DE2953223T1 - B-GLUCURONIDASE ACTIVITY AND / OR PH-DEPENDENT PHARMACEUTICALS AND THEIR METHODS OF PRODUCTION AND USE FOR SELECTIVE TREATMENT OF DISEASES - Google Patents

Description

-X-

DESCRIPTION

The present invention relates to the treatment of tumors which have β-glucuronidase activity with the aid of glucuronides with toxic aglycones (deglycon _: glucuronidized residues), in particular an improvement of such a method which prevents damage to the kidneys. The toxic aglycones can contain a nitrile group. The invention also relates to the treatment of infections with certain bacteria which have ß-glucuronidase activity. The invention also relates to a new class of glucuronides whose activity as aglycones or whose water solubility is pH-dependent, as well as the process for producing such glucuronides. The invention also relates to new, nitrile-containing glucuronides and, finally, a diagnostic urine-analytical determination by means of which the presence of tumors with β-glucuronidase activity can be determined.

There are a number of publications that deal with deal with the general concept that the direct delivery of an agent that is toxic to tumor cells, directly to tumors with ß-glucunoridase activity Conjugation of the agent with glucuronic acid is to be achieved. These include publications by Von Ardenne, M. et al .: Agressologie, T ?, 5, 261-264- (1976), the GDR patent 122 386, DT-OS 22 12 014-, publication by Sweeney et al .: Cancer Research, 31_, 4-77-4-78, (1971), Baba et al .: Gann, 69, 283-284- (1978) and Ball, CR .: Biochem. Pharm., 23, 3171-3177 (1974).

In the work of Von Ardenne are widely used proposed many types of aglycones associated with glucuronic

030 601/0045

acid can be conjugated and at the site of the tumor; are effective. These generally include alkylating groups, antimetabolites, cytotoxins, membrane-active (lytic) groups, glycolysis stimulators, respiratory inhibitors, ■ inorganic and organic acids and substances that stop the cell cycle. The GDR patent also suggests many such combinations, including 5-i ¹ luorouracil-glucuronide, methotrexate-glucuronide, 6-mercaptopurine-glucuronide, anilinsenfgas-glucuronide and many others. The DT-OS also mentions a large number of glucuronides. Sweeney's work relates to the anti-tumor effects of mycophenolic acid-ß-D-glucuronides, Baba in turn to that of 5-fluorouracil-O-ß-D-glucuronide and Ball to the anti-tumor effects of p-hydroxyanilinfgas-glucuronide.

It has also been reported that the selectivity of this transport mechanism can be improved by acidifying the tumor cells. The aforementioned work by Von Ardenne and the GDR patent clearly recognize the importance and feasibility of acidifying tumor cells when using the glucuronide mechanism. Von Ardenne mentions a process which produces a pH difference of at least one pH unit and can consequently be used as a basis for the selective treatment. It refers to the achievement of steady-state conditions after hyperacidity, the pH of the brain being 7.0 and that of the tumor tissue around 5.5 to 6.0. See also Von Ardenne, M. et al .: Pharmazie, 52 (2) : 74-75 (1977).

Bicker, U., Nature, 252: 726-727 (1974-) specifically mentions that the lysosoraal enzyme ß-glucuronidase has an optimum pH of 5.2 and that to achieve an anti-tumor

Effectiveness of glucuronides the pK approximately through the administration of glucose needs to be lowered. He describes experiments that suggest that acidification is caused by: Glucose is necessary to undergo significant deconjugation. To achieve (cleavage) of glucuronides.

Even with overacidification of the tumor cells according to known However, procedures such as, for example, the administration of glucose are still a problem for other organs and tissues of the body which physiologically have a high β-glucuronidase activity, also the release toxic aglycones and damage healthy tissues as a result. This problem poses especially with regard to the kidneys, which normally have an acidic pH environment.

In GB-PS 788 855 the use of mandelonitrile-ß-D-glucuronic acid was suggested in the treatment of malignant tumors as ß-glucuronidase in malignant Degenerate tissues predominate and selectively mandelonitrile-D-glucuronic acid will attack the site of the malignant tumor with elimination of hydrogen cyanide. The US PS 2,985,664 also refers to mandelonitrile-ß-D-glucuronic acid and a method for producing them. These compounds were identified by the applicants of the above Patents called latriles.

However, it has not been found that any of the methods of manufacture set forth in the patents cited above this connection is reproducible. Try prunasin to oxidize yield the glucuronide of mandelic acid because the CN group is unstable. Try using mandelonitrile Glucuronic acid or glucuronolactone or tetra-acetylglucuronolactone halide to condense failed because mandelonitrile tends to polymerize.

0 30 601 / 004S

A publication by Fenselau, C. et al., Science ,: Γ 198 (4317) : 625-627 (1977) entitled "Mandelonitrilfi-Glucuronoide: Synthesis and Charactization" confirms that the synthesis described in the original patents; "" could not be reproduced. This publication also confirms that although the compound mandelonitrile-:. β-D-glucuronide was originally given the name Lätril ·, """however, this compound does not appear in the Mexican preparations sold under the name Lätril, the predominant component of these pharmaceutical preparations currently sold under the name Lätril from amygdalin, which can easily be produced from natural substances such as the kernels of apricots, almonds, and other members of the Prunus family, but amygdalin cannot be broken down by ß-glucuronidase.

Fenselau's work shares a method for biosynthesis of mandelonitrile-ß-D-glucuronic acid with. Although this A laboratory-scale method of making the compound may be satisfactory, such a biosynthesis would be undoubtedly very difficult and expensive to use in industry.

The problems associated with chemical synthesis of mandelonitrile-ß-D-glucuronic acid also exist for that Synthesis of each glucuronide of an aglycone, which is a strong electron acceptor, since the glucuronide in the course is deconjugated (hydrolyzed) using the classical method.

Diagnosis must be made before using glucuronide treatment of tumors with ß-glucuronidase activity. The previous Publications (e.g. Sweeney, as mentioned above) suggest taking a biopsy for determination ■ '"■■. -0 30601/0045

γί-

the presence of the ß-glucuronidsse. It would be desirable, the presence of tumors with such β-glucuronidase activity to be able to determine by a simple urine test.

The aim of the present invention is to improve the treatment of malignant tumors, with the disadvantages of known methods are overcome, in particular an improved method for treating malignant tumors with high ß-glucuronidase activity, being selective toxic effects on tumor cells are released, but healthy tissues are not damaged. Further is goal of the present invention an improved method wherein the tumor cells selectively with nitrile-containing compounds treated while simultaneously receiving therapy to avoid the possibility of cyanide poisoning im rest of the body is operated.

Further objects of the present invention are new compounds and pharmaceutical mixtures with a very high low toxicity for the whole organism, but very high selective toxicity for tumor cells, in particular Tumor cells with high ß-glucuronidase activity. Another object of the invention is a diagnostic method under Use of radioactive isotopes to selectively label tumor cells so that both primary tumor cells as well as metastases can be precisely localized. Another aim of the invention is a method for the preparation of the compound which can be used in the treatment method according to the invention, in particular for the production of mandelonitrile-ß-D-glucuronic acid by total chemical synthesis.

Another object of the present invention is a method of examining urine to diagnose the presence of

030801/0045

Tumors with ß-glucuronidase activity. Another goal of the present invention is a method and pharmaceutical compositions for treating infections with Bacteria which have ß-glucuronidase activity.

It has been found that the selectivity of glucuronide compounds against tumors can be increased considerably and the possible deconjugation of the toxic aglycones in normal parts of the body can be reduced considerably by administering an alkalizing agent to the patient before the administration or at the same time as the administration of the glucuronide maintains the pH of the rest of the body at a value of about 7, ⁷ I-. It is known that at a pH of 7.4 and above the ß-glucuronidase activity is practically zero. Therefore, the administration of alkalizing agents such as bicarbonates or other basic salts will substantially reduce and eliminate the β-glucuronidase activity which is normally found in certain healthy tissues such as the kidneys, spleen and liver. Such administration of alkalizing agents is the. However, do not reduce the acidity of the tumor cells themselves, as a result of the physiologically low pH of the tumor cells, the mechanism of previous acidification and the significant reduced blood flow in the tumorous areas, as well as other mechanisms. In the known literature the idea even emerges that bicarbonate actually increases the acidity of tumor cells (Gullino, PM, et al., JNCI, 54, 6 ; 857-869 (1965).

Since the ß-glucuronidase activity of the tumor cells is increased by acidification and the ß-glucuronidase activity of the remaining body, particularly the kidneys, is essentially eliminated by alkalizing, the toxic ones become Aglycones only at the site of the tumor itself due to the

030601/0045

Deconjugation of glucuronides under the action of ß-glucuronidase released. Without the alkalization step - Significant amounts of toxic material were released in the kidneys, for example, and the toxins thus released were seen Aglycons could cause significant damage to these organs. Therefore you can only use the procedure of the present invention, glucuronides of compounds that are toxic to tumor cells clinically with a large Degree of security to be used. The greater the toxicity of the aglycones, the more important it becomes Alkalization step.

Another aspect of the present invention lies in the use of certain new glucuronide compounds, which is particularly suitable for use in the present invention because of the significant pH gradient between the tumor cells and the surrounding healthy tissue. If the aglycon is more active at lower pH or is apolar under acidic conditions and only becomes polar under alkaline conditions, i.e. if the aglycon is in pH ranges above, about 7 water soluble and at pH ranges below 7 fat soluble, then the selectivity becomes of the method according to the invention further increased. When using these new compounds, the aglycon becomes, itself when deconjugation takes place anywhere in the body, be water-soluble due to the alkaline pH and off quickly flushed out of the system. In the lower pH range of the acidic tumor cells, however, this becomes Aglycon quickly attached to the tumor cells and not brought into solution and washed out. Even if one Should some amount of the aglycone be removed from the site of the tumor cells, it immediately becomes alkaline Milieu come and therefore become water-soluble and are quickly flushed out of the body.

030601/0045

Examples of the new glucuronides in this category are 2,4-Dinitrophenol-ß-D-glucuronic acid, 4 ~ chloro-m-cresol-ß-D-glucuronic acid, ^ -, e-Dinitro-o-cresol-ß-D-glucuic acid, .-; ^ -Chlor-J ^ -xylsnol-ß-D-glucuronic acid, chlorothymol-ß-D-glucuronic acid, 2-phenyl-6-chlorophenol-ß-D-glucuronic acid, 5-chloro-7-iodo-8-quinolinol ~ ß-D-glucuronic acid and podophyllotoxin-ß-D-glucuronic acid. Chloro-ro-cresol-ß-D-glucuronic acid is of particular interest as it is their toxic. Actually effective in an alkaline environment loses.

These new compounds add to their anti-tumor efficacy as well as all other glucuronide compounds with cytotoxic aglycones also have an antibacterial effect, especially against types of bacteria with glucuronidase activity. For example, it is known that streptococci, Staphylococci and Escherichia coli bacteria have ß-glucuronidase activity. If that's why the Glucuronides come in contact with these bacteria, they are deconjugated and the cyt ο toxic aglycones are raised the bacteria develop their toxic effect.

It has been described that the pH optimum of the bacterial ß-Glucuronic3ase higher than the pH optimum of ß-Glucuronidase from normal, healthy internal organs such as liver, kidney, spleen, etc. Therefore, when the Body according to the inventive method the ß-glucuronidase activity the internal organs are essentially turned off while that of the bacteria, though they are alkalized, will still be present no. The administered glucuronide is then deconjugated to its active form only at the site of the infection. Because tumor cells should not be treated in this application, no acidification step is necessary.

03 06 01/0 0A5

-AO-

While the above-mentioned glucuronide compounds for use are preferred in the process according to the invention. ,, It should be noted that the glucuronides: any drug that is effective against tumors, including that proposed in the known literature, with greater advantage in the process of the present ~ Invention can be used since their selectivity is increased by the alkalization step. Examples for those compounds, some of which are already known, and which are used in the present invention although they are currently not known to differ in toxicity or solubility as a function of pH are about 5-i'luorouracil-O-ß-D-glucuronic acid, p-hydroxyanilinflgas-ß-D-glucuronic acid, Methotrexate-ß-D-glucuronic acid, fluoxuridine-ß-D-glucuronic acid, cytarabine-D-glucuronic acid, Melphalan-ß-D-glucuronic acid, hydroxyurea-ß-D-glucuronic acid, Adriamycin-ß-D-glucuronic acid, thiouracil-ß-D-glucuronic acid, chlorophenol-ß-D-glucuronic acid, Methacrylonitrile-ß-D-glucuronic acid, fluoroacetic acid-ß-D-glucuronic acid, and the same.

Other preferred forms of glucuronide for use in the present invention are those containing their aglycones unfold their toxic effect on cancer cells on the cell membrane. Me anti-tumor toxicity of many common drugs with anti-tumor activity requires that these penetrate to the nucleus or to the mitochondria within the cell. In earlier chemotherapy procedures for cancer treatment, the drugs had to be procured that way be that they only attack cancer cells and not all cells in the body with which they came in contact. This is the reason why the development of antineoplastic drugs has been pursued with particular emphasis so far, which intervene in cell division. Many of these drugs actually have to go into the core of the cancer cell.

Ü30 6Ö1 / 0 045

-4Λ-

urge to be effective. For such drugs, however, it must therefore always be keen to ensure that they are transported without change through the membrane of the cancer cell ■ "before they can exert their toxic ^effects.

With the help of the method of the present invention it is not important that the toxicity of the agent is only directed against cancer cells, in contrast to all healthy cells in the human body, namely due to the fact that with the aid of the method according to the invention of the aglycon only at the location of the cancer cell is released. Accordingly, a particularly useful aglycone is one that counteracts its toxicity Cells unfold by attacking the cell membrane itself. That way you don't need to pay attention direct the mechanism of transport of the drug across the cell membrane. In addition, the attack on the Membrane changes the nature of this membrane and thus the antigenic properties of the cell. That's why this will The host's immune system will contribute to the action of the toxic agent and rid the host of these cells. Accordingly, a much lower dose is needed.

Examples of aglycones that have this effect, are phenol and cresol. Particularly effective glucuronides for use in the method of the invention include therefore phenol-ß-D-glucuronic acid and cresol-ß-D-glucuronic acid.

There can also be further steps to increase the ß-glucuronidase activity be undertaken at the location of the tumor cells. One method of accomplishing this is by raising the temperature of the toxic cells at the time of treatment. This can be done by

03060 1/0045

Al-

the temperature of the entire body is raised, for example by using a pyrogenic drug, or by raising the temperature only in the area of the toxic cells, for example by microwave radiation or electric current. Increasing the temperature increases the ß-glucunidase activity and thereby the effectiveness of the deconjugation of the glucuronides. It is known that an increase in the temper8ti
vity increased by 50 % .

Increase in temperature of 3 C the ß-glucuronidase activity

Examples of well-known pyrogenic drugs are etiocholanolone, progesterone, dinitrophenol, and dinitrocresol like that. Both dinitrophenol and dinitrocresol are also cytotoxic, as discussed in more detail below will. Therefore, the use of these compounds is preferred, especially when administered as a glucuronide will. As a result, when the glucuronide is deconjugated at the site of the tumor, the aglycon not only to denature the cytoplasmic protein, but also to raise the temperature directly in the area of the tumor cells and thus greatly increases the effectiveness of further deconjugation.

The local hyperthermia in the area of the suspicious tumor cells is opposed to general hyperthermia preferred, since the latter also has the ß-glucuronidase activity would increase in healthy cells. However, because of the alkalization step, this is not a major one Problem. In the case of the application of local hyperthermia, this offers an additional degree of benefit Assurance that the glucuronides are only deconjugated at the tumor site. The application of a to the location of the Microwave treatment of suspicious tumors is one way to achieve local hyperthermia. Because of the different electrical resistance of tumor cells

030601 / Ό045

Another method of achieving a certain level of local hyperthermia is to use a
low electric current through the body.

Another method for increasing the ß-glucuronidase activity selectively at the location of the tumor cells is the administration of estrogen to female patients or testosterone to male patients. It has been reported that
these compounds increase the ß-glucuronidase activity in
Increase trophoblast cells. Certain tumor cells are known to be trophoblastic. This method
so would be particularly useful with such cells.
The alkalization procedure would prevent damage to healthy trophoblastic cells.

Another aspect of the present invention relates to the method of making the glucuronides. It has been found that it is impossible to prepare conjugates of glucuronic acid by the classical methods' if the aglycon is a strong electron acceptor, since these compounds are first methyl esters of the
Glucuronic acid must be produced and it is not possible to use the classical method to produce the methyl ester
to convert without deconjugating the aglycone to acid.

While barium methoxide was suggested for this purpose in a related process in US Pat. No. 2,985,664, it has been found that barium methoxide does not serve this purpose. However, it has been found that when using barium hydroxide, the methyl ester of the aglycon des
Glucuronide can be converted to the barium salt, and that the barium salt can be converted to the free acid by the use of sulfuric acid without Dekonjugatio.n the glucuronide. In addition, the acetyl protective groups are removed in the same step, which eliminates the need for a separate step for this.

U 3 U ö 0 1/0 0 A S

You know this new reaction step with the use of barium hydroxide in the chemical synthesis of: Mandelonitrile-ß-D-glucuronic acid can be used. He however, it fails when attempting mandelonitrile-ß-D-glucuronic acid to synthesize because when trying to conjugate "; of methyl (tri-0-acetyl-ß (-D-glucopyranosyl) haliduronate with mandelonitrile the latter is more likely to polymerize than to form hemiacetal with the. Glucuronic acid tends.

The process for the synthesis of mandelonitrile-ß-D-glucuronic acid according to the present invention first comprises the conversion of mandelic acid to mandelic acid amide Reaction with gaseous ammonia. The mandelic acid amide is treated with methyl (tri ~ O-acetyl- £ X-D-glucopyranosyl) bromiduronate with the preparation of the methyl ester of mandelic acid amide-triacetylglucuronic acid implemented. This compound can then be mixed with acetic anhydride to convert the mandelic acid amide to mandelonitrile. the Treatment with barium hydroxide and sulfuric acid supplies Mandelonitrile-ß-D-glucuronic acid.

Another aspect of the present invention is that it offers additional security that healthy tissues of the body are protected against possible release of hydrogen cyanide from nitrile-containing aglycones. This safety aspect is advantageous in addition to the aspects already mentioned with regard to the pH adjustment. Even with protection against deconjugation of glucuronide in areas of the body other than tumors, concerns of possible cyanide poisoning have been raised when nitrile-containing glucuronides are used. In Schmidt, ES, et al., JAMA, 239 (10) : 9 ^ 5-9 '+? (1978), with laetrile (amygdalin) readily available, it was predicted that

030601/0 045

A5-

Cyanide poisoning would increase. It is unknown, whether it is the entire nitrile-containing aglycon, mandelonitrile, which is causing the toxic effect on tumor cells:; or whether it is hydrogen cyanide, which is released during the decomposition of mandelonitrile. However, there were Theories developed that it is the total nitrile-containing aglycone that has the toxic effect on the Tumor cells exercises. This is why it is important to protect the rest of the body against the possible release of hydrogen cyanide to protect from the nitrile-containing aglycones. According to the present invention, this is achieved by that when using aglycones containing nitrile, sodium thiosulphate is administered at the same time. It is well known that sodium thiosulfate is an antidote to cyanide poisoning. Sodium thiosulphate converts in the presence of the enzyme rhodanase cyanide into sodium thiocyanide.

It is believed that the co-administration of sodium thiosulfate reduces the toxicity of the aglycone at the site of the. Tumor cells are not affected for two reasons: First, even in the presence of rhodanase, sodium thiosulfate will not affect the mandelonitrile molecule itself. Therefore, if it is the entire mandelonitrile molecule that is toxic to the cancer cells, then the presence of sodium thiosulfate will not affect this toxicity. Furthermore, even if it were the hydrogen cyanide which would be toxic to the cancer cells when released at the site of these cells, it has been stated in the known literature that cancer cells do not contain rhodanase. For this see Lupo, M. et el., "Critical Review of Studies on Malignant Diseases" Minerva Med. 67 (30) : 1973-1981. (1976). For this reason, the simultaneous administration of sodium thiosulfate will protect normal cells against cyanide poisoning, but the attack of the cyanide on the tumor

030601/0 045

do not affect cells.

With regard to the relative lack of toxicity of glucuronide compounds, as well as with respect to the mechanism according to which the the present _invention: toxic aglycone is only released at the tumor site, and also due to the inventive protection against a possible Cyanwasserstofffreisetzung to other parts of the body, it is entirely possible to use glucuronides from other toxic nitrile-containing aglycones in the method of the present invention. One such compound is methacrylonitrile-ß-D-glucuronic acid.

Due to the acid-alkaline gradient between tumor cells and the rest of the body, which is after the procedure the present invention can be achieved, it is possible to use certain compounds that cytoplasmic proteins denature or directly affect the energy supply of the cells without changing them first conjugate with glucuronic acid. However, this can only happen if it is a connection whose effectiveness or solubility is pH-dependent. Examples of such compounds are 2,4-dinitro-phenol, Chlorine-m-cresol, 4,6-dinitro-o-cresol, 4-chloro-3,5-xylanol, Chlorothymol, 2-phenyl-6-chlorophenol, 5-chloro-7-ood-8-quinolinol and podophyllotoxin. The direct use of these compounds without prior conjugation with glueuronic acid would be particularly beneficial in treating tumors with no demonstrated β-glucuronidase activity.

Another aspect of the present invention relates to the extremely high tumor selectivity according to this invention is to be achieved. Because of this selectivity, if one or more of the atoms of the Aglycons exchanged for a radioactive isotope

0 1/0045

local radioactivity will be achieved. This method is not only important for diagnostic purposes, to find the tumor and its metastases, but it can also be used, if an isotope with a β-radiation effect is selected, for local radiation treatment on; V site of the present tumor to be applied. The use of radioactive isotopes is particularly important when an aglycon is used, which is known to be apolar in acidic conditions and polar in alkaline conditions. If it has this quality, then the aglycon at the site of the possible tumor is not only enriched due to the ß-glucuronidase activity, but also due to its insolubility in water at the site of the tumor. At the same time, the compounds with the radioactive isotopes are washed out of the rest of the body. The use of p-iodophenol-ß-D-glucuronic acid produces an aglycon, p-iodophenol, which meets these requirements. A radioactive isotope of iodine can be used as the iodine component of this compound. Preferential

131 133

wisely .J is used for marking and J for treatment, as the former has a larger proportion of gamma radiation, while the latter emits more ß-radiation. To the iodine from the immigration to the thyroid gland · hold, premedication with non-radioactive Lugol ¹ shear solution to saturate the thyroid gland can be used.

Another compound that can easily be radiolabelled is the glucuronide of phenylsulfazole, whereby a radioactive sulfur atom can be used.

This compound does not migrate into the thyroid and the aglycon is not soluble in water.

Before treating patients in accordance with the present invention, ^ AJ-i ^ gffi ^ W ^ v ^ fi ¹¹ »class of each in

The tumor type in question has a high level of ß-glucuronidase activity having. This can be done in a number of ways. One way is to determine the ß-glucuronidase activity in tumor cells obtained through a biopsy. If such a test is positive, then; the pharmaceutical mixtures of the present invention can be administered.

A second method is to administer a Glucuronide, the aglycon of which has been labeled with a radioactive isotope. If you have a full body scan then finds the radioisotope in certain guarantees of the body is enriched, then this will not only indicate the location of the tumor, but also a Indicate that the tumor has sufficient ß-glucuronidase activity to deconjugate the glucuronide. After this determination can be a suitable Amount of the glucuronide of choice to be administered. There is no tumor or the tumors are of a type without ß-glucuronidase activity, then you will not find any accumulation of the radioisotope in the body, since the alkalization step according to the invention the entire usual ß-glucuronidase activity is eliminated and the isotope passes through the body. »

Another method of diagnosing tumors that can be treated using the present invention, consists of examining the urine for the presence of free glucuronic acid. While glucuronides usually do present in the urine is the presence of free glucuronic acid in the urine, and especially the presence of increasing amounts of glucuronic acid in the determination over a period of several days meaningful indication of the presence of tumors with ß-glucuronidase activity. One has the hypothesis

0 30 80 1/0 0A5

found that the presence of free glucuronic acid in the ^"urine of cancer patients by the action of .beta.-glucuronidase in the cancer cells 8UF the intercellular bridges:; i:. and connective tissue caused, the glucuronic acid is a" Eeaktionsprodukt this activity because the intercellular bridges and the connective tissue consist of polymers whose

Component is glucuronic acid, and the known substrates "-

for the enzyme ß-glucuronidase.

A method of distinguishing between free glucuronic acid of conjugated glucuronides in urine is ... another consideration of the present invention. Both glucuronides and glucuronic acid make a chromogenic Complex with tetraborate in concentrated sulfuric acid, which with m-hydroxydiphenyl to form a colored water-soluble complex reacts. If lead acetate is added at an alkaline pH, the glucose

ronide from and the addition of ditizone (dithiosemicarbazone) creates a stable complex with; the excess lead. Accordingly, one can make an optical reading that the amounts of total glucuronides and free glucuronic acid after the addition of tetraborate and m-hydroxydiphenyl. A second reading can then be taken after the conjugated glucuronides and excess lead removed from the aqueous phase by adding basic lead acetate and adding Ditizon. Alternatively to this Conjugated glucuronides can also be removed by reaction with barium hydroxide. The addition of Barium hydroxide in the urine sample is used to precipitate conjugated glucuronides, but not free glucuronic acid to lead. After centrifugation and filtration, the conjugated glucuronides are eliminated and it only the free glucuronic acid remains. You can now read off the amount of free glucuronic acid. This alternative

• Approach bypasses the need to use Ditizon.

030601/0045

Although many glucuronide compounds with aglycones that are toxic to cancer cells are theoretically in the literature described, very few have actually been manufactured to date. This is because they are very difficult to synthesize, especially if: the aglycone is a strong electron acceptor. The improved methods of the present invention obviate this problem and allow the preparation of conjugates of glucuronic acid for almost every type of aglycon. The standard procedures can be used to prepare the methyl esters of triecetylglucuronic acid conjugates are applied, it however, it is often very difficult from the triacetyl methyl ester to get to the glucuronic acid conjugate. This problem has been addressed by treatment according to the method of the present invention Invention solved.

The glucuronides according to the present invention for use in the method according to the present invention can be synthesized from methyl ctri-O-acetyl-βf-D-glucopyranosyl bromide) uronate, which is the active glucuronic acid and according to the information of Bollenback, GN, et al. , J. Am. Ohem. Soc. '77: 3310 (1955). This compound is condensed with the aglycon in a solution of quinoline, phenol, methyl cyanide or methyl nitrite under catalysis by silver oxide or silver carbonate. Another method of condensation uses sodium or potassium hydroxide as a condensing agent in aqueous acetone solution. The reaction scheme is as follows:

030601/0 045

COOCH

OAc

+ RAW

COOCH,

AcO

(D

OAc

wherein ROH is the desired aglycone.

Palis the methyl ester of glucuronide is desired the .Acetylschutzgruppe by anhydrous sodium methoxide or anhydrous barium methoxide can be removed according to the following reaction:

COOCH

COOCH

CH ₃ ONa CH ₃ OBa

OAc

- R

(II)

The acid can be produced by making the triacetyl methyl ester with barium hydroxide to produce the barium salt with the following reaction:

COOCH-

COOBa

Ba (OH)

OAc

(III)

This barium salt of glucuronide precipitates. An equi-

030.601 / 0045

molar solution of sulfuric acid releases aas free glucuronide free according to the following reaction equation:

COOBa

H ₂ SO ₄

COOH

HO

The following examples illustrate the invention.

example 1 Synthesis of 2,4-dinitrophenol-β-D-glucuronic acid

Methyl (2,3,4-tri-O-acetyl-ör-D-glucopyranosyl bromide ronate was prepared according to the method of Bollenback, GN, et al., J. Am. C.hem. Soc. 7Z- 3310 (1955) 4 g of methyl (tri-O-acetyl-0 (~ D-glucopyranolsyl bromide) uronate in 80 ml of acetone and 8.9 g of 2,4-dinitrophenol were treated with 9 ml of 5N potassium hydroxide and the solution was 2M st kept at 25 ° C., then diluted with 3 volumes of chloroform. The chloroform-acetone layer was washed with water and dried. Separation of the solvent and double recrystallization from acetone gave the methyl-2,3,4-tri-O-acetyl-β-D -glucopyranosyluronate of 2,4- dinitrophenol.

The compound free acid was prepared by adding 2,4-dinitrophenyl-methyl (tri-0-8cetyl-β-D-glucopyranosyl bromide) uronate reacted with the half molar amount of barium hydroxide to produce the barium salt. This barium salt of glucuronide precipitated out as a white, amorphous precipitate. An equimolar solution of Sulfuric acid released the free glucuronide. distillation the supernatant gave light, yellow-brown crystals

$ 030601/004

43-

with a Pp 179-180 ⁰ C. This compound was incubated with beta-glucuronidase to yield 2.4 ~ dinitro-phenol, thus confirming that the final product was in fact 2.4 ~ dinitrophenol ~ ß-D-glucuronic acid.

The other glucuronides according to the invention, for example chloro-m-cresol-ß-D-glucuronic acid, 4,6-dinitro-o-cresol-ß-D-glucuronic acid, 4-chloro-1-4 -xylanol-ß-D-glucuronic acid, chlorothymol -ß-D-glucuronic acid, 2-phenyl-6-chlorophenol-ß-D-glucuronic acid, 5-chloro-7-; iodo-8-quinolinol-ß-D-glucuronic acid and podophyllotoxin-ß-D-glucuronic acid, as well as p-Iodophenol-ß-D-glucuronic acid and phenylsulfazole-ß-D-glucuronic acid, can be prepared in a similar manner by adding a stoichiometric excess of the aglycone with the methyl (tri-0-acetyl-OC-D-glucopyranosyl bromide) uronate is reacted in 5 ^N potassium hydroxide and the reaction solution is kept at tree temperature for 24 hours. The solution is then diluted with 3 times the volume of chloroform and the chloroform acetone layer is washed with water and dried. After the solvent has been separated off, the crystals obtained are treated with a half-molar amount of barium hydroxide to produce the barium salt, which is then reacted with an equimolar solution of sulfuric acid to produce the free glucuronide.

The free acid form of glucuronide or a salt thereof ionized under the conditions of use provide the preferred forms of compounds for use according to the present invention. However, pharmaceutically acceptable esters can also be used, although in most cases one would expect their activity to be a little lower, due to their relatively low affinity for ß-glucuronidase. This is especially true of aglycones, which are strong electron acceptors. Accordingly, when using

030601 / Ό0 A 5-

the term "glucuronide compound" as used herein Description and in the claims below not only the free glucuronic acid of the conjugate but also pharma Zeutisch useful salts and esters thereof, as mentioned above, to understand '.

Example 2 Synthesis of mandelonitrile-ß-D-glucuronic acid

Handelonitrile-ß-D-glucuronic acid can be synthesized according to the present invention from methyl (tri-0-acetyl - # - D-glucopyranosyl bromide) uronate, which is the active form of glucuronic acid, and can be synthesized according to the instructions of Bollenback, GN, et al., J. Am. Chem. Soc. 77: 33 "1O (1955). Since this compound cannot be conjugated directly with mandelonitrile, mandelic acid amide is formed first. This compound is prepared by blowing gaseous ammonia into mandelic acid at ⁰ ° C., according to the following reaction equation:

HO - C - COOH

HO-C-CONH2

0 ⁰ C NH,

The mandelic acid amide is converted into methyl (tri-O-acetyl- <v-D-glucopyranosyl) bromiduronate in a solution of phenol under catalysis by a small amount of silver oxide introduced. In addition to phenol, quinoline, methyl nitrile or methyl cyanide can also be used as solvents.

03DSTM / OfUS

16-

In addition, silver carbonate can also be used as a catalyst. Another method of condensation uses sodium or potassium hydroxide as condensation - · " medium in aqueous acetone solution. A stoichiometric ■ Excess mandelic acid amide is preferably used. -, The reaction solution will last 24 h or until completion.; the reaction was kept at room temperature. The response is shown in the following equation:

COOCH

HO-C- CONH-

COOCH

^A c °

r- O -

- CONH,

(II)

The above solution is then mixed with acetic anhydride in a molar ratio of 1: 1 and heated to 70 ° C. for 30 minutes heated so that the mandelic acid amide is converted to mandelonitrile according to the following reaction:

COOCH

O - C - CON ¹ H

COOCH

O -C - 'CN

OAc

(III)

OAc

The acid is made by adding the triacetyl methyl ester, obtained in reaction (III) with a Ί / 2 molar amount of 0.5 η barium hydroxide converts, which is slowly added to the solution with the formation of a white precipitate. Preferably an excess of barium hydroxide is added until no further precipitation occurs. The reaction can be represented as follows:

COOCH.

- O - C - CN

OAc

Ba (OH)

(IV)

The addition of 0.5 ^N sulfuric acid in the same volume, followed by cooling in ice water for 20 minutes, releases the free glucuronides according to the following reaction equation:

COOBa ₁

- O - C - CN

OH

COOH

1-0-C-CN

+ BaSO

(V)

30 601 / ti UL S.

- 36 -

The mixture is then filtered and the supernatant is im Vacuum dried and recrystallized from ether.

Example 3 Synthesis of methacrylonitrile-B-D-glucuronic acid

Methacrylonitrile-ß-D-glucuronic acid or other glucuronides of nitrile-containing cytotoxic compounds can according to the present invention in a similar manner as in Example 2, although the step of converting the methacrylonitrile to: - methacrylamide before condensation with methyl (tri-O-acetyl- • ^ -D-glucopyranosyl bromide) uronate is not necessary, since methacrylonitrile does not have the same problem of polymerization as with mandelonitrile offers. In general, the preferred method is direct condensation of the aglycone in it, a stoichiometric excess of the aglycone (in the pail of methacrylonitrile ^ ß-D-glucuronic acid Methacrylonitrile) with the methyl (tri-O-acetyl - «- D-glucopyranosyl bromide) uronate to implement in 5 η potassium hydroxide and the reaction solution for 24 st Maintain room temperature. The solution is then diluted 3 times the volume of chloroform and the chloroform acetone layer is washed with water and dried. After removal of the solvent, the obtained Crystals treated with a half molar amount of barium hydroxide, thereby producing the barium salt, which then with an equimolar solution of sulfuric acid is implemented with the position of the free glucuronide.

Example 4 Acute intravenous toxicity to rabbits from Mandelo

nitrile-ß-D-glucuronic acid

NZW rabbits in the weight range from 2000 to 3200 g for . '. 030601/0045

female and 2200 to 5800 g for males. Mandelonitrile-ß-D-glucuronic acid solution injected intravenously. Rabbits injected with saline only served as controls. The mandelonitrile-ß-D-glucuronic acid solution contained 10 % mandelonitrile.

Over a 14-day observation period, mortality rates were determined and signs of poisoning registered. Table I gives the observations.

Table I - Mortality data for groups of rabbits (2 per group) with intravenous injection of DMBG solution. preliminary experiment

Dosage death rate

ml / kg number of deaths /

Body weight, number of people treated

animals

0.25 0/2

0.5 V2

1.0 .2 / 2

2.0 2/2

Λ, Ο 2/2

The results of the preliminary test, as given in Table I, show that the mean lethal intravenous Dose (LD-50) in the range from 0.23 to 2 ml per kg of body weight lay.

The dosage was then extended to larger groups of rabbits (5 male and 5 female per group), to determine the mean lethal dose more precisely. Table II gives the mortality data for the larger ones

030601/0045

-X-

Group on,

Table II - Mortality rate of rabbits after intravenous injection of DMBG solution, main experiment, weight range female animals 2000 to 3200 g, male animals 2200 to 5800 g.

Dosage mortality time after administration / kg pro rate compliance

Body weight number of deaths number of hours weight cases / number of animals traded animals

Male
lich 0.44
0.66 0/5
2/5 1.0 3/5 1.5 4/5 2.25 5/5 woman
lich 0.44
0.66 0/5
2/5 1.0 5/5 1.5 5/5 2.25 5/5

<3 <3 <3 <3

<3 <3 <3

Signs of post-treatment reaction that were seen 2 minutes after dosing included ataxia and paralysis. Two minutes later, some animals in the high dose group died. All deaths in all Groups happened within 3 hours of administration. The surviving animals showed no clinical signs Symptoms during the following 14 days. Autopsy of all animals showed. no clear gross pathological, Changes.

The acute mean lethal intravenous dose (LD-50) and its 95 % confidence intervals, which were calculated using the method of Weil, CS., Biometrics, 8: 249 (1952),

for rabbits for mandelonitrile-ß-D-glucuronic acid in 10 # solution is calculated as follows: 0.84187 (0.78087-0.90287) ml / kg body weight for male animals. 0.6873 (0.64417-0.73043) ml / kg body weight for females. ^:

From the above data it can be assumed that the maximum harmless dose of the order of magnitude of 0.44 ml / kg body weight and one can assume that this limit should not be exceeded during therapy in the human organism.

Prior to therapeutic treatment with compounds according to the present invention, the presence of a Tumors with high ß-glucuronidase activity can be diagnosed. The safest way to permanently backup whether a tumor with high ß-glucuronidase activity is to perform a biopsy and the obtained Test tumor cells for their ß-glucuronidase activity. This is natural for most types of Tumors not feasible. Another way of diagnosing the presence of tumors with β-glucuronidase activity is to do a urine test to determine the presence of free glucuronic acid. Normal Patients show 200 to 400 mg of free glucuronic acid in their urine per 24 hours. Cancer patients with well-developed Tumors which have ß-glucuronidase activity become more than 2000 to 7000 mg per 24 hours free Have glucuronic acid. Accordingly, using the assay of the present invention is the detection of more than 400 mg per 24 hours of free glucuronic acid is an excellent indication of the presence of tumors high ß-glucuronidase activity.

iQ 1

A negative result of this urine test is not inevitable the presence of tumors with ß-glucuronidase activity exclude, since tumors in their initials. ' Stages even though they have ß-glucuronidase activity, may not release enough free glucuronic acid to produce a positive urine test result to effect. Therefore, the urine test should be repeated, and if there is an increasing amount of free glucuronic acid is found, this is a further indication of the presence of a tumor with β-glucuronidase activity. An example of the method for determining the amount of free glucuronic acid in urine is given in the following example 5 given.

Example 5 Test for glucuronic acid in the urine

Both glucuronide and glucuronic acid with tetraborate and concentrated sulfuric acid result in a chroraogenic Complex which reacts with m-hydroxydiphenyl to form a colored, water-soluble complex. Furthermore, glucuronides with basic lead acetate precipitate at pH 8, while free glucuronic acid from lead acetate is not affected. By complexing the excess lead with dithiocarbazone, a more stable one is formed Complex with lead, which can be removed, whereby the free glucuronic acid is retained.

0.1 η ammonium hydroxide was added to 10 ml of a urine sample until a pH of 8 was reached. An excess A saturated solution of basic lead acetate was then added, causing the precipitation of the conjugated .Glucuronide was achieved. The sample was centrifuged and the supernatant was separated. 2 ml of the supernatant were then mixed with 10 ml of 10 # dithiocarbazone (Ditizon)

0 3 0 6 0 1/0045

egg-

Treated in chloroform to remove the excess lead remove. After waiting until the deposition was complete, the aqueous phase was separated off. 1.2 ml of sodium tetraborate in concentrated sulfuric acid were added to 2 ml of the aqueous phase. The mixture was mixed well in a test tube and ground ice chilled. The test tube was then 5 minutes th heated in boiling water and immediately cooled in ice until it turned cold. Then became 20 microliters 0.15 # m-hydroxydiphenyl in 0.5 # sodium hydroxide added. After 5 minutes the optical density was read at a wavelength of 520 nm. the The value obtained here corresponded to the amount of free glucuronic acid that was present in the urine.

The total amount of free and conjugated glucuronic acid is easily determined by directly using the sample Tetraborate and hydroxydiphenyl treated without first removing the free "glucuronides. Reading at one wavelength of 520 nra gave the total amount of conjugated Glucuronides and free glucuronic acid present in the sample.

Example 5A Test for glucuronic acid in the urine

0.1 η ammonium hydroxide was added to 10 ml of a urine sample until a pH of 8 was reached. Then became a Excess of barium hydroxide was added, causing a filing of conjugated glucuronides. the Sample was then centrifuged and the supernatant filtered. To 0.2 ml of the filtered supernatant was added 1.2 ml Sodium tetraborate added in concentrated sulfuric acid. The mixture was mixed well in a test tube and then chilled in ground ice. The test tube

0 3 0 6 0 1 / Q 0 & S

was then heated in boiling water for 5 minutes and immediately chilled in ice until cold. 20 microliters of 0.15 # m-hydroxydiphenyl in 0.5 tiger sodium hydroxide were then added. After 5 minutes, the optical density was read at a wavelength of 520 nm. the The reading shows the amount of free glucuronic acid that is present in the urine.

The relative amount of the total of conjugated glucuronides and free glucuronic acid present in the urine can be determined exactly as described in Example 5 above.

Example 6 Method of Administration of Glucuronide Therapy

After a tumor with ß-glucuronidase activity has been secured in the patient, the first step of treatment is the administration of a dose of glucose, for example in the form of 100 g honey, glucose or other sugar. About 1 hour later, an intravenous infusion of a solution is started which contains about 10 % glucose and 60 milliequivalents of sodium bicarbonate in distilled water. About 1 liter is given, provided there are no contraindications, and the urine pH is checked to determine that it has reached a pH of about 7.4. This indicates that the system has been alkalized and that it is now safe to administer the glucuronide. Another liter of the same glucose bicarbonate solution is then added, which also contains the desired amount of glucuronide. This is repeated daily as needed. If a glucuronide of a nitrile-containing cytotoxic aglycone is used, 50 ml of a solution of sodium thio-

• 0 30 601 / 00A5. . '

sulfate administered, preferably by slow intravenous infusion. The sodium thiosulfate is preferably in the Glucose bicarbonate glucuronide solution containing intra-; venous infusion. However, this gift can also be continued at a later date in order to make a greater one Achieve security area.

If there are contraindications to the administration of bicarbonate, an antacid can then be administered orally. The key criterion is that the urine pH is around 7.4 reached and remains at this value during treatment;

The overacidification of the tumor cells is caused by hyperglycemic conditions in the patient. Therefore everyone can any hyperglycemic agents are used as acidifying agents, for example fructose, galactose, Lactose or glucagon. It should also be noted that these hyperglycemic conditions are known to any Way can be effected. For example, if the patient is diabetic, then one can have these conditions by decreasing the amount of insulin administered.

Any agent that raises the urine pH to around 7.4 can be used as an alkalizing agent, including of sodium or potassium bicarbonate or citrate or other basic salts or antacids. Although they are preferably given intravenously, they can also be given by administered orally.

If in the following description and claims of a pH of "about 7.4" in relation to the pH level, that is maintained in the rest of the body, so it should be clear that a pH level is something

> 030 601/0045

15-

above or below 7.4- can be used, although it isn't is preferred. If the pH falls below 7.4, the ß-glucuronidase activity decreases closed (until the pH optimum is reached). Furthermore, below a pH of 7.0, the remainder of the Body should not be alkaline but acidic. About 7.4 takes the risk of alkalosis without a substantial one further decrease in ß-glucuronidase activity. A pH of 7.4- is preferred because it is physiological pH acts and this cannot be harmful to the body, it is also known that the ß-glucuronidase activity is essentially zero in healthy organs at this pH.

The dosage of the glucuronides should be monitored to all side effects are due to the massive release of toxins caused by the dying cancer cells will avoid. It can be advantageous to start the treatment with glucuronides in short cycles of a few days, between which a few days is paused to carry out, so that any toxins from dying cancer cells released, can leave the body before treatment continues.

In addition to intravenous administration, the glucuronides can be administered by any type of parenteral administration. However, the glucuronides should not be administered orally as ß-glucuronidase is known to be present in the digestive tract. However, the sodium thiosulfate can To be given orally, if a suitable enteral coating is provided to prevent gastric release prevented.

The amount of glucuronide to be administered to any particular patient must be determined empirically and is dependent on the conditions dns ^ «+ - ienten

030 60 1 / OO4 5

be different. One can give relatively small amounts of glucuronide first and then the daily dose gradually let it increase if no adverse effects are found. Of course, the maximum can Safe dose as found in routine animal toxicity studies was determined never to be exceeded.

It is clear that any tumor cells with ß-glucuronidase activity can be treated according to the present invention, while the other organs of the Body can be protected by the alkalization step. Tumors known to have ß-glucuronidase activity are e.g. solid breast tumors and their metastases, bronchogenic carcinomas and their metastases, as well as lymphotne. It is also known to be among the neoplasties that do not have high ß-glucuronidase activity and which therefore cannot be treated according to the present invention, which belongs to leukemia. It must be noted, however, that this list is by no means intended to be exhaustive and that. in the loading: many other tumors with β-glcuruonidase activity were known from the literature are mentioned. However, regardless of whether it is already known whether any particular tumor is a Having β-glucuronidase activity can do so by the various methods discussed in the present specification diagnostic methods are determined, and if the tumor is found to have ß-glucuronidase activity then the therapeutic treatment according to the present invention can be effectively applied.

If it is desired to induce hyperthermia to increase the ß-glucuronidase activity, then a Method chosen, according to which the temperature as much as possible without the risk of damage to healthy body

030601 / € 045

parts, such as the eyes, is increased. An increase of about 2 ° G for whole body hyperthermia or of about 4.5> ° C in the case of local hyperthermia is preferred. The hyperthermia should be timed so that it lasts for about 1 hour at the time of the greatest glucuronide concentration at the tumor site. If, for example, local microwave treatment is chosen, this should be started about 1/2 hour after the start of the intravenous glucuronide infusion and continued for about 1 hour. The correct dosage of known pyrogens to achieve the desired level of hyperthermia is known to those skilled in the art and can easily be determined empirically. For example, a dose of about 30 mg per day would be suitable for dinitrophenol.

Estrogen or are administered testosterone, so a dose of 5 to 15 mg P ^r o kg of body weight per day, the desired excitation would ß-glucuronidase activity ensure.

Example 7 Method for the administration of radioisotopes

If an aglycon marked with a radioactive isotope is to be administered, the marking can be carried out by any of known procedures happen. Relatively small amounts of these can be used for diagnostic purposes alone labeled glucuronides. They are otherwise given in the same way as in Example 6 is set out for unlabeled glucuronides. Scanning the body to determine if any of the radiolabeled aglycones is retained by the body indicate whether a tumor with ß-glucuronidase activity is present and will also show where the tumor is or metastases of the tumor can be found. How

030 60 1 / 00AS

already mentioned above, gamma rays are isotopes, such as iodine, particularly suitable for this purpose.

The radiolabelled glucuronides can also be used for the Radiation therapy can be used in situ, especially if an isotope with a high proportion of ß-radiation is present,

1 35

is applied, such as - ^ iodine. This gives double that Effect that the cancer cells are attacked not only with the toxic aglycones but also with the ß-radiation will. Again the method of administration will be the same as outlined in Example 6.

Another area of application of the present invention is the use of boron-containing aglycones. It is already known that when bombarding Eoratoms with Neutrons are split into lithium with the emission of positrons. If the boron atoms at this point are attached to tumor cells, the positrons will be quickly absorbed by the tumor tissue and for it be lethal. This method will be of outstanding use when the boron atoms are formed according to the method of present invention are concentrated exclusively in the tumor cells.

Example 8 Method of delivering pH dependent therapy

If the tumor cells become over-acidic according to the method set out in Example 6 and the healthy tissues are alkalized there will be an acidic pH gradient between the tumor cells and the healthy cells. Therefore, compounds, their activity or solubility can be pH-dependent be administered directly without first being conjugated with a glucuronide. These connections become selective in the acidified tumor cells

030601/0 045

attack without affecting the rest of the body with a cause damage to alkaline pH levels.

As in the procedure of Example 6, the patient is first given an oral dose of a hyperglycemic agent such as 100 grams of honey, glucose or other sugar. About 1 hour later, an intravenous infusion of a solution of approx. 10 % glucose and 60 milliequivalents of sodium bicarbonate in distilled water is started. You give about 1 liter, provided there are no contraindications, and the pH _{> of} the urine is checked to make sure that a pH of about 7.4 has been reached. This shows that the system is alkalized and that it is now safe to administer the acidic compound. Another liter of the same glucose bicarbonate solution is added, which, however, now also includes the desired amount of the compound active in the acid. This can be repeated daily if necessary.

Compounds such as 2,4-dinitrophenol, 4,6-dinitro-o-cresol, 4-chloro-3,5-xylanol, chlorothymol, 2-phenyl-6-chlorophenol, 5-chloro-7-iodo-8-quinolinol and podophyllotoxin, are water-soluble at alkaline pH values and fat-soluble at acidic pH values. Therefore -these connections are made, if they are administered in the manner outlined above, do not cause any significant damage to healthy tissues, because they are washed out of the system relatively quickly. At the location of the tumor tissue with acidic pH, however, these Connections, 8US of the aqueous solution occur and their exert cytotoxic or energy supply impairing effects on the tumor cells.

Compounds such as chlorine-m-cresol, as well as 4,6-dinitro-o-cresol, 4-chloro-3,5-xylanol, chlorothymol and

030601/0045

2-Phenyl-6-chlorophenol are more active at lower pH. Therefore the administration of these compounds together with acidification of the tumor cells and alkalization of the rest Body will be even less harmful to healthy tissue since their activity is at the pH of healthy tissue is decreased.

The dosage of the non-glucuronide compounds according to this embodiment of the present invention are generally somewhat lower than those of the corresponding glucuronides, since the glucuronide form of the compounds is much less toxic than the free compounds. The exact dosage has to be empirically dependent determined by the patient's conditions. Relatively small doses should be given first then increase gradually daily if no adverse effects are noticed. Of course, the maximum is allowed The safe dose, as determined in routine animal experiments, must never be exceeded.

Other acidifying and alkalizing agents can also be used, as. earlier with reference to the glucuronide embodiment of the invention can be used in conjunction with this embodiment.

Example 9

Method of antibacterial therapy

Glucuronide can be used to treat bacterial infections, if they are Bacteria act, one of which has ß-glucuronidase activity is known. Examples of such bacteria are streptococci, staphylococci and Escherichia coli. The Treatment method for such bacterial infections is similar to that set forth in Example 6, except

030 601/00 AS

that no acidification is necessary. This is the pell because bacterial ß-glucuronidase at higher ' pH values than the ß-glucuronidase of normal, healthy internal organs is effective. In addition, such a would Acidification step does not affect the pH of the bacteria, as its mechanism is specific for tumor cells is.

The first step in antibacterial treatment is an intravenous infusion of distilled water with 60 milliequivalents sodium bicarbonate. One gives approx. 1 liter and the pH of the urine is checked so that you can determine when it reaches a pH of approx. 7.4- has. Then add another liter of the same bicarbonate solution, which, however, also contains the desired amount of glucuronide. This treatment can be used if necessary to be repeated daily.

The alkalizing agent can also be administered orally and any agent can be used which alkalizes the body to such an extent that the pH of the Urine is approx. 7.4-. The glucuronide should not be administered orally but it can be given by any type of parenteral administration.

Certain known antibacterial drugs with adverse side effects can also be used as glucuronides in accordance with the method of the present invention in order to reduce these adverse effects or turn it off. For example, chloramphenicol is known to cause bone marrow depression, which does not take place if the glucuronide is used. Neomycin is a well known antibiotic because of its Toxicity cannot be used internally. It can however, for the treatment of infections caused by bacteria

0 30601/00 k p

with high ß-glucuronidase activity administered orally v / earth, when first conjugated to glucuronic acid.

The diagnostic procedure with radioisotope-labeled Aglycones, which has already been discussed above with regard to tumor diagnosis, can also be used to determine the The presence and localization of bacterial infections should be used. For example, one can Give the radioactively labeled glucuronides to patients who complain of pain in the appendix area. If there is no accumulation of the isotope in the area in question, then inflammation by bacteria can occur with ß-glucuronidase activity can be excluded as the cause of the pain. In most cases an inflammation of the appendix is based on infection with bacteria with ß-glucuronidase activity. Other forms of application Such a diagnostic procedure could easily be found by the person skilled in the art.

In addition to the glucuronide compounds mentioned here, all known conjugable antibiotics can be used against ß-glucuronidase-containing infectious agents with Glucuronic acid can be conjugated. This has the advantage of reducing the amount of free antibiotic that is in the circulation circulated, can be largely reduced. The only antibiotic that is released is on site released from infection. Therefore lower doses can be given. Accordingly, the Glucuronides of the present invention serve as internally administered local antibiotics. Because of the well-known ß-Glucuronidase activity in the digestive tract should not be given orally during any type of glucuronide psrenteral administration is permitted.

030601 / 004S

If the antibiotic aglycon is known to be none Has an effect on the kidneys, the alkalinization step can be omitted. Many antibiotics are, however

known to be nephrotoxic to a certain extent and therefore the alkalization step is important to protect the kidneys.

Example 10 Biosynthesis of mandelonitrile-ß-D-glucuronic acid

22 ml of a solution of 5 % mandelonitrile (benzaldehyde cyanohydrin) in propylene glycol are prepared and this solution is administered intramuscularly to a donkey or a goat. The 24-hour urine is collected and acidified with acetic acid to a pH of 4. The urine is then filtered through a fiberglass filter and the piltrate is treated using one of the following three different methods:

A. A saturated solution of lead acetate is added to the filtrate. The white precipitate that appears is collected by centrifugation and filtered. The filtrate is made alkaline with ammonia to a pH of 8 and then a saturated solution of basic lead acetate is added. The precipitate is with cold water washed and gaseous HpS is blown in, the black precipitate of lead sulfide is separated. The filtrate is then reduced to 1/3 des Confined in volume. A brown paste is obtained which is dissolved in absolute alcohol and left to stand overnight will. The solution is filtered and the filtrate is placed in a vacuum and ether is added. Mandelonitrile-D-glucuronic acid is crystallized from the ethereal solution.

0 306KM / 004S

B. The urine is made with hydrochloric acid on a pH 4 and acidified through a 'fiberglass filter filtered. The solution is then dried in vacuo and the residue is dissolved in ether and from the ethereal · see solution recrystallized.

C. 0.1 η barium hydroxide in aqueous solution is the Urine added. The white precipitate of the barium salt of mandelonitrile glucuronide is then placed in cold water washed and stirred and 0.1 η sulfuric acid is added. An insoluble residue of barium sulfate is removed and the supernatant is dried in vacuo and then recrystallized from the ethereal solution. ■

Because mandelonitrile is very toxic and only a very low one The following semi-biosynthetic method can be used:

20 g of mandelic acid amide (2-hydroxybenzamide) are added to the A goat or donkey feed is mixed and the animal's urine is collected over 24 hours. The mandelic acid amide glucuronide is separated by one of the methods described above. Then icetic anhydride is added and the glucuronide (2,3,4-triacetate-glucopyranose-mandelonitrile) is precipitated with barium hydroxide. The barium is then removed with sulfuric acid and the glucuronide is im Vacuum as described above, isolated.

It will be apparent to those skilled in the art that various changes the illustrated method can be carried out without departing from the scope of the invention, and that the invention is not limited to the content of the description.

030601/0045

Claims (1)

MÜLLBR'IiOItiS · DBUFEL · SCHÖN · HEITTKL PATENT LAWYERS ο - DR. WOLFGANG MÜLLEH-ΒΟηέ (PATENT ADVOCATE FROM 1927 - 1975) DR. PAUL DEUFEL. DIPL.-CH EM. DR. ALFRED SCHÖN. DIPL.-CHEM. WEHNER HERTEL, DIPL. -PHYS. APPROVED REPRESENTATIVES AT THE EUROPEAN PAVfTNTAMT HEPHESENTATIVES 0EFORE THE EUROPEAN ΡΛ7ΕΝΤ OFHiE MANDATAIRES AGREES PRES L ¹ OFKiCE EUhOHLEN DHS UfIEVEI ?. dt / n - S 3240 zh * June 1980 ADOLF W. SCHWIMMER, Savyon / Israel, IRWIN S. SCHWARTZ, Tel Aviv / Israel, and DAVID RUBIN, Jerusalem / Israel ß-glucuronidase activity and / or pH-dependent drugs, Process for their production and their use for the selective treatment of diseases PATENT CLAIMS: 1. Improvement of the method for the treatment of tumors with higher ß-glucuronidase activity than the surrounding one Tissue by acidifying the tumor cells, followed by the administration of an effective anti-tumor dose of an Glucuronide compound, the aglycon of which is responsible for the tunior cells is toxic, with the ß-glucuronidase activity of the acidic tumor cells causing the deconjugation of the glucuronide compound causes at the location of the tumor cells and the local release of the aglycone, characterized in that the Tumor selectivity of the procedure improves and the risk 030601/0045 MUNICH 8β-SIEBERTSTn-A-POSI ~ .. ~~. _m xj x-oxjt AT TEI .. (089) 474005 ■ TELEX B-S4 U- dei 'injury is reduced by non-tumor tissues, by administering to the patient an alkalinizing agent in an amount sufficient that the pH level of _the: is held nichttumorösen tissue of the patient during the Glucuronidbeb.8ndlung at about 7 »^. 2. The method according to claim 1, characterized in that the alkalizing agent is an alkali bicarbonate or - Includes citrate. 3. The method according to claim 1, characterized in that the aglycon has a higher toxic effect in acidic Environment than in an alkaline environment or that it is soluble in water in an alkaline environment and in an acidic environment is insoluble in water or only poorly soluble in water. 4. Procedure according to. Claim 3 »characterized in that that the gulcuronide compound is 2,4-dinitrophenol-ß-D-glucuronic acid, 4-chloro-m-cresol-ß-D-glucuronic acid, 4,6-dinitro-o-cresol-ß-D-glucuronic acid, 4-chloro-3,5-xylanolß-D-glucuronic acid, Chlorothymol-ß-D-glucuronic acid, 5-chloro-7-iodo-8-quinolinol-ß-D-glucuronic acid, Podophyllotoxin-ß-D-glucuronic acid, 2-phenyl-6-chlorophenol-ß-D-glucuronic acid, p-iodophenol-ß-D-glucuronic acid and / or phenylsulfazoleß-D-glucuronic acid is used. 5. The method according to claim 3, characterized in that an aglycon is used which is a radioisotope contains, which is capable of emitting radiation that ensures a radiotherapeutic effectiveness in situ. 6. The method according to claim 5 _5, characterized in that ice radiation uses essentially ß-radiation 030801/0045 7. The method according to claim 5 »characterized in that 131 13 "
that as glucuronide I- or ß-D-glucuronic acid is used. 131 133
that p-iodophenol labeled as glucuronide I or ^! 8. The method according to claim 5, characterized in that 35 that as glucuronide S-labeled phenylsulfazole-ß-D-glucuronic acid is used. 9. The method according to claim 1, characterized in that the acidification of the tumor cells is carried out, by administering a hyperglycemic agent in an amount sufficient to over-acidify the tumor cells. 10. The method according to claim 9, characterized in that the hyperglycemic agent is glucose, fructose, Galactose, lactose and / or glucagon is used. 11. The method according to claim 9, characterized in that the hyperglycemic agent orally or intravenously is administered. 12. The method according to claim 1, characterized in that the alkalizing agent is administered orally or intravenously will. 13. The method according to claim 1, characterized in that that the alkalizing agent is given before the administration of the glucuronide compound, said glucuronide compound administered after the pK value of the The patient's urine is measured at approximately 7.4, and that the administration of said alkalizing compound continued during administration of the glucuronide compound will. 030601/0045 -H- 14. The method according to claim 1, characterized in that the glucuronide compound by intravenous infusion is administered. " 15. Method according to claim 14, characterized in that said alkalizing agent is contained in the solution for '"intravenous infusion. '. '. 16. The method according to claim 9, characterized in that that the glucuronide compound is administered by intravenous infusion and said hyperglycemic Agent and the alkalizing agent are contained in the solution for intravenous infusion. 17. The method according to claim 1, characterized in that that the glucuronide compound is given orally in a form that prevents the release of the compound in the stomach. 18. The method according to claim 1, characterized in that the glucuronide compound is phenol-ß-D-glucuronic acid and / or cresol-ß-D-glucuronic acid can be used. 19. The method according to claim 1, characterized in that that a glucuronide compound, the aglycon of which contains nitrile, is used, and that further to the patient a dose of sodium thiosulphate is administered which is intended for Effect as an antidote against cyanide poisoning is sufficient. 20. The method according to claim I9, characterized in that that sodium thiosulfate in a dose of about 50 ml one 25% solution is administered. 21. The method according to claim 19, characterized in that 0 3 060 1 / 00A5 that the glucuronide used is mandelonitrile-ß-D-glucuronic acid will. 22. The method according to claim 19 »characterized in that - that as the glucuronide methacrylonitrile-ß-D-glucuronic acid is turned. 23. The method according to claim 1, characterized in that that, in addition, hyperthermia is induced at least at the site of the tumor to be treated, which is sufficient, that at this point the ß-glucuronidase activity is essential is increased without affecting the overall well-being of the patient, at least at the moment. the maximum glucuronide concentration on the tumor is significantly impaired. 24. The method according to claim 23, characterized in that that the hyperthermia locally on the tumor by administration of the glucuronide of a pyrogen, by microwave treatment or induced by applying an electric current through the body. 25. The method according to claim 1, characterized in that that estrogen or testosterone is also administered simultaneously with the administration of said glucuronide. 26. A method for the treatment of tumors with lower pH levels than in the surrounding tissue, characterized in that that the tumor cells are acidified, the patient an alkalizing agent in a sufficient amount given that the pH of the non-tumorous tissue is kept at a pH level of approximately 7 »4, and that a compound is administered which is toxic to the tumor cells only at a pH below 7 or which is toxic to the tumor cells and in water only to one pH below 7 is insoluble. 030601/0045 27. The method according to claim 26, characterized "," - that the compound 2,4-dinitrophenol, chlorine-tn-cresol, -2,6-dinitro-o-cresol, 4 - ChIOr ^, 5 ~ xylanol, chlorothymol , " 2-phenyl-6-chlorophenol, 5-chloro-7-iodo-8-quinolinol and / ', or podophyllotoxin is given. _ 28. Method of treating infections caused by. Bacteria with ß-glucuronidase activity are caused, characterized in that the patient receives an alkalizing Agent is given in an amount sufficient to keep the pH of the patient's internal tissues at one Value of about 7 »4 is kept and that the patient a glucuronide compound, the aglycone of which is toxic to the bacteria, is given in an antibacterially effective amount where the ß-glucuronidase activity of the bacteria deconjugates the glucuronide at the site of the infection and thus causes the release of the aglycone. 29. The method according to claim 28, characterized in, that strep, staph, and Escherichia coli infections are treated. 30. The method according to claim 29, characterized in that a glucuronide compound is used, the Aglycon contains nitrile, and that also the patient a sufficient amount of sodium thiosulfate is given that this can serve as an antidote to cyanide poisoning. 31. Compound containing the glucuronide of a cytotoxic Contains aglycons, characterized in that they have a greater toxic effect in an acidic environment than in alkaline medium unfolds, or that they are water-soluble in an alkaline medium and water-insoluble in an acidic medium or is only poorly soluble in water. 52. Compound according to claim 3Ί »namely 2,4-DinitiO-phenol-ß-D-glucuronic acid, Chlorine-m-cresol-ß-D-glucuronic acid, 4-, 6-dinitro-o-cresol-ß-D-glucuronic acid, 4 ~ chloro-3,5-xylanol-ß-D ~ glucuronic acid, Chlorothymol-ß-D-glucuronic acid, P-phenyl-ö-chlorophenol-ß-D-glucuronic acid, 5-chloro-7-iodo-8-quinolinol-ß-D-glucuronic acid, Podophyllotoxin-ß-D-glucuronic acid, p-Iodphenol-ß-D-glucuronic acid or phenylsulfazole-ß-D-glucuronic acid. 33 · Methacrylonitrile-ß-D-glucuronic acid. 34-. Method of diagnosing the presence of tumor cells with ß-glucuronidase activity, characterized in that that the tumor cells are acidified, the patient an alkalizing agent in a sufficient amount given that the pH of the patient's non-tumorous tissues is kept at a value of about 7 »4-, a Glucuronidverbindungen is administered, the aglycone of which contains a radioisotope, which for the emission of a sufficient Radiation is capable of being detected in a full body scan, and. that in the patient -> is made in full-body scan, with the presence and localization of any immobilized therein radioactive aglycons is determined. 35 · Procedure for determining the amount of free glucuronic acid in the urine, characterized in that the urine sample with a saturated solution of basic lead acetate treated in an amount sufficient to cause the precipitation of any glucuronides present to be the precipitate was separated from the supernatant and a sufficient amount of dithiocarbazone was added to the supernatant is so that excess lead is removed that the aqueous phase is separated, you a tetraborate compound in concentrated sulfuric acid to form a chromo- U30601 / 004S Sl- genes complex is added and that finally the Product of previous steps with m-hydroxydiphenyl to produce a colored., water-soluble complex is implemented, the color intensity of the amount of free Glucuronic acid of a sample is directly correlated. 36. Method for determining the amount of free glucuronic acid in urine, characterized in that the urine sample treated with barium hydroxide in an amount sufficient that the precipitate is conjugated to present Glucuroniden is effected, the precipitate is separated from the supernatant and the supernatant is a tetraborate compound in concentrated sulfuric acid is added to form a chromogenic complex, and that Finally, the product of the previous steps is reacted with m-hydroxydiphenyl, so that a colored, water-soluble complex is generated whose color intensity is directly related to the amount of free glucuronic acid in the sample is correlated. 37 · Process for the preparation of conjugates of free Glucuronic acid with aglycones, which are strong electron acceptors, characterized in that the aglycon with Methyl (tri-O-acetyl-öf-D-glucopyranosyl) halogenuronate with formation of the methyl ester of aglycon-tri-O-acetylß-D-glucuronic acid is condensed, a sufficient amount of barium hydroxide is added to the product of this condensation that a precipitate is generated, this precipitate is separated and in a sufficient amount Sulfuric acid is treated until the precipitation of barium sulfate it is completed that the supernatant of the product of this reaction step is removed and dried, so that the conjugate of said aglycone with free glucuronic acid is obtained. 030601/00 ^ 5 63- 38. A process for the production of mandelonitrile-D-glucuronic acid, characterized in that mandelic acid amide with methyl (tri-0-acetyl- <XD ~ glucopyranosyl) haliduronate to form the methyl ester of mandelic acid aniide-tri-O-acetyl-ß-D-glucuronic acid is condensed, the product of this condensation reaction with acetic acid ^; anhydride is reacted at a temperature and over a period of time which is sufficient that the methyl ester of mandelonitrile-tri-O-acetyl-ß-D-glucuronic acid is generated, the product of this reaction step is mixed with a sufficient amount of barium hydroxide that a white precipitate is produced arises, this precipitate is separated off and treated with a sufficient amount of sulfuric acid until the precipitation of barium sulfate is complete, that finally the supernatant of the product of this reaction step is separated off and dried, so that mandelonitrile-ß-D-glucuronic acid is obtained. 39 · Method for diagnosing the presence of bacteria with ß-glucuronidase activity, characterized in that that the patient is administered an alkalizing compound in an amount necessary for maintenance a pH value of the patient of approx. 7 * 4 is sufficient, the Patients are administered a glucuronide compound, the aglycone of which contains a radioisotope that causes emission is capable of sufficient radiation that it can be detected by a whole-body scan, and that Finally, a full-body scan is performed on the patient, sp that the presence and the localization any radioactive aglycone immobilized therein can be determined. 4-0. Pharmaceutical mixture, characterized in that it is an aqueous solution of an anti-tumor agent Amount of a glucuronide compound, the aglycon of which is used for tumor 030601 / 00A5 cells, a hyperglycemic agent in an amount sufficient to make tumor cells acidic after administration, and an alkalizing one Means in an amount sufficient that the pH of the non-tumorous tissues of the patient after administration is held at about 7 »4. 41. Pharmaceutical mixture according to claim 40, characterized in that it comprises a glucuronide compound, the aglycon of which has a higher toxic effect in an acidic medium than in an alkaline medium, or which is water-soluble in an alkaline medium and water-insoluble or only sparingly water-soluble in an acidic medium. 42. Pharmaceutical mixture according to claim 40, characterized in that the aglycon of said glucuronide compound Contains nitrile and that it also contains sodium thiosulphate comprises in an amount sufficient that it can serve as an antidote to cyanide poisoning. 43. Pharmaceutical mixture according to claim 42, characterized characterized in that the glucuronide compound mandelonitrile-ß-D-glucuronic acid or methacrylonitrile-ß-D-glucuronic acid is. 30601/0045 .VS ⁴ A: