Treatment of Cancer with Phlorizin and Its Derivatives

Europaisches Patentamt J) European Patent Office © Publication number: 0 291 151 Office europeen des brevets A1

EUROPEAN PATENT APPLICATION

(^) Application number: 88302189.1 © int.Ci.4:A61K 31/70

(§) Date of filing: 14.03.88

©> Priority: 18.03.87 US 27413 Applicant: LeVeen, Harry H. 321 Confederate Circle @ Date of publication of application: Charleston, SC 29407(US) 17.11.88 Bulletin 88/46 Applicant: LeVeen, Robert F. © Designated Contracting States: 312 Lombard Street OE FR GB GR IT NL SE Philadelphia Pennsylvania 19147(US)

Applicant: Leveen, Eric G. 141 South Battery Charleston South Carolina 29401 (US)

Inventor: LeVeen, Harry H. 321 Confederate Circle Charleston, SC 29407(US) Inventor: LeVeen, Robert F. 312 Lombard Street Philadelphia Pennsylvania 19147(US) Inventor: Leveen, Eric G. 141 South Battery Charleston South Carolina 29401 (US)

Representative: McCall, John Douglas et al W.P. THOMPSON & CO. Coopers Building Church Street Liverpool L1 3AB(GB)

® Treatment of cancer with phlorizin and its derivatives.

© Malignant, neoplastic cells are treated by inhibit- t—ing glucose transport into the cell by administering ^one or more compounds selected from phlorizin, phloretin, phlorizin glucoronide, 4-deoxyphloretin-2- iflD-glucose, and cytochalasin-B, preferably while con- r" currently administering adjunct therapy such as heat, ^radiation or chemotherapy. In this manner, the cells Ojare prevented from growing or repairing the damage M caused by the adjunct therapy, which can be admin- Qistered in dosages that would otherwise be non- lethal if used alone. Q. Ill

Xerox Copy Centre 0 291 151

TREATMENT OF CANCER WITH PHLORIZIN AND ITS DERIVATIVES

The present invention relates to a method, substantiation for the latter explanation that a great- compounds and compositions for inhibiting glucose er number of receptor sites are responsible. transport across cell membranes, and more specifi- The rate of glucose transport across cells has cally to a method, compounds and compositions been shown to be directly related to the growth by which the growth of malignant neoplastic cells 5 potential of the cell. Glucose is a requirement for can be halted and such cells kept from repairing energy needs related to synthesis, as well as for and rejuvenating themselves. In a preferred form actual structural requirements in the synthesis of the invention is particularly concerned with a com- macromolecules (J Nat Can Inst. 62:3, January bined treatment whereby malignant cells are made 1979). Because malignant cells exhibit an extensive more susceptible to lethal damage from exposure w augmentation of glucose transport, they are under- to otherwise non-lethal doses of energy and anti- standably much more sensitive to drugs which tumor agents. inhibit glucose transport than are normal cells. The Researchers have long sought for a biochemi- magnitude of abnormal carbohydrate metabolism of cal difference by which malignant cells could be malignant cells increases the competition for glu- distinguished from normal cells. The work of War- 15 cose which develops between rapidly growing tu- burg in the early part of the twentieth century mors and the host's normal cells. Warburg men- (Warburg, The Metabolism of Tumors, Richard R. tions that the glycolysis of tumor cells can be so Smith & Co., New York, 1931) focused attention on rapid as to reduce the blood sugar in diabetic the differences in the way that cancer cells metab- patients. For example, the blood sugar concentra- olize glucose. Using a concept advanced by Pas- 20 tion in rats with a rapidly growing sarcoma remains teur, Warburg considered that cancer cells ferment normal until the tumor/body weight ratio increases while normal cells respire. The arguments ad- above .15. At ratios of .31 or greater, hypol- vanced by Warburg (Annual Review of Biochem- glycemia occurrs. Liver glycogen declines at high istry. 33:1, 1964) seemed to characterize the ma- tumor/body weight ratios. Gluconeogenesis from jority of rapidly growing malignant cells, and the 25 lactate increases thirty fold over autogenous occasional instance where the metabolism of the gluconeogenesis from endogenous alanine (Cancer tumor does not strictly fit Warburg's criteria is Res. 40:1699, 1980). Similar findings have been limited to very slow growing cancers which do not made in patients with rapidly progressive malignant produce death by cachexia and widespread metas- disease (Cancer Res. 39:1968, 1979; Cancer 33:66, tasis. 30 1974). Obviously, cancers sequestrate glucose and However, some recent knowledge with respect glucose utilization in cancer patients is extremely to the regulation of the energy metabolism in the high. The high potential for the malignant tumor to cell was unknown to Warburg. For instance, the metabolize glucose has even caused it to be re- Crabtree effect (inhibition of respiration by ferred to as a glucose trap (Acta Chir Scan [Suppl] glycolysis) 'is observed in practically all cells 35 498:141, 1980). (Biochema et Biophysica Acta 591:209, 1980). The This excessive glucose turnover in malignant addition of glucose to anaerobic suspensions of patients has again focused attention on the role of glucose starved malignant cells causes a burst of glucose metabolism in cancer cachexia. One con- respiration and glycolysis with lactate production sequence of the anaerobic metabolism of glucose which results in an inhibition of both respiration and 40 is the release of lactate into the circulating blood. glycolysis to values below those observed prior to The lactic acid is transported to the liver by the the addition of glucose. circulating blood. The liver converts the lactic acid The rate limiting factor in glucose metabolism to glucose thus completing the Cori cycle. The which determines the quantity of lactate formed in conversion of lactate to glucose is energy consum- the cell has been shown to be dependent upon the es ing and has been estimated to account for 10% rate at which glucose is transported across the cell increase in energy expenditure (Cancer Res. membrane. This knowledge suggests that the bio- 37:2336, 1977). The production of lactate may be chemical defect in cancer cells does not reside in so excessive in patients with cancer that with im- fermentation or respiration; but, rather, the primary pairment of liver physiology as with extensive liver defect must be the increased rate of glucose trans- so metastasis, lactic acidosis may occur (Cancer port across the cell membrane. This must be re- 47:2026, 1981). The severe carbohydrate drain lated either to an enhancement of the transport causes excessive gluconeogenesis which further mechanism or an actual increase in the receptor depletes the cancer patient (SA Med. J. 59:518, sites for glucose transport which are present in the 1981) (Ann NY Acad Sci, 72:103, 1980). membrane of the cell wall; and there is excellent Glycogen synthesized from glucose is abun- 0 291 151

