Note: Descriptions are shown in the official language in which they were submitted.
1~8~86
--1--
~-Glucuronidase Activity and/or p~-Dependent Pharmaceuticals
and Their Methods of Production and Use for Selective
-
Treatment of Diseases
Technical Field
5lhe present invention relates to the treatment of
tumors exhibiting ~-glucuronidase activity by means of glu-
curonides having toxic aglycones and, more particularly, to
an improvement of such processes w~ich eliminàtes damage to
the kidneys. The toxic aglycones may incorporate a nitrile
group. The invention further relates to the treatment of
certain bacterial infections having ~-glucuronidase acti-
vity. The present invention further relates to a new class
of glucuronides whose aglycone's activity or water solu-
bility is pH dependent as well as the method of preparation
of such glucuronides. The present invention still further
relates to a novel nitrile-containing glucuronide. Finally,
the present invention also relates to a diagnostic
urinalysis test by which the presence of tumors having
~-glucuronidase activity can be determined.
Background Art
There have been many reports in the prior art relat-
ing to the general concept of providing direct transport
of an agent which is toxic to tumor cells directly to tumors
having B-glucuronidase acti~ity by conjugating the agent
with glucuronic acid. Among such reports are Von Ardenne,
M. et al. Agressologie, 1976, 17, 5, 261-264; East Ger~an
patent 122,386; German Offenlegungschrift 22 12 014; Swee-
ney et al, Cancer Research, 31, 477-478, ~pril 1971; Baba
et al, Gann, 69, 283-2841 1978; and Ball, C.R., Biochem.
Pharm., 23, 3171-3177 (1974~.
The Von Ardenne reference suggests broadly many
types of aglycones which may be conjugatedto glucuronic
,~ .
~,
,
.
.- -.- , . , -. , .
11~8~86
--2--
acid and will be active at the tumor site. These include,
broadly, alkylating groups, antimetabolites, cytotoxins,
membrane-active (lytic) groups, glycolysis stimulators,
respiration inhibitors, inorganic and organic acids and
cell cycle stoppers. The ~ast German patent also suggests
many such combinations including 5-fluorouracil-glucuronide,
methotrexate-glucuronide, 6-mercaptopurene-glucuronide,
aniline mustard-glucuronide and many others. The Offenle-
gungsschrift also mentions a large number of glucuronides.
The Sweeney article relates to the anti-tumor activity of
mycophenolic acid-~-D-glucuronides, Baba relates to the
anti-tumor activity of 5-fluorouracil-O-~-D-glucuronide,
and Ball relates to the anti-tumor activity of p-hydroxy-
aniline mustard glucuronide.
It has also been reported that the selectivity of
this transport mechanism can be improved by hyperacidifica-
tion of the tumor cells. The Von Ardenne reference supra,
as well as the East German patent, ~learly recognize the
importance and the feasibility of hyperacidification of the
tumor cells when using the glucuronide mechanism. The Von
Ardenne reference speaks of a method that yields a pH dif-
ference of at least 1 pH unit and may therefore by used as
a basis for selectivity. It refers to reaching steady
state conditions after hyperacidification in which the brain
pH is 7.0 and the tumor tissue pH is approximately 5.5 to
6Ø Note also Von Ardenne, M. et al, Pharmazie, 32 (2):
74-75, 1977.
B.cker, U., Nature, 252, December 20-27, 1974, pp.
726-727, particularly notes that lysosomal enzyme ~-glucuro-
nidase has an optimum p~ of 5.2 and that for anti-tumor ac-
tivity of glucuronides, the pH must be lowered such as by
the administration of glucose. Experiments are detailed
which indicate that the hyperacidification by glucose is
necessary in order to obtain significant deconjugation of
glucuronides.
11481;~86
--3--
Even with hyperacidification of the tumor cells by
known methods as, for example, glucose administration,
however J there is still a problem in that otner organs
and tissues of the body whic'n have a naturally occurring
high ~-glucuronidase activity, will also release the
toxic aglycones and thereby cause da~age to healthy
tissues. This is most particularly a problem with regard
to the kidney which normally has an acid pH environment.
It has been suggested in British patent 788,855 that
mandelonitrile-~-D-glucuronic acid may be used in the
treatment of malignant tumors as ~-glucuronidase is preva-
lent in malignant tissues and will selectively attack man-
delonitrile-~-D-glucuronic acid at the site of the malig-
nant tumors to split off hydrogen cyanide. U. S. patent
2,985,664 is also related to mandelonitrile-~-D-glucuronic
acid and a method of producing same. These compounds have
been named Laetrile by the patentees of the above-mentioned
patents.
It has been discovered, however, that none of the
mothods of producing this compound set forth in the above-
mentioned patents are reproducible. The present inventor
has discovered that attempts to oxidize prunasin produce
the glucuronide of mandelic acid because the CN group is
unstable. Attempts to condense mandelonitrile with
glucuronic acid or glucuronolactone or tetra-acetyl-glucu-
ronolactone halogenide failed because the mandelonitrile
tends to polymerize.
An article by Fenselau, C. et al in Science,
198 (4317) 625-627, 1977, entitled "Mandelonitrile ~-Glucuron-
ide: Synthesis and Characterization" cGnfirms that thesynthesis described in the original patents has not been
reproduced. This article also confirms that while it was
mandelonitrile-~-D-glucuronide which was originally given
the name Laetrile, this compound does not appear in the
~148(;~86
--4--
Mexican preparations marketed as Laetrile. The major
component of preparations currently marketed as Laetrile
is amygdalin which may be easily prepared from natural
source material, such as kernels of apricots, almonds,
S and other members of the Prunus family. However,
amygdalin cannot be split by the enzyme ~-glucuronidase.
The Fenselau reference teaches a method for the
biosynthesis of mandelonitrile ~-D-glucuronic acid.
While this method may be satisfactory for producing
laboratory amounts of the compound, such a biosynthetic
process would no doubt be very difficult and costly to
commercialize.
The problems involved in the chemical synthesis of
mandelonitrile ~-D-glucuronic acid also exist for the
synthesis of any glucuronide the aglycone of which is a
strong electron acceptor. This is because the glucuronide
will become deconjugated (hydrolyzed) in the course of
the classical process.
Before using glucuronide treatment, there must be
a diagnosis of tumors having ~-glucuronidase activity.
The prior art (for example, Sweeney, supra) suggests taking
a biopsy to determine such ~-glucuronidase presence. It
would be desirable to be able to detect the presence of
such ~-glucuronidase activity tumors by a simple urine test.
Disclosure of Invention
Accordingly, it is an object of the present invention
to overcome the deficiencies of the prior art.
It is another object of the present invention to
provide for the improved treatment of malignant tumors.
It is a further object of the present invention to
provide an improved process for the treatment of malignant
tumors having high ~-glucuronidase activity.
It is still ~nother o~ject of the present invention
to provide such an improved process which is selectively
toxic to tumor cells, but does not harm healthy tissue.
1~48~86
-5 -
It is yet another object of the present invention
to provide such an improved process in which the tumor cells
are selectively treated with nitrile-containing compounds
with concurrent therapy to avoid the possibility of cyanide
poisoning in the rest of t'ne body.
It is still another object of the present invention
to provide new compounds and pharmaceutical compositions
which have very low toxicity to the organism as a whole but
very high selective toxicity toward tumor cells, and
particularly tumor cells having high ~-glucuronidase activity.
It is another object of the present invention to
provide a diagnostic method using radioactive isotopes to
selectively label tumor cells so that both primary tumor
cells and metastases can be precisely located.
