U.S. patent application number 11/273723 was filed with the patent office on 2006-08-10 for administration of a thiol-based chemoprotectant compound.
This patent application is currently assigned to Oregon Health & Science University. Invention is credited to Leslie Muldoon, Edward A. Neuwelt, Michael A. Pagel.
Application Number | 20060177523 11/273723 |
Document ID | / |
Family ID | 26895307 |
Filed Date | 2006-08-10 |
United States Patent
Application |
20060177523 |
Kind Code |
A1 |
Neuwelt; Edward A. ; et
al. |
August 10, 2006 |
Administration of a thiol-based chemoprotectant compound
Abstract
A method of administration of a thiol-based chemoprotectant
agent including NAC (N-acetylcysteine) and STS (sodium thiosulfate)
that markedly affects biodistribution and protects against injury
from diagnostic or therapeutic intra-arterial procedures. A method
for treating or mitigating the side effects of cytotoxic cancer
therapy for tumors located in the head or neck and brain tumors.
The thiol-based chemoprotectant agent is administered
intra-arterially with rapid and first pass uptake in organs and
tissues other than the liver.
Inventors: |
Neuwelt; Edward A.;
(Portland, OR) ; Muldoon; Leslie; (Tigard, OR)
; Pagel; Michael A.; (Milwaukee, OR) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Oregon Health & Science
University
Portland
OR
Government of The United States, d/b/a The Department of
Veterans Affairs
Baltimore
MD
|
Family ID: |
26895307 |
Appl. No.: |
11/273723 |
Filed: |
November 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10257879 |
May 16, 2003 |
7022315 |
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PCT/US01/40624 |
Apr 26, 2001 |
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11273723 |
Nov 14, 2005 |
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60199936 |
Apr 26, 2000 |
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60229870 |
Aug 30, 2000 |
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Current U.S.
Class: |
424/711 ;
514/15.1; 514/19.3; 514/21.9; 514/562 |
Current CPC
Class: |
A61K 33/243 20190101;
A61P 39/00 20180101; A61K 31/195 20130101; A61K 38/063 20130101;
A61K 31/198 20130101; A61K 9/08 20130101; Y10S 514/922 20130101;
A61K 33/04 20130101; A61P 43/00 20180101; A61P 35/00 20180101; A61K
31/661 20130101; A61K 9/0019 20130101; A61K 31/282 20130101; A61K
31/198 20130101; A61K 2300/00 20130101; A61K 31/661 20130101; A61K
2300/00 20130101; A61K 38/063 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/711 ;
514/562; 514/018 |
International
Class: |
A61K 38/05 20060101
A61K038/05; A61K 31/198 20060101 A61K031/198; A61K 33/04 20060101
A61K033/04 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The present invention was partially supported under NIH
grant No. NS 33618 and by The Department of Veterans Affairs merit
review grant. The United States Government may have certain rights
in this invention.
Claims
1-48. (canceled)
49. A method for treating or mitigating myelosuppression,
comprising: administering intraarterially a high-dose thiol based
chemoprotectant agent.
50. The method for treating or mitigating myelosuppression of claim
49 wherein the high-dose thiol based chemoprotectant agent is from
about 400 mg/m.sup.2 to about 1200 mg/m.sup.2 per procedure.
51. The method for treating or mitigating myelosuppression of claim
49 wherein the high-dose thiol based chemoprotectant agent is
selected from at least one of the group consisting of N-acetyl
cysteine (NAC), sodium thiosulfate (STS), GSH ethyl ester,
D-methionine, and Ethyol.
52-76. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 10/257,879 filed on May 16, 2003, which claims
priority to PCT/USO1/040624 having an international filing date of
Apr. 26, 2001, which claims the benefit of the filing dates under
35 U.S.C. .sctn. 119(e) to U.S. Provisional Application No.
60/199,936 filed on Apr. 26, 2000 and U.S. Provisional Application
No. 60/229,870 filed on Aug. 30, 2000, which are hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] The present invention is based, in part, upon the discovery
that a method of administration of N-acetylcysteine (NAC) markedly
affects its effective biodistribution. The present invention
provides a method for treating or mitigating the side effects,
including organ damage, of cytotoxic cancer therapy for tumors
located in the head or neck. Additionally, NAC or other thiols can
be administered concurrently with, before or after, intra-arterial
procedures and provides protective affects to prevent or diminish
organ damage.
[0004] N-acetylcysteine (NAC) is an analog of cysteine. When NAC is
administered to a mammal it is deacylated and enters a cellular
synthetic pathway for the production of glutathione. Glutathione is
involved in the cellular pathways influencing a tumor's resistivity
to cytotoxic drugs. The cytotoxic properties of chemotherapeutic
drugs can be enhanced by pretreatment with buthionine sulfoximine
(BSO) thereby reducing intracellular glutathione. However,
reduction of intracellular gluthionine will potentiate systemic
toxicities associated with chemotherapeutic drugs. Thus, this
procedure is dose-limiting. For protection, the glutathione levels
of "normal" cells have to be reestablished if BSO is used to
potentiate the cytotoxic properties of cytotoxic cancer therapies
(Kamer et al., Cancer Res. 47:1593-1597, 1987; Ozols et al.,
Biochem. Pharm. 36:147-153, 1987; McLellan et al., Carcinogenesis
17:2099-2106, 1995; and Shattuck et al., J. Parenteral Enteral
Nutrition 24:228-233, 1998). It may be possible to reduce the bone
marrow toxicity of chemotherapeutic drugs by using
sulfur-containing chemoprotective agents (thio, thiol, and
thioether compounds) to mimic one or many of the activities of
glutathione such as conjugation, free radical scavenging, and drug
efflux via the multidrug resistance associated proteins. NAC and
other thiol agents such as STS have early detoxifying activity not
related to the later increase in glutathione levels. These early
detoxifying effects occur because the thiols themselves mimic some
actions of glutathione such as free radical scavenging,
anti-oxidant activity, chemical conjugation, and activation of
efflux pumps.
[0005] A potential problem with any chemoprotectant is the
possibility of deactivating the anti-tumor effect of the
chemotherapy or radiation therapy. The goal of chemoprotection is
to reduce unwanted toxicities of chemotherapy or radiotherapy
without affecting efficacy.
[0006] For brain tumor chemotherapy, one must attempt to increase
the delivery of chemotherapy to the brain tumor and block the
delivery of the chemoprotective agent. Additionally, one will want
to target the chemoprotectant agent to the bone marrow to protect
against myelosuppression and to liver, kidney and lung to prevent
organ toxicity. Therefore, there is a need in the art to improve
pharmacokinetics and biodistribution of chemoprotectant agents so
that they will be more effective when delivered in a
tissue-specific manner. Preferably, delivery is maximized to the
bone marrow, chest and abdomen organs while minimized to the
brain.
[0007] There are more than 10 specific active transport systems
that transport compounds from the blood to the brain. Otherwise,
substances, such as chemoprotectants can only nominally penetrate
this barrier by passive diffusion. Brain tumors are particularly
difficult to treat because the blood-brain barrier is an anatomical
structure that limits the egress of constituents in the blood to
the brain. Thus, brain tumors often respond poorly to
chemotherapeutic drugs. There have been many attempts to try to
increase brain bioavailability of various drug compounds to brain
tissue. One technique uses osmotic BBB modification by
administering mannitol through the internal carotid artery (Neuwelt
et al., Cancer Res. 45:2827-2833, 1985). This technique is useful
for administering the chemotherapeutic methotrexate to experimental
brain tumors that would otherwise be inaccessible to this drug (it
poorly crosses the BBB). The osmotic shrinkage caused by
intracarotid mannitol administration allowed for temporary BBB
disruption and increased tumor delivery of the methotrexate. Thus,
a temporary disruption of the barrier functions of the BBB can be
induced by a sugar, such as mannitol, and cause higher brain
concentrations of a drug compound that would not otherwise have
crossed the BBB. This BBB opening technique has also been
investigated with other chemotherapeutic drugs (Neuwelt et al.,
Proc. Natl. Acad. Sci. USA 79:4420-4423, 1982; Fortin D. McCormich
Cl, Remsen LG, Nixon R, Neuwelt EA, "Unexpected neurotoxicity of
etoposide phosphate when given in combination with other
chemotherapeutic agents after blood-brain barrier modification
using propofol for general anesthesia in a rat model," Neurosurgery
47:199-207, 2000).
[0008] An example of chemoprotection is a drug neutralization
technique described in U.S. Pat. No. 5,124,146 wherein excess toxic
drug compounds are "mopped up" or bound by a binding or
neutralizing agent not able to penetrate the blood brain barrier.
This technique requires precise timing as to when the drug
neutralizing agent is administered.
[0009] There are several thiol-based chemoprotectant agents that
contain a thio, thiol, aminothiol or thioester moiety. Several
thiol-based chemoprotectant agents have been shown to provide
protection against at least some of the systemic toxicities caused
by alkylating chemotherapeutics. The thiol based chemoprotective
agents include N-acetyl cysteine (NAC), sodium thiosulfate (STS),
GSH ethyl ester, D-methionine, and thiol amifostine (Ethyol or
WR2721). NAC is currently marketed in the United States under an
orphan indication for oral and intravenous (i.v.) administration
for overdosing with acetaminophen. NAC has also been shown to be a
chemoprotectant when administered in combination with a vanadate
compound (U.S. Pat. No. 5,843,481; and Yarbo (ed) Semin. Oncol. 10
[Suppl 1]56-61, 1983). Ethyol is also marketed in the United States
under the generic name of Amifostine. GSH ethyl ester is an
experimental thiol not yet marketed for clinical use, but is
representative of the class of thiols that is converted directly to
glutathione.
[0010] In addition, NAC has been shown to be a mucoregulatory drug
used for the treatment of chronic bronchitis (Grassi and Morandini,
Eur. J. Clin. Pharmacol. 9:393-396, 1976; Multicenter Study Group,
Eur. J. Respir. Dis. 61: [Suppl.]93-108, 1980; and Borman et al.,
Eur. J Respir. Dis. 64:405-415, 1983).
[0011] In plasma, NAC can be present in its intact, reduced forms
as well as in various oxidized forms. It can be oxidized to a
disulfide by reacting with other low molecular weight thiols, such
as cysteine and glutathione. NAC can be oxidized by reacting the
thiol groups of plasma proteins. When administered intravenously,
the brain levels of NAC are <5%. Yet, NAC does cross the BBB if
given by an intra-arterial route of administration. NAC is rapidly
cleared from plasma via the liver and kidney. Moreover, NAC does
not show neurotoxic properties.
[0012] There are bioanalytical methods for the determination of NAC
in plasma, including Cotgreave and Moldeus, Biopharm. Drug Disp.
8:365-375, 1987; and Johansson and Westerlund, J. Chromatogr.
385:343-356, 1986 that also permit a determination of other forms
of NAC. Moreover, cysteine and cystine have been identified as
major metabolites of NAC. The excreted urinary product is inorganic
sulfate together with small amounts of taurine and unchanged NAC.
According to the label indications for NAC manufactured by
(American Regent Laboratories Shirley, NY), vials of NAC are
produced as a sterile solution for oral administration diluted with
water or soft drinks.
[0013] Another thiol-containing chemoprotectant is sodium
thiosulfate (STS). Its chemical formula is Na.sub.2S.sub.2O.sub.3
and it has been used clinically for cyanide poisoning and for
nephrotoxicitiy caused by cisplatin. STS is cleared rapidly from
circulation primarily by the kidney. The plasma half life after a
bolus injection is about 17 minutes. STS can also inactivate
platinum agents through covalent binding to platinum agents at a
molar excess >40:1 (STS:platinum). With i.v. administration of
STS, the brain levels of STS are <5% of blood showing poor brain
localization. Neurotoxic side effects, in the form of seizures, may
occur when brain levels of STS are enhanced through i.a.
administration within 30 min of BBB disruption.
[0014] Diagnostic or therapeutic procedures involving
intra-arterial catheterization can cause a variety of organ
toxicities, complications and side effects from injuries. For
example, placement of an arterial catheter can dislodge plaques
from artery walls that can lodge elsewhere in the vasculature
causing ischemia. Ischemia increases the presence of free radicals
and leads to cell death. As another example, nephrotoxicity of
radiographic contrast agents can lead to acute renal failure even
when measures are taken to reduce toxic effects. As a third
example, intra-arterial catheterization is used during angioplasty
procedures wherein a balloon catheter is inserted into the arterial
circulation and then threaded (with radiographic contrast agents
for visualization) to a site of occlusion. In dilating the occluded
artery, various forms of tissue damage and inflammatory reactions
(e.g., restenosis) can occur including ischemic tissue injury.
[0015] Specifically, toxic side effects of intra-arterial
catheterization and infusion of radiographic contrast agents
prolong hospital stays, add to the cost of medical care, and can be
fatal. The incidence of radiographic-contrast-agent-induced acute
renal failure, currently estimated to be as high as 50 percent
among patients with diabetes mellitus and preexisting renal disease
who receive contrast agents, is likely to remain high as the use of
invasive intra-arterial procedures to diagnose and treat complex
disease continues to grow.
[0016] Radiographic contrast agents are used in medical imaging.
Medical imaging is the production of images of internal organs and
tissues by the application of nonsurgical techniques. Contrast
agents are chemicals used to enhance the image, and to increase
contrast between the target organ and surrounding tissues.
Prevention or mitigation of renal failure after the administration
of a radiographic contrast agent has been notably difficult.
Calcium-channel antagonists, adenosine antagonists, and dopamine
have all been used without convincing evidence of benefit.
[0017] Tepel et al. proposed the oral administration of
approximately 1200 mg of N-acetylcysteine per day, given orally in
divided doses on the day before and on the day of the
administration of the radiographic contrast agent. (Tepel et al.,
New England J. Med., Jul. 20, 2000). Oral administration allegedly
prevented the expected decline in renal function in all patients
with moderate renal insufficiency, and therefore high risk, who
were undergoing computed tomography.
[0018] NAC has been used successfully to ameliorate the toxic
effects of a variety of experimentally or clinically induced
ischemia-reperfusion syndromes of the heart, kidney, lung, and
liver. In each of these syndromes, it is thought that the activity
of NAC is related to its action as a free-radical scavenger, or as
a reactive sulphydryl compound that increases the reducing capacity
of the cell. The specific mechanism of NAC to prevent the
nephrotoxic effects of contrast agents is not known.
[0019] Therefore, there is a need in the art to find better ways to
use thiol-based chemoprotectants, such as NAC and STS and to take
advantage of their pharmacokinetic properties. There is also a need
in the art to find better, higher dose cytotoxic treatment regimens
for head and neck as well as brain tumors that avoid dose-limiting
due to side effects.
[0020] There is a need in the art for a compound that can be used
with intra-arterial catheterization procedures to reduce organ
toxicity. Diabetic patients with markedly reduced renal function,
in whom coronary angiography is often delayed because of the
considerable risks to renal function entailed by angiography, may
particularly be benefited by targeted delivery of a protectant
agent. Additionally, there is a need in the art for a low cost
compound which is generally available, easy to administer and has
limited side effects. There is a need in the art for a compound and
a method of administration of the compound that can be used to
reduce or eliminate tissue damage caused by intra-arterial
procedures.