dantly stored in cancer ceils (Cancer 19:98, 1966); ceptor sites. Both compounds will inhibit glucose however, the glycogen content decreases during transport but it takes a higher concentration of the exponential phase of tissue growth. Brain tu- phlorizin in non sodium dependent transfer sites. mors for example contain five times as much gly- Because of its low toxicity and the fact that cogen as small mammals brains (J. Neurochem. 5 phlorizin is more water soluble it has been the drug 29:959, 1977). This further supports the concept of choice in spite of the fact that for some transfer that increased glucose transport is a significant sites phloretin may be more active. This has also requirement for the rapidly growing cancer cell. caused the does of phlorezin to sometimes be Slow growing tumors contain more glycogen than raised beyond the dosage indicating a complete more rapidly growing tumors which utilize the glu- 10 block of glucose transfer in the kidneys. Work on cose more swiftly (Can. Res. 41:1165, 1981). phloretin indicates that more water soluble deriva- These factors support the concept that cancer cells tives of phloretin are possible without the loss of transfer glucose more swiftly than do normal cells. pharmacological activity. These energy related revelations have also These compounds are especially effective in turned attention to the glucose metabolism of grow- 75 preventing glucose transport across malignant cell ing cancer cells as a mechanism for the control of membranes, thereby suppressing the growth of cancer growth. Lonidamine has been found to be a cancer especially when used in conjunction with selective inhibitor of aerobic glycolysis in urine chemotherapy, radiation, heat or other techniques. tumor cells (J Nat Can Ins. 66:497, 1981). Dactylirin The pharmaceutical composition according to the is a new antibiotic which has a potential anticancer 20 invention comprises phlorizin, its glucuronide, effect since it influences the energy yielding car- cytochalasin-B or 4-deoxyphloretin-2-D-glucose, bohydrate mechanisms which function in malignant preferably together with a pharmaceutically accept- cells (Can Res. 39:4242, October 1979). able carrier. These compositions may be solid or In accordance with the present invention a pro- liquid and can be used in forms currently used in cess and compositions are provided for treating 25 medicine such as tablets, capsules, syrups and malignant neoplasms such as cancers and sar- injectable preparations. Because of its poor absorp- comas in mammals by inhibiting glucose entry into tion, the compositions of the invention is preferably the malignant cell to thereby render the cell more administered parenterally, dissolved in a suitable susceptible to the injurious effect of chemotherapy, carrier such as 1/2 normal saline . Orally accept- radiation, heat or anoxia and prevent repair or 30 able carriers are those currently used in medicine growth of the cell. Glucose entry into the malignant such as calcium carbonate, starch, lactose, talc, cell is inhibited by administering effective amounts magnesium stearate, gum acacia, aqueous alcohol, of phlorizin, its glucuronide, 4-deoxyphoretin-2-D- glycol or oil solutions or suspensions. glucoside, phloretin or cytochalasin B or a com- While sufficient phlorizin or its analogs should bination of these compounds either alone or in 35 be given to saturate glucose binding sites in the combination with Lonidamine or other tumor cells, excess dosage is simply excreted in chemotherapeutic agents such as bleomycin or the urine. mitomycin C. Phloretin is the aglucone portion of The dosage required to effectively inhibit can- the phlorezin molecule and also inhibits glucose cer growth is easily determined since the patient transport across the cells. Different tissue differ in 40 acts as his own bioassay. The concentration which their responsiveness to phloretin as compared to makes the tumor cells impermeable to glucose, phlorezin. For instance, phlorezin is much more makes other normal cells in the body also im- effective at blocking transfer across the kidney permeable to glucose. The extent to which glucose tubules and intestinal mucosa than is phloretin. On transfer has been blocked can be assessed by the other hand, phloretin is much more effective in 45 measuring the concentration of glucose in the blocking transfer of glucose across red blood cells. urine. Urine is formed by glomerular filtration. The It has been postulated that phloretin blocks non glomerular filtrate contains all the constituents of sodium dependent glucose transport more effec- blood except protein. As the glomerular filtrate tively than does phlorezin. Glucose transport passes down the renal tubules, the proximal tubule across renal tubular cells and intestinal mucosa is so reabsorbs glucose. Therefore, normal urine con- sodium dependent. If sodium transport is blocked, tains no glucose unless there is some impairment glucose transport will not be inhibited by phlorezin. of reabsorption or the blood concentration of glu- Many tissues exhibit both type of receptors. For cose exceeds the renal tubules ability to reabsorb instance, there are both sodium dependent and glucose as occurs in diabetes mellitus. When the non sodium dependent transfer sites in the capil- 55 dosage is adequate to prevent glucose entry into lary endothelium of the blood brain barrier. In tis- cells, the proximal tubular cells can no longer ab- sues with such mixed type of receptors. Phlorezin sorb glucose and glucosuria occurs. When total and phloretin are mutually competitive for the re- blocking of glucose absorption into the cell occurs, 0 291 151 glucose then appears in the urine in almost the istration of a total dosage of about 200-1000 mg of same concentration that it is present in the serum phlorizin or the indicated derivative per kilogram of depending on the degree of water absorption of the body weight is an adequate dose in most patients. urine. The correct dosage of the composition of the One need not fear giving an excessive quantity of invention can be determined by measuring the 5 phlorizin, however, since this substance has proven glucose clearance, which approaches 125 cc's per to be non-toxic and is rapidly excreted in the urine. minute and which is the same as the xylose and The effect of phlorizin is dependent on a critical inulin clearance. The creatinine clearance is a good concentration in the extracellular fluid which will substitute since it is only slightly higher than the completely block all of the receptors sites for glu- inulin clearance. 10 cose. Clinical experience has shown that, the com- The composition is generally administered dur- position of the invention is preferably administered ing intervals of chemotherapy or radiation therapy in a continuous drip, approximately 1 mg per kilo of by heating, radiation or chemotherapeutic drugs. body weight per hour as a maintenance dose after Heat therapy can be administered by total phlorizination to completely abolish glucose 75 radiofrequency thermotherapy. Complete entry into cells. An initial loading dose of 4-6 mg phlorizination for twenty-four hours prior to therapy per kilo of body weight is usually desirable to reduces cellular glycogen levels and renders the completely phlorizinize a patient as evidenced by therapy more effective. This dose is sufficient to the failure of the tubular cells to absorb glucose. reduce the blood glucose concentration to low lev- This can be given by a slow push or over a ten 20 els and to partly or completely deplete cellular minute interval. The duration of the effects last glucose. Further inhibition of glucose entry into the approximately one to one and one-half hours when cancer cell is extended to the time of patient ther- administered intravenously as a single dose. With apy by heat, chemotherapy or radiation and contin- oral therapy, much of the dose appears in the ued for 24 hours post therapy. The patient should stool. That glucose reabsorption in the kidney is 25 be carefully observed for salt depletion. If for any completely blocked can be determined by compar- reason it is necessary to interrupt the therapy, it ing clearance of glucose to the clearance of xylose can be counteracted with glucose infusions. after administration. Although not required for effective treatment in Xylose clearance is determined by taking the accordance with this invention, the effectiveness of concentration of xylose in the blood and measuring 30 phlorizin or its indicated derivatives or analogs can the total amount of xylose excreted in a time period be enchanced by the inclusion of a divided by the plasma concentration and the num- chemotherapeutic agent such as lonidamine, ber of minutes of collection. This will determine bleomycin or mytomycin in amounts of 50 to 500 how many cc's of blood were completely cleared mgs per kilo of body weight. of xylose every minute. Xylose is a non-metabo- 35 For I.V. infusion of phlorizin ten grams of lized sugar which is not reabsorbed by the renal phlorizin or the indicated analogs are dissolved in tubules. When glucose entry into cells is com- 20 cc's of 95% ethyl alcohol which is then mixed pletely blocked, glucose clearance and xylose with 930 cc's of warm or hot 0.5 normal sodium clearance are almost the same. Other substances . chloride solution to which one ampule of sodium can be used in place of xylose such as inulin or 40 bicarbonate (50 mq in 50 ml H2O) has been added. sorbitol. This mixture may require heating until it is warm The glucose clearance approaches 125 cc's enough to fully dissolve the phlorizin. After initial per minute when the patient is completely administering of the loading dose, the infusion is phiorozinized. This is easily obtained by measuring slowed to 10-15 cc per hour depending on body the quantity of glucose excreted, divided by the 45 weight. serum glucose concentration and the number of While not wishing to be bound to a particular minutes over which the urine was collected. Urinary theory, it has been postulated that phlorizin and and serum glucose concentrations can approach phloretin and their indicated analogs and deriva- one another but the concentration in the urine is tives act on cells by attachment of the glucosyl unit always higher unless water reabsorption from the 50 to the glucose binding sites in the transfer site tubules is minimal which can occur at high glucose while the phloretin group has a high affinity for a concentrations since osmotic diuresis occurs and cellular binding site adjacent the entrance of the the tubules are unable to do osmotic work. When glucose site. The aglucone unit attaches to the the glucose clearance is identical to a substance sites with great affinity while glucose occupies the that is filtered by the glomerulus but not secreted 55 enzyme site (J Physol. 169:229, 1963). Phlorizin is or reabsorbed by the tubules (such as inulin or easily disassociated from the receptor site and its xylose), glucose utilization is completely blocked. effect on glucose transport is not lasting. Also, Generally, it has been found that the admin- since it is a competitive inhibitor of glucose, high 0 291 151 concentrations of glucose tend to displace phlorizin to complete necrosis with heat applied by from the binding site. The aglucone portion of the radiofrequency thermotherapy. Necrosis of normal molecule attaches to the surface of the cell and tissue was not seen on microscopic section in spite exerts its action on the cell surface rather than in of the fact that tumor tissue intimately infiltrating the interior of the cell (Harvey Lectures 59:53, 5 normal muscle tissue was found to be completely 1961). destroyed. Insulin and glucose enhance transport of glu- Phlorizin is useful by itself and in combination cose across the cell membrane. Their administra- with other agents affecting carbohydrate metabo- tion produce an increased concentration of gly- lism since it deprives the tumor cell of primary cogen in muscle tissues such as the diaphragm. w energy source. This effect is accentuated by tem- This increases in glycogen stimulated by insulin perature elevation. 5-thio-glucose was not found to and glucose can be prevented by the in vitro or in enhance the effect of phlorizin on tissue necrosis vivo administration of phlorizin (Harvey Lectures with heat or by itself. This is explained by the fact 56:63, 1961). This is strong evidence that phlorizin that phlorizin blocks glucose entry into the cell and prevents glucose entry into the cells. Blocking the 75 similarly blocks the entrance of thio-glucose into entry into the renal tubular cell leads to glucosuria the cell allowing thio-glucose to be completely in phlorizinized animals and humans. The action of spilled into the urine. When radioactive gold thio- phlorizin on the excretion of glucose in humans is glucose is administered to growing mice, the gold identical to the action of phlorizin on the excretion thio-glucose is concentrated in the satiation center of glucose in other lower animals (J Clin Invest. 20 of the brain and animals become obese. The de- 12:1083, 1933). Phlorizin is relatively non-toxic and struction of this center renders the mice hyper- has been administered to man parenterally and by phagic (Am J Physio. 226:574, 1974). When mouth in wafer form. Doses as high as 15 grams phlorizin is administered to these animals, glucose have been administered in a single oral dose (J transport is inhibited thus preventing a lethal collec- Clin Invest. 13:749, 1934). It has been given intra- 25 tion of gold-thio-glucose to concentrate in the re- venously and subcutaneously to humans. Phlorizin gion of the hypothalmus. Therefore, phlorizin pro- by inhibiting glucose entry into the renal tubular tects by preventing the entrance of gold thio-glu- cell causes glycosuria but since it also inhibits cose into the hypothalmic cells (Nutri Reviews glucose entry into intestinal mucosal cells, absorp- 33:23, 1975). tion from the intestines is inhibited and sugar may 30 In addition to animal tests, tests have also been be present in the feces. performed on tumor cells (mastoma) growing in In accordance with the present invention, stud- vitro. When glucose absorption was blocked, cell ies have been directed toward the in vivo observa- growth in tissue culture was completely sup- tion of the effect of large dosages of phlorizin, 5- pressed. thio-d-glucose and lonidamine both by themselves 35 The survival of cells heat-treated in the ab- and in combination with each other in Erlich car- sence of nutrients is greatly reduced compared to cinoma transplanted to the back of Swiss mice. those heat treated with nutrients and the ability of These tumors were observed with drug therapy in cells to repair heat damage is impaired. The state the above combination with and without localized of the cell cycle also depends on nutriment supply hyperthermia developed by radiofrequency dielec- 40 and oxygen availability. The center of a cancer tric heating of the localized tumor sites. Tumor contains only cells in the synthetic phase, while the temperatures were measured during therapy for hyperemic edge of cancerous tissue exhibits rapid varying periods of time. Temperatures from 39° to growth and good viability. Cells in the central pla- 44° were achieved in the tumors as measured by teau phase are more susceptible to thermal de- thermocouples placed into the center of the tumor. 45 struction than are the cells in the proliferative In addition, animals were treated for two days and phase (Radiol. 113:207, 1974). Hypoxic cells are then sacrificed. The tumors were then excised for also more susceptible to heat which again im- histology. Phlorizin alone enhanced the ability of plicates the role of metabolism in ameliorating cell otherwise non-lethal dosages of heat to produce destruction by heat. (Radiol. 117:477, 1975). tumor necrosis and the addition of lonidamine and 50 Glucose is essential for DNA synthesis through other chemotherapeutic agents slightly increased the pentose phosphate pathway (SA Med J, P. 518, the effect further. Phlorizin with heat is very de- April 4, 1981). Since ribose-5-phosphate is essen- structive and it is unnecessary to achieve the high tial for DNA and RNA synthesis, any damage to lethal temperatures to kill carcinoma in animals DNA caused by radiation and chemotherapy can- when used without phlorizin. The tumor destruction 55 not be repaired if glucose entry into the cell is was complete with low temperatures in the prevented. Increased glucose transport occurs in lonidamine plus phlorizin treated animals. Animals cancer cells because their anaerobic metabolism is treated with phlorizin alone also showed extensive less effective than aerobic metabolism. Malignant 0 291 151 10 cells are totally dependent on glucose since malig- nal wall, transverse colon and hepatic region. The nant cells deprived of glucose are unable to main- tumor was not possible to completely resect at any tain their ATP levels for periods longer than four surgery and each time the lesion was more exten- hours whereas normal cells have no difficulty in sive. The patient again became jaundiced and an- maintaining their ATP levels in the absence of 5 other laparotomy with debulking was performed. A glucose (Biochem et Biophysica Res Comm. T-tube was placed in the common duct and a 82:787, 1978). The repair of hyperthermic damage gastroenterostomy was performed. is totally dependent on metabolic processes in The patient was treated with antibiotics for which the metabolism of glucose plays an impor- cholangitis and infection of abdominal wall wound. tant role. The lethal thermal damage to malignant w She was then started on phlorizin given continu- cells caused by two one-hour heating periods at ously for 12-24 hours with local heat. Systemic 44° C has been quantitated. temperature rose to 40°C. Vinblastin and Heating is most lethal when no interval be- Mitomycin C were given I.V. in very small doses. tween the one hour heating periods is allowed. If The entire tumor mass became necrotic. Massive the interval between the heatings is greater than 75 necrosis of necrotic tumor became liquified and four hours (bringing the temperature back to 37°C infected and have required multiple drainages with between heatings), additional killing over and above catheters placed by radiographers under x-ray con- that which occurs with a single heating is not trol. Bowel wall which was replaced by nectrotic observed since the thermal damage is completely tumor tissue has communicated with a large in- repaired by the metabolic processes of the cell 20 traabdominal abcess which has been drained. during the four hour interval between heatings. Sepsis is now being controlled. CT scans show Nonetheless, if the temperature between heatings multiple areas of tumor liquification and cavitation. is dropped to 0°C instead of 37° , repair of thermal The patient's survival with large necrotic masses is damage is prevent and the killing is identical to a questionable but patient is being supported and steady two hour heating. This demonstrates the 25 sepsis seems under control. strong role of metabolism in repairing thermal dam- age (Cancer Res. 36:1035, 1976). Chemotherapy and radiation damage to cells is Example 2: characterized by single and double breaks in the DNA chain. The repair of this damage is also a 30 metabolic process (Radio. 123:475, 1977) requiring A 51 year old female had a low anterior resec- the metabolism of glucose. tion for cancer of rectum. At time of surgery, he- Phlorizin, and equivalents by prohibiting glu- patic metastases were discovered. Had full course cose entry into the interior of the cell, impairs the of radiation to pelvis, post surgery. She was treated vitality, metabolism, and repair and injury to cells 35 with 5 FU, Mitomycin C and Novotrome without caused by outside forces and changes sublethal response. Was started on 8-12 hour phlorizin plus cellular damage to lethal damage thus enhancing the same chemotherapeutic agents. Made an im- the effect of chemotherapy, radiation and heat. mediate response with regression of tumor and no The following examples further illustrate and further bowel obstruction. support the clinical efficacy of the present inven- 40 tion: Example 3:

45 A 57 year old male had a melanoma over right scapula removed. There was a local recurrence A thin female 57 years of age entered with and a local infection with paraincisional melanosis. sarcoma of duodenum resected 3 years prior. Pa- This was removed with lymph nodes. Pathological tient was jaundiced and work up indicated obstruc- diagnoses was Clark level two melanoma. 7 years tion of common bile duct. Tumor was debulked so later, a cerebral recurrence was found. There were around porta hepatis and cholecystojejunostomy two lesions in frontal lobes and one in parietal area. was done. Patient was then given life time dose of Was treated with radiation without response. All radiation and chemotherapy. Pathology report of therapy was then abandoned and the patient was patient was a leiomyosarcoma. About one year told he had 60 days to live. later, the tumor recurred in the umbilical area re- 55 The patient was started on small doses of quiring further debulking at which time large portion Vincrestine, methyl CCNU and received phlorizin of abdominal mass was resected. by bolus 5 grams and mild heat to heat by RFTT. Further debulking was done from the abdomi- There was a 50% reduction of lesion by NMR 11 0 291 151 12

scan. NMR scan of brain was negative. He had a motherapy were also administered I.V. Patient re- recurrance and was treated but chemotherapy sponded with disappearance of complete gastric omitted. The lesion did not respond and patient outlet obstruction. She is now on full diet and the died. Fistula has spontaneously closed.

Die 4:

46 year old female developed a melanoma of 10 Patient has carcinoma of breast and previous the right leg during pregnancy. An excision of mastectomy but experienced a large recurrence in primary with lymph node desection and removal the axilla. Because of a generalized vasculitis poor- was done. Pathology reported that lymph nodes ly understood she did not receive chemotherapy were positive for melanoma. Metastases appeared but only received phlorizin and heat treatment. She in head and iungs. Patient was placed on Vin- 75 was given a bolus containing 2.5 grams of phlorizin cristine and Precarbizone. Severe pain in abdomen and 75 watts of R.F.T.T. to axilla. Lesion dried up probably caused by hemorrhage into a liver mets. and CT scan was negative. She was treated with a bolus of 5 grams of phlorizin and decadron to lower any possible in- flammatory response from necrotic tumor tissue 20 along with RFTT of 50 watts to head and the chemotherapy which had previously been unsuc- cessful. Vincristine Methyl CCNU and procarbozine 34 year old male had a resection of sigmoid was restarted in small doses. The lesion in her and descending colon for cancer of colon. Patho- brain completely resolved on CT scan. The liver 25 logical report described perivascular invasion, and abdominal diseases had increased and patient lymph node invasion and diffuse periotoneal seed- died in June of liver and abdominal disease without ing. All lymph nodes could not be removed. Was any recurrence in the head. treated with 5FU Mitomycin and a bolus of phlorizin. Now has normal CT scan of liver and 30 abdomen. C.E.A. 2.8 and has remained tumor free Example 5: for 1 year.

38 year old female developed a melanoma of left axilla which was widely excised with adjacent 35 lymph nodes. Lymph nodes were negative for me- tastases. She developed plural effusion with mul- 54 year old female with cancer of breast am- tiple nodules in left lung. Received 50.0 mg 5 F.U. putated and was placed on chemotherapy and and 5 mg. mitomycin C intraplurally. Heated with tomoxifen and local radiation. There was a metas- 100 watts R.F.T.T.. Given vincrestine procarbozine 40 tases to the 4th ventricle of the brain on CT scan. CCNU but did not respond. Chest was drained Patient told no treatment possible and was sent to again in September. Received same treatment plus a terminal cancer hospital. phlorizin over 12 hours. Responded and now is Was treated with mild heat, phlorizin and de- tumor free for past 1 1 months. cadron without chemotherapy. CT scan now nega- 45 tive and patient remains well.

A 39 year old woman presented with total so obstruction from stomach and a large bowel fistula 62 year old executive had adenocarcinoma of from previous surgery. She was unable to take any lung with metastases to brain. Three separate le- food by mouth. She underwent treatment with sions appeared in brain on CT and NMR scans. R.F.T.T. to area of obstruction after 8 hours of Brain was radiated without response. Lung also continuous phlorizin infusion. Small doses of che- 55 radiated without response. Patient was started on 13 0 291 151 14

5FU mytomycin and phlorizin. CEA was 8.2. Scan motherapy. Phlorizin was added by bolus to re- has improved and only 1 small lesion is now visible gime. Patient progressed very slowly. Eight hours in brain and 2 have disappeared. Lung lesion has of phlorizin was instituted with chemotherapy. Tu- disappeared. No other soft tissue metastases. mor regressed by 80%.

Die 11.

Primary tumor in colon was resected. Patient w 58 year old female had a right colon resection underwent liver resection for right hepatic meta- for carcinoma. The cancer had perforated the bow- static colon cancer. No other tumor seen. Devel- el wall and there was peritoneal seeding visable. All oped recurrent bowel obstruction and peritoneal of the tumor could not be resected. CEA was 13.9 seeding. Repeated bowel obstructions respond Was treated postoperatively with 5 FU Mitomycin C each time to chemotherapy and phlorizin. 75 and phlorizin infusion plus mild heat. Last CT scan Laparotomy with a colostomy slowed massive tu- was negative for tumor and C.E.A. remains at low mor regression. Has improved long term survival. level of 3.0.

Example 12: 20 Example 16:

56 year old man had cancer of rectum re- 80 year old female with massive liposarcoma sected. Recurrance three years later in abdomen of left lower leg. Patient has had repeated resec- was treated with a bolus, heat mitromycin and 5FU. 25 tions of tumor mass of left lower leg. Patient was Patient developed renal shut down from obstructed injected with mitomycin C and Vinblastin with ureters. There were liver metastases and abdomi- phlorizin. Because of the shortage of phlorizin, it nal tumor present. Patient was given 8-12 hour was injected intralesionally and a tourniquet applied phlorizin plus chemotherapy as above and has to prevent reabsorption. The lesion has resolved responded with tumor regression and ureter is no 30 with a 90% regression in tumor size. Requires longer obstructed. occasional therapy with heat RFTT and chemo- therapy but has not required further surgery.

35 Example 17:

78 year old female had anterior resection for cancer of rectum. She developed a recurrence one 48 year old female who had upper Q.I. discom- year later with radiation to pelvis. No response fort. She had been working with x-rays for 20 occured and the bowel was . obstructed so a 40 years. Exploratory laparotomy revealed cancer of laparotomy was done and an end colostomy per- stomach with diffuse metastases over peritoneum. formed. Liver metastases were found. Laparotomy The patient gradually deteriorated and developed and exenteration was done for colovesical fistula. partial bowel obstruction. She received 8-12 hours Biopsies were all positive for cancer post exen- phlorizin infusion plus 5FU and mitomycin C and teration. Patient was treated with 8 hour infusion of 45 RFTT to area of obstruction. Obstruction resolved phlorizin. Chemotherapy and heat were adminis- and went home to Texas. Returned 6 weeks later tered. All biopsies turned negative and there was with another obstruction. Retreated. Mass reduced no evidence of pelvic cancer. Pelvic biopsies were in size and obstruction overcome. Responds well to negative. Liver metastases remain stable. treatments. 50

Example. 18:

Nasopharangeal cancer treated by radiation 55 54 year old obese female with metastases hy- and chemotherapy. Pathology was a poorly dif- pernephroma to brain, scalp and lungs. Primary ferentiated squamous cell carcinoma. Lesion tumor had been removed some years previously. spread to both sides of neck. Progressed on che- Patient had failed chemotherapy and was dying. 15 0 291 151 16

Phlorizin given in bolus of 2 grams with Lonidamine 2. A method for treatment of malignant neo- I.V. Heat given to head with conductive R.F.T.T. of plastic cells in a human being, characterised by 50-100 watts. Necrosis of tumor occurred imme- inhibiting glucose transport into said cells by ad- diately and patient went into acute cerebral edema ministering to said human being an effective requiring immediate neurosurgical decompression. 5 amount of a compound or compounds selected The necrotic tumor was scooped out from brain. from the group consisting of phlorizin, phlorizin Pathology report confirmed that dead tumor tissue giucoronide, phloretin, 4-deoxyphloretin-2-D-glu- had caused acute edema. Tumor of scalp also cose, and cytochalasin-B. became necrotic and disappeared leaving a scar 3. A method for treatment of malignant neo- biopsy free of tumor. CT scan now confirm that io plastic cells in humans, characterised by admin- tumor in head had disappeared. Patient now alive istering one or more compounds which are effec- and well. tive to inhibit glucose transport in the cells while subjecting said cells to additional therapy in the form of chemotherapy, thermal or radiation therapy. 75 4. A compound or mixture of compounds for the treatment of malignant neoplastic cells in hu- mans, characterised in that said compound or com- 37 year old male with massive liver cancer. pounds comprises one or more compounds which The biopsies disclosed a carcinoid tumor but a are effective to inhibit glucose transport in the cells. search for the primary tumor did not disclose any 20 5. A compound or compounds for inhibiting intestinal primary. He was operated upon and the glucose transport into malignant neoplastic cells in tumor proved to be completely unresectible. Pa- humans, characterised in that said compound or ' tient was placed on high doses of chemotherapy compounds comprises one or more compounds both before and after surgery but it failed com- selected from phlorizin, phloretin, phlorizin pletely. 25 giucoronide, 4-deoxyphloretin-2-D-glucose, and Patient had received phlorizin by bolus plus cytochalasin-B. previously failed chemotherapy and lonidamine but 6. A compound or compounds as claimed in responded only slowly. Therefore, he was switched claim 4 or 5, characterised in that said compound to long term phlorizin over 12 to 24 hours with the or compounds are administered in combination same chemotherapy and heat regime and has re- 30 with a chemotherapeutic agent selected from the sponded to long infusions of phlorizin. The long group consisting of a lonidamine, bleomycin and infusion technique depleted the cell of glycogen mytomycin, 5-fluorouricil, and cysplatinum. and made it impossible for damage of cancer cell 7. A compound or compounds as claimed in to be repaired. The results have been dramatic with any one of claims 4 to 6, characterised in that said marked response in weight gain, strength, well be- 35 compound or compounds is administered in a total ing. Tumor size has decreased significantly on amount of about 200 to 1000 mg per kilogram of direct palpation and CT scan. CT scan shows body weight of said human being. necroses of hepatic metastases. 8. A compound or compounds as claimed in The above noted examples represent numer- any one of claims 4 to 7, characterized in that said ous examples of treatment of patients with phlorizin 40 compound or compounds is administered intrave- taken from varours medical records showing pa- nously. tient treatment. 9. A compound or compounds as claimed in Having disclosed a preferred embodiment of any one of claims 4 to 8, characterised in that said the present invention, it is understood that changes compound or compounds is administered initially may be made in the disclosed invention as set 45 as a dosage sufficient to block glucose transport forth in the following claims. into said cells followed by a continuous mainten- ance dosage. 10. A compound or compounds as claimed in Claims claim 9, characterised in that said initial dosage is so about 4-6 mg per kilogram of body weight and said 1. A method for inhibiting glucose transport into maintenance dose is administered at a rate of malignant neopiastic cells, characterized by admin- about 1 mg per hour per kilogram of body weight. istering an effective amount of a compound or 11. A compound or compounds as claimed in compounds selected from the group consisting of any one of claims 4 to 10, characterised in that phlorizin, phloretin, phiorizin giucoronide, 4- 55 administering of said compound or compounds is deoxyphloretin-2-D-glucose, and cytochalasin-B to done concurrently with subjecting said cells to ad- a human being. ditional therapy in the form of chemotherapy, ther- mal or radiation therapy. 17 0 291151 18

12. A compound or compounds as claimed in any one of claims 4 to 11, characterised in that said one or more compounds comprises phlorizin, and/or phloretin in an amount effective to inhibit glucose transport in the neoplastic cells and is 5 administered along with one or more additional compounds selected from the group consisting of lonidamine, bleomycin and mytomycin while sub- jecting said cells to additional therapy in the form of chemotherapy, thermal or radiation therapy. w 13. A compound or compounds as claimed in claim 12, characterised in that the dosage of said one or more compounds is an effective amount of at least 200-1000 mg per kg of body weight and the dosage of said additional one or more com- ?s pounds is 50 to 500 mg per kilo of body weight. 14. A compound as claimed in any one of claims 4 to 11, characterised in that said com' pound is phloretin in an effective amount to inhibit glucose transport in the cells and said cells are 20 concurrently subjected to thermal treatment. 15. A compound as claimed in claim 14, characterised in that said phloretin is administered in combination with a chemotherapeutic agent se- lected from the group consisting of lonidamine, 25 bleomycin and mytomycin, 5-flurouricil, and cisplatinum. 16. A compound or compounds as claimed in any one of claims 4 to 15, characterised in that said one or more compounds is administered along 30 with one or more additional therapeutically effective compounds while subjecting said cells to additional therapy in the form of chemotherapy, thermal or radiation therapy. 17. Use of a compound or compounds as 35 claimed in any one of claims 4 to 16, in manufac- turing a medicament for treatment of malignant neoplastic cells in humans. 1 8. A medicament for use in treatment of ma- lignant neoplastic cells, characterised by compris- 40 ing a compound or compounds as claimed in any one of claims 4 to 16.

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10 PARTIAL European Patent EUROPEAN SEARCH REPORT Application number J> Which under Rule 45 of the Eur°Pean Patent Convention Office EP 88 30 2189 shall be considered, for the purposes of subsequent proceedings, as the European search report

DOCUMENTS CONSIDERED TO BE RELEVANT Cateaorv Citation of document with indication, where appropriate. Relevant CLASSIFICATION OF THE of relevant passages t0C|alm APPLICATION (Int. CI.")

X DIALOG INFORMATION SERVICES, Data . C1 T ,..._„ Base 72: EMBASE 82-88 A bl K 31/70 Accession nr. 86199919 DELICADO, E. et al. : "Effects of insulin on glucose transporters and metabolic patterns in Harding- Passey melanoma cells" & CANCER RES. (U.S.A.), 1986, 46/8 (3762-3767) * Abstract * 1-2,4, 5,17-' 18

X DIALOG INFORMATION SERVICES, Data Base 72: EMBASE 82-88 Accession nr. 86187256 KASAHARA, M. et al. : "Decrease in glucose transport activity of Friend erythroleukemia -cell — caused by dimethylsulf oxide , a S™SK differentiation-inducing reagent" & BIOCHIM. BIOPHYS. ACTA (Netherlands), 1986, 856/3 (615- „ c. v 623 / A 61 K INCOMPLETE SEARCH The Search Division considers that the present European patent application does not comply with the provisions of the European Patent Convention to such an extent that it is not possible to carry out a meaningful search into the state of the art on the basis of some of the claims. Claims searched completely: 4 — 18 Claims searched incompletely: 1 — 3 Claims not searched: Reason for the li m itatio n of the search : Method for treatment of the human or animal body by surgery or therapy (See art. 52(4) of the European Patent Convention)

Place of search Date of completion of the search Examiner The Hague 20-07-1988 SCARPONI CATEGORY OF CITED DOCUMENTS T : theory or principle underlying the invention v E : earlier patent document, but published on, or X : particularly_*.,,, relevant .., if taken, alone. after the filing date Y: particularly relevant if combined with another D: document cited in the application document of the same category |_ : document cited for other reasons A : technological background 0. non-written disclosure &: member of the same patent family, corresponding P : intermediate document document European Patent Application n'_'rnber PARTIAL EUROPEAN Of,lce SEARCH REPORT EP 88 30 2189 -2- DOCUMENTS CONSIDERED TO BE RELEVANT CLASSIFICATION OF THE APPLICATION (Int. Cl. <) Category C. tat. on ol oocument with indication, where appropriate of relevant Relevant passages to ciaim * ! Abstract * 1-2,4, 5,17- 18

X DIALOG INFORMATION SERVICES, Data Base 173: EMBASE 74-79 Accession nr. 77012446 WALUM, E. et al. : "Kinetics of 2 - deoxy-D-glucose transport into cultured mouse neuroblastoraa cells" & EXP. CELL RES. (U.S.A.), 1976 97/1 (15-22) * Abstract * 1-2,4, TECHNICAL FIELDS 5,17- SEARCHED (Int CM) 18

X US-A-4 565 806 (K.M.E. SETALA) * Claims * 1-6, 11-12, 16-18

X EP-A-0 172 721 (H.H. LEVEEN et al. ) * Whole document * 1-6, 11-12, 16-18

EPO Form 1=05.3 06.78