It is yet another object of the present invention
to provide a process of preparin~ the compounds which may
be used in such processes of treatment.
It is still another object of the present invention
to provide a process for preparing mandelonitrile ~oD-gllu-
curonic acid by totally chemical synthesis.
It is still another object of the present inventionto provide a method for testing the urine to determine the
presence of t~mors having ~-glucuronidase activity.
It is still another object of the present invention
to provide a process and compositior.s for the treatment of
bacterial infections when the bacteria exhibit ~-glucuroni-
dase activity.
These and other objects of the present invention will
be better understood from a reading of the following summary
and the detailed description of the present invention.
It has now been found that the selectivity of glucu-
ronide compounds toward tumors can be greatly increased and
the possible deconJugation of the tox~c aglycones in
normal parts of the body can be greatly minimized by admin-
istering to the patient, prior to or simultaneously with
-:
1148~86
--6--
administration of the glucuronide, an alkalinizing agent
which will maintain the pH of the rest of the body at a
pH of about 7.4. It is known that at a p~. of 7.4 and
above ~-glucuronidase activity is substantially nil.
Thus, the administration of alkalinizing agents such
as bicarbonates or other basic salts will substantially
decrease and eliminate ~-glucuronidase activity which
naturally occurs in certain healthy tissues such as the
kidneys, spleen, and liver. Such an administration of
alkalinizing agent will not diminish the acidity of the
tumor cells themselves, however, in view of the naturally
low pH of the tumor cells, the mechanism of prior hyper-
acidification, and the lack of substantial blood perfusion
through the tumor areas, as well as other possible
mechanisms. It has been suggested in the literature, in
fact, that bicarbonate will actually increase the acidity
of the cancer cells. Gullino, P.M., et al, J.N.C.I.,
34, 6, 857-869 (1965).
Since the ~-glucuronidase activity of the tumor
cells will be enhanced by acidification, and the ~-glucuro-
nidase activity of the rest of the body, particularly of
the kidneys, will be substantially eliminated by alkalini-
zation, the toxic aglycones will only be released at the
tumor site itself due to deconjugation of the glucuronides
by the action of ~-glucuronidase. Without the alkaliniza-
tion step, substantial amounts of toxic materials may be
released, for example, in the kidneys, and the toxic agly-
cones so released may cause substantial damage to these
organs. Thus, only through the use of the present inven-
tion can glucuronides of compounds toxic to tumor cellsbe used clinically with a great degree of safety. The
greater the toxicity of the aglyco~es, the more important
is the alkalinization step.
A further feature of the present invention is the
use of certain novel glucuronide compounds which are parti-
1~4~86
--7--
cularly suitable for use in the present invention becauseof the significant pH differential between the tumor cells
and surrounding healthy tissue. If the aglycone is more
active at lower pH, or non-polar in acid condition and
becoming polar only in alkaline condition, i.e., the
aglycone is water-soluble at pH ranges above about 7 and
lipid-soluble at pH ranges below 7, then the selectivity
of the present invention is further increased. Using
these new compounds, even if there is deconjugation else-
where in the body, the aglycone will be water-soluble due
to the alkaline pH and be washed out of the system quickly.
However, in the low pH range of the hyperacidified tumor
cells, the aglycone will actually become attached to the
tumor cells and will not become solubilized and washed
away. Even if some amount of aglycone becomes removed
from the locus of the tumor cells, they will immediately
come into an alkaline environment and thus become water
soluble and be quickly swept from the body.
Among the novel glucuronides in this category are
2,4-dinitrophenol-~-D-glucuronic acid; 4-chloro-m-cresol-~-
D-glucuronic acid; 4,6-dinitro-o-cresol-~-D-glucuronic
acid; 4-chloro-3,5-xylanol-~-D-glucuronic acid; chlorothymol-
~-D-glucuronic acid; 2-phenyl-6-chlorophenol-~-D-glucuronic
acid; 5-chloro-7iodo-8-quinolinol-~-D-glucuronic acid; and
podophyllotoxin-~-D-glucuronic acid. The chloro-m-cresol-~-
D-glucuronic acid is of particular interest as it actuall~
loses its toxic activity in an alkaline environment.
Aside from the anti-tumor utility, these novel com-
pounds, and any other glucuronide compounds having cytotoxic
aglycones, also have an anti-bacterial activity, particularly
against those types of bacteria having glucuronidase
activity. It is known, for example, that streptococcus,
staphylococcus and E. coli bacteria have ~-g~ucuronidase
activity. Therefore, if the glucuronides come into contact
with these bacteria, they will become deronjugated and the
1148~86
--8--
cytotoxic aglycones will be toxic to the bacteria.
It has been reported that the optimum p~. of bacte-
rial ~-glucuronidase is higner than the optimum p~ of the
~-glucuronidase of normal healthy internal organs, such
as liver, spleen, kidney, etc. Therefore, upon alkaliniza-
tion of the body in accordance with the method discussed
hereinabove, the ~-glucuronidase activity of the organs
will be substantially eliminated, while that of the
bacteria, although alkalinzed, will still ~e present. The
administered glucuronide will then only be deconjugated
to its active form at the site of the infection. Since
tumor cells are not being treated for this utility, no
hyperacidification step is necessary
While the glucuronide compounds discussed herein-
above are preferred for use in the process of the presentinvention, it should be understood that the glucuronides
of any anti-tumor drug, including those previously suggested
in the prior art as being useful,may be used to greater
advantage in the process of the present invention since the
selectivity thereof will be increased by the alkalinization
step. Non-limiting examples of compounds, some of which
may have been known, which may also be used in tne present
invention, even though th~y have no presently known differ-
entiation of toxicity or solubility which is p~ dependent,
include 5-fluorouraciI-O-~-D-glucuronic acid; p-hydroxy-
aniline mustard-~-D-glucuronic acid; methotrexate-~-D-glucu-
ronic acid; floxuridine-~-D-glucuronic acid; cytarabine-
~-D-glucuronic acid; melphalan-~-D-glucuronic acid; hydroxy-
urea-~-D-glucuronic acid; adriamycin-~-D-glucuronic acid;
thiouracil-~-D-glucuronic acid; chlorophenol-~-D-glucuronic
acid; methacrylonitrile-~-D-glucuronic acid; fluoroacetic
acid-~-D-glucuronic acid; etc.
Other preferred ~orms of glucuronide for use in the
present invention are ones the aglycones of whic~ exert
their toxic effect on the cancer cells at the cell membrane.
~4~3~86
g
The anti-tumor toxicity of many conventional anti-cancer
drugs requires that they penetrate to t'ae nucleus or the
mitochondria within the cell. In prior cancer treatment
chemotherapy processes the dru~s had to be designed to
attack only cancer cells and not all of the other cells of
the body with which they come into contact. This is why
particular,efforts have been made in the past to develop
anti-neoplastic drugs which interfere with cell division.
Many of these drugs must actually enter the nucleus of the
cancer cell to be effective. For such drugs, therefore,
one must always be concerned that they be transported with-
out change through the membrane of the cancer cell before
they can exert their toxic effects.
By means of the process of the present invention it
is not important that the toxicity of the agent be directed
only at cancer cells as opposed to all of the healthy cells
of the human body, in view of,the fact that by means of the
process of the present invention the aglycone is only
released at the cancer site. Accordingly, a particularly
useful aglycone is one which exerts its cell toxicity by
,attacking the cell membrane itself. In this way one need
not be concerned with the transfer mechanism of the drug
through the membrane. Furthermore, by attacking t'ne membrane
the nature of the membrane is changed and the antigenic
properties of the cells are changed. Therefore the immuni-
logical system of the host will aid the toxic agent in rid-
ding the host of these cells. Accordingly a m~lch lower
dose need be used.
Examples of aglycones which exert this effect include
phenol and cresol. Therefore particularly useful glucuronides
for use in the process of the present invention include
phenol-~-D-glucuronic acid and cresol-~-D-glucuronic acid.
Other steps for increasing ~-glucuronidase activity
at the tumor~cells may also be undertaken. One method of
doing this is to elevate the temperature of the toxic cells
'
1~481;~86
-10-
at the tim~ o~ treatment. This may be done by elevating the
temperature of the entire body such as by use of a pyrogenic
drug or by elevating the temperature soley in the area of the
toxic cells, such as by microwave radiation or electrical
current. Raising of the temperature increases ~-glucuron-
idase activity thereby-increasing the efficiency of the
deconjugation of the glucuroni.des. It is known that an
elevation of temperature of 3C increases ~-glucuronidase
activity by 50%.
Known pyrogenic drugs include etiocholanolone,
progesterone, dinitrophenol, dinitrocresol, etc. Both
dinitrophenol and dinitrocresol are also cytotoxic, as
will be discussed hereinbelow. Therefore the use of
these compounds are preferred, especially when administered
as the glucuronide. This gives the result that when the
glucuronide is deconjugated at the tumor site the aglycone
will act not only to denature the cytoplasmic protein but
also to raise the temperature directly in the region of the
tumor cells thus greatly increasing the efficiency of
further deco~jugation. .
~ch~
Local ~ ~ lothcrmia in the region of suspected tumor
cells is preferred to general hyperthermia because general
hyperthermia will also increase the ~-glucuronidase
activity in healthy cells. However, because of the alka-
linization step this is not a major problem. If the hyper-
thermia is local, then this provides an additional degree
of certainty that the glucuronides will only become
deconjugated at the tumor site. The application of micro-
wave treatment directed at the suspected tumor site is
one way to achieve local hyperthermia. Due to the
different electrical resistence o tutnor cells, another
method of achieving some degree of local hyperthermia is
by administering a low electrical current through the body.
.~ '` ' . ' ~ `:
.
1148~86
-11-
A further manner of increasing ~-glucurondase
acitivity selectively at tumor cells is by administration
of estro&en to female patients or testosterone to male
patients. It has been reported that these compounds induce
~-glucuronidase activity in trophoblastic cells. Certain
tumor cells are known to be trophoblastic; t'nis method
would thus be particularly useful for those cells. The
alkalinization step would prevent damage to healthy
trophoblastic cells.
Another feature of the present invention relates
to the process of preparing the glucuronides. It has been
discovered that it is impossible to prepare conjugates of
glucuronic acid by the classical methods when the aglycone
is a strong electron acceptor, as these compounds must
first be prepared-as the metnyl ester of the glucuronic
acid and it is not possible by the classical methods to
convert the methyl ester to the acid without deconjugating
the aglycone. While barium methoxide has been suggested
for this purpose in a related process in U. ~. patent No.
2,985,664, it has been discovered that barium methoxide
will not work. However, it has now been discovered that
if barium hydroxide is used, the methyl ester of the agly-
cone of the glucuronide may be converted to the barium salt,
and the barium salt may be converted to the free acid by
the use of sulfuric acid without deconjugation of the glu-
curonide. Moreover, removal of the acetyl protecting
groups is accomplished in the same step, thus eliminating
the need of a separate step to accomplish this function.
This novel step using barium hydroxide may also be
used in the chemical synthesis of mandelonitrile ~-D-glu-
curonic acid. However, this process will fail when
attempting to synthesize mandelonitrile ~-D-glucuronic acid
because when attempting to condense the methyl (tri-0-acetyl
~-D-glucopyranosyl) halide-uronate with mandelonit~ile, the
,
.
.
- .;.
~ ; .
114~86
-12 -
mandelonitrile will tend to polymerize rather than to
create the hemi-acetal bond with the glucuronic acid.
The method of synthesis of mandelonitrile ~-D-glucuronic
acid in accordance with the present invention comprises
first converting mandelic acid to mandelic amide by reaction
with gaseous ammonia. The mandelic amide is then reacted
with the methyl (tri-O-acetyl ~-D-glucopyranosyl) bromide-
uronate to produce the`methyl ester of the mandelic amide
triacetyl glucuronic acid. This compound may then be mixed
with acetic anhydride to convert the mandelic amide to
mandelonitrile. Treatment with barium hydroxide and
sulfuric acid will produce the mandelonitrile ~-D-glucuronic
acid.
Another feature of the present invention resides in
an additional safety feature by which the healthy tissues
of the body are protected against possible release of
hydrogen cyanide from nitrile-containing aglycones. This
feature is preferably in addition to the feature disclosed
hereinabove with respect to pH adjustment. Even with such
protection against deconjugation of the glucuronide at
areas of the body other than tumors, concern has been
expressed about possible cyanide poisoning when using nitrile-
containing glucuronides. For example, in Schmidt, E.S.,
et al. J.A.M.A. 239 (10):943-7, 6 March 78, it was predicted
that there will be an increased incidence of cyanide poison-
ing in man as Laetrile (amygdalin) becomes more readily
available. It is not known whether it is the entire
nitrile-containing aglycone, mandelonitrile, which exerts
the toxic effect on the tumor cells, or whether it is the
hydrogen cyanide which is released upon the decomposition of
mandelonitrile. It is theorized, however, that it is the
entire nitrile-containing aglycone which exerts the toxic
e~fect on the tumor cells. ~herefore, it is important to
protect the rest of the body against possible release of
hydrogen cyanide from the nitrile-containing aglycones.
:
1148~86
-13-
This is accomplished in accordance with the present
invention by the concurrent administration of sodium
thiosulfate when glucuronides of nitrile-containing
aglycones are used. It i9 well known that sodium
thiosulfate is an antidote for cyanide poisoning.
Sodium thiosulfate in the presence of the enzyme
rhodanase conve ts hydrogen cyanide to sodium thiocy-
anate.
It is believed that the concurrent administration
of sodium-thiosulfate will not affect the toxicity of
the aglycone at the cancer site for two reasons. First,,
even in the presence of rhodanase, sodium thiosulfate
wilI not affect the mandelonitrile molecule itself.
Therefore, if it is the entire mandelonitrile molecule
which is toxic to the cancer cells, then the presence
of sodium thiosulfate will not affect this toxicity. '
Furthermore, even if it is the hydrogen cyanide which is
toxic to the cancer cells when released at the site of
the cancer cells, it has been suggested in the literature
that cancer cells do not contain rhodanase. See Lupo,
M. et al, "Critical Review of Studies on Malignant
Diseases," Minerva Med. 67 (30) 1973-1981, 1976. There-
fore, the concurrent administration of sodium thicsulfate
will protect normal cells against cyanide poisoning but
will not affect the attack of the cyanide'on the tumor
cells.
In v~w of the relative lack of toxicity of glucuro-
nide compounds, and in view of the mechanism of the present
invention by which the toxic aglycone is released only at
the tumor site,-and further in view of the protection of
the present invention against possible hydrogen cyanide
release at other parts of the body, it is entirely possible
to use glucuronides of other toxic nitrile-contalning
agly~ones in the process of the present invention. One
- ~ .
.
~8 ~8 6
-14-
such compound is methacrylonitrile ~-D-glucuronic acid.
Because of the acid-alkaline differentiation
be~ween the tumor cells and the rest of the body achiev-
able by the process of thepresent invention, it is pos-
sible to use certain compounds which denature cytoplasmicproteins or affect the energy supply of the cells directly.
without first conjugating with the glucuronic acid. This
can only be done, however, if the compound is one whose
activity or solubility is pH-dependent. Examples of such
compounds are 2,4-dinitro-phenol; chloro-m-cresol; 4,6-
dinitro-o-cresol; 4-chloro-3,5-xylanol; chlorothymol;
2-phenyl-6-chlorop-nenol; 5-chloro-7-iodo-8-quinolinol; and
podophyllotoxin. The use of these compounds directly
without first conjugating with glucuronic acid would be
particularly useful in treating tumors with no demonstrated
~-glucuronidase activity.
Another feature of the present invention is related
to the extremely high tumor selectivity which is achievable
in accordance with the present invention. In view of the
selectivity, if one or more of the atoms of the aglycone is
exchanged with a radioactive isotope, a local radioactivity
can be exerted. This method is not only important for
diagnostic purposes to trace the tumor and its metastases,
but if an isotope is chosen with ~-radiation activity, then
this method may also be used for local radiation treatment
at the cancer site. This use of radioactive isotopes is
particularly important when using an aglycone which is
known to be non-polar in acid condition and polar in alka-
line condition. When this quality exists, the aglycone is
accumulated at the cancer site not only because of the
~-glucuronidase activity, but also because of its insolubil-
ity in water at the cancer site. At the same time, t.he
compounds with the radioactive isotopes are washed away from
the rest of the body. The use of p-iodophenol ~-D-glu-
curonic acid produces an aglycone, p-iodophenol, which
fulfils these demands. A radioactive isotope of iodine can
~481;~36
-15 -
be used as the iodine cons~ituent of this compound. It
is preferable to use ~ 3 II for labelling and ~ 3 3 I for
treatment, as the former is richer in gamma radiation
while the latter is richer in beta radiation. In order
to prevent the iodine from migrating to the thyroid gland,
premedication with non-radioactive Lugol's solution may
be used for saturating the thyroid gland.
Another compound which can be easily radioactive
labelled is the glucuronide of phenylsulfazole. A radio-
active sulfur atom can be used. This compound does notmigrate to the thyroid gland, and the aglycone is not
soluble in water.
Before treatment of patients in accordance with
the present invention, it should be ascertained that the
particular type of tumor involved has high ~-glucuronidase
activity. This may be done in a number of ways. One way
is to assay tumor cells obtained in a biopsy for ~-glucuroni-
dase activity. If such a test is positive, then the pharma-
ceutical compositions of the present invention may be
administered.
A second method is the administration of a glucu-
ronide whose aglycone has been labelled with a radio-active
isotope. If upon a full body scan it is found that the
radioisotope is accumulated at any specific areas of the
body, then this will indicate not only the location of the
tumor but the fact that the tumor has sufficient ~-glucu-
ronidase activity to deconjugate the glucuronide. After
this has been determined, the appropriate amount of the
glucuronide of choice may be administered. If there are no
tumors present, or if the tumors are of the type which do
not have ~-glucuronidase activity, then there will be no
accumulation of radioisotope in the body as the alkaliniza-
tion step of the present invention eliminates all usual
~-glucuronidase activity and the isotope will be passed
through the body.
11~8~86
-16 -
Another method of diagnosing tumors which are
treatable by means of the present invention is to test
for the presence of free glucuronic acid in the urine.
While the presence of glucuronides in the urine is common,
the presence of free glucuronic acid in the urine, and
particularly the presence of increasing amounts of
glucuronic acid when tested over a period of several
days, is a potent indication of the presence of tumors
with ~-glucuronidase activity. It is hypothesized that
the presence of free glucuronic ~cid in the urine
in the cancer patients is caused by the action of ~-glucu-
ronidase in the cancer cells on the intercellular filaments
and connective tissue. Glucuronic acid is a reaction
product of such activity because the intercellular
filaments and connective tissue are composed of polymers of
which glucuronic acid is an element and which
are known substrates for the enzyme ~-glucuronidase.
A method of distinguishing free glucuronic acid
from conjugated glucuronides in the urine is another feature
of the present invention. Both glucuronides and glucuronic
acid give a chromogenic complex with tetraborate in concen-
trated sulfuric acid which reacts with m-hydroxydiphenyl
to create a colored water-soluble complex. When lead
acetate is added at an alkaline pH, the glucuronides
precipitate and the addition of ditizone (dithiosemicarbizone)
makes a stable complex with the excess lead. Accordingly,
an optical reading may be taken representative of the
amounts of total glucuronides and free glucuronic acid after
tetraborate and m-hydroxydiphenyl have been added. A
second reading may then be taken after the conjugated
glucuronides and excess lead have been removed from the
aqueous phase by the addition of basic lead acetate and
after ditizone has been added. Alternatively, the conju-
gated glucuronides can be removed by reaction with bar;um
hydroxide. The addition of barium hydroxide to the urine
sample will cause pFecipitation of the conjugated glucuron-
,
~1481386-17 -
ides but not of the free glucuronic acid. After
centrifugation and filtration the conjugated glucuronides
are eliminated and what remains is only the free glucuronic
acid. A reading representative of the amount of free
glucuronic acid may then be taken. This alternative
procedure bypasses the necessity of the use of ditizone.
Best Mode for Carrying Out the Invention
While many glucuronide compounds having aglycones
which are toxic to cancer cells have been described
theoretically in the literature, very few have actually
been produced. This is because they are very difficult to
synthesize, particularly when t'ne aglycone is a strong
electron acceptor. The improved method of the present
invention avoids the problem and permits the production
of conjugates of glucuronic acid of almost any type of
aglycone. The standard methods can be used to form the
methyl ester of the triacetyl glucuronic acid conjugate~
but it is often quite difficult to go from the triacetyl
methyl ester to the glucuronic acid conjugate. This
problem has been solved by treatment in accordance with
the process of the present invention.
The glucuronides in accordance with the present
invention and for use in ~he process of the present
invention, may be synthesized from methyl (tri-O-acetyl-
~-D-gluco-pyranosyl bromide)-uronate which is the active
glucuronic acid and is formed in accordance with the
teachings of Bollenback, G.N., et al, J. Am. Chem. Soc.
77,3310, (1955). This compound is condensed with the
aglycone in a solution of quinoline, phenol, methyl
cyanide or methyl nitrite catalyzed by silver oxide or
silver carbonate. Another method of condensation is to
use sodiu~ or potassium hydroxide as the condensing
agent in aqueous acetone solution. The reaction scheme
is illustrated as follows:
. ,
`` 1148~86
-18-
COt lCH3 COOCH3
AFO AcO
OAc OAc
wherein ROH is the desired aglycone.
If the methyl ester of the glucuronide is desired,
the protecting acetic acid groups may be removed by
anhydrous sodium methoxide or anhydrous barium methoxide
in accordance with the following reaction:
COOCH3 . `
~0 - R ~ ~- O - - R (Il)
AcO
C~c OH
The acid may be produced by reacting the triacetyl
methyl ester with barium hydroxide to produce the barium
1~ salt in accordance with the following reaction:
tCOOCH3 COOBa~
B AcO~ Ba ~OH) 2 ~ ~$~ (III)
OAc OH -
This barium salt of the glucuronide pxecipitates. An
equimolar solution of sulfuric acid releases the free glucur-
onide according to the following reaction:
COOBa j~ C:OOH
HOK~ NO~
OH
..jt
11~8V86
-19-
Example I shows the preparation of 2,4-dinitro
phenol-~-D-glucuronic acid.
Example I - Synthesis of 2,4-Dinitrophenol-g-D-~lucuronic
cid .
Methyl-(2,3,4-tri-0-acetyl-~-D-glucopyranosyl
bromide)-uronate was prepared in accordance with the process
of Bollenback, G.N., et al, J. Am. Chem.__oc. 77, 3310
(1955). Four grams of methyl (tri-0-acetyl-~-D-glucopyranosyl
bromide)-uronate in acetone (80 ml) and 8.9g 2,4-dinitro-
phenol were treated with 5N potassium hydroxide (9 ml) and
the solution kept at 25C for 24 hours, then diluted with
3 volumes chloroform. The chloroform-acetone layer was
washed with water and dried. Removal of the solvent and
two recrystallizations from acetone yielded the methyl-2,3,4-
tri-O-acetyl-~-D-glucopyranosyl uronate of 2,4-dinitro-
phenol.
The free acid form of the compound was formed by
treating the 2,4-dinitrophenyl-methyl(tri-0-acetyl-~-D-glu-
copyranosyl bromide)-uronate with a one-half molar amount
of barium hydroxide to produce the barium salt. This barium
salt of the glucuronide precipitates as a white amorphous
material. An equimolar solution of H2S04 releases the free
glucuronide. Distillation of the supernatant yielded
bright yellow-brown crystals having a melting point of
179-180C. This compound was incubated with ~-glucuronidase
and produced 2,4-dinitrophenol, thus confirming that the
final product is indeed 2,4-dinitrophenol-~-D-glucuronic
acid.
The other glucuronides in accordance with the present
invention, e.g. chloro-m-cresol-~-D-glucuronic
acid; 4,6-dinitro-o-cresol-~-D-glucuronic acid; 4-chloro-
3,5,-xylanol-~-D-glucuronic acid; chlorothymol-~-D-glucuro-
nic acid; 2-phenyl-6-chlorophenol-~-D-glucuronic a~id; 5-
chloro-7-iodo-8-quinolinol-~-D-glucuronic acid; and podo-
phyllotoxin-~ D-glucuronic acid, as well as p-iodophenol-~-
D-glucuronic acid and phenylsulfazole-~-D-glucuronic acid,
may be made in a similar manner by reacting a stoichiometric
`" 1148~86
-20-
excess of the aglycone with the methyl-(tri-0-acetyl-~-D-glu-
copyranosyl bromide)-uronate in 5 normal potassium hydroxide
and maintaining the reaction solution at room temperature
for 24 hours. The solution is then diluted with 3 volumes
chloroform and the chloroform-acetone layer washed with
water and dried. After removal of the solvent, the crystals
which are obtained are treated with a one half molar amount
of barium hydroxide to produce the barium salt which is then
treated with an equimolar solution of sulfuric acid to pro-
duce the free glucuronide.
The free acid form of the glucuronide, or a saltthereof which will ionize at the conditions of use, is the
preferred form of the compounds to be used in accordance
with the present invention. However, pharmaceutically ac-
ceptable esters may also be used, although in most cases itwould be expected that their activity would be somewhat lower
due to their relatively lower affinity to ~-glucuronidase.
This is particularly true with respect to aglycones which
are strong electron acceptors. Accordingly, whenever the
term "glucuronide compound" is used in the present specifi-
cation and claims it is understood to include not onLy the
free glucuronic acid form of the conjugate but also pharma-
ceutically acceptable salts and esters thereof as discussed
hereinabove, both in this and subsequent examples.
Example II - SYnthesis of Mandelonitrile~ -D-~lucuronic Acid
Mandelonitrile ~-D-glucuronic acid may be synthesized,
in accordance with the present invention, from methyl
- (tri-O-acetyl-~-D-glucopyranosyl bromide)-uronate, which is
the active form of glucuronic acid, and may be produced in
accordance with the teachings of Bollenback, G.N., et al,
J. Am. Chem. Soc. 77, 3310, (1955). Since this compound
cannot be directly conjugated with mandelonitrile, mandelic
amide is first formed. This compound is ~ormed by bubbling
gaseous NH3 into mandelic acid at 0C as illustrated in
reaction:
1~48~8
21
H H
HO - C - COOM HO - C - CONH2
~ 0'C ~
The mandelic amide is introduced to the ~ethyl (tri-
O-acetyl ~-D-glucopyranosyl) bromide uronate in a solution
of phenol catalyzed by a small catalytic amount of silver
5 oxide. Besides phenol, there may be used, as solvent,
quinoline, methyl nitrile or methyl cyanide. Silver car-
bonate may also be used as the catalyst. Another method
of condensation is to use sodium or potassium hydroxide
as the condensing agent in aqueous acetone solution. A
10 stoichiometric excess of mandelic amide is preferably used.
The reaction solution is maintained at room temperature
for 24 hours or until the reaction is complete. The
reaction is illustrated as follows:
H - H
' 1, ' I ~
C:OOCH ~ COOCH - - COWH
3 HO - C - CONH2 3 _ 2
- AcO~
OAC
~5 The above solution is then mixed with acetic anhydr-
ide in 1:1 molar ratio and heated to 70C for 30 minutes
in order to convert the mandelic amide to the mandelo-
nitrile in accordance with the following reaction:
.
. .
,
:, ~
!
1148~86
-22-
H
H COOCE~
COOCH
3 - O - C - C~l
AcO op":: AcO OAc .
The acid is produced by reaction of the triacetyL
methyl ester obtained by reaction (III) with a 1/2 molar
amount of 0.5 N barium hydroxide which is added slowly to
this solution to form a white precipitate. Preferably an
excess of barium hydroxide is added until there is no
more precipitation. The reaction can be illustrated as
follows:
- H H
COOCM3 I COOBa~
--O - C -- CN --O-C-CN
~ ~ ¦ sa~o~2 ~ ~ ~ (IV)
AcO HO
OAc OH
,,: ... . ....
, . . .
,~ .
,
1~48~86
-23-
The addition of 0.5N sulfuric acid, volume to volume,
then ~ooling in ice water for 20 minutes, relases the free
glucuronides according to the following reaction:
COOBa~
COOH
O - C - CN O- C-CN
¦~-- ¦ H2S04 1/ o~ ~ BaSO
110~ N~
OH . ,i
~j The mixture is then filtered and the supernatant is
dried in vacuum and crystallized from ether. Il
~xample III - Synthesis of Methacrylonitrile ~-D-Glucuronic
Acid
Methacrylonitrile ~-D-glucuronic acid or other
glucuronides of nitrile~containing cytotoxic compounds
may be produced in accordance with the present invention
in a manner gimilar to that disclosed in Example Ii
though the step of converting the methacrylonitrile ~o
methacrylamide prior to condensation with methyl(tri-0-
acetyl-a-D-glucopyranosyl bromide)-uronate will not be
necessary as there is not the same polymerization problem
with methacrylonitrile as there is with mandelonitrile.
In general, the preferred proc,ess when condensing the
B aglycone directly, is to re ~ the stoichiometric
excess of the aglycone (methacrylonitrile in the case of
methacrylonitrile ~-D-glucuronic acid) with the methyl (tri-
0-acetyl~ glucopyranosyl bromide)-uronate in 5 N potas-
sium hydroxide and maintaining the reaction solution at
room temperature for 24 hours. The solution is then diluted
with 3 volumes chloroform and the chloroform-acetone layer
.- :
. ; `~, '
, ' ' ':
:~ '
1148~86
-24-
washed with water and dried. After removal of the solvent,
the crystals which are obtained are treated with a one half
molar amount of barium hydro~ide to produce the barium salt
which is then treated with an equimolar solution of sul-
furic acid to produce the free glucuronide.
Exam~le IV - Acute Intravenous Toxocity to Rabbits
of Mandelonitrile ~-D-Glucuronic Acid
NZW rabbits in the weight range of 2,000 to 3,200 g
for females and 2,200 to 3,800 g for males were injected
intravenously with mandelonitrile ~-D-glucuronic acid
solution. Rabbits injected with saline alone served as
the control. The mandelonitrile ~-D-glucuronic acid solution
contained 10% mandelonitrile.
During the 14 day observation period a record was
kept of all mortalities and signs of toxicity. Table I
gives the range finding screen.
Table I - Mortality Data for Groups of Rabbits (2 per Group)
Intravenously injected with DMBG Solution.
Ran~e Finding Screen
20 Dosage Mortality Ratio
mllkg no. of deàths/
no dosed
.
0.25 0/2
0 5 1/2
1.0 2/2
2.0 2/2
25 4.0 2/2
The results of the preliminary range finding tests
as shown in Table I indicated that the median lethal
intravenous dose (LD-50) was in the region of 0.23 - 2 ml
per kg body weight.
Dosing was then extended to larger groups of rabbits
(5 males and 5 females per group) in order to locate the
median lethal dose more precisely. Table II gives mortality
data for this larger group.
'
'
' -
- -
`` ~14~3086
-25-
Table II - Mortality Ratio of Rabbits Intravenously Injected
with DMBG Solution. Full Scale Test - Weight
range: Females 2,000-3,200 g, Males 2,200-3,800 g.
Dosage Mortality ratio Time of death after dosing
ml/kg No. of deaths/
No. dosed No. of animals No of hours
Males 0.44 0/5
0.66 2/5 2 < 3
1.0 3/5 3 < 3
1.5 4/5 4 < '3
- 2.25 5l5 5 < 3
Females 0.44 0/5
0.66 2/5 2 < 3
1.0 5/5 5 < 3
1.5 5/5 5 < 3
2.25 5l5 5 ~ 3
Signs of reaction to treatment, observed 2 minutes
after dosing, incIuded ataxia and paralysis. Two minutes
later a few animals of the high dose group died. All the
deaths of all the groups occurred within 3 hours after
dosing. The animals which survived did not show any clini-
cal symptoms during the following 14 days. Autopsy of all
animals did not show clear gross pathological changes.
The acute median lethal intravenous dose (LD 50)
and its 95% confidence limints calculated by the method of
i Weil, Ç.~. 1952, Biometrics, 8:249, to rabbits of
1 2 mQV~Jon.
J ; mandc~o nitrilc ~-D-glucuronic acid 10% solution are calcu-
lated to be:
Males: 0.84187 (0.78087-0.90287) ml/kg body weight
Females: 0.6873 (0.64417-0.73043) ml/kg body weight
- From the above data, it is believed that the maximum
safe dose is on the order of 0.44 ml/kg body weight, and
it is believed that this limit should not be exceeded in
human therapy.
:.
.. . . . ~ , .
. . , . ~ .
-: ~ .. ...
'
1~48086
-26-
Prior to therapeutic treatment with compounds of
the presen~ invention, the presence of tumor having high
~-glucuronidase activity must be diagnosed. The most posi-
tive way to definitively ascertain whether a tumor is
present having high ~-glucuronidase activity is to conduct a
biopsy and to assay the tumor cells obtained for ~-glucu-
ronidase activity. This, of course, is not feasible for
most kinds of tumor. Another way to diagnose for the
presence of tumors having ~-glucuronidase activity, is to
conduct a urine test in order to determine the presence of
free glucuronic acid. Normal patients show between 200 to
400 mg per 24 hours of free glucuronic acid in the urine.
Cancer patients with well developed tumors which have
~-glucuronidase acitivity will show greater than 2,000 to
7,000 mg per 24 hours free glucuronic acid. Accordingly,
using the test of the present invention, if substantially
more than 400 mg per 24 hours of free glucuronic acid is
shown, then this is an excellent indication of the presence
of tumors having high ~-glucuronidase activity.
A negative indication on this urine test will not
conclusively rule out the presence of tumors having
~-glucuronidase activity, because tumors in their initial
stages, though they might have ~-glucuronidase activity, might
not release sufficient free glucuronic acid to cause a
positive reading of the urine. Therefore, the urine test
should be repeated, and if an increasing amount of free
glucuronic acid is found, then this is another indication
of the presence of a tumor having ~-glucuronidase activity.
An example of the method of determining the amount of free
glucuronic acid in the urine is given in Example V .
-~xample V - Test for;Glucuronic ~cid in ~Jrine
Both glucuronides and glucuronic acid give a chromo-
genic comp~ex with tetraborate and concentrated sulfuric
acid which reacts with m-hydroxydiphenyl to create a colored
water-soluble complex. Furthermore, glucuronides precipi-
.,
1148~86
-27-
tate with basic lead acetate when pH is 8, while the free
glucuronic acid is not affected by the lead acetate.
Complexing the excess lead with dithiocarbizone forms a
stable complex with lead which can be removed, thus
leaving free glucuronic acid.
To 10 cc of a urine sample 0.1 N ammonium'nydroxide
is added until a pH of 8 is reached. An excess of saturated
solution of basic lead acetate is then added causing pre-
cipitation of the conjugated glucuronides. The sample is
then centrifuged and the supernatent separated. Two cc of
the supernatant is then treated with 10 cc of 10% dithio-
carbizone (ditizone) in chloroform in order to remove the
excess lead. After waiting until the separation is
complete, the aqueous phase is separated. To 0.2 cc of the
aqueous phase is added 1.2 cc of sodium tetraborate in
concentrated H2SO4. The mixture is mixed well in a test
tube and chilled in crushed ice. The test tube is then
heated for 5 minutes in boiling water and immediately
cooled in ice until it becomes cold. Twenty microliters of
0.15% m-hydroxydiphenyl in 0.5% NaOH is then added. After
waiting S minutes, the optical density is read at a wave-
length of 5200 A. The reading obtained represents the amount
of free glucuronic acid present in the urine.
The total amount of free and conjugated glucuronic
acid is simply determined by directly treating the sample
with tetraborate and hydroxydiphenyl, without first,removing
the free glucuronides. Reading at a wavelength of 5200 A
will give the indication of the total amount of conjugated
glucuronides and free glucuronic acid which is present.
Example ~A - Test for Glucuronic Acid in Urine
. . .
To 10 cc of a urine sample 0.1 N ammonium hydroxide
is added until a pH of 8 is reached. An excess of barium
hydroxide is then added causing precipitation of the
conjugated glucuronides. The sample is then centrifuged and
the supernatant filtered. To 0.2 cc of the filtered super-
natant is added 1.2 cc of sodium tetraborate in concentrated
.
~148086
-28-
H2SO4. The mixture is mixed well in a test tube and
chilled in crushed ice. The test tube is then heated
for 5 minutes in boilin~ water and immediately cooled
in ice until it becomes cold. Twenty microliters of
0.15% m-hydroxydiphenyl in 0.5% NaOH is then added.
After waiting 5 minutes, the optical density is read
at a wavelength of 5200 A. The reading obtained
represents the amount of free glucuronic acid present
in the urine.
The relative amount of the total of conjugated
glucuronides and free glucuronic acid which is present
may be read in the same manner as set forth hereinabove
in Example V.
Example VI - Method of Administration of Glucuronide
Therapy
After it has been determined that the patient has
a tumor with ~-glucuronidase activity, the first step of
the treatment is to give him a dose of glucose as, for
éxample, 100 g of honey, glucose or other sugar. Approxi-
mately 1 hour laterj an intravenous drip is begun of asolution in distilled water containing approximately 10%
glucose and 60 milli-equivalents sodium bicarbonate.
Approximately 1 liter is administered, assuming no contra-
indications, and the pH of the urine is checked to determine
that it has reached a pH of approximately 7.4. This will
establish that the system has become alkalinized and it is
now safe to administer the glucuronide. Another liter of
the same glucose-bicarbonate solution, but also including
the desired amount of glucuronide, is then administered.
This is repeated daily as needed. ~en a glucuronide of a
nitrile-containing cytotoxic aglycone is being used,
immediately before, during or after administration of the
glucuronide,50 cc of a 25% solution of sodium thiosulfate
is administered, preferably intravenously by slow drip.
The sodium thiosulfate is preferably included in the
glucose-bicarbonate-glucuronide solution which is being
dripped intravenously. However, it may also be continued
afterward for a greater margin of safety.
. . , , . j . ............ , .... . . . .. ,., ." . ~ ,. ,
.
1148086
-29 -
If there are contraindications for the administra-
tion of bicarbonate, then antacid may be orally administered.
The important criterion is that the pH of the urine become
approximately 7.4 and remain so during treatment.
The hyperacidification of the tumor cells is caused
by a hyperglycemic condition in the patient. Therefore
any hyperglycemic agent may be used as the hyperacidifica-
tion agent, as for example, fructose, galactose, lactose or
glucagon. Furthermore, it should be understood that this
hyperglycemic condition may be effected in any known manner.
For example, if the patient is diabetic then the condition
can be brought about by decreasing the insulin administra-
tion.
Any agent which will raise the pH of the urine to
approximately 7.4 can be used as the alkalinizing agent,
including sodium or potassium bicarbonate or citrate, or
other basic salts or antacids. While it is preferred that
these be administered intravenously, they may be administer-
ed orally~
When the term "approximately 7.4" is used in the
present specification and claims, with respect to the pH
level to be maintained in the rest of the body, it should be
understood that a pH level slightly above or below 7.4 may
be used, although not preferred. As the pH decreases from
7.4 the ~-glucuronidase activity increases (until the
optimal pH is reached). Furthermore, below pH 7.0 the rest of the
body will not be alkaline but will be acid. Above 7.4 the
danger of alkalosis increases without any substantial further
decrease in i~-glucuronidase activity. A pH level of 7.4
is preferred as this is physiological pH and cannot be
harmful to the body, and it is known that the ~-glucuronidase
activity in healthy organs is substantially nil at this pH
level.
The dosage of the glucuronides should be monitored to
avoid any side effects due to the massive release of toxins
caused by the dying cancer cells. It may be preferable to
.
.
1~48~86
-30-
to treat with glucuronides in short courses of several
days, leaving several days in between, to allow any toxins
released by the dying cancer cells to leave the body before
the further treatment continues.
Besides intravenous administration, the glucuronides
may be administered by any means of parenteral administration.
However, the glucuronides should not be administered orally
as it is known that ~-glucuronidase is present in the digestive
tract. The sodium thiosulfate, however, can be administered
orally if a proper enteric coating is provided to avoid
release in the stomach.
The amount of glucuronide to be administered to any
given patient must be determined empirically and will
differ depending on the condition of the patient. Relatively
small amounts of glucuronide can be administered at first
with steadily increasing daily dosages if no adverse effects
are noted. Of course, the maximum safe toxicity dosage as
determined in routine animal toxicity tests should never be
exceeded.
It is clear that any tumor cells having ~-glucuroni-
dase activity may be treatable in accordance with the present
invention with the remaining organs of the body being
protected by the alkalinization step. Tumors which are known
to have ~-glucuronidase activity include solid breast tumors
and their metastases, bronchogenic carcinoma and its metatases,
and lymphomas. It is also known that neoplasms that do not
have high ~-glucuronidase activity, and therefore cannot be
treated in accordance with the present invention, include
leukemia. It must be understood, however, that this list is
not meant to be complete, and that the prior art is aware of
many other tumors that have ~-glucuronidase activity. However,
whether or not the art is presently aware that any ~iven tumor
has ~-glucuronidase activity, this can be determined by any o~
the various methods of diagnosis discussed in the present speci-
fication and if it is determined that the tumor does have~-glucuronidase activity, the therapeutic treatment of the
present invention can be effectively used.
86
-31-
~ nen it is desired to induc~ hyperthermia to increase
~-glucuronidase activity, a method should be selected by
which the te~perature is raised as much as possible without
risking damage to healthy portions of the body, such as
the eyes. An increase of about 2C for whole body hyper-
thermia and as much as 4.5C for local hyperthermia is
preferred. The hyperthermia should be timed to last about
an hour at the time of greatest glucuramide concentration at
the tumor site. For example, when local microwave treatment
is selected, it should begin about one half hour after
commencement of the intravenous glucuronimide drip and bé~
continued for about an hour. The proper dosage of known
pyrogens to achieve the desired degree of hyperthermia would
be known to those skilled in the art or could be easily
empirically determined. A dosage of about 30 mg/day for
dinitraphenol, for example, would be apt.
When estrogen or testosterone are to be administered,
a dosage of 5-15 mg/body wt/day would provide the desired
inducement of ~-glucuronidase activity.
Example VII - Method of A~ninistration of Radioisotopes
If an aglycone labelled with a radioactive isotope
is to be administered, the labelling may be accomplished by
~ny method known per se. For diagnostic purposes only,
- relatively small amounts of these labelled glucuronides may
be administered. They are otherwise administered in the
same manner as set forth in Example VI for non-labelled
glucuronides. Scanning of the body to determine whether
any of the radio-labelled aglycone is retained by the body
will indicate whether a tumor is present having ~-gluduroni-
dase activity and will also indicate where the tumor or anymetastases thereof may be found. As noted above, gamma
ray emitting isotopes, such as 1 3 lI, are par~icularly
suitable for this purpose.
The radio-labelled glucuronides may aIso be used for
in situ radiation therapy, particularly if an isotope is
"` 114~ 86
-32 -
used having high beta-radiation activity, such as 133I.
This wîll give the dual effect of attacking the cancer
cells not only with the toxic aglycones but also with
the beta-radiation. Again, the method of administration
will be the same as set forth in Example VI.
Another utility for the present invention is the
use of the boron-containing aglycone. It is already known
that if boron atoms are bombarded with neutrons, they
will break into lithium with the consequent release of
positrons. If the boron atoms are attached to tumor
tissue at the time, the positrons will be abruptly absorbed
by the tumor tissue which uill be lethal thereto. This
process will have outstanding utility when the boron atoms
are concentrated exclusively at the tumor cells in
accordance with the process of the present invention.
Example VIII - Method of Administration of pH DePendent
Therapy
If the tumor cells are hyperacidified and the healthy
tissue alkalinized in accordance with the method set forth
in Example VI, an acid-alkaline pH differential will be
created between the tumor cells and healthy cells. Thus,
compounds whose activity or solubility is pH-dependent
may be administered directly, without first conjugating with
a glucuronide. Such compounds will selectively attack the
acidified tumor cells without harming the remainder of the
body which has an alkaline pH.
As in the method of Example VI, the patient is first
given an oral dose of hyperglycaemic agent, such as 100 g
of honey, glucose or other sugar. Approximately 1 hour
later, an intravenous drip is begun of a solution in
distilled water containing approximately 10% glucose and
60 milliequivalents sodium bicarbonate. Appro~imately l
liter is administered, assuming no contraindications, and
the pH of the urine is checked to determine that it has
reached a pH of appro~imately 7.4. This will establish
. '
.
' :~
" ~148~86
-33-
that the system has become alkalinized and it is now
safe to administer the acid-active compound. Another
liter o the same glucose-bicarbonate solution, but also in-
cluding the desired amount of acid-active compound, is
then administered. This is repeated daily as neededO
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 podophyllo-
toxin are all water soluble at alkaline pH's and lipid-sol-
uble at acid pH's. Therefore, if the compounds are adminis-
tered in the manner discussed above they will not create
substantial harm to healthy tissue because they will be
washed through the system relatively quickly. At the site
of tumor tissue with acid pH, however, these compounds will
come out of water solution and exert their cytotoxic or
energy-supply effecting action 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. There-
fore, administration of these compounds with concomitanthyperacidification of the tumor cells and alkalinization of
the remainder of the body will be even less harmful to
healthy tissue, as their activity is dimished at the pH
of the healthy tissue.
The dosage of the non-glucuronide compounds in
accordance with this embodiment of the present invention will
generally be somewhat less than the corresponding glucuro-
nides as the glucuronide form of the compounds is substan-
tially less toxic than the free compounds. The precise
dosage must be determined empirically depending on the
condition of the patient. Relatively small doses should be
administered at first with steadily increasing daily dosage
if no adverse effects are noted. Of course, the maximum
safe toxicity dosage as determined in routine animal toxicity
tests should never be exceeded.
~148~86
-34-
Alternative acidifying and alkalinizing agents, as
discussed hereinabove with respect to the glucuronide em-
bodiment, may also be used with the present embodiment.
Example IX - Method of Anti-Bacterial Administration
Glucuronide administration may be used in the treat-
ment of bacterial infections if the bacteria involved are
known to have ~-glucuronidase activity. Examples of such
bacteria are streptococci, staphylococci, and E. coli. The
method of treatment of such bacterial infections will be
similar to the method set forth in Example VI except that
no hyperacidification will be necessary. This is so be-
cause bacterial ~-glucuronidase is active at higher pH
levels than ~-glucuronidase of normal healthy internal
organs. Furthermore, such a hyperacidification step would
not affect the pH of the bacteria as its mechanism is spe-
cific to tumor cells.
The first step in antibacterial administration is an
intravenous drip of distilled water and 60 milliequivalents
sodium bicarbonate. Approximately one liter is administered
and the pH of the urine is checked to determine that it has
reached a pH of approximately 7.4. Another liter of the
same bicarbonate solution, but also including the desired
amount of glucuronide, is then administered in the same
manner. This treatment may be repeated daily if necessary.
The alkalinizing agent may also be orally adminis-
tered and any agent may be used that will alkalinize the
body to an extent such that the pH of the urine becomes
approximately 7.4. The ~lucuronide should not be administered
orally but it may be administered by any means of parenteral
administration.
Certain known anti-bacterial drugs having adverse
side-effects may also be administered as glucuronides in
accordance with the method of the present invention in order
to reduce or eliminate these adverse effects. For example,
35 chloroamphenicol is kno~n to have a bone marrow depression
effect which will not take place if the glucuronide is used.
Neomycin is a known antibacterial which cannot be administered
11~8~86
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internally because of its toxicity. However, it can
be orally administered for the treatment of infections
of bacteria having high 3-glucuronidase activity if
first conjugated to glucuronic acid.
The radioisotope-labelled aglycone diagnostic
procedure ~discu~sed hereinabove with respect to tumor diagnosis
may also be used to determine the existance and location
of bacterial infections. For example, a patient complaining
of pain in the area of the appendix can receive the radio-
labelled glucuronides. If no accumulation of isotope is
found in the area then inflammation caused by bacteria with
~-glucuronidase activity as a cause of the pain can be
ruled out. In most instances inflammation in appendicitis
is due to infection by bacteria with ~-glucuronidase activity.
Other use of such a diagnostic procedure would be obvious
to those skilled in this art.
Besides the glucuronide compounds discussed herein-
above, any known conjugatable antibiotic may be conjugated
with glucuronic acid for use against ~-glucuronidase contain-
ing infections. This has the advantage of greatly diminish-
ing the amount of free antibiotic circulating in the blood
stream. The only antibiotic which is released will be
released at the site of the infection. Therefore much
smaller dosages may be given. Accordingly, the glucuronides
of the present invention can serve as an internally adminis-
tered local antibiotic. Because of the known ~-glucuronidase
activity in the digestion tract, no glucuronide should be
administered orally, although any mode of parenteral
administration is permissible.
If the antibiotic aglycone is known not to have any
effect on the kidneys, then the alkalinization step can be
eliminated. Many antibiotics, however, are known to be
nephrotoxic to some extent and thus the alkalinization step
is important to protect the kidneys.
,, ~ .
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Example ~ - Biosynthesis of Mandelonitrile ~-D-Glucuronic
Acid
A 22 cc solution of 5% mandelonitrile (benzaldehyde
cyanohydrin) in propylene-glycol is prepared and an intra-
muscular injection of this solution is given to a donkeyor a goat. The 24 hr. urine is collected and acidified
with acetic acid until the pH becomes 4. The urine is
then filtered through a fiberglass filter and the filtrate
is treated in any one of the following three different
ways:
A. A saturated solution of lead acetate is added to
the filtrate. The white precipitate that appears is
separated by centri~uge and filtered. The filtrate is
alkalined with NH3 to pH 8 and then a saturated solution
of basic lead acetate is added. The precipitate is washed
with colder water and gaseous H2S is bubbled into it, the
black precipitate of lead sulfide being separated. The
filtrate is put into a vacuum until the volume is reduced
to one third. A brown paste is achieved which is dissolved
in absolute alcohol and kep overnight. The solution is
filtered and the filtrate is vacuumized and ether added.
The mandelonitrile ~-D-glucuronic acid is crystallized
from the ether solution.
B. The urine is acidified with hydrochloric acid
to pH 4 and filtered through a fiberglass filter. After-
wards, the solution is dried in a vacuum state and the
residue is dissolved in ether and recrystallized from the
ether solution.
C. 0.1 N barium hydroxide water solution is added
to the urine. The white precipitate of the barium salt
of the mandelonitrile glucuronide is then washed in cold
water and stirred and 0.1 N sulfuric acid is added. An
insoluble solution of barium sulfate is removed and the
supernatant vacuum dried and then recrystallized from
ether solution.
1~48~6
Since mandelonitrile is very toxic and only a very
small amount can be administered, the following semi-bio-
synthetic procedure may be used,
20 g mandelic amide (2-hydroxybenzamide) is mixed
with goat or donkey food and the urine is collected for 24
hours. The mandelic amide glucuronide is separated by any
of the methods described hereinabove. Acetic anhydride is
then added and the glucuronide (2,3,4-triacetate glucopyra-
nose mandelonitrile) is precipitated with barium hydroxide.
The barium is removed with sulfuric acid and the glucuro-
nide is recovered in vacuum as describjed hereinabove.
It will be obvious to those skilled in the art that
various changes may be made without departing from the
seope of the invention and the invention is not to be
eonsidered limited to what is described in the specification.
