U.S. patent application number 09/428375 was filed with the patent office on 2002-08-08 for method and composition for treating prostate cancer.
Invention is credited to GOKCEN, MUHARREM.
Application Number | 20020106359 09/428375 |
Document ID | / |
Family ID | 23698630 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020106359 |
Kind Code |
A1 |
GOKCEN, MUHARREM |
August 8, 2002 |
METHOD AND COMPOSITION FOR TREATING PROSTATE CANCER
Abstract
A method of treating prostate cancer in a living mammal includes
local administration of a composition that includes a
therapeutically effective concentration of collagenase. In one
embodiment, a method of treating prostate cancer in a living mammal
includes local administration of a composition that includes a
therapeutically effective concentration of collagenase and at least
one of a glycosidase, a protease, a nuclease, a lipase, an
esterase, a plasminogen activator, a streptokinase, and
combinations thereof. Preferably a glycosidase, such as, for
example, hyaluronidase, is administered. Compositions used in
methods for treating prostate cancer can also include or be
administered with calcium ions, a nonionic surfactant, such as, for
example, Triton.RTM. X-100, and an antibiotic, such as, for
example, gentamicin. Another method of treating prostate cancer in
a living mammal includes activating PSA in vivo by, for example,
locally administering calcium ions.
Inventors: |
GOKCEN, MUHARREM;
(MINNEAPOLIS, MN) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Family ID: |
23698630 |
Appl. No.: |
09/428375 |
Filed: |
October 28, 1999 |
Current U.S.
Class: |
424/94.2 ;
424/94.6; 435/4; 436/64 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 38/4886 20130101; A61P 13/08 20180101; A61K 38/4886 20130101;
A61K 38/4886 20130101; A61K 33/06 20130101; A61K 2300/00 20130101;
A61K 38/47 20130101; A61K 31/70 20130101; A61K 38/47 20130101; A61K
31/70 20130101; A61K 33/06 20130101; A61K 38/4886 20130101; A61P
35/00 20180101; A61K 38/164 20130101 |
Class at
Publication: |
424/94.2 ;
424/94.6; 435/4; 436/64 |
International
Class: |
A61K 038/46; C12Q
001/00; G01N 033/48; A61K 038/54 |
Claims
We claim:
1. A method of treating prostate cancer in a mammal comprising
local administration of a composition having a therapeutically
effective concentration of collagenase.
2. The method of claim 1, further comprising administering a
glycosidase, a protease, a nuclease, a lipase, an esterase, a
streptokinase, or a combination thereof.
3. The method of claim 2, wherein the glycosidase comprises
hyaluronidase, neuraminidase, lysozyme, amylase, or a combination
thereof.
4. The method of claim 2, wherein the protease comprises elastase,
trypsin, chymotrypsin, pronase, dispase, bromelin, clostripain,
thermolysin, subtilisin, papain, chymopapain, fibrinolysin,
serrathiopeptidase, pancreatin, cathepsin-G, plasminogen activator,
PMN leukocyte serine protease, or a combination thereof.
5. The method of claim 2, wherein the nuclease comprises DNase I,
Rnase, or a combination thereof.
6. The method of claim 2, wherein the lipase comprises
phospholipase.
7. The method of claim 2, wherein the esterase comprises
cholesterol esterase.
8. The method of claim 2, comprising administering hyaluronidase
trypsin, chymotrypsin, pronase, elastase, DNase I, dispase,
fibrinolysin, or a combination thereof.
9. The method of claim 8, comprising administering
hyaluronidase.
10. The method of claim 9, wherein the composition comprises
collagenase at a concentration of about 250 to 250,000 U/ml and
hyaluronidase at a concentration of about 160 to 160,000 U/ml.
11. The method of claim 10, wherein composition comprises
hyaluronidase at a concentration of about 1,600 to 16,000 U/ml.
12. The method of claim 1, wherein the composition further
comprises an effective concentration of a nonionic surfactant.
13. The method of claim 12, wherein the nonionic surfactant
comprises an ethylene oxide ester of a C10-C20 fatty acid, an
ethylene oxide ester of a C8-C22 alkyl alcohol, or a combination
thereof.
14. The method of claim 12, wherein the nonionic surfactant
comprises Triton.RTM. X-100.
15. The method of claim 1, wherein the composition further
comprises an effective concentration of an antibiotic.
16. The method of claim 15, wherein the antibiotic comprises
gentamicin.
17. The method of claim 1, wherein the composition further
comprises an effective concentration of calcium ion.
18. The method of claim 17, wherein the composition comprises
CaCl.sub.2.
19. The method of claim 1, wherein the composition further
comprises a hyaluronidase, a Triton.RTM. X-100, a gentamicin, and a
calcium ion.
20. The method of claim 1, wherein local administration comprises
intraprostatic injection.
21. The method of claim 20, wherein the intraprostatic injection
comprises intralesional injection.
22. The method of claim 20, wherein the intraprostatic injection
comprises transurethral injection.
23. A method of treating prostate cancer in a living mammal
comprising local administration of a composition comprising a
therapeutically effective concentration of collagenase in
combination with a glycosidase, a protease, a nuclease, a lipase,
an esterase, a streptokinase, or combination thereof.
24. A method of treating prostate cancer in a living mammal
comprising activating PSA in vivo.
25. The method of claim 24, wherein activating PSA in vivo
comprises administering calcium ion.
26. The method of claim 25, further comprising locally
administering a therapeutically effective concentration of
collagenase.
27. The method of claim 26, further comprising administering a
nonionic surfactant an antibiotic, or a combination thereof.
28. The method of claim 26, further comprising administering a
glycosidase, a protease, a nuclease, a lipase, an esterase, a
streptokinase, or a combination thereof.
29. The method of claim 26, wherein the glycosidase comprises
hyaluronidase, neuraminidase, lysozyme, amylase, or a combination
thereof.
30. The method of claim 28, wherein the protease comprises
elastase, trypsin, chymotrypsin, pronase, dispase, bromelin,
clostripain, thermolysin, subtilisin, papain, chymopapain,
fibrinolysin, serrathiopeptidase, pancreatin, cathepsin-G,
plasminogen activator, PMN leukocyte serine protease, or a
combination thereof.
31. The method of claim 24, wherein local administration comprises
direct intraprostatic injection.
32. The method of claim 24, further comprising eliciting a host
immune response.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to a method for treating
prostate cancer by administering collagenase alone or in
combination with a glycosidase, a protease, a nuclease, a lipase,
an esterase, a streptokinase, or a combination thereof. A typical
embodiment includes administering a composition including a mixture
of collagenase, hyaluronidase, a nonionic surfactant, an
antibiotic, and calcium ions.
BACKGROUND OF THE INVENTION
[0002] Approximately ten million American men are believed to have
prostate cancer today. Although fewer than 3% of men with the
disease die from it, prostate cancer still is the second most
common cause of cancer death among men. The cancer usually is
localized in the prostate, but in some cases, the cancer is not
diagnosed until it has metastasized to the bone, kidneys, or the
brain.
[0003] Yearly screening for the disease increases the likelihood of
early detection, especially prior to the disease metastasizing.
Such screening usually involves a digital rectal exam and a
prostate-specific-antigen (PSA) blood test. Other screening methods
include ultrasound imaging, radionucleid scan, and biopsy.
[0004] The PSA blood test has revolutionized the early diagnosis of
prostate cancer and the effectiveness of treatment. PSA is a
proteolytic enzyme in the family of serine proteases and one of the
most abundant proteins in the prostate secretions. PSA is
synthesized in the ductal epithelium and prostatic acini and is
located within the cell in cytoplasmic granules and vesicles, rough
endoplasmic reticulum, vacuoles, secretory granules, and lyosomal
dense bodies. PSA is secreted into the lumina of the prostatic
ducts where it becomes a component of seminal plasma. To reach
blood serum, PSA diffuses from luminal cells through the epithelial
basement membrane and prostatic stroma and either passes through
the capillary basement membrane and epithelial cells or into the
lymphatics. Once in the bloodstream, the majority of PSA forms
complexes with .alpha.-1-antichymotrypsin (PSA-ACT) and
.alpha.-1-macroglobulin, while small quantities remain free (free
PSA). Free PSA levels are usually elevated in instances of prostate
cancer.
[0005] Once prostate cancer is diagnosed, a suitable method of
treatment must be determined and then administered. Current methods
of treatment include radical prostatectomy, radiation, and hormonal
suppression. To determine the appropriate method of treatment,
factors such as the age of the patient and severity of the disease
are often considered. The disease generally is more aggressive for
younger patients. Any tumor greater than 0.5 cc is typically
considered clinically significant. The preferred treatment for
localized prostate cancer is radical prostatectomy. This treatment
may result in death, incontinence, impotence, rectal injury, or
pulmonary emboli.
[0006] Thus, it is desirable to provide improved methods of
treatment for prostate cancer that reduce the likelihood of one or
more of these unpleasant side effects. In particular, it is
desirable to provide improved methods of treatment that reduce the
likelihood of the treatment rendering the patient impotent.
SUMMARY OF THE INVENTION
[0007] The current invention is directed to a method of treating
prostate cancer in a living mammal. In one aspect, the treatment is
directed to activating PSA in vivo. PSA in or near prostate can be
activated by one of several methods, such as by local
administration of calcium ion preferably in combination with
collagenase and, optionally, another hydrolase such as
hyaluronidase.
[0008] PSA is a proteolytic enzyme, which is one of the most
abundant proteins in the prostate secretion. Activated PSA may
degrade prostate cancerous tumors by, for example, interfering with
the initial growth stage of a tumor and/or interfering with the
angiogenesis stage. The initial stage of prostate cancer growth
involves the tumor growing to about the size of a pea. The tumor
cannot get any larger unless it can form its own blood vessels
around and inside the tumor, a process known as angiogenesis.
Angiogenesis involves sending chemical signals to surrounding blood
vessels that erode the vessel walls, sending capillaries toward the
tumor. By interfering with angiogenesis, the tumor cannot grow
beyond its initial size. In one aspect, PSA may be activated by
administering calcium ions.
[0009] In another aspect, a method of treating prostate cancer is
directed to administering a composition that includes a
therapeutically effective concentration of a collagenase.
Collagenase may degrade prostate cancerous tumors by, for example,
supporting connective tissue around and inside of the cancerous
tumor including angiogenesis-generated capillaries.
[0010] In another aspect, a method of the invention is directed to
local administration of a therapeutically effective concentration
of collagenase in combination with a glycosidase, a protease, a
nuclease, a lipase, an esterase, a streptokinase, or a combination
thereof. Preferably a glycosidase, such as, for example,
hyaluronidase, is administered with collagenase.
[0011] Compositions used in methods of the invention may also
include or be administered in combination with calcium ions, a
nonionic surfactant, such as, for example, Triton.RTM. X-100, and
an antibiotic, such as, for example, gentamicin.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention provides a method for treating
prostate cancer. In accordance with the present invention, it has
been found that compositions containing at least one hydrolytic
enzyme can be used to treat prostate cancer. "Treatment" and
"treating" as used herein include preventing, inhibiting, curing,
and alleviating prostate cancer or symptoms thereof and preventing
or alleviating the metastasis of prostate cancer.
[0013] Alleviating prostate cancer includes degrading a prostate
tumor, for example, breaking down the structural integrity or
connective tissue of a prostate tumor, such that the tumor size is
reduced when compared to the tumor size before treatment. Curing
prostate cancer includes degrading a prostate tumor such that a
tumor cannot be detected after treatment. The tumor may be reduced
in size or become undetectable, for example, by atrophying from
lack of blood supply or by being attacked or degraded by one or
more components administered according to a method of the
invention.
[0014] Alleviating metastasis of prostate cancer includes reducing
the rate at which the prostate cancer spreads to other organs.
Preventing metastasis of prostate cancer includes preventing the
prostate cancer from spreading outside of the prostate.
[0015] A treatment according to the invention may be carried out by
administering a therapeutically effective concentration of a
composition including collagenase in an amount effective for
alleviating, curing, inhibiting, or preventing prostate cancer or
preventing or alleviating the metastasis of prostate cancer.
[0016] The composition including collagenase is locally
administered to the prostate. Local administration includes
delivering the composition into or near the prostate and/or
cancerous tumor. Local administration further includes surrounding
the prostate and/or cancerous tumor with the composition or
applying the composition to the surface of the prostate and/or
cancerous tumor. Cancerous tumor includes prostate cancer,
cancerous cells, and the like.
[0017] Another treatment according to the invention may be carried
out by activating PSA in vivo. Activating PSA in vivo includes
activating PSA, typically the mammal's endogenous PSA, in and/or
near the prostate. PSA is a proteolytic enzyme that, upon being
activated, can facilitate dissolution or degradation of a prostate
tumor. In one embodiment, PSA can be activated by locally
administering calcium ions to the prostate.
[0018] Although the methods of the invention are not meant to be
limited to a single theory, one theory directed to how the methods
of the invention treat prostate cancer will be described. In
initial stages, a cancerous tumor grows to about the size of a pea.
Through angiogenesis the tumor then grows larger (i.e., the tumor
grows blood vessels around and inside itself by sending chemical
signals to surrounding blood vessels to send capillaries toward the
tumor). It is believed that the treatments of the invention
interfere with this development.
[0019] PSA is a serine protease having a molecular weight of about
33 kilodaltons and is one of the most abundant proteins in the
prostate secretion. It is believed that by activating PSA, PSA can
partially or completely dissolve or degrade tumor cells in the
prostate. This process can be particularly useful at the initial
growth stage of a tumor as well as at the angiogenesis stage. PSA
can be activated by, for example, administering calcium ions.
[0020] Collagenase is a metalloprotease. According to one theory,
the activity of collagenase may be increased in the presence of a
small amount of proteolytic enzyme--i.e., the amount of proteolytic
enzyme present should not be so high that the proteolytic enzyme
digests the collagenase to such an extent that the collagenase
becomes inactive. PSA secreted into the prostate may be sufficient
to increase the activity of collagenase. By increasing the activity
of collagenase, the activity of collagenase is raised from a level
that may be ineffective for cleaving sufficient substrate (e.g.,
degrading connective tissue) to a level that may be effective for
cleaving sufficient substrate. Then collagenase, in the presence of
PSA, for example, can degrade collagen or connective tissue, tumor
blood vessels, and/or basement membranes of the tumor cells. As a
result, the tumor blood supply is reduced or eliminated, causing
the tumor cells to die. This may also result in a person's immune
system further attacking the tumor and aiding the alleviation or
cure of prostate cancer or the alleviation or prevention of
metastasis of prostate cancer.
[0021] In methods of the invention, collagenase can also be
administered in combination with other proteases, enzymes, or
proteins suitable for increasing the activity of collagenase or for
aiding in the degradation of a tumor. For example, collagenase can
be administered in combination with a protease, a glycosidase, a
nuclease, a lipase, an esterase, a streptokinase, or a combination
thereof.
[0022] Glycosidases include any enzyme that catalyzes the
hydrolysis of glycosidic linkages. Suitable examples include
hyaluronidase, neuraminidase, amylase, and lysozyme. Preferably the
glycosidase includes hyaluronidase.
[0023] Proteases include any enzyme that catalyzes the hydrolysis
of one or more peptide bonds in a protein or peptide, such as, for
example, carboxypeptidases, aminopeptidases, and endopeptidases.
More specific suitable examples include elastase, trypsin,
chymotrypsin, pronase, dispase, bromelin, clostripain, thermolysin,
subtilisin, papain, chymopapain, fibrinolysin, serrathiopeptidase,
pancreatin, cathepsin-G, plasminogen activator, and PMN leukocyte
serine protease.
[0024] Nucleases include any enzyme that catalyzes the hydrolysis
of ester linkages in nucleic acids, such as, for example,
ribonuclease and deoxyribonuclease. Suitable examples include DNase
I and RNase.
[0025] Esterases includes any enzyme that catalyzes the hydrolysis
of an ester. A suitable example includes cholesterol esterase.
[0026] Lipases include any enzyme that catalyzes the hydrolysis of
acylglycerol carboxylic esters. A suitable example includes
phospholipase.
[0027] Streptokinases include proteins that form a complex with
plasminogen that then catalyzes the activation of plasminogen to
plasmin.
[0028] The compositions can also include or be administered in
combination with calcium ions, a surfactant, and/or an
antibiotic.
[0029] The components of the composition--collagenase, additional
protease, protein, or enzyme, calcium ions, surfactant, and
antibiotic--can be administered alone, sequentially, or preferably,
combined with one another in the form of a liquid pharmaceutical
unit dosage form suitable for local administration. The dose of the
composition administered can vary over a wide range as can readily
be determined by the clinician. The preferred dosage for obtaining
the desired therapeutic objective can vary depending on the age of
the patient, nature and severity of disease, potency of the
composition, and route of administration.
[0030] Preferably, the compositions suitable for use in methods of
the invention include collagenase and at least one of a
glycosidase, preferably hyaluronidase; a protease, preferably
trypsin, chymotrypsin, pronase, elastase, dispase, or fibrinolysin;
or a nuclease, preferably DNase I. More preferably, the
compositions include collagenase and a glycosidase, preferably
hyaluronidase. Compositions suitable for use in methods of the
invention have been described in U.S. Pat. No. 5,116,615 issued to
Gokcen et al., incorporated herein by reference.
[0031] Collagenase
[0032] Bacterial collagenase, for example, Clostridium
hystolyticum, EC 3.4.24.3, is a well-characterized,
commercially-available enzyme that degrades collagen into small
peptides by hydrolysis at several sites along the triple helix.
Collagenase contains Zn.sup.+2 in its active site and requires
Ca.sup.+2 for binding to its substrate and for achieving the
conformation necessary for full catalytic activity.
[0033] Intravenous injections of collagenase have shown a very low
degree of danger to experimental animals. In mice the IV LD-50 of
crude collagenase has been shown to be 300 mg/kg body weight. Oral
solutions of collagenase in water have been proven to be nontoxic
at doses as high as 8,000 mg/kg body weight. The acute IV LD-50 in
rats has been shown to be 1272 U/kg for collagenase.
[0034] Commercial preparations of bacterial collagenase often
include small amounts of contaminating proteases, peptidases,
mucopolysaccharidases, and glycosidases including: clostripain,
trypsin, and a caseinase-like aminopeptidase. Clostripain is the
most abundant contaminating enzyme in crude collagenase
preparations. Clostripain contains an essential SH group, is
activated by cysteine, and is inhibited by sulfhydryl binding
agents. Clostripain also has trypsin-like specificity. Crude
collagenase preparations with small amounts of contaminating
enzymes such as trypsin and clostripain are often more effective
than highly purified preparations of collagenase, suggesting that a
possible combined action of the multiple enzymes or proteases aids
in the treatment of prostate cancer. These enzymes and/or proteases
can help activate collagen and/or can help degrade the tumor by
catalyzing the hydrolysis of bonds important to the growth or
structural integrity of the tumor.
[0035] Crude collagenase preparations are especially useful for
treatment when combined with other proteins and/or enzymes, such
as, for example, a glycosidase, a protease, a nuclease, a lipase,
an esterase, a streptokinase, or a combination thereof.
[0036] Glycosidases
[0037] Examples of glycosidases suitable for use in methods of the
invention include hyaluronidase, neuraminidase, lysozyme, and
amylase. Preferably the glycosidase is hyaluronidase.
[0038] Hyaluronidase (hyaluronate-4-glycanhydrolase) is a known
enzyme that catalyzes the degradation of hyaluronic acid (an acidic
mucopolysaccharide) into disaccharides, tetrasaccharides, or a
mixture of both. Hyaluronidase derived from ovine testes (EC
2.1.1.35) is preferred.
[0039] In animals, the intravenous injections of 75,000
International Units of hyaluronidase results in no significant
change in blood pressure, respiration, body temperature, or renal
function. Hyaluronidase is typically not injected into areas of
known infection.
[0040] Amylase includes enzymes, such as, for example,
.alpha.-amylase or .beta.-amylase, able to hydrolyze O-glucosyl
linkages in starch, glycogen, and related polysaccharides.
[0041] Neuraminidase degrades 2,3-, 2,6-, and 2,8-glucosidic
linkages joining terminal nonreducing N- and O-acylneuraminyl
residues to galactose, N-acetylhexosamine, or N- or
O-acylneuraminyl residues to galactose, N-acetylhexosamine, or N-
or O-acylated neuraminyl residues in oligosaccharides,
glycoproteins, or glycolipids.
[0042] Lysozyme hydrolyzes .beta.-1,4-linkages between
N-acetylmuramic acid and 2-acetamido-2-deoxy-D-glucose residues in
peptidoglycan heteropolymers. Lysozyme is identified by EC
3.21.17.
[0043] Proteases
[0044] Examples of proteases suitable for use in methods of the
invention include elastase, trypsin, chymotrypsin, pronase,
dispase, bromelin, clostripain, thermolysin, subtilisin, papain,
chymopapain, fibrinolysin, serrathiopeptidase, pancreatin,
cathepsin-G, plasminogen activator, and PMN leukocyte serine
protease.
[0045] Elastase includes any protease that hydrolyzes elastin. One
example is pancreatic elastase identified by EC 3.4.21.36.
Pancreatic elastase catalyzes the hydrolysis of proteins, including
elastin, with preferential cleavage at Ala-Xaa.
[0046] Trypsin is a serine endoprotease that preferentially cleaves
at Arg-Xaa and Lys-Xaa. Trypsin is identified by EC 3.4.21.4.
[0047] Chymotrypsin is a serine endopeptidase identified by EC
3.4.21.1.
[0048] Pronase (a registered trademark of Calbiochem/Behring, La
Jolla, Calif.) is a mixture of various exo-and endoproteases,
obtained from Streptomyces. It is able to hydrolyze virtually any
protein almost completely to free amino acids.
[0049] Dispase is a neutral metalloprotease typically obtained from
Bacillus polymyxa.
[0050] Bromelin is a cysteine protease that has broad specificity
and is identified by EC 3.4.22.32.
[0051] Clostripain is a cysteine protease that preferentially
cleaves Arg-Xaa and Arg-Pro bonds and is identified by EC
3.4.22.8.
[0052] Thermolysin is a metalloendoprotease that preferentially
cleaves Xaa-Leu and Xaa-Phe and is characterized as EC
3.4.24.27.
[0053] Subtilisin is a serine endoprotease that catalyzes the
hydrolysis of proteins with broad specificity for peptide bonds and
is identified by EC 3.4.21.62.
[0054] Papain is a cysteine endopeptidase that preferentially
hydrolyzes peptide bonds at the carbonyl end of Arg, Lys, Phe
residues and is identified by EC 3.4.22.2. Papain also has
esterase, thioesterase, transamidase, and transesterase
activity.
[0055] Chymopapain is an endoprotease with specificity similar to
that of papain. Cymopapain is identified by EC 3.4.22.6.
[0056] Fibrinolysin is also known as plasmin, which is a serine
protease that converts insoluble fibrin of a blood clot into
soluble products. It is formed from plasminogen by proteolysis and
preferentially cleaves Lys-Xaa and Arg-Xaa. Fibrinolysin is
identified by EC 3.4.21.7.
[0057] Cathepsin G is a glycoprotein serine endoprotease with a
specificity similar to that of chymotrypsin. This protease is
identified by EC 3.4.21.20.
[0058] Plasminogen activators include any serine protease that
converts plasminogen into plasmin.
[0059] PMN leukocyte serine protease includes a serine protease
found in PMN leukocytes.
[0060] Other proteases include but are not limited to pancreatin
and serrathiopeptidase.
[0061] Nucleases, Esterases, and Lipases
[0062] Examples of nucleases suitable for use in methods of the
invention include DNase I and RNase. DNase I (Deoxyribonuclease I)
is an enzyme that catalyzes the endonucleolytic cleavage of DNA
into 5'-phosphodinucleotide and 5'-phosphooligonuleotide end
products. DNase I is identified by EC 3.1.21.1. RNase is any of a
group of nuclease enzymes that cleave phosphodiester bonds and
chains of RNA.
[0063] Examples of esterases suitable for use in methods of the
invention include cholesterol esterase.
[0064] Examples of lipases suitable for use in methods of the
invention include phospholipase. Phospholipase is an enzyme that
catalyzes the hydrolysis of a glycerophospholipid. Phospholipases
have been subdivided into types A.sub.1, A.sub.2, B, C, and D.
[0065] Calcium Ions
[0066] Calcium ions can also be administered to treat prostate
cancer. Calcium ions can serve to activate both collagenase and/or
PSA. The calcium ions can be provided by, for example, calcium
chloride. The concentration of calcium- ions is typically between
about 1 mM and 50 mM, preferably between about 10 mM and 50 mM, and
more preferably about 20 mM.
[0067] According to one treatment, activated PSA can degrade or
dissolve a prostate tumor and interfere with initial tumor growth
and/or angiogenesis. According to another treatment, collagenase
can be administered in combination with PSA being activated, and
the activated PSA can increase the proteolytic activity of
collagenase. Collagenase can, in turn, degrade a prostate tumor.
According to another treatment, collagenase can be administered in
combination with calcium ions and/or a protease, an enzyme, a
protein, a nonionic surfactant, or an antibiotic to treat prostate
cancer.
[0068] Nonionic Surfactants
[0069] Compositions suitable for use in methods of the present
invention also can include or be administered in combination with a
nonionic surfactant. A surfactant aids in the solubilization and
lysis of prostatic cancer tissue. Examples of suitable surfactants
include alkylphenylpolyoxethylene surfactants such as Triton.RTM.
X-100 (octylphenoxypolyethoxyethanol, which is an
octylphenylpolyoxyethylene oxide available from Rohm and Haas,
Philadelphia, Pa.) and other polyoxyalkylene-based nonionic
surfactants such as Tween.RTM. 20/80 (Atlas Chemical), Genapol
X-080/100/150, C-100 (Hoechst AG), Thesit (Destin-Werk GMBH), Brij
35, Lubrol PX (ICI Americas), Pluronic F-127 (Wyandotte Chemicals
Corp.), Nonidet P-20/40 (Shell Oil Corp.), Igepal CO-630/710 (GAF),
Surfonic N-95 (Jefferson), Tergitol NP-27 (Union Carbide). Other
suitable surfactants include the condensation products of ethylene
oxide with partial fatty acid esters of sorbitol and sorbitol
anhydride, such as, for example, the Tween.RTM. series (Atlas
Chemical) wherein the molar ratio of ethylene oxide to alcohol is
within the range of about 15:1 to 25:1 with the fatty acid
component comprised of laurate, stearate, or oleate
(C.sub.10-C.sub.20).
[0070] Other nonionic surfactants that-can be employed-in the
present composition include the ethylene oxide esters of
C.sub.6-C.sub.12 alkyl phenols such as nonylphenoxy polyoxyethylene
ether. Particularly useful are the esters prepared by condensing 8
to 12 moles of ethylene oxide with nonylphenol. Commercially
available detergents of this type include the Igepal CO series (GAF
Corp.).
[0071] Additional useful nonionic surfactants can include the
condensation products of ethylene oxide with a hydrophobic
polyoxyalkylene base such as propylene oxide condensed with
propylene glycol. Compounds of this type include the commercially
available surfactants Pluronic F-127, Pluronic PX, and Pluronic
L-62 (Wyandotte Corp.).
[0072] Further useful nonionic surfactants include the condensation
products of C.sub.8-C.sub.22 alkyl alcohols containing 2 to 50
moles of ethylene oxide per mole of alcohol. Detergents of this
type include the condensation products of C.sub.10-C.sub.20 fatty
alkyl alcohols containing 3 to 45 moles of ethylene oxide per mole
of alcohol. These compounds are commercially available as the
Poly-Tergent SLF series (Olin Chemicals) or the Tergitol series
(Union Carbide).
[0073] The performance of suitable surfactants can be effected by,
for example, pH, temperature, ionic strength, and surfactant
concentration.
[0074] Surfactants are preferably added to the present composition
at a concentration of about 0.1% to 10% by volume of the
composition. More preferably, the surfactants are present at a
concentration of about 0.5% to 5% by volume.
[0075] Antibiotics
[0076] Compositions suitable for use in methods of the invention
also can include or be administered in combination with an
antibiotic. Local administration to the prostate, such as by, for
example, direct injection of the prostate, is generally a safe,
simple, and effective means of introducing the composition into the
body. Yet some local administration techniques can be associated
with the risk of acquiring bacterial infection that can lead to,
for example, fever, bacteriuria, and bacteremia.
[0077] Antibiotics usually relieve the symptoms of acute prostatic
infections promptly. However, no antimicrobial agent is effective
against all pathogenic urinary tract microorganisms. Each has its
own spectrum of activity against one or a variety of species. The
therapeutic agents for curing bacterial prostatitis preferably are
highly lipid soluble; possess basic pKa; show minimal binding to
plasma proteins; and are bactericidal against the common gram
negative uropathogens.
[0078] It is preferred that the present enzyme composition include
a suitable antimicrobial agent to prevent or reduce the incidence
of bacterial infection that can be associated with the present
injection method. Antibiotics used typically provide adequate
protection against the commonly encountered bacterial strains of
uropathogens including: Escherichia coli, Streptococcus faecalis,
Proteus/Pseudomonas spp., and coagulase-positive Staphylococcus.
The antibiotic is preferably selected to not substantially inhibit
the enzymatic activity of the composition.
[0079] The antibiotics of the present claimed composition,
preferably gentamicin or trimethoprim/sulfamethoxazole, can be
selected from the groups of antibiotics that exhibit the
appropriate spectrum of activity against the commonly encountered
bacterial strains of uropathogens including: penicillins
(penicillin G, penicillin V, benzathine penicillin); amino
penicillins (ampicillin, amoxicillin); carboxy penicillins
(carbenicillin, piperacillin, mezlocillin); penicillinase resistant
penicillins (methicillin, oxacillin, nafcillin); cephalosporins
(cephalexin, cephalothin, cefotaxime, cephazolin); aminoglycosides
(streptomycin, neomycin, kanamycin, tobramycin, amikacin,
netilmicin, sisomicin); tetracyclines (doxycycline, minocycline,
tetracycline), polymyxins (polymyxin B & E); sulfonamides
(sulfisoxazole, sulfasuxidine); fluoroquinolones(ciprofloxacin,
norfloxalin); basic macrolides (erythiomycin, oleandomycin);
lincomycin; clindamycin; chloramphenicol; nitrofurantoin; and
nalidixic acid.
[0080] Examples of antibiotics suitable for use in methods of the
invention include gentamicin sulfate (Garamicin.RTM., Schering
Corp., Kenilworth, N.J.), trimethoprim/sulfamethoxazole
(Septra.RTM., Burroughs Wellcome, Research Triangle Park, N.C.),
nitrofurantoin, nalidixic acid, tobramycin, amikacin, and
netilmicin sulfate.
[0081] Preferably gentamicin sulfate or
trimethoprim/sulfamethoxazole are administered. The administration
of trimethoprim/sulfamethoxazole currently is the drug of first
choice in the treatment of bacterial prostatitis. Recent studies
have indicated that a single dose of gentamicin, trimethoprim/
sulfamethoxazole, or netilmicin sulfate is as effective as longer
treatment in the prevention of postoperative bacterial urinary
tract infections.
[0082] Trimethoprim is a lipid-soluble base with limited binding to
plasma proteins, and typically shows prostatic tissue: serum levels
of 2:1 to 3:1. Trimethoprim/sulfamethoxazole (TMP/SMX) produces
therapeutic levels in the urine and prostatic secretions with an
appropriate antibacterial spectrum of activity. Recommended therapy
with TMP/SMX involves dosages-of 160 mg TMP and 800 mg SMX,
orallytwice a day for 30 days. Should TMP/SMX not be tolerated
(allergies), gentamicin therapy is recommended.
[0083] The antibiotic generally is present in the composition at a
concentration of about 0.15 to 150 .mu.g/ml. The preferred
antibiotic, gentamicin sulfate, is present in the composition at a
concentration of 1.5 to 150 .mu.g/ml, preferably 10 to 25 .mu.g/ml.
Alternatively, for those patients who may be allergic to
aminoglycosides in general, and gentamicin in particular, the
combination drug trimethoprim/sulfamethoxaz- ole can replace
gentamicin as the preferred antibiotic of the present claimed
composition. Trimethoprim is preferably present at a concentration
of 1 to 10 .mu.g/ml, more preferably 5 to 10 .mu.g/ml.
Sulfamethoxazole is preferably present at a concentration of 30 to
105 .mu.g/ml, more preferably 50 to 105 .mu.g/ml.
[0084] Preparation and Testing of an Illustrated Composition
[0085] The present invention discloses as a preferred embodiment an
aqueous composition containing a safe and therapeutically effective
concentration of the hydrolytic enzymes collagenase and
hyaluronidase, the detergent Triton.RTM. X-100, and the antibiotic
gentamicin for local administration. It is preferred that the
disclosed composition be prepared as a relatively concentrated
solution of hydrolytic enzymes in a relatively small volume.
[0086] It is preferable that the claimed composition be provided in
a unit dosage form suitable for intraprostatic injection. The
composition can be administered to the patient as an injectable
dosage of a solution or suspension of the compounds in a
physiologically acceptable liquid diluent, such as pyrogen-free
saline. For example, vials containing a lyophilisate of the
composition can be prepared such that a sterile aliquot of the
composition can be reconstituted and withdrawn as a
pharmaceutically acceptable aqueous solution for injection into
living mammals. A preferred unit dose contains 250 to 250,000 U/ml
collagenase; 160 to 160,000 U/ml hyaluronidase; 0.1% to 10%
nonionic surfactant; and 0.15 to 150 .mu.g/ml antibiotic. More
preferably a unit dose contains 2,500 to 25,000 U/ml of
collagenase; 1,600 to 16,000 U/ml of hyaluronidase; 0.5 to 5%
nonionic surfactant; and 15 to 150 .mu.g/ml antibiotic.
[0087] Compositions of the present invention were prepared.
Collagenase (Sigma Chemical Co., St. Louis, Mo.) and hyaluronidase
(Boehringer/Mannheim Corp., Indianapolis, Ind.) were obtained as
lyophilisates and reconstituted with citrate-buffered saline
containing 20 mM to 50 mM CaCl.sub.2 (CBSCa) to the desired
concentration. Collagenase, obtained from Clostridium hystolyticum,
was chromatographically purified and contained small contaminating
amounts of the enzymes clostripain, trypsin, and caseinase.
Hyaluronidase, obtained from ovine testes, was also purified
chromatographically. All enzyme activities were expressed as
international units per mg.
[0088] The enzymes are stable when stored as lyophilisates at
4.degree. C. However, it is advantageous to prevent access of
moisture to lyophilized enzymes. For example, cold vials of
lyophilized enzyme are typically first be warmed to room
temperature before being opened. Dilute reconstituted solutions of
enzymes are typically stored at 4.degree. C., protected from light,
and placed in an ice-bath when working at the bench.
[0089] Freshly distilled, deionized, sterile water is preferred for
the reconstitution of enzymes and preparation of buffers used for
injectable solutions. In the preferred embodiment, the buffer
solution used is 0.05 M citrate-buffered saline (CBS, pH 6.7),
containing an adequate amount (preferably 0.01 M to 0.05 M, more
preferably 0.02 M to 0.05 M) of calcium ions to activate the
collagenase. It is recognized that any suitable buffer solution
such as Ringers saline or tris-buffered saline can be used.
However, the buffer typically contains sufficient calcium ions to
activate collagenase, and typically does not contain calcium
chelators such as EDTA or other inhibitors of enzyme activity such
as cysteine.
[0090] A preferred buffer exhibits a physiological pH, which can
range from about 6.5 to 7.5, with pH 6.7 to 7.0 preferred. The
saline, e.g., sodium chloride, concentration is preferably about
0.1 M to 0.2 M with about 0.15 M to 0.2 M most preferred.
Similarly, the concentration of citrate is preferably about 0.02 M
to 0.1 M with 0.05 M to 0.1 M most preferred.
[0091] A preparation of 0.05 M CBS+20 mM CaCl.sub.2 (pH 6.7) is
composed of 550 mg sodium citrate, 190 mg NaOH, and 876 mg NaCl
dissolved in 100 ml sterile, pyrogen-free, deionized H.sub.2O. The
solution was adjusted to a pH of 6.7 with 3 ml of 1 N NaOH, and 294
mg CaCl.sub.2 was added. The surfactant Triton.RTM. X-100
(Malinckrodt, Paris, Ky.) and the antibiotic gentamicin (Sigma
Chemical Co.) were added to obtain appropriate final
concentrations. Tritong X-100 exhibits a density of 1.082 g/ml at
20.degree. C. (924 Tl/g at 20.degree. C.). Gentamicin is included
so as to obtain a final concentration of 150 .mu.g/ml by adding 1.5
ml of a 10 mg/ml (15 mg) sterile solution of the antibiotic to 100
ml of the mixture.
[0092] The resulting solution can be purified and sterilized by
standard techniques. A solution (5 ml) of the enzymes collagenase
and hyaluronidase (0.1% to 10%) in citrate buffered saline (pH 6.7)
containing Triton.RTM. X-100 (0.1% to 10%), the antibiotic
gentamicin (1.5 to 150 .mu.g/ml), and CaCl.sub.2 (20 mM) is
prepared in pyrogen-free water, and passed over a 1 ml column
(Detoxi-Gel.RTM.) to remove potential endotoxins. The final step in
the preparation of a pharmaceutically acceptable solution involves
the passage of the composition through a certified sterile,
nonpyrogenic microporous polysulfone filter with a pore size of 0.2
micrometer. Low protein binding filter membranes composed of
polysulfones show significantly less protein absorption than
comparable cellulose acetate/nitrate membrane filters. Filtration
through a 0.2 mm sterile filter affords protection against
contamination with microorganisms. Additionally, filtration
minimizes the risks to patients posed by insoluble particulates or
microaggregates.
[0093] Reconstitution and preparation of pharmaceutically
acceptable solutions for parenteral use in humans is performed
routinely in hospital pharmacies as standard practice. Solutions of
collagenase/hyaluronidase/T- riton.RTM. X-100/gentamicin (CHTG) in
0.05 M citrate-buffered saline containing 20 mM CaCl.sub.2 (CBSCa
6.7) ranging in concentration from 0.1% to 10% are stable for 2
weeks when stored at 4.degree. C. and remain highly effective in
solubilizing human and canine prostatic tissue. The toxicity of the
composition is not believed greater than the toxicities of the
individual components, which are known.
[0094] Collagenase (EC 3.4.24.3) derived from Clostridium
hystolyticum was purchased from Sigma Chemical Company (Type XI,
Product # C-7657, Lot Numbers: 96F-6801 and 96F-6838; Type XI-S,
Product # C-4785, Lot Number: 17F-6814). Lot to lot variations of
enzyme activity in terms of collagenase U/mg and levels of
contaminating enzymes were observed and ranged from: 1910 to 2450
U/mg collagenase, 0.86 to 1.4 U/mg clostripain, 40 to 85 U/mg
caseinase, and 0.05 to 0.52 U/mg trypsin.
[0095] One unit of collagenase activity is defined as the amount of
collagenase that will release peptides from native collagen,
equivalent in ninhydrin color, to 1 micromole of L-leucine in five
hours at pH 7.4 at 37.degree. C. in the presence of calcium ions.
One unit of clostripain will hydrolyze 1 micromole of
N-alpha-benzoyl-L-arginine ethyl ester (BAEE) per minute at pH 7.6
at 25.degree. C. in the presence of 2.5 mM dithiothreitol. One unit
of caseinase (nonspecific protease) will hydrolyze casein to
produce color equivalent to 1 micromole (181 Tg) of L-tyrosine in
five hours at pH 7.5 and 37.degree. C. (color by Folin-Ciocalteu
reagent). One unit of trypsin activity will hydrolyze 1 micromole
of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) per minute at pH
7.6 and 37.degree. C.
[0096] The activities of collagenase and any additional enzymes can
be determined according to methods known in the art. Examples of
collagenase and hyaluronidase assays are disclosed in U.S. Pat. No.
5,116,615 issued to Gokcen et al., incorporated herein by
reference.
[0097] Compositions are essentially free of endotoxins, which are
pyrogenic lipopolysaccharide components of gram-negative bacteria
that are known to have potent adverse effects in humans and
animals.
[0098] Methods of Delivery
[0099] One method of the invention is directed to activating PSA in
vivo. In one embodiment, PSA can be activated in vivo by, for
example, locally administering a therapeutically effective
concentration of calcium ions. PSA can be activated in vivo in
combination with locally administering a therapeutically effective
amount of collagenase, which can be administered in combination
with a selected protease, protein, or enzyme, calcium ions, a
surfactant, and/or an antibiotic.
[0100] Another method of the invention is directed to administering
an aqueous parenteral composition suitable for treating prostate
cancer. Suitable compositions are described above and include a
therapeutically effective amount of collagenase and optionally
include one or more of the following: an above-identified selected
protease, protein, or enzyme, calcium ions, a surfactant, and an
antibiotic. In a preferred embodiment, the composition includes
collagenase, hyaluronidase, calcium ions, a surfactant, and an
antibiotic. The calcium ions can be supplied by, for example,
CaCl.sub.2. The surfactant can be, for example, Triton.RTM. X-100,
and the antibiotic can be, for example, gentamicin.
[0101] The composition is locally administered to the prostate.
Local administration includes delivering the composition into or
near the prostate and/or cancerous cells or tumor, preferably into
the tumor. Local administration also includes surrounding the
prostate and/or cancerous tumor with the composition or applying
the composition to the surface of the prostate and/or cancerous
tumor. For example, the composition can be locally administered by
direct intraprostatic injection or by infusion into the prostate.
Small volumes, such as, for example, about 5 cc or less, are
preferably injected. Larger volumes, such as, for example, greater
than about 5 cc, are preferably transfused slowly over, for
example, about 15 to 20 minutes.
[0102] To verify the accuracy of the administration--i.e., whether
the composition is administered near or into the cancerous cells or
tumor--the urologist typically has a good mapping of the prostate
from, for example, ultrasonic imaging, which can be obtained during
the diagnostic work-up and prostate biopsy.
[0103] The composition is typically administered such that local
administration of therapeutically effective amounts of the
composition result in treating prostate cancer. Preferably the
amount is effective to degrade the prostate tumor, either curing or
alleviating prostate cancer.
[0104] In one embodiment, the composition is locally administered
by intraprostatic injections. Intraprostatic injections are carried
out by inserting a long, fine needle into the prostate under
digital rectal control and/or ultrasonic guidance. The injections
are usually done under local anesthesia, and the injection solution
can be diluted with iodocaine. During the injection, the needle can
be frequently relocated in order to obtain the best possible
distribution of the composition. Several routes of injection are
available for the introduction of the disclosed composition to the
prostate.
[0105] The preferred route of administration is by means of
transurethral intraprostatic (intralesional) injection. The
transurethral technique is immediately preceded by catheterization.
The volume of the composition injected typically varies from 1 to
50 cc.
[0106] To optimize the effectiveness of the injected composition,
it can be desirable to dilate the prostatic urethra with an
inflatable balloon. The cystoscopically inserted balloon inhibits
the immediate egress of the injected enzyme solution through the
porous duct system that empties into the urethra. The advantage of
this route of injection is that the method allows for direct
cystoscopic visualization of the nodular areas of pathology and for
the placement of a high concentration of the composition at the
desired location without the risk of metabolic inactivation. The
pain and discomfort experienced by patients during direct injection
of the prostate typically are minimal and comparable to
intramuscular injections.
[0107] Alternatively, the transperineal or transrectal routes of
prostatic injection can be used. The transperineal route of
injection involves the placement of 22 g.times.20 cm aspiration
biopsy needle through the perineum into the prostate guided by
ultrasound and/or digital palpation. Again, 1 to 50 cc of the
disclosed composition is typically injected into each lateral lobe
of the prostate. The injections are generally done under local
anesthesia. During injection, the needle is frequently relocated to
obtain the best possible distribution of the composition. The
position of the needle can be guided by ultrasound while kept under
constant digital rectal control. The transperineal route of
injection can be a better alternative than either the transurethral
or transrectal routes in terms of reducing potential complications
due to postinjection bacterial infection.
[0108] To reduce the incidence of bacterial infection that can be
associated with transperineal intraprostatic injection, aseptic
injection techniques are recommended and are well known to those
skilled in the art. Any one of a variety of standard bactericidal
preparations such as Phiso-Hex.RTM., Betadine.RTM.,
povidone-iodine, or chlorhexidine applied to the skin of the
perineum provides adequate pre-injection antibacterial protection.
With sterile urine, adequate skin preparation, and sterile
technique, the entire procedure should have a low rate of
infectious complication.
[0109] The transrectal route allows needle introduction through the
rectal wall and injection of the prostate while performing digital
rectal palpation. Injection via the transrectal route is performed
with a slightly curved 22 g.times.20 cm flexible aspiration biopsy
needle. The use of a Franzen needle guide (Precision Dynamics, San
Fernando, Calif.) allows the needle to be safely directed into a
suspected lesion under ultrasonic and/or tactile guidance
techniques. The sterilized prostate needle guide is placed on a
gloved index finger. A finger cot is placed over the needle guide.
The index finger and needle guide are inserted into the rectum and
suspected lesions of the prostate are palpated. The needle is
inserted through the guide and advanced into the tissue.
Approximately 1 to 50 cc of the solution can be injected into the
lateral lobes of the prostate. In order to inject sufficient
material, the needle can be moved back and forth three to five
times. An anesthetic jelly can be applied before injection to
reduce pain during needle puncture.
[0110] Upon injection, the prostatic lobe swells, increases in
size, and becomes turgid. Injected fluid forced through the veins
at the site of injection can induce wide spread venospasm
associated with microinfarcts. Acute urinary retention can occur in
the immediate post injection period. Fluid injected into the
prostate fills the alveoli of the gland at the site of injection
and can rupture through the walls of adjacent alveoli entering the
prostatic urethra via the alveolar ducts. As much as 1/3 to 1/2 of
the injected fluid can ultimately reach the prostatic urethra.
[0111] A bolus injection of more than 5 cc of the composition into
the body of the in situ prostate can result in reflexive smooth
muscle contraction causing the therapeutic enzyme solution to be
rapidly emptied through the porous ducts, away from the target
tissue, and into the urethra. The force of the injection can cause
rupture of prostatic tissue at the site of injection. The injected
fluid can gain access to the ducts of the glandular alveolar system
and completely fill the gland. Once the gland is filled, the fluid
takes the path of least resistance and flows to the urethra.
[0112] Injection fluid can gain access to the prostatic circulation
and be responsible for scattered minute areas of infarction. Enzyme
induced thrombophlebitis of the veins can be responsible for the
appearance of widespread hemorrhagic infarctions. About 1/5 of the
prostatic injection fluid can enter the general circulation as
demonstrated by experiments involving excretion of methylene blue
by the kidneys. The subcapsular and periurethral zones of the
prostate are more vascular in nature and can enhance the flow of
the fluid into the blood stream. Injections of India ink carbon
particles into the prostate resulted in no gross or microscopically
detectable particles in the lymphatics of the pelvis. Prostatic
injection fluid can also reach the surface of the prostate and
periprostatic tissue through the point of needle entry.
[0113] Radiographs taken immediately after intraprostatic injection
of radio-opaque microemulsions of barium sulfate have shown fluid
leakage beneath the prostatic capsule and escape into the bladder.
Occasionally, the fluid has been seen to leak outside the capsule
as well.
[0114] The injected lobule of the prostate undergoes necrosis
mainly due to the enzymatic action of the injected composition and
partly due to the shear force of the fluid injected under pressure.
Part of the fluid that leaks back through the point of needle entry
can cause thrombosis of surface vessels and be responsible for
adhesions with adjacent viscera. The quantity of fluid that leaks
back depends upon the local pressure and is likely to increase with
the force of the injection. Injected fluid passing through the
periurethral veins can cause their inflammation and thrombosis,
which in turn can result in necrosis and sloughing of the urethral
epithelium.
[0115] Urethral and periurethral solubilization is expected to
occur around the entire circumference of the prostatic urethra
which can lead to denudation of the urethral epithelium.
Histopathologic changes associated with injection of the tissue
solubilizing enzymes include those due to fluid escape along ducts
of the gland resulting in damage to the ducts and surrounding
alveoli. The fluid that leaks back through the point of needle
entry can affect the capsular vessels and smooth muscle
fibromuscular stroma situated there.
[0116] Direct localized injection of the prostate results in a high
concentration of therapeutic enzymes at the very focus of the
problem without the risk of metabolic inactivation. However,
intraprostatic injections during acute exacerbations of infection
are not recommended because of the danger of general dissemination
of the infection and possible septicemia. Hematuria and hemospermia
can be present for some weeks following the injection.
[0117] Depending upon the patient being treated, the
therapeutically effective dose of the composition administered can
range from 1 cc to 50 cc preferably containing 160 to 160,000 U/ml
hyaluronidase; 250 to 250,000 U/ml collagenase; 0.1 to 10% nonionic
surfactant preferably Triton.RTM. X-100; and 0.15 to 150 .mu.g/ml
antibiotic, preferably gentamicin. More preferably, a dose includes
2,500 to 25,000 U/ml collagenase; 1,600 to 16,000 U/ml
hyaluronidase; 0.5 to 5% nonionic surfactant, preferably
Triton.RTM. X-100; and 15 to 150 .mu.g/ml antibiotic, preferably
gentamicin. These dosage ranges represent quantities of the various
components of the composition that are estimated to be
therapeutically effective for treating prostate cancer. But the
dose of the composition can vary depending on the age of the
patient, nature and severity of disease, potency of the
composition, and route of administration. Treatment regimens
encompassed by a preferred embodiment of the present invention
employ the intraprostatic injection of safe and effective amounts
of the preferred composition to cause the regression of prostate
cancer. The injections can be administered in daily, weekly, or
monthly injection protocols until the therapeutically desired
result is obtained.
[0118] In some embodiments, to determine the appropriate dosage,
the size of the tumor is estimated because a larger tumor can
require a higher concentration of the composition suitable for use
in methods of the invention. For example, for a tumor smaller than
7 g, a single bolus dose of the composition is typically
administered. A bolus dose can be 5 cc and preferably contains
250-250,000 U/ml collagenase, 160-160,000 U/ml hyaluronidase, 0.1%
to 10% nonionic surfactant, 0.15 to 150 .mu.g/ml antibiotic, and 20
mM to 50 mM CaCl.sub.2 in citrate buffer (pH 6.7). The bolus dose
is preferably injected slowly into the tumor, for example, over
several minutes. This injection can be followed by additional
injections if necessary. For example, this treatment can be
administered again in, for example, a week or a month or can be
administered repeatedly, for example, weekly or monthly.
[0119] For a tumor between 7 and 15 g, a first bolus dose is
typically followed by a larger dose of the composition. For
example, the 5 cc bolus dose described above is first administered.
Then, a second dose of the same composition in 25 to 45 cc is
slowly administered over, for example, about 15 to 20 minutes.
These injections can be succeeded by still another dose if
necessary, but a total volume of 50 cc for all doses is typically
not exceeded. For example, this treatment can be administered again
in, for example, a week or a month or can be administered
repeatedly, for example, weekly or monthly.
[0120] For a tumor larger than 15 g, a dose of up to about 50 cc
can be administered by first administering a bolus dose followed by
a larger dose as described above.
[0121] Other Methods of Delivery
[0122] If problems are encountered that limit or inhibit the
therapeutic effects of the present composition, alternative means
of delivery can be used. For instance, the effectiveness of enzyme
therapy can be limited by the short circulating half-lives of
exogenously administered enzymes, by the development of
immunological responses to foreign protein, by inhibition from
antiproteinase effectors (I-1-antitrypsin, I-2-macroglobulin), or
by the inability to specifically target the enzymes to nodular
areas of pathology.
[0123] A number of different carrier systems can be utilized to
carry the enzymatic composition to the desired site in the
prostate. In general, a suitable carrier guides the therapeutic
agent to its target without loss of specificity or reactivity. The
carrier is preferably capable of linkage with the therapeutic
enzymes and remaining as a complex until delivery is completed. The
carrier preferably avoids triggering the immune defense mechanisms
resulting in biodegradation or inactivation of the present
composition.
[0124] The composition can be administered as a depot formulation
that permits sustained release, prevents access to general
circulation, and increases the prostate-specific localization of
the composition. Such a formulation can be provided as a
slow-release implant or can be microencapsulated or attached to a
biodegradable polymer or a prostate-specific immunoglobulin.
[0125] The use of antibodies as an enzyme composition carrier
system can be desirable. The use of antibodies as carrier systems
for the delivery of therapeutic agents to specific tissues exploits
the antibody's unique ability to recognize and bind to targeted
antigens. Also, antibodies carrying therapeutic reagents can be
more effectively localized in tissues that are highly vascular in
nature or are undergoing neovascularization. Additionally,
cocktails of immuno-enzyme conjugates recognizing different cell
types with different specificities can be useful.
[0126] Tissue-specific monoclonal antibodies can be produced that
better define the antigenic cellular targets to be localized. The
use of F(ab) or F(ab').sup.2 fragments can improve localization
properties. Antibodies containing the F(c) fragment tend to be
specifically localized over a longer time period than F(ab) or
F(ab').sup.2 fragments that exhibit accelerated clearance
mechanisms. The recent development of human-mouse (chimeric)
monoclonal antibodies can have therapeutic applications and provide
advantages over conventional monoclonal antibodies of murine
derivation. Human-mouse antibodies exhibit a wide range and high
degree of specificity. Chimeric antibodies are less likely than
conventional mouse monoclonal antibodies to elicit an immune
reaction when injected into humans. The preparation and use of
human monoclonal antibodies as carriers can further reduce the
degree of immune response in the recipient to the introduction of
foreign proteins. The antiglobulin response can also be controlled
by such factors as method of antibody preparation, dosage, and
route of injection.
[0127] The purpose of immuno-targeted enzyme therapy is to deliver
an effective concentration of enzymes to a tissue specific site of
activity, reduce toxicity to nearby normal tissues, and thereby
increase the therapeutic index. Enzymes can be coupled to
monoclonal antibodies that bind the enzyme covalently yet do not
affect the enzyme's catalytic activity. Enzymes that are coupled to
tissue specific monoclonal antibodies can be able to achieve a
higher degree of specific localization in the targeted tissue than
native enzymes while maintaining their proteolytic activity.
[0128] Other specific localization concepts include
zymogen-antibody conjugates (trypsinogen) or enzyme-antibody
conjugates (collagenase, hyaluronidase, elastase, DNase) that
retain both enzyme and antibody activity. Enzymes can be
encapsulated in lipoprotein, red blood cell (RBC) ghosts,
polylactic acid, and other biodegradable membranes or synthetic
microcapsules containing prostate-specific antibodies to increase
and maintain specific targeting, localization, and activity of the
solubilizing proteases in prostatic tissue.
[0129] The administration of collagenase and hyaluronidase can have
immunologic consequences as repeated injections can result in the
development of antibody titers and the accompanying risk of
anaphylaxis or other less serious hypersensitivity reactions.
Additionally, the presence of specific antibodies to collagenase or
hyaluronidase can inhibit enzyme activity. Potential immunologic
problems could occur if the active enzyme is recognized as foreign
by the recipient's immune system. Antibodies can be produced
against the enzymes and inactivate or precipitate the enzyme. Use
of enzymes of human origin or those produced by recombinant
techniques can minimize these potential immunologic
complications.
[0130] Strategies that avoid the immune surveillance system involve
methods of entrapment of enzyme preparations in biodegradable
vesicles that protect the enzyme activity yet facilitate specific
delivery. Targeting to specific sites of cellular pathology can be
accomplished by attaching tissue specific proteins (monoclonal
antibodies) to these vesicles. Enzymes can also be encapsulated in
liposomes or other biodegradable microcapsules and subsequently
attached to tissue-specific monoclonal antibodies for specific
localization purposes.
[0131] Liposomes are small spheres of concentric phospholipid
bilayers containing an aqueous phase that have been shown to be
useful as carrier systems. Current liposome preparation techniques
permit the incorporation of a variety of drugs, hormones, or
enzymes into either phase. Monoclonal antibodies can be
incorporated into the outer layers of liposomes and provide
increased specificity of delivery for the liposome-contained
therapeutic agent.
[0132] The entrapment of enzymes in synthetic microcapsules or
biodegradable vesicles can provide a valuable method of specific
delivery in addition to protecting the enzyme from physiologic
inactivation and preventing immune complications. Various forms of
membrane encapsulation techniques are available for the entrapment
of enzymes including: erythrocyte ghosts; synthetic polymeric
microcapsules; and lipid vesicles (liposomes) composed of
cholesterol, lecithin, and phosphatidic acid. Use of the
recipient's own erythrocytes to deliver active enzyme can avoid the
potential immunologic and physiologic problems resulting from
enzyme administration in synthetic carriers (e.g., liposomes and
microcapsules).
[0133] The covalent attachment of polyethylene glycol (PEG) to
enzymes renders these proteins nonimmunogenic, can extend their
circulating half-life, provides a means of escape from inhibition
by naturally occurring enzyme inhibitors, and can result in
enhanced enzyme activity with decreased autodigestion. The
attachment of PEG to proteins is simple and yields homogeneous
reaction products that can be purified by ultrafiltration.
[0134] The invention is further elaborated by the representative
examples that follow. Such examples are not meant to be
limiting.
EXAMPLES
Example 1
[0135] A First Case Study of a Man with Localized Prostate
Cancer
[0136] A 70-year-old male is diagnosed with localized prostate
cancer (i.e., no metastasis can be detected by means of
radionucleid scan and CAT scans). The estimated size of the tumor
in the prostate is 7 grams. The location of the tumor is in the
right lateral lobe as seen in the sonogram. The PSA level in the
blood is 40 ng/ml.
[0137] The composition for injection was prepared as previously
described (Preparation and Testing of Composition), and contained
12,500 U/ml collagenase; 7,500 U/ml hyaluronidase; 1%(v/v)
Triton.RTM. X-100; 150 Tg/ml gentamicin; and 40 mmol CaCl.sub.2 in
0.05 M citrated-buffered saline (Ph 6.7). The final composition is
drawn into a syringe ready for injection (5 cc).
[0138] The patient is prepared for a cystoscopy procedure using
local anesthesia. A cystoscope is placed into the urethra and the
injection needle is inserted into the lesion (the tumor) and is
monitored by the ultrasound imaging. Once the needle is secured, 5
cc of the injectable composition is given slowly over a period of
about 4 to 5 minutes. During the injection, vital signs are
monitored for symptoms of toxic, allergic, or other adverse
reactions.
[0139] The patient's recovery is normal, and no signs or symptoms
of any adverse reactions to the injected composition are observed.
The clinical progress is uneventful. The blood PSA level gradually
declines over a period of three months to normal levels (4 ng/ml or
less) and remains normal. After 5 years, the patient is diagnosed
as free of prostate cancer.
Example 2
[0140] A Second Case Study of a Man with Localized Prostate
Cancer
[0141] A 65-year-old male is diagnosed with localized prostate
cancer (i.e., no metastasis can be detected by means of
radionucleid scan and CAT scans). The estimated size of the tumor
is 15 grams and the total prostate weight is 90 grams. The PSA
level in the blood is 200 ng/ml. The tumor is located in the right
lobe and is one solid nodule.
[0142] The composition for injection is prepared as previously
described (Preparation and Testing of Composition). It contained
2,400 U/ml collagenase; 1,600 U/ml hyaluronidase; 1%(v/v)
Triton.RTM. X-100; 150 Tg/ml gentamicin; and 120 mmole CaCl.sub.2
in 0.05 M citratebuffered saline (pH 6.7). The total volume of
injection is 30 cc.
[0143] Under the guidance of a cystoscope, the composition is
injected into the tumor. First, the bolus dose of 5 cc is injected
slowly. Next, the remaining 25 cc volume is infused slowly over a
period of about 15 to 20 minutes. Upon completion of injection,
vital signs are monitored for symptoms of toxic, allergic, or other
adverse reactions.
[0144] The patient's recovery and the postinjection progress is
uneventful. The blood PSA level gradually declines to normal levels
(4 ng/ml or less) over a period of three months and remains normal.
After 5 years, the patient is diagnosed as free of prostate
cancer.
Example 3
[0145] A Third Case Study of a Man with Localized Prostate
Cancer
[0146] A 72-year-old male is diagnosed with localized prostate
cancer (i.e., no metastasis can be detected by means of
radionucleid scan and CAT scans). The estimated size of the tumor
is about 9 grams, and the distribution is rather diffuse. The PSA
level in the blood is 30 ng/ml.
[0147] A composition including 50 mM calcium-chloride in 0.05 M
citrate-buffered saline (pH 6.7) is prepared in a volume of 10 cc.
Under the guidance of a cystoscope, the calcium-chloride
composition is injected into multiple sites of the prostate. Upon
completion of the injection, vital signs are monitored for symptoms
of toxic, allergic, or other adverse reactions. Approximately one
month later, about 75% of the tumor mass has been degraded.
Subsequently, a second injection of 25 mM calcium chloride in 0.05
M citrate-buffered saline is administered intralesionally in a
volume of about 5 cc. After three years, the patient is clinically
free of cancer, and the patient's PSA levels are within normal
limits.
[0148] It should be noted that, as used in this specification and
the appended claims, the singular forms "a," "an," and "the"
include plural referents unless the content clearly dictates
otherwise. Thus, for example, reference to a composition containing
"a compound" includes a mixture of two or more compounds.
[0149] The invention has been described with reference to various
specific and preferred embodiments and techniques. It should be
understood, however, that many variations and modifications can be
made while remaining within the spirit and scope of the
invention.
* * * * *