[0021] Additionally, there is a need in the art to find better ways
to use thiol-based radiographic protectants, such as NAC and STS
(sodium thiosulfate) and to take advantage of their pharmacokinetic
properties. There is also a need in the art to find an agent
protective against intra-arterial catheterization-induced
reductions in organ function. These and other problems of the prior
art are solved by the present method and pharmaceutical
composition.
SUMMARY OF THE INVENTION
[0022] The present invention provides a method and compound for
locally administering a thiol based chemoprotectant to treat or
mitigate the side effects of cytotoxic cancer therapy for brain
tumors located in the head or neck. Further, the present invention
provides a method and compound for locally administering a
thiol-based chemoprotectant to an organ or tissue to protect
against injury from diagnostic or therapeutic intra-arterial
procedures. The method includes administering a thiol-based
chemoprotectant agent intra-arterially in conjunction with, before,
or after administration of a cytotoxic agent.
[0023] In one embodiment, the cytotoxic agent is a cancer
chemotherapeutic agent. The cytotoxic agent is dose-limited due to
myelosuppressive effects systemically. The cancer chemotherapeutic
agent is selected from the group consisting of cis-platinum
compounds, taxanes (e.g., paclitaxel), steroid derivatives,
anti-metabolites, vinca alkaloids, adriamycin and doxorubicin,
etoposide, arsenic derivatives, intercalating agents, alkylating
agents (such as melphalan) and combinations thereof. The cytotoxic
agent is administered within eight hours (before, during or after)
of the thiol-based chemoprotectant agent administration. In yet
another embodiment, when the tumor is located in the head or neck
or brain, the cytotoxic agent is administered such that the
majority of the dose is directed to the head or neck region.
[0024] Preferably, the thiol-based chemoprotectant agent is a
compound selected from the group consisting of N-acetyl cysteine
(NAC), sodium thiosulfate (STS), GSH ethyl ester, D-methionine,
Ethyol, and combinations thereof. In one embodiment, the
thiol-based chemoprotectant agent is administered in a pyrogen-free
sterile solution by a catheterization procedure via a catheter
having a tip that is located in the descending aorta. In yet
another embodiment, the dose of the thiol-based chemoprotectant
agent per procedure is from about 200 mg/m.sup.2 to about 40
g/m.sup.2. Most preferably, the dose of NAC agent per procedure is
from about 400 mg/m.sup.2 to about 1200 mg/m.sup.2 and the dose of
STS is from about 5 g/m.sup.2 to about 40 g/m.sup.2.
[0025] In one embodiment, the intra-arterial catheter is positioned
in an artery providing blood flow to a potential site or organ of
injury. In yet another embodiment, the intra-arterial
administration is at a site in the descending aorta. In yet another
embodiment, the injury is caused by injecting a cytotoxic agent
including a radiographic contrast agent using an intra-arterial
catheter.
[0026] In yet another embodiment, the thiol-based chemoprotectant
agent is administered in a pyrogen-free, non-oxidized sterile
solution having a reducing agent, a buffer to maintain pH at or
near physiologic pH and a metal chelating agent to bind metal ions
that can catalyze oxidation of the thiol-based chemoprotectant
agent. Preferably, the reducing agent is selected from the group
consisting of vitamin E, tocopherol, dithiotreitol,
mercaptoethanol, glutathione, and combinations thereof. Preferably,
the buffer is one that is relatively non-toxic and can maintain a
pH of between 6 and 8 (e.g., phosphate buffer, Tris buffer).
Preferably, the thiol-based chemoprotectant agent is stored in a
vial having a blanket of an inert gas. Most preferably, the inert
gas is selected from the group consisting of argon, helium,
nitrogen and mixtures thereof.
[0027] The present invention further provides a pharmaceutical
composition for treatment to protect against injury from diagnostic
or therapeutic intra-arterial procedures, and for head and neck
brain tumors. The compound includes a first agent for
intra-arterial administration and a second agent administered
intra-arterially.
[0028] Preferably the second agent is a thiol-based chemoprotectant
agent. In one embodiment, the second agent is administered to the
descending aorta or further downstream.
[0029] The first agent is administered to a carotid or vertebral
artery. The first agent is a radiographic contrast agent delivered
intra-arterially to position a catheter. In one embodiment, the
first agent is administered within eight hours (before, during or
after) of the second agent. The second agent is administered
immediately after intra-arterial catheterization, prior to
radiographic contrast agent, to within eight hours of the
radiographic contrast agent.
[0030] In one embodiment, the second agent is administered in a
pyrogen-free, sterile solution. In further embodiments, the
solution is non-oxidized and has a reducing agent, a buffer to
maintain pH at or near physiologic pH and a metal chelating agent
to bind up metal ions that can catalyze oxidation of the
thiol-based chemoprotectant agent. Preferably, the reducing agent
is selected from the group consisting of vitamin E, tocopherol,
dithiothreitol, mercaptoethanol, glutathione, and combinations
thereof. Preferably, the buffer is one that is relatively non-toxic
and can maintain a pH of between 6 and 8 (e.g., phosphate buffer,
Tris buffer).
[0031] In one embodiment, the thiol-based chemoprotectant agent is
stored in a vial having a blanket of an inert gas. Most preferably,
the inert gas is selected from the group consisting of argon,
helium, nitrogen and mixtures thereof. Preferably, the thiol-based
chemoprotectant agent is a compound selected from the group
consisting of N-acetyl cysteine (NAC), sodium thiosulfate (STS),
GSH ethyl ester, D-methionine, Ethyol, and combinations thereof.
Preferably, the dose of the thiol-based chemoprotectant agent per
procedure is in the range of 200 mg/m.sup.2 to 2000 mg/m.sup.2. In
a further preferred embodiment, the dose of NAC per procedure is in
the range of 400 mg/m.sup.2 to 1200 mg/m.sup.2.
[0032] Further, one advantage of NAC is that it is protective
against multiple intra-arterial procedure toxicities, including but
not limited to those caused by radiographic contrast agents.
[0033] The methods and compounds will best be understood by
reference to the following detailed description of the preferred
embodiment, taken in conjunction with the accompanying drawings.
The discussion below is descriptive, illustrative and exemplary and
is not to be taken as limiting the scope defined by any appended
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows an anatomical diagram of major arteries and the
top level for placing the catheter for administration of the
thiol-based chemoprotectant agent.
[0035] FIG. 2 (A-C) shows the effect of bone marrow recovery and
lower nadirs using the left carotid aortic infusion inventive
method with chemotherapy and the chemoprotectant NAC. Specifically,
FIG. 2A shows the effect on white blood cells, FIG. 2B the effect
on platelets and FIG. 2C the effect on granulocytes.
[0036] FIG. 3A shows dose/response for N-acetylcysteine
chemoprotection. FIG. 3B shows dose/response for D-methionine
chemoprotection. Cytotoxicity was assessed in cultured LX-1 SCLC
cells, 1.times.10.sup.4 cells per well in 96 well plates, using the
WST colorometric assay. Cells were treated with approximately 90%
lethal dose of chemotherapy (melphalan =20 .mu.g/ml, carboplatin
=200 .mu.g/ml, cisplatin =20 .mu.g/ml). Chemoprotectant was added
at the indicated concentration of N-acetylcysteine shown in FIG. 3A
or D-methionine shown in FIG. 3B either alone (n) or immediately
following chemotherapy. Data are expressed as the percentage of
live cells compared to untreated control samples (without
chemotherapy) and each point represents the mean .+-.standard
deviation of 4 wells.
[0037] FIGS. 4A and 4B show cytoenhancement and chemoprotection.
FIG. 4A shows the effect of BSO and N-acetylcysteine on Carboplatin
cytotoxicity. FIG. 4B shows the effect of BSO and N-acetylcysteine
on etoposide phosphate cytotoxicity. Cytotoxicity was assessed in
cultured LX-1 SCLC cells, 1.times.10.sup.4 cells per well in 96
well plates, using the WST colorometric assay. BSO cytoenhancement
consisted of preincubation at 100 .mu.M BSO for 18 hours. The
chemoprotective agent was added immediately after chemotherapy. The
experimental conditions were dose/responses for chemotherapy
(carboplatin or etoposide phosphate) alone (m), BSO cytoenhancement
(), N-acetylcysteine rescue, (1000 .mu.g/ml N-acetylcysteine, n ),
or BSO cytoenhancement and N-acetylcysteine rescue (u). Data are
expressed as the percentage of live cells compared to untreated
control samples without chemotherapy and each point represents the
mean .+-. standard deviation of 4 wells.
[0038] FIG. 5 shows cytoenhancement and chemoprotection in
fibroblasts. Cytotoxicity was assessed in GM294 human fibroblasts,
1.times.10.sup.4 cells per well in 96 well plates, using the WST
colorometric assay. Cells were pretreated with or without BSO, 100
.mu.M for 18 hours prior to addition of chemotherapeutics
(melphalan =10 .mu.g/ml, carboplatin =100 .mu.g/ml, cisplatin =7.5
.mu.g/ml, etoposide phosphate 100 .mu.g/ml) either alone (open
bar), or with N-acetylcysteine rescue, (1000 .mu.g/ml
N-acetylcysteine, striped bar), BSO cytoenhancement (black bar), or
BSO cytoenhancement and N-acetylcysteine rescue (cross hatched
bar). Data are expressed as the percentage of live cells compared
to control samples (without chemotherapy) and each point represents
the mean .+-. s.d. of 4 wells.
[0039] FIG. 6 shows time dependence for rescue of chemotherapy
cytotoxicity. Chemotherapy cytotoxicity was assessed in cultured
LX-1 SCLC cells (1.times.10.sup.4 cells/well in 96 well plates)
using the WST colorometric assay. Cells were treated with melphalan
at 20 .mu.g/ml, carboplatin 200 .mu.g/ml, cisplatin 10 .mu.g/ml, or
etoposide phosphate 200 .mu.g/ml. Cells then received either no
protectant (open bars), or sodium thiosulfate, 2000 .mu.g/ml, added
immediately (striped bars), 2 hours (black bars) or 4 hours (cross
hatched bars) after chemotherapy. Data are expressed as the
percentage of live cells compared to control samples (without
chemotherapy) and each point represents the mean .+-. standard
deviation of 4 wells.
[0040] FIGS. 7A and 7B show the effect of cytoenhancement and
chemoprotection on apoptosis. FIG. 7A shows caspase-2 enzymatic
activity. FIG. 7B shows TUNEL staining for DNA fragmentation.
Apoptosis was assessed in cultured LX-1 SCLC cells pretreated for
18 hours with or without 100 .mu.M BSO. The experimental conditions
were no addition (open bars), melphalan (10 .mu.g/ml, striped bars)
or melphalan (10 .mu.g/ml) plus N-acetylcysteine (1000 .mu.g/ml)
(cross hatched bars) for 20 h prior to harvest. Caspase-2 activity
is expressed as percentage activity in untreated control samples
(mean .+-.0 standard deviation, n =3). TUNEL staining is expressed
as the percentage of cells showing positive staining.
[0041] FIG. 8 shows the results of NAC delivery to rat brain.
Radiolabeled NAC in combination with unlabeled low dose NAC (140
mg/kg) or high dose NAC (1200 mg/kg) was administered to rats with
the following routes of infusion: i.v. (open bars),
intra-arterially into the right carotid artery (striped bars),
intra-arterial via the left carotid artery with left internal
artery occlusion (aortic infusion) (black bars), and intra-arterial
(right carotid) with BBBD (cross hatched bars). Radiolabel in
tissue homogenates is expressed as the mean and standard error of
the % administered dose of 14C-NAC per gram of tissue (n =3 rats
per group). Significant differences from i.v. delivery are
indicated by *P<0.05 and **P<0.001.
[0042] FIG. 9 represents tests showing the effect of NAC route of
administration on mortality +/-BSO. A Kaplan-Meier product limit
analysis was used to evaluate the mortality due to chemoprotection
with NAC. Rats were treated with or without BSO (10 mg/kg i.p.
b.i.d..times.3, black bars) prior to treatment with chemotherapy
(Carboplatin 200 mg/m2, etoposide phosphate 100 mg/m2, melphalan 10
mg/m2). Chemoprotection consisted of NAC (1200 mg/m2) or NAC (1000
mg/m2) plus STS (8 g/m2) given either i.v. (n=17 BBSO, n=8+BSO) or
by aortic infusion (n=19 BBSO, n=28 +BSO). P=0.0014 by Wilcoxon
analysis.
[0043] FIGS. 10A-10D show the results of tests on chemoprotection
for chemotherapy-induced myelosuppression. Rats received tri-drug
chemotherapy (Carboplatin 200 mg/m2, etoposide phosphate 100 mg/m2,
melphalan 10 mg/m2), with (triangles, squares) or without (circles)
chemoprotection. Blood counts were determined prior to
chemotherapy, at the blood nadir (6 days), and in the recovery
phase (9 days after treatment). Chemoprotection was with NAC (1200
mg/kg) administered via aortic infusion 30 min prior to
chemotherapy (triangles) or NAC prior to chemotherapy and STS (8
g/m2) immediately after chemotherapy (squares). In FIG. 10B and
FIG. 10D animals received BSO (10 mg/kg i.p..times.3 d) prior to
treatment with chemotherapy. Panel A shows granulocyte counts
without BSO FIG. 10B shows granulocyte counts with BSO (mean
+/-SEM, n=6 per group) FIG. 10C shows platlet count without BSO
FIG. 10D shows platlet count with BSO. Significant difference from
the no protectant groups were determined by
Wilcoxon/Kruskal-Walliis rank sums tests (* p<0.05, **
p<0.01).
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present method includes administration of at least two
different agents for treating brain or head and neck tumors. The
first agent is a cytotoxic agent. In one example, the cytotoxic
agent is radiation or a chemotherapeutic agent. The radiation or
chemotherapeutic agent generally has a dose-limiting systemic side
effect of myelosuppression. If myelosuppression is too severe, it
is life threatening as the patient is unable to generate enough
white blood cells of multiple lineages to coordinate immune
surveillance function for defending against pathogen attack.
Therefore, any ability to reduce bone marrow toxicity or
myelosuppression will allow for greater and more effective
administration of the cytotoxic agent.
[0045] Current treatment to reduce bone marrow side effects include
recombinant growth factors that are lineage-specific. Such growth
factors have included EPO (erythropoietin) for red cells and G-CSF
(granulocyte colony stimulating factor) or GM-CSF (granulocyte
macrophage colony stimulating factor) for various lineages of
infection-fighting white cells. In addition, TPO (thrombopoietin)
is in clinical trials for augmenting a platelet response in
myelosuppressed patients. However, such growth factors act to
stimulate lineage specific precursor cells to divide and mature
down lineage-specific paths. Thus, the use of growth factors
results in a more rapid recovery from bone marrow toxicity but does
not generally reduce the nadir of toxicity. Such growth factors
have been able to allow a patient to tolerate a greater number of
cytotoxic treatments (where myelosuppression is a limiting
toxicity), but generally not higher doses of the cytotoxic agent
administered.
[0046] The method allows for greater doses of the cytotoxic agent
directed to head and neck tumors. Specifically, the cytotoxic agent
(if it is a chemical compound or combination of compounds) is
administered intra-arterially such that it is directed initially to
the head and neck circulation. Thus, the highest concentration of
cytotoxic agent is direct to the location of the tumor to be
treated. By contrast, iv administration provides for the same
concentration of cytotoxic agent in the bone marrow, where side
effects happen, as systemic dose.
