, Eric G. 115099q; Columbus, Ohio, US; A.R. KOLBER 321 Confederate Circle et al.: "Evidence for carrier mediated trans- Charleston South Carolina 29407(US) port of monosaccharides In the Ehrllch as- Inventor: LeVeen, Robert F. cites tumor cell" 312 Lombard Street Philadelphia Pennsylvania 19147(US) CHEMICAL ABSTRACTS, vol. 73, no. 25, De- 00 cember 21, 1970, page 223, ref. no. 129240q, Columbus, Ohio, US; M.I. ILINICH et al.: © Representative: McCall, John Douglas et al "Effect of glucose and ATP on the resplra- W.P. THOMPSON & CO. Coopers Building CM tlon of ascitic cancerous cells In the pres- Church Street ence of phlorizin and ouabain" Liverpool L1 3AB (GB) CM

Note: Within nine months from the publication of the mention of the grant of the European patent, any person ® may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition CL shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee LU has been paid (Art. 99(1) European patent convention). Rank Xerox (UK) Business Services (3. 10/3.5x/3.0. 1) EP 0 172 721 B1

DIALOG INFORMATION SERVICES, Data Base, EMBASE 74-79, ref. no. 77012446 E.WALUM et al.: "Kinetics of 2-deoxy-D-glucose transport system In the human K-562 chronic myelogenous leukemia-derived cell"

DIALOG INFORMATION SERVICES, Data Base, EMBASE 80-81, ref. no. 81246326 J.C. DOZIER et al.: "The hexose trnasport system In the human K-562 chronic myelogenous leukemia-derived cell"

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Description glucose to lactic acid. Chemical Abstracts 73 129240q, 1970 de- This application relates to treatment of cancer scribes the effect of glucose and ATP on the and in particular provides a method of inhibiting the respiration of ascitic cancerous cells in the pres- growth of tumors (cancer) cells by blocking glu- 5 ence of phlorizin and ouabain. cose transport across the cell membrane. Chemical Abstracts, 67 115099q, 1967 de- Many cancer cells are characterized by their scribed evidence for carrier-medicated transport of excessive glycolysis of glucose whereby glucose is monosaccharide in the Ehrlich ascites tumor cells converted to lactic acids (fermentation) whereas and concludes that phlorizin inhibited sugar trans- normal cells break glucose down to carbon dioxide io port. and water (respiration) as described by Warburg in EMBASE 74-79, 77012446 describes kinetics "The Metabolism of Tumors" published by Richard of 2-deoxy-D-glucose transport into cultured mouse R. Smith Inc., New York, 1931. The rate limiting neuroblastoma cells. Inhibitors such as phloretin factor in this excessive tumor glycolysis is depen- and phlorizin were used to show that the transport dent on the rate at which glucose can be trans- 75 process involving the take up of glucose by these ported across the cell membrane. This had led cells was passive. some investigators to believe that the primary bio- EMBASE 80-81, 81246326 describes the hex- chemical defect is the increased rate of glucose ose transport system is the human K-562 chronic transport across the cell membrane. The intracel- myelogenous lukemia-derived cell. It showed that lular transfer of glucose is directly related to the 20 D-glucose, phloretin and phlorizin competitively in- growth potential of the cancer cell. (J. Nat. Cancer hibited the transport of 3-0-methyl-glucose. Inst. 62: 3, January 1979). Blocking of glucose In accordance with one aspect of the present transport across the cancer cell membrane de- invention there is provided a product containing a prives the cancer cell of its energy needs. The compound or compounds for inhibiting glucose glucose analogue 5-thio-D-glucose, which blocks 25 transport into malignant neoplastic cells a phar- anaerobic glycolysis, has been shown by Kim et al, maceutical^ suitable carrier therefore and a Science, Volume 200, pages 206 and 207 to sen- chemotherapeutic agent as a combined preparation sitize tumor cells to heat which raises the meta- for simultaneous, separate or sequential use in bolic rate and oxygen requirement of cancer cells cancer therapy. as well as normal cells so that their energy require- 30 In accordance with another aspect of the inven- ments exceed their energy supply thus causing tion there is provided a pharmaceutical composition destruction of the cancer cell. for the treatment of cancer comprising: Phlorizin is a glucose, 1- [2-(BD- a) a compound or compounds for inhibiting glu- glucopyranosyloxy)-4,6-dihydroxyphenyl]-3-(4- cose transport into malignant neoplastic cells, hydroxyphenyl)-1-propanone, which has been 35 b) a pharmaceutically acceptable carrier, and known and investigated both in humans and ani- c) a chemotherapeutic agent. mals for many years, Benedict et al, Proceedings The invention is also directed to inclusion of of the Society for Experimental Biology and Medi- Lonidamine, which has been found to have a syn- cine, Volume 11, pages 134-136(1913-14). U.S. ergistic effect with phlorizin and heat on cancer Patent No. 3,523,937, issued August 11, 1970, de- 40 cells. Lonidamine and its effects as a selective scribes phlorizin analogues and their usefulness in inhibitor of aerobic glycolysis in murine tumor cells the elimination of glucose from animals. Phlorizin is is described in J. Nat. Can. Inst. 66, page 497 known to block the entry of glucose into cells, and (1981). The administration of phlorizin and it has now been found to be able to block glucose Lonidamine need not be simultaneous although this entry into cancer cells as well. 45 is preferred for ease of administration. Cancer cells unlike normal cells require both These compounds are effective in suppressing glucose and oxygen to satisfy energy needs. The the growth of cancer even in the absence of hyper- blocking of glucose entry impedes vital processes thermia. The pharmaceutical composition according of the cancer cell and at elevated temperatures to the invention comprises phlorizin, its glucuronide becomes lethal for cancer cells. By contrast, heat 50 or 4-deoxyphloretin-2-D-glucose and a and the reduction of its glucose supply is well chemotherapeutic agent such as Lonidamine to- tolerated by normal tissues. Body tissues can sat- gether with a pharmaceutically acceptable carrier. isfy energy needs by the metabolism of fatty acids These compositions may be solid or liquid and can in the presence of oxygen as the sole energy be used in forms currently used in medicine such source. The oxygen tension in cancerous growths 55 as tablets, capsules, syrups and injectable prepara- is extremely low and is inadequate for the metabo- tions. Because of its poor absorption, it is prefer- lism of fat in cancer cells. Hence, the tumor derives ably administered parenterally dissolved in pro- most of its energy by the anaerobic breakdown of pylene glycol. Orally acceptable carriers are those

3 3 EP 0 172 721 B1 4 currently used in medicine such as calcium car- concentration and the number of minutes of collec- bonate, starch, lactose, talc, magnesium stearate, tion. This will determine how many ml of blood gum acacia, aqueous alcohol, glycol or oil solutions were completely cleared each minute of xylose. or suspensions. Xylose is a nonmetabolized sugar which is not The daily dosage to be administered depends 5 reabsorbed by the tubules. The glucose clearance on the lean body mass, diet and carbohydrate and the xylose clearance become identical when tolerance of subject in need of treatment. Gen- complete phlorinization has been obtained. Other erally, it has been found suitable to administer 200 substances can be used in place of xylose such as mg to 1000 mg per kg of body weight per day of insulin or sorbitol. The clearance of these sub- phlorizin or one of the above two derivatives io stances which are filtered but not secreted or reab- Excess phlorizin is excreted in the urine. Dos- sorbed is approximately 125 ml per minute. This age of phlorizin required to effectively influence value is called the filtration rate since it is a mea- cancer is consequently easily determined since the sure of glomerulus filtration in one minute. The patient acts as his own bioassay. In other words, a glucose clearance approaches 125 ml per minute concentration of phlorizin which makes the tumor is when the patient is completely phlorizined. This is cells impermeable to glucose, makes all other cells easily determined by measuring the quantity of all over the body impermeable to glucose cells. glucose excreted divided by the serum glucose How well glucose is prevented from entering concentration and the number of minutes over the cancer cell can be assessed by measuring which the urine was collected. The urinary and concentration of glucose in the urine. Urine is 20 serum glucose clearance become close to one formed by glomerular filtration. The glomerular fil- another, but the concentration in the urine is always trate contains all the constituents of blood except higher, unless water reabsorbtion from the tubules protein. As the glomerular filtrate passes down the is minimal, which can occur at high glucose con- renal tubules, the proximal tubule reabsorbs glu- centrations. When the glucose concentration is cose. Therefore, normal urine contains no glucose 25 identical to a substance that is filtered by the unless there is some impairment of reabsorption, glomerulus but not secreted or reabsorbed by the such as occurs in diabetes mellitus. When the tubules (such as insulin or xylose), the patient is dosage of phlorizin is adequate to prevent glucose completely phlorinized. entry into cells, the proximal tubular cells can no In practice the administration of 500 mg of longer absorb glucose. Glucose will then appear in 30 phlorizin in propylene glycol or carbowax per kilo the urine almost at the same concentration that it is of body weight given intramuscularly is usually present in the serum. Hence, the effect of phlorizin more than adequate dose in most patients. One on glucose metabolism can be monitored by the need not fear giving an excessive quantity of urinary concentration of glucose, and the correct phlorizin since this substance has proven to be dosage determined. A satisfactory concentration is 35 relatively non-toxic and is rapidly excreted in the achieved in the blood stream when all of the glu- urine. The effect of phlorizin is dependent on a cose clearances in the urine approaches the xylose concentration in the extracted fluid which will com- or creatinine clearance. In man, the creatinine pletely block all of the receptors sites for glucose. clearance can be used. Clinical experience has In animals or humans fed or injected with shown that approximately 100 mg per kilo of body 40 phlorizin, ketosis occurs and the respiratory weight is intravenously required to completely quotient of the entire body shifts to that of fat phlorizinize the patient as evidenced by the failure becoming as low as 0.65. Nonetheless, glucose is of the tubular cells to absorb any glucose. The still required for the metabolism of cancer cells, effects of phlorizin last approximately one to one and hence the metabolism of tumor cells is selec- and one-half hours when administered intravenous- 45 tively depressed. At this level the cancer cells are ly. A better means of administration is to inject the sensitized to treatment with heat and other modalit- material at doses of approximately 500 mg per kilo ies. intramuscularly. Although the phlorizin can be giv- Treatment with phlorizin can also be effectively en by mouth, it requires approximately 400 mg per combined with treatment of cancer using kilo to completely phlorizinize the patient, and 50 Lonidamine. The Lonidamine is preferably admin- much of the dose appears in the stools. That istered at a dosage of 50 mg to 500 mg per kilo of glucose reabsorbtion in the kidney is completely body weight per day. blocked can be determined by comparing clear- When referring to phlorizin, above, and in the ance of glucose to the clearance of xylose after the following description it should be understood that administration of xylose. The xylose clearance is 55 phlorizin glucuronide and 4-deoxyphloretin-2-D- determined by taking the concentration of xylose in glucoside can be substituted in equivalent amounts the blood and measuring the total amount of xylose with comparable results. excreted (in a timed period) divided by the plasma

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A patient with lung cancer is treated with 7. A pharmaceutical composition for the treat- radiofrequency thermotherapy preceded by treatment of cancer comprising: ment with phlorizin in accordance with the present a) a compound or compounds for inhibiting invention, as follows. The phlorizin is dissolved in glucose transport into malignant neoplastic ethylene glycol at a concentration of 500 mg per 5 cells, cc. After fasting overnight the patient is given 300 b) a pharmaceutically acceptable carrier, mg of phlorizin per kilo body weight intramuscular- and ly which induces glycosuria. The urinary sugar c) a chemotherapeutic agent. reaches a maximum in about fifteen to twenty minutes at which point radiofrequency ther- w Patentanspruche motherapy is commenced. R.F. treatment is contin- ued to raise the tumor temperatures to approxi- 1. Produkt enthaltend eine Verbindung oder Ver- mately 44 °C. The dose of phlorizin is repeated in bindungen zum Inhibieren des Glucosetrans- three hours, and glycosuria persists for a total of ports in bosartige neoplastische Zellen, einen eight hours. The glycosuria condition is used as an is pharmazeutisch geeigneten Carrier dafur und indicator to show that sufficient concentration of ein Chemotherapeutikum als kombiniertes Pra- phlorizin has been reached in the tissue, on the parat zur simultanen, separaten oder sequen- assumption that the tumor cells are affected as the tiellen Verwendung in der Krebstherapie. kidney cells and are also unable to transfer glucose. Treatment is repeated, as above, as required. 20 2. Produkt nach Anspruch 1, in dem die Verbin- Phlorizin and the two derivatives can also be dung oder Verbindungen zum Inhibieren des used to accentuate response to chemotherapy with Glucosetransports in bosartige neoplastische drugs, ouch as adriamycin. These drugs are used Zellen aus Phlorizin, Phlorizin-glucuronid und after phlorizination as described above in connec- 4-deoxyphloretin-2-D-glucosid gewahlt ist/sind. tion with the use of radiofrequency thermotherapy. 25 3. Produkt nach Anspruch 1 oder 2, worin das Claims Chemotherapeutikum Lonidamin umfa/St.

1. A product containing a compound or com- 4. Produkt nach einem der vorhergehenden An- pounds for inhibiting glucose transport into ma- 30 spruche in Form von Dosierungseinheiten. lignant neoplastic cells a pharmaceutically suitable carrier therefore and a chemotherapeutic 5. Produkt nach einem der vorhergehenden An- agent as a combined preparation for simulta- spruche, das in einer zur Verabreichung durch neous, separate or sequential use in cancer intramuskulare Injektion geeigneten Form vor- therapy. 35 liegt.

2. A product as claimed in claim 1, in which the 6. Produkt nach einem der vorhergehenden An- compound or compounds for inhibiting glucose spruche, das in einer zur Verabreichung durch transport into malignant neoplastic cells is/are intravenose Injektion geeigneten Form vorliegt. selected from phlorizin, phlorizin glucuronide 40 and 4-deoxyphloretin-2-D-glucoside. 7. Pharmazeutische Zusammensetzung zur Be- handlung von Krebs umfassend: 3. A product as claimed in claim 1 or 2, wherein a) eine Verbindung oder Verbindungen zum the chemotherapeutic agent comprises Inhibieren des Glucosetransports in bosarti- lonidamine. 45 ge neoplastische Zellen, b) einen pharmazeutisch akzeptablen Car- 4. A product as claimed in any of the preceding rier und claims in unit dosage form. c) ein Chemotherapeutikum.

5. A product as claimed in any one of the pre- 50 Revendications ceding claims, which is in a form suitable for administration by intramuscular injection. 1. Un produit contenant un compose ou des composes pour inhiber le transport du glucose 6. A product as claimed in any one of the pre- dans les cellules neoplastiques malignes, un ceding claims which is in a form suitable for 55 excipient approprie pharmaceutiquement pour administration by intravenous injection. cela, et un agent chimiotherapique en tant que preparation combinee pour un emploi simulta- ne, separe ou sequentiel en therapeutique can-

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cereuse.

2. Un produit comme revendique a la revendica- tion 1, dans lequel le compose ou les composes pour I'inhibition du transport du glucose 5 dans les cellules neoplastiques malignes est/sont choisis parmi la phlorizine, le glucuronide de phlorizine et le 4-desoxyphloretine-2- D-glucoside. 10 3. Un produit comme revendique a la revendica- tion 1 ou 2, caracterise en ce que I'agent chimiotherapique comprend de la lonidamine.

4. Un produit comme revendique dans I'une des is revendications precedentes, sous forme de dose unitaire.

5. Un produit comme revendique dans I'une quel- conque des revendications precedentes, qui 20 est sous une forme appropriee pour I'administration par injection intramusculaire.

6. Un produit comme revendique dans I'une quel- conque des revendications precedentes qui est 25 sous une forme appropriee pour I'administration par injection intraveineuse.

7. Une composition pharmaceutique pour le trai- tement du cancer qui comprend : 30 a) un ou des composes pour I'inhibition du transport du glucose dans les cellules neoplastiques malignes, b) un excipient pharmaceutiquement acceptable et 35 c) un agent chimiotherapique.