SE461983B - CONJUGATOR TRIGLUCURONIC ACID AND A CYTOXIC AGLYCON, PROCEDURES FOR ITS PREPARATION AND A PHARMACEUTICAL COMPOSITION THEREOF - Google Patents

Description

461 983 2 mycophenolic acid B-D-glucuronides, Baba refers to antitumor activity of 5-fluorouracil-O-β-D-glucuronide and Ball refers to anti-tumor activity of p-hydroxy-aniline-mustard-glucuronide.

It has also been reported that the selectivity of these transport mechanisms can be improved by hypersurgery of the tumor cells. The article by von Ardenne above as well as the East German patent clearly recognize the importance and benefit of hypersurgery of the tumor cells when the glucuronide mechanisms were used. The Von Ardenne reference refers to a process which gives a pH difference of at least one pH unit and can therefore be used as a basis for selectivity. It is intended to achieve a state of continuity after hypersurgery in which the pH of the brain is 7.0 and the pH of the tumor tissue is about 5.5 to 6.0. See also von Ardenne, M. and co-workers, Pharmazie, 32 (2): 74-75, (1977).

Bicker, U., Nature, 252, December 20-27, (1974), p. 726-727, notes in particular that lysosomal enzyme B-glucuronidase has an optimal pH at 5.2 and that in order to obtain the anti-tumor activity of glucuronides, the pH value must be lowered, for example by administering glucose. The experiments are detailed and suggest that hypersurgery with glucose is necessary to obtain a significant separation of the glucuronides. However, even in hypersurgery of the tumor cells in a known manner, for example by glucose administration, there is still a problem in that other organs and tissues in the body have a naturally present high beta-glucuronidase activity which also releases the toxic glycones and thereby causes damage to healthy tissue. This is especially a problem with regard to the kidneys, which normally have an acidic environment.

It has been proposed in British Patent 788,855 that mandelonitrile-beta-D-glucuronic acid can be used in the treatment of malignant tumors as beta-glucuronidase is predominant in malignant tissue and selectively attacks mandelonitrile-beta-D-glucuronic acid at the site of the malignant tumors and thus hydrogen cyanide. U.S. Pat. No. 2,985,664 also relates to mandelonitrile-beta-D-glucuronic acid and a process for its preparation.

However, it has been discovered that none of the processes for the preparation of these compounds set forth in the above W) 3,461,983 patents are reproducible. The present inventors have discovered that attempts to oxidize prunasin yield the glucuronide of mandelic acid because the CN group is unstable. Attempts to condense mandelonitrile with glucuronic acid or glucuronolactone or tetraacetyl-glucuronactone halide fail because mandelonitrile tends to polymerize.

An article by Fenselau, C and colleagues in Science, 198 (4317) 625-627, (1977) entitled "Mandelonitrile-beta-Glucuronide: Synthesis and Characterization" confirms that syntheses described in the original patent could not be reproduced. This article confirms that although mandelonitrile beta-D-glucuronide was originally given the name Laetrile, this compound is not present in the Mexican preparation marketed as Laetrile.

The main component of the preparations now marketed as Laetrile is amygdalin, which can be easily made from natural materials such as apricot kernels, almonds and other species of the genus Prunus.

However, amygdalin cannot be cleaved by the enzyme beta-glucuronidase.

The Fenselau process teaches a process for the biosynthesis of mandelonitrile-beta-D-glucuronic acid. Although this process is satisfactory for the preparation of laboratory quantities of the compound, a biosynthetic preparation would undoubtedly be very difficult and expensive to make commercially.

The problems with the chemical synthesis of mandelonitrile-beta-D-glucuronic acid also exist in the synthesis of any glucuronide where the aglycone is a strong electron acceptor. This is because the glucuronide is deconjugated (hydrolyzed) during the course of the classical procedure.

The basic object of the present invention is to overcome the disadvantages of the prior art and to enable improved treatment of malignant tumors, in particular tumors with high beta-glucuronidase activity. The object of the invention is thereby to enable such an improved treatment which is selectively toxic to tumor cells, but does not damage healthy tissue.

To this end, the invention aims to provide novel compounds and pharmaceutical compositions which have very low toxicity to the organism as a whole but are very selectively toxic to tumor cells and in particular tumor cells with high beta-glucuronidase activity.

Furthermore, the invention intends to provide a process for the preparation of these new compounds.

It has been found that the selectivity of glucuronide compounds against tumors can be greatly increased and the possible secretion of the toxic aglycones in normal parts of the body can be greatly reduced by administration to the patient before or simultaneously with administration of the glucuronide with an alkalizing agent. , which maintains the pH of the rest of the body at a pH of about 7.4. It is known that at a pH of 7.4 and above, the beta-glucuronidase activity is essentially non-existent. Thus, administration of alkalizing agents such as bicarbonates or other basic salts will substantially reduce or eliminate the beta-glucuronidase activity which normally occurs in certain healthy tissues, such as kidneys, spleen and liver. However, such administration of alkalizing agents does not reduce the acidity of the tumor cells themselves due to the naturally low pH of the tumor cells, the mechanism of the previous hyperacidification and the absence of significant blood flow in the tumor areas, as well as other possible mechanisms. In fact, it has been suggested in the literature that bicarbonate actually increases the acidity of cancer cells, Gullino, P.M. and co-workers, J.N.C.I., 34, 6, 857-869 (1965).

Because the beta-glucurinidase activity in tumor cells is enhanced by acidification and the beta-glucuronidase activity in the rest of the body, especially in the kidneys, is mainly eliminated by alkalinization, the toxic glycons are released only at the tumor point itself due to the glucuronidase deconjugation of beta-glucuronides. Without the alkalinization step, substantial amounts of toxic material can be released, for example, into the kidneys, and the toxic aglycones released in this way can cause significant damage to these organs. Thus, only through the alkalization can glucuronides of compounds toxic to tumor cells be used clinically with a high degree of safety.

The greater the toxicity of the aglycones, the more significant the alkalization step. The present invention relates primarily to certain novel glucuronide compounds which are particularly suitable for use as above due to the significant differences in pH between tumor cells and surrounding healthy tissue. If the aglycones are more active at lower pH or non-polar in acidic conditions and become polar only in alkaline conditions, e.g. in that the aglycone is water-soluble at a pH range above about 7 and lipid-soluble at a pH range below 7, the selectivity as above will be further increased. When using these new compounds, even if they are deconjugated elsewhere in the body, the aglycone will be water soluble due to the alkaline pH and quickly washed out of the system. In the lower pH ranges of hyper-acidified tumor cells, on the other hand, the aglycones will attack the tumor cells and not be dissolved or washed away. Even if a certain amount of aglycones are removed from the site of the tumor cells, they will immediately become water-soluble in an alkaline environment and are quickly removed from the body. The novel compounds of the invention are 2,4-dinitrophenol-beta-D-glucuronic acid; chloromycresol-beta-D-glucuronic acid; 4,6-dinitro-o-cresol-beta-D-glucuronic acid; 4-chloro-3,5-xylanol-beta-D-glucuronic acid; chlorotymol-beta-D-glucuronic acid; 2-phenyl-6-chlorophenol-beta-D-glucuronic acid; 5-Chloro-7-iodo-8-quinolinol-beta-D-glucuronic acid and podophyllotoxin-beta-D-glucuronic acid. Chloro-m-cresol-beta-D-glucuronic acid is of particular interest because it loses its toxic activity in an alkaline environment.

In addition to their usefulness as anti-tumor agents, these novel compounds and any other glucuronide compound with cytotoxic aglycones also have antibacterial activity, especially against those types of bacteria which have glucuronidase activity.

For example, streptococci, staphylococci and E. coli bacteria are known to have beta-glucuronidase activity. Therefore, if the glucuronides come in contact with these bacteria, they become cleaved and the cytotoxic aglycones become toxic to the bacteria.

It has also been reported that the optimum pH for bacterial beta-glucuronidase is higher than the optimum pH for beta-glucuronidase in normally healthy internal organs, such as liver, spleen and kidney, etc. Therefore, in alkalinization, ringing of the body in accordance with the above, the beta-glucuronidase activity in the organs is essentially eliminated, although that of the bacteria independent of the alkalinization will still be present. The administered glucuronide is only cleaved to its active form at the site of infection. Because tumor cells are not treated in this use, no hypersurgery step is necessary.

When using the compounds of the present invention, it is not important that the toxicity of the agent be directed only to the cancer cells in contrast to all healthy cells in the body, since in the process according to the invention the aglycones are only released where the cancer is present.

Consequently, a particularly useful aglycone is one that exercises through it. There is no need to worry about the transfer mechanism of the drug through the membranes. their cell toxicity by attacking the cell membranes themselves.

By attacking the membranes, the nature of the membranes and the antigenic properties of the cells further change. Therefore, the immune system of the host will help the toxicant to release the host from such cells. Consequently, a much lower dose is required. Examples of aglycones which exert this effect include phenol and cresol.

Other measures to increase the beta-glucuronidase activity of the tumor cells may also be taken. One way is to raise the temperature of the toxic cells at the time of treatment.

This can be done by raising the temperature throughout the body by using pyrogenic drugs or by raising the temperature only in the tumor cell area, such as by microwave radiation or electric current. Elevation of the temperature increases the beta-glucuronidase activity and thereby increases the efficiency in the deconjugation of the glucuronides. It is known that an increase in temperature of 3 ° C increases beta-glucuronidase activity by 50%.

Known pyrogenic drugs include ethiocholanolone, progesterone, dinitrophenol, dinitrocresol, etc. Both dinitrophenol and dinitrocresol are also cytotoxic as will be shown below. Therefore, the use of these compounds is preferred, especially if they are administered as the glucuronide. This gives the result that when the glucuronide is deconjugated at the tumor site, the aglycone not only acts to denature the cytoplastic protein but also raises the temperature directly in the area of the tumor cells and thus greatly increases the efficiency of further deconjugation.

Local hyperthermia in the area of suspected tumor cells is preferred over general hyperthermia because general hyperthermia also increases beta-glucuronidase activity in healthy cells. However, due to the alkalization step, this is not a major problem. If hyperthermia is local, this provides an additional degree of certainty that the glucuronides are only cleaved at the tumor site. Microwaves directed at the suspected tumor site are a way to achieve local hyperthermia. Depending on the different electrical resistance in tumor cells, another procedure to achieve a certain degree of local hyperthermia is to administer a low electrical current through the body.

A further way to selectively increase beta-glucuronidase activity in tumor cells is by administering estrogen to female patients or testosterone to male patients. These compounds have been reported to induce beta-glucuronidase activity in trophoblastic cells. Some tumor cells are known to be trophoblastic, so this method is particularly useful for such cells. The alkalization step prevents damage to healthy trophoblastic cells.

The present invention further relates to a process for the preparation of the glucuronides. It has been found that it is impossible to produce glucuronic acid conjugates by classical methods when the aglycone is a strong electron acceptor, due to the fact that these compounds must be prepared as the methyl ester of the glucuronic acid and that it is not possible with previously known processes to convert the methyl ester to the acid without cleaving the aglycone. Although barium methoxide has been proposed for this purpose in a similar process according to U.S. Pat. No. 2,985,664, it has been found that barium methoxide is not useful. It has now been found that if barium hydroxide is used, the methyl ester of the aglycone of the glucuronide can be converted to the barium salt and the barium salt can be converted to the free acid by using sulfuric acid without deconjugating the glucuronide. Furthermore, the deprotection of the acetyl group takes place in the same step, of a separate step for carrying out this function. thereby eliminating the need Other applications of the invention are associated with the extremely high tumor selectivity that can be achieved. Depending on the selectivity, local radioactivity can be exerted if one or more of the atoms in the aglycones are exchanged for a radioactive isotope. This method is not only important for diagnostic purposes to detect tumors and their metastases, but if the isotope is selected with beta-radiation activity, the method can also be used for local radiotherapy of the cancer site. This use of radioactive isotopes is especially important when using an aglycone which is known to be acidic solution and polar in alkaline solution. When this property exists, the aglycone can accumulate at the cancer site not only due to the beta-glucuronidase activity but also due to its insolubility in water at the cancer site, while the compounds with the radioactive isotope are washed away from the rest of the body. . The use of p-iodophenol-beta-D-glucuronic acid provides an aglycone, p-iodiphenol, which meets these requirements. A radioactive isotope of iodine can be used as an iodine constituent in this compound. It is preferred to use l3lI for labeling: and l33I for treatment, stronger gamma radiation and that while the former has a later a stronger beta- To prevent iodine from migrating to the thyroid gland, a pretreatment with non-radioactive Lugol solution can be used to saturate the thyroid gland.

Other compounds which can be easily labeled radioactively are the glucuronide of phenylsulfazole. A radioactive sulfur atom can radiate. be used. This compound does not travel to the thyroid gland and the aglycone is not water soluble.

Prior to treatment of patients with compounds of the present invention, it must be established that the particular type of tumor involved has high beta-glucuronidase activity. This can be done in a number of different ways. One way is to test tumor cells obtained by biopsy for beta-glucuronidase activity. If such a test is positive, then a pharmaceutical composition of the present invention can be administered.

A second method is the administration of a glucuronide whose aglycone is labeled with a radioactive isotope. If a whole-body scan shows that the radioisotope accumulates in a specific area of the body, this indicates not only the location of the tumor, but also the fact that the tumor has sufficient beta-glucuronidase activity to cleave the glucuronide. Once determined, an appropriate amount of the selected glucuronide is administered. If no tumor is present or if the tumors are of such a nature that they have no beta-glucuronidase activity, no accumulation of the radioisotope occurs in the body, due to the fact that the alkanization step of the present invention eliminates all normal beta-glucuronidase activity, and isotopes pass through the body¿ Another method of diagnosing tumors which are treatable with compounds of the invention is to test for the presence of free glucuronic acids in the urine. Although the presence of glucuronides in urine is common, the presence of free glucuronic acids in the urine and especially the presence of increasing amounts of glucuronic acid during test periods of several days is an effective means of determining the presence of tumors with beta-glucuronidase activity. It is believed that the presence of free glucuronic acids in the urine of cancer patients is caused by the action of beta-glucuronidase in the cancer cells on intercellular fibers and connective tissue. Glucuronic acid is a reaction product of such activity due to the fact that the intercellular fibers and connective tissue are composed of polymers in which glucuronic acid is a component and which are known substrates for the enzyme beta-ghkunxüdhs.

Preferred Embodiment of the Invention Although many glucuronide compounds with aglycones that are toxic to cancer cells have been theoretically described in the literature, few have actually been prepared. This is because they are very difficult to synthesize, especially since the aglycone is a strong electron acceptor. The improved process of the present invention avoids the problem and enables the production of glucuronic acid conjugates from almost any type of aglycone. Standard procedures can be used to form the methyl ester of triacetyl-glucuronic acid conjugate, but it is often difficult to get from the triacetylmethyl ester to the glucuronic acid conjugate. This problem has been solved by treatment in accordance with the method of the present invention.

The glucuronides of the present invention can be synthesized from methyl (tri-O-acetyl-alpha-D-glucopyranosyl bromide) uronate, which is the active glucuronic acid and formed according to J.Am.

Chem.Soc. 77, 3310, (1955). This compound is condensed with what is stated by Bollenback, G.N. and co-workers, aglycone in a solution of quinoline, phenol, methyl cyanide or methyl nitral catalyzed by silver oxide or silver carbonate.

Other methods of condensation are to use sodium or potassium hydroxide as a condensing agent in an aqueous acetone solution. The reaction scheme is illustrated below: (I) -OAC OA: where ROH is the desired aglycone.

If the methyl ester of the glucuronide is desired, the protecting acetic acid group can be removed by means of anhydrous sodium methoxide or anhydrous barium methoxide in accordance with the following reactants COOCH3 COOCHB CH 0Na _, _ ° "" n cuso ' can be prepared by reacting the triacatylmethyl ester with barium hydroxide to form barium hydroxide in accordance with the following reaction: .nl- 461 983 11 'coocas cooeaä ° o-- R' o - a - Ba - ç fl (III) Aco OH. 0Ac. OH This barium salt of the glucuronide precipitates. An equimolar solution of sulfuric acid releases the glucuronide according to the following reaction: CO0Baä con o__. Example 2 shows the preparation of 2,4-dinitro-phenol-beta-D-glucuronic acid.

Example I - found by 2,4-dinitrophenol-beta-D-glucuronic acid Methyl- (2,3,4-tri-U-acetyl-alpha-D-glucopyranosyl bromide) -uronate was prepared according to the procedure of Bollenback, oN , and co-workers, J. Am. chem. soc. 77, 3310 (1955).

Four grams of methyl (tri-U-acetyl-alpha-D-glucopyranosyl bromide) uronate in acetone (80 ml) and 8.9 g of 2,4-dinitrophenol were treated with 5N potassium hydroxide (9 ml) and the solution was kept at 2506 for 24 hours, after which it was diluted with 3 volumes of chloroform. The chloroform-acetone phase was washed with water and dried. Removal of the solvent and two recrystallizations from acetone gave methyl 2,3,4-tri-O-acetyl-beta-D-glucopyranosyl uronate of 2,4-dinitro-phenol.

The free acid form of the compound was formed by treating 2,4-dinitrophenyl-methyl (tri-β-acetyl-beta-D-glucopyranosyl bromide) -uronate with a half molar amount of barium hydroxide to produce the barium salt. This barium salt of the glucuronide precipitated as a white amofft material. An equimolar 461 985 l2 solution of HZSD4 releases the free glucuronide. Distillation of the supernatant gave light yellow-brown crystals, m.p. 179-18D ° C. This compound was incubated with beta-glucuronidase to give 2,4-dinitrophenol, which confirmed that the final product was in fact 2,4-dinitrophenol-beta-Qeglucuronic acid.

Other glucuronides in accordance with the present invention, e.g. chloro-m-cresol-beta-D-glucuronic acid; 4,6-dinitro-o-cresol-beta-D-glucuronic acid; 4-chloro-3,5-xylanol-beta-D-glucuronic acid; chlorotymol-beta-D-glucuronic acid; 2-phenyl-6-chlorophenyl-beta-D-glucuronic acid; 5-Chloro-7-iodo-8-quinolinol-beta-D-glucuronic acid and podo-phyllotoxin-beta-D-glucuronic acid as well as p-iodophenyl-beta-D-glucuronic acid and phenylsulfazole-beta-Dpglucuronic acid can be prepared on similarly by reacting stoichiometric amounts of aglycones with methyl (tri-D-acetyl-alpha-D-glucopyranosyl bromide) uranium in 5-normal potassium hydroxide and maintaining the reaction solution at room temperature for 24 hours. The solution was then diluted with 3 volumes of chloroform and the chloroform-acetone phase was washed with water and dried. After removal of the solvent, crystals were obtained, which were treated with a half molar amount of barium hydroxide to form the barium salt, which was then treated with an equimolar amount of sulfuric acid to form the free glucuronide.

The free acid form of the glucuronide, or a salt thereof, which ionizes under conditions of use, is the preferred form of the compound for use in accordance with the present invention. However, pharmaceutically acceptable esters can also be used, although in most cases it would be agreed that their activity would be slightly lower due to the relatively low affinity for beta-glucuronidase. This is especially true with respect to aglycones which are strong electron acceptors. Accordingly, by the term "glucuronide compound" used in the present specification and claims, is intended to include not only the free glucuronic acid form of the conjugate but also pharmaceutically acceptable salts and esters thereof as discussed above in both this and subsequent examples.

Example II - Method of administering glucuronide therapy Once it has been determined that the patient has a tumor with beta-glucuronidase activity, the first step in the treatment is to give him a dose of glucose, for example 100 g of honey, glucose or other sugar. Approximately 1 hour later, intravenous drip of a solution of distilled water containing approximately 10ß glucose and 60 milliequivalents of sodium bicarbonate is started. Approximately 1 liter is administered provided that there are no contraindications and the pH value in the urine is tested to determine that it has reached a pH value of approximately 7.4.

This is achieved when the system has been alkalinized and now the system is safe for administration of glucuronide. Another liter of the same glucose bicarbonate solution is added, but together with the desired amount of glucuronide.

This treatment is repeated daily for as long as necessary.

When a glucuronide with nitrile-containing cytotoxic aglycone is used, before, during and after administration of the glucuronide, 50 ml of 25% sodium thiosulphate solution is administered, preferably by intravenous drip. The sodium thiosulfate is preferably included in the glucose bicarbonate glucuronide solution which is added by intravenous drip.

However, it can also continue after to obtain a larger safety margin.

If there are contraindications to the administration of bicarbonate, antacids can be administered orally. An essential criterion is that the pH value in the urine becomes about 7.4 and is maintained during the treatment.

The hypersurgery of the tumor cells is caused by a hyperglycemic condition in the patient. Therefore, any hyperglycemic agent can be used as a hypersensitive agent, for example fructose, galactose, lactose or glucagon. Furthermore, it is obvious that this hyperglycemic condition can be achieved in any other known way. For example, if the patient is diabetic, the condition can be achieved by reducing insulin administration.

Any agent that raises the pH of the urine to about 7.4 can be used as an alkalizing agent, including sodium or potassium bicarbonate or citrate or other basic salts or antacids. Although it is preferred that these be administered intravenously, they may be administered orally.

When the term "approximately 7.4" is used in the present specification and requirements with respect to the pH maintained in the rest of the body, it must be understood that pH levels slightly above or below 7.4 may be used, although this is not preferred. As the pH decreases from 7.4, the beta-glucuronidase activity increases (until an optimal pH is reached). Below pH 7, the rest of the body will not be alkaline but acidic. above 7.4 increases the risk of alkalosis without significantly further decrease in beta-glucuronidase activity. A pH of 7.4 is preferred because it is a physiological pH and can not be harmful to the body, and it is known that the beta-glucuronidase activity of healthy organs is essentially non-existent at this pH. -value.

The doses of glucuronides should öwuväæm fi t'æü: umàdka side effects due to massive release of toxins caused by dying cancer cells. It is preferred to treat with glucuronides in short sessions of a few days, after which several days may go without treatment to allow all toxins released from dying cancer cells to leave the body before further treatment continues.

In addition to intravenous administration, the glucuronides may be administered by any means for parenteral administration.

However, the glucuronides should not be administered orally because the beta-glucuronidase is known to be present in the digestive tract. However, the sodium thiosulphate can be administered orally if a suitable coating prevents it from being released into the stomach.

The amount of glucuronide to be administered to a particular patient is determined empirically and differs depending on the patient's condition. Relatively small amounts of glucuronide _ can be administered from the beginning with a regular increase in the daily dose if no adverse effects are observed.

Of course, the maximum safe dose determined in routine animal testing should never be exceeded.

It is clear that any tumor cell with beta-glucuronidase activity can be treated in accordance with the present invention with other organs of the body protected by the alkalization step. Tumors which are known to have beta-glucuronidase activity include solid breast tumors and their metastases, bronchogenic carcinomas and their metastases and lymphomas. It is also known that neoplasms that do not have high beta-glucuronidase activity and therefore cannot be treated in accordance with the present invention include leukemia. However, it is clear that this list is not intended to be exhaustive and that what is previously known is that many other tumors have beta-glucuronidase activity.

However, whether or not it is known presently that a given tumor has beta-glucuronidase activity, this can be determined by any of the various diagnostic methods discussed in the present specification, and whether it is determined that the tumor has beta-glucuronidase activity. glucuronidase activity, the therapeutic treatment of the present invention can be effectively used.

If it is desirable to induce hyperthermia to increase beta-glucuronidase activity, a method should be chosen in which the temperature is raised as much as possible without risk to healthy parts of the body, such as the eyes. An increase of about 20 in hyperthermia of the whole body and as much as 461 985 16 4.5 ° C in local hyperthermia is preferred. Hyperthermia should be maintained for about 1 hour at the time of the highest glucuramide concentration at the tumor site. When local microwave therapy is chosen, for example, it should start] / 2 hours after the insertion of the intravenous glucuronimide drop and continue for about 1 hour. An appropriate dose of known pyrogenic agents to achieve the desired degree of hyperthermia is known to those skilled in the art or can be readily determined experimentally.

A dose of about 30 mg / day for dinitraphenol, for example, is suitable.

When estrogen or testrogen is administered, a dose of 5 - 15 mg / body weight / day provides the desired induction of beta-glucuronidase activity.

Example III - Procedure For administration of radioisotopes If an aglycone labeled with a radioactive isotope is to be administered, the labeling can be performed in a manner known per se.

For diagnostic purposes, relatively small amounts of these labeled glucuronides are administered. Incidentally, they are administered in the same manner as in Example VI for unlabeled glucuronides; Scanning of the body to determine whether any of the radiolabeled glycone is retained by the body indicates that a tumor present has beta-glucuronidase activity and also indicates where the tumor or metastases thereof can be found. As stated above, gamma-ray isotopes such as 131I are suitable for this purpose.

The radiolabeled glucuronides can also be used for in situ radiation therapy, especially if an isotope with high beta-radiation activity is used, such as 331. This gives a double effect in the attack on the cancer cells in that not only the toxic aglyliorxemautan can also affect the beta radiation. on the cells. Again, the method of administration is the same as set forth in Example VI. Another use for the present invention is the use of boron-containing aglycones. It is already known that boron atoms bombarded with neutrons decompose in lithium and at the same time release positrons. If the boron atoms are attached to the tumor tissue during neutron irradiation, the positrons are rapidly absorbed by the tumor tissue and are lethal to it.

This method has excellent utility when the boron atoms are concentrated in the tumor cells in accordance with the method of the present invention.

Example: Vf - Procedure For Administration of pH Dependent Therapy Dm the tumor cells are hypersuritated and the Healthy Tissue alkalinized according to the procedure set forth in Example VI, an acid-base pH differential is created between the tumor cells and the Healthy cells. Thus, compounds whose activity or solubility is pH-related can be administered directly without first being linked to a glucuronide. Such compounds will selectively attack the acidified tumor cell without damaging the remainder of the body, which has an alkaline pH.

As indicated in Example VI, the patient first receives an oral dose of a hyperglycemic agent, such as 100 g of honey, sucrose or other sugar. Approximately 1 hour later, an intravenous drip of a solution in distilled water containing about 10% glucose and 60 milliequivalents of sodium bicarbonate begins. Approximately 1 liter is administered provided that no contraindications are present and the pH value in the urine is tested To determine when it has reached a pH value of about 7.4. This shows that the system has been alkalinized and that it is now safe to administer the acid-active compound. An additional liter of the same glucose bicarbonate solution, which also contains the desired amount of acid-active compound, is administered. This treatment is repeated daily for as long as needed. Compounds such as 2,4-dinitrophenul; 4,6-dinitr @ - @ - kresu1; 4-chloro-3,5-xylanyl clunotymol; 2_feny1_5_klDr0fenu1; 5-K1gr0-7-iodo-8-quinolin-1-ol and podophyllotoxin are all water-soluble at alkaline pH and lipid-soluble at acidic pH. Therefore, if the compounds are administered in the manner set forth above, they have no significant detrimental effect on healthy tissue because they are washed out of the system relatively quickly. At such points where tumor tissue occurs with acidic pH, however, the compounds come out of the aqueous solution and exert their cytotoxic or energy supply affecting effect on the tumor cells.

Compounds such as chloro-m-cresol as well as 4,6-dinitro-o-cresol, 4-chloro-3,5-xylanol, chlorothymol and 2-phenyl-6-chlorophenol are more active at lower pH. Therefore, administration of these compounds with simultaneous hypersurgery of the tumor cells and alkalinization of the rest of the body will be less harmful to healthy tissue, as their activity decreases at the pH values in healthy tissue.

The dose of the non-glucuronidic compound in accordance with this embodiment of the present invention is generally slightly lower than that of the corresponding glucuronide, since the glucuronide is substantially less toxic than the free compound. The exact dose must be determined empirically depending on the patient's condition. Relatively small doses should be administered from the beginning with regularly increasing daily doses if no adverse effects are noted. Of course, the maximum safe dose, which is determined by routine testing on animals, should never be exceeded.

Alternative acidifying and alkalizing agents discussed above with respect to the glucuronide embodiment may also be used in accordance with the present embodiment.

Example X7 - Method of Anti-Bacterial Administration Glucuronide administration can be used in the treatment of bacterial infections if the bacteria involved are known to have a beta-glucuronidase activity. Examples of such bacteria are streptococci, staphylococci and E. coli. The procedure for treating such bacterial infections is similar to that of Example VI except that no hypersurgery is necessary. This is because the bacterial beta-glucuronidase is active at a higher pH value than the beta-glucuronidase in normally healthy internal organs. Furthermore, such a hypersurgery step would not affect the pH of the bacteria, since it is a mechanism specific to thin cells.

The first step in antibacterial administration is an intravenous drip of distilled water and 60 milliequivalents of sodium bicarbonate. Approximately one liter is administered and the pH value in the urine is tested to determine when it has reached a pH value of about 7.4. An additional liter of bicarbonate solution is added but comprising the desired amount of glucuronide, which is administered in the same manner.

The treatment can be repeated daily if necessary.

The di-alkalinizing agent can also be administered orally and any agent can be used which alkalinizes the body to such an extent that the pH of the urine becomes about 7.4. Glucuronide should not be administered orally but may be administered parenterally.

Certain known antibacterial agents, with undesirable side effects, may be administered as glucuronides in accordance with the method of the present invention to reduce or eliminate these undesirable effects. For example, chloramphenicol is known to have a bone marrow-damaging effect, which does not occur if glucuronide is used. Neomycin is a known antibacterial agent, which cannot be administered internally due to its toxicity. However, it can be administered orally for the treatment of infections by bacteria with high beta-glucuronidase activity if it first binds to glucuronic acid. 4651 9233 The radioisotope-labeled aglycone diagnostic methods discussed above with respect to tumor diagnostics can also be used to determine the existence and location of bacterial infections. For example, a patient suffering from pain in the appendix area may receive radiolabelled glucuronides. If no accumulation of isotopes is obtained in the area, inflammation caused by bacteria with beta-glucuronidase activity as the cause of the pain can be written off. In most cases, appendicitis inflammation is due to bacteria with beta-glucuronidase activity. Other uses of such diagnostic procedures will be apparent to those skilled in the art.

In addition to glucuronidate compounds, each known antibiotic that can be bound with glucuronic acid used against beta-glucuronidase-containing infections was discussed above. This has the advantage of greatly reducing the amount of free antibiotics circulating in the bloodstream. The only amount of antibiotic released is released at the site of infection. Therefore, much smaller doses can be given. Accordingly, the glucuronides of the present invention can serve as internally administered, local antibiotics. Due to the known bsta-glucuronidase activity in the digestive tract, the glucuronides should not be administered orally, while any type of parenteral administration is possible.

If the antibiotic aglycones are known to have no effect on the kidneys, the alkalization step can be omitted. However, many antibiotics are known to be nephrotoxic to some extent and thus the alkalinization step is important for protecting the kidneys.

Claims (3)

A compound in the form of a conjugate of free glucuronic acid and a cytoxic aglycone, which has a greater toxic effect in an acidic environment than in an alkaline environment and / or is water-soluble in an alkaline environment and water-insoluble or only slightly water-soluble in an acidic environment, or of a pharmaceutically acceptable ionizable salt of said glucuronic acid conjugate, the compound being 2,4-dinitrophenol-beta-D-glucuronic acid; chloro-m-cresol-beta-D-glucuronic acid; 4,6-dinitro- o-cresol-beta-D-glucuronic acid; 4-chloro-3,5-xylanol-beta-D-glucuronic acid; chlorotymol-beta-D-glucuronic acid; 2-phenyl-6-chlorophenol-beta-D-glucuronic acid; podophyllotoxin-beta-D-glucuronic acid; p-iodophenol-beta-D-glucuronic acid or phenylsulfazole-beta-D-glucuronic acid. A process for the preparation of a conjugate of free glucuronic acid and an aglycone, which is a strong electron acceptor, wherein the aglycone is condensed in a manner known per se with methyl- (tri-O-acetyl-alpha-D-glucopyranosyl) -halide- uronate to form the methyl ester of aglycone-tri-O-acetyl-beta-D-glucuronic acid, characterized in that barium hydroxide is added to said methyl ester so as to obtain a precipitate of the barium salt of the glucuronide, which precipitate is removed and treated with sulfuric acid , so that a precipitate of barium sulphate is obtained, after which the supernatant liquid is separated from the barium sulphate precipitate and dried, whereby the conjugate of free glucuronic acid and the aglycone according to claim 1 is obtained. Pharmaceutical composition, characterized in that it is an aqueous solution of a compound according to claim 1; a hyperglycemic agent for hypersurgery of tumor cells during administration of the composition; and an alkalizing agent for maintaining the pH level of non-tumor tissue in the patient at about 7.4 when administering the composition.