[0047] Thiol-based chemoprotectant agents are nonspecific
chemoprotectant agents. They are not specific to "normal" tissue or
cells but can protect both normal and tumor tissue. Earlier
attempts to utilize the chemoprotectant properties of thiol-based
chemoprotectant agents have failed due to the fact that they were
administered either orally or systemically, they were rapidly
metabolized, and protected both normal and tumor tissue. Systemic
administration includes iv.
[0048] The method includes utilizing a spatial two-compartment
pharmacokinetic model which results in a general tissue first pass
effect to prevent significant or chemoprotectant doses of
thiol-based chemoprotectant agents from gaining general systemic
circulation through the venous circulatory system. The method
utilizes only one pass going to tissues below the level of the
heart in order to effect a chemoprotectant effect. Therefore, head
and neck tumors are treatable through regionalization of doses of
the cytotoxic agent to the brain or head and neck where the tumor
tissue is located and doses of the chemoprotectant to general
tissues below the level of the heart where the majority of bone
marrow tissue is located.
[0049] The ability of a thiol-based chemoprotectant agent to show a
first pass effect through non-liver tissue was surprising. Once any
thiol-based chemoprotectant agent that is not tissue-absorbed gains
access to the venous circulation, it will be cleared through a
liver first pass or rapidly removed through renal clearance. When
administered not according to the method described, NAC is actively
transported across the BBB. An example of spatial
compartmentalization is the administration of a thiol-based
chemoprotectant agent into the descending aorta or lower preventing
any significant chemoprotectant concentrations of the thiol-based
chemoprotectant agent from ever reaching the brain or head or neck
region where the tumor tissue is located. Spatial
compartmentalization is facilitated by the rapid tissue uptake of
the thiol-based chemoprotectant agent. Spatial compartmentalization
is needed in order to achieve any meaningful therapeutic benefit
with a thiol-based chemoprotectant agent. Without spatial
compartmentalization, the undesirable result of the thiol-based
chemoprotectant agent protecting the tumor tissue will occur. In
the case of NAC as the thiol-based chemoprotectant agent, passage
of the BBB by NAC will restore glutathione levels to the brain
tumor, thereby increasing the tumor's resistance to cytotoxic
drugs.
[0050] The ability to set up a two-compartment pharmacokinetic
model was discovered through a series of experiments using in vitro
and in vivo models and administering both chemotherapeutic agents
and the thiol-based chemoprotectant agents NAC and STS. However, it
should be noted that radiation therapy can be similarly localized
or even more easily localized than chemotherapeutic agents. Tissue
culture pharmacological studies have shown that treatment with NAC
and STS can reduce cell killing by the chemotherapeutics melphalan,
carboplatin, and cisplatin. The in vivo results show that localized
NAC or STS administration resulted in less myelosuppression and
faster recovery from chemotherapy. Moreover, synergistic results
were observed with the combination of thiol-based chemoprotectant
agent NAC and STS providing evidence for a combination of
thiol-based chemoprotectant agents to enhance the protective role
when the combination of thiol-based chemoprotectant agents is
administered according to the inventive process.
[0051] As shown in FIG. 1, in another embodiment of the method, a
thiol-based chemoprotectant agent is administered intra-arterially
such that it is directed systemically. This provides the highest
concentration of thiol-based chemoprotectant agent to the location
of organ damage, for example the kidneys, to protect against
reduction of renal function. Oral administration, by contrast,
provides for general systemic administration with the concentration
of protective agent going elsewhere in the body, mainly the liver,
and requiring higher dosages so as to provide a sufficient dose at
the kidney (often the site of organ damage for radiographic
contrast agents). In addition, the thiol-based chemoprotectant
agent is not specific to normal tissue or cells but can protect
both normal and tumor tissue.
[0052] The method is based upon results obtained utilizing a
spatial two-compartment pharmacokinetic model that resulted in a
general tissue first pass effect to prevent significant or
radiographic-protectant doses of thiol-based
radiographic-protectant agents (specifically illustrated are NAC
and STS) from gaining general systemic circulation through the
venous circulatory system. Thus, there was a surprising need for
only one pass going to tissues of the renal system. This result
prevented decreased renal function through regionalization of doses
of the radiographic agent to the area where radiography is to be
performed and doses of the protectant to the renal system.
[0053] As shown in FIG. 1, a catheter was inserted into the
circulatory system generally via the femoral artery. In the first
embodiment, a catheter is inserted into the body and administers a
dosage of radiographic contrast agent into the body. An effective
dosage of the thiol-based protective agent, preferably NAC, is
administered at any time from immediately after the intra-arterial
catheterization, preferably before contrast agent, to within about
eight (8) hours after the administration of the radiographic
contrast agent. The catheter for delivery of the radiographic
protective agent is preferably inserted into the arterial system
downstream of the aorta and directed in the mesenteric artery
system. The thiol-based protective agent is introduced into the
body at any time within about 5 hours after administration of the
radiographic contrast agent to reduce or eliminate renal failure or
decrease in renal function associated with administration of a
renal contrast agent.
[0054] In a further embodiment, a catheter is inserted into the
circulatory system (e.g., femoral artery) and administers an
effective dosage of a thiol-based protective agent into the body. A
radiographic contrast agent is administered by the same catheter
and is used to position the catheter to the appropriate place for
the therapeutic or diagnostic procedure. The thiol-based protective
agent is introduced into the body, generally via the same arterial
catheter, at any time within about 5 hours before or after
administration of the radiographic contrast agent. Preferably, the
thiol-based protective is administered via the arterial catheter
one or a plurality of times during the procedure.
Pharmaceutical Formulations
[0055] Techniques for the formulation and administration of the
compounds of the instant application may be found in "Remington's
Pharmaceutical Sciences" Mack Publishing Co., Easton, Pa., latest
addition. Suitable routes of administration are intra-arterial.
[0056] The compositions and compounds of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, emulsifying, encapsulating,
entrapping, or lyophilizing processes. Pharmaceutical compositions
for use in accordance with the present invention thus may be
formulated in conventional manner using one or more physiologically
acceptable carriers comprising excipients and auxiliaries that
facilitate processing of the active compounds into preparations,
which can be used pharmaceutically. Proper formulation is dependent
upon the route of administration chosen.
[0057] For injection, the compounds of the invention may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers, such as Hank's solution, Ringer's solution, or
physiological saline buffer. The compounds may be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulary agents such as suspending, stabilizing
and/or dispersing agents.
[0058] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0059] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use. In one embodiment, a reducing agent
or an anti-oxidant agent is added to the formulation of the
thiol-based protective agent to prevent oxidation of the
thiol-based protective agent. The antioxidant may include, but is
not limited to, vitamin E, tocopherol, dithiotreitol,
mercaptoethanol, glutathione. In one embodiment, an inert or
non-oxidizing gas is added to a vial for intra-arterial
administration. Examples of such gasses are nitrogen, argon,
helium, and combinations/mixtures thereof.
[0060] A therapeutically effective dose refers to that amount of
the compound that results in a reduction in the development or
severity of myelosuppression. In another embodiment, toxicity and
therapeutic efficacy of such compounds can be determined by
standard pharmaceutical, pharmacological, and toxicological
procedures in cell cultures or experimental animals, e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio between LD.sub.50and ED.sub.50. Compounds
that exhibit high therapeutic indices are preferred. The data
obtained from cell culture assays or animal studies can be used in
formulating a range of dosage for use in humans. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition.
(See e.g. Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1).
[0061] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
[0062] According to the method, a thiol-based chemoprotectant agent
is administered intra-arterially in order for systemic tissues to
be exposed to an initial dose of the thiol-based chemoprotectant
agent in high enough concentration to provide a chemoprotective
effect and to bypass the venous circulation and be eliminated by
the liver. NAC is actively transported across the BBB. In one
embodiment, to prevent access to the brain, after a transfemoral
carotid/vertebral artery catheter is placed to perfuse the brain,
the catheter can then be subsequently retracted and placed in the
descending aorta for NAC infusion, thus performing a single
surgical procedure with one catheter. This allows for minimal risk
from arterial catheter procedures only and for high concentrations
of NAC to be delivered to peripheral tissues and organs but not to
brain and head.
Synthesis
[0063] Each thiol-based chemoprotectant agent, such as NAC or STS,
can be synthesized by. convention methods and are commercially
available as a sterile solution.
EXAMPLE 1
[0064] This example shows the results of an in vivo experiment in
rats having a catheter implanted in the descending aorta for NAC
administration. The rats were set up for drug administration by
pushing a catheter forward past the junction of the external and
internal carotid arteries (toward the aorta), and temporarily
sealing off the internal carotid for good measure so nothing goes
to the brain (left carotid aortic infusion method). In patients
where entry is via the femoral artery and the catheter is threaded
through the aorta to get to the brain in the first place, one would
just pull the catheter back to the aorta to do the NAC
infusion.
[0065] Rats were treated with carboplatin (200 mg/m.sup.2),
melphalan (10 mg/m.sup.2 ) and etoposide phosphate (100
mg/m.sup.2). In the NAC animals, NAC was infused with the left
carotid aortic infusion method immediately after the
chemotherapeutic agents, at concentrations ranging from 400-1200
mg/m.sup.2. White blood cells (wbc) and platelets (plt) were
counted at baseline before the experiment and at 6 and 9-11 days
after treatment with the chemotherapeutic agents. The animals that
did not receive NAC were tested on day 9-10 while the NAC animals
were tested on day 10-11. Counts are in 1000 s per .mu.l blood.
These data in the initial experiment shown in Table 1 provide
initial results without a white blood cell effect. The lack of
white blood cell effect was not repeated as there were significant
effects shown below. TABLE-US-00001 TABLE 1 chemo alone wbc base
wbc d6 wbc d9/10 plt base plt d6 plt d9/10 mean 11.4 0.8 3.2 778 63
164 +/-sd +/-4.1 +/-0.2 +/-1.6 +/-233 +/-59 +/-91 number 8 4 3 7 4
3 chem + NAC wbc base wbc d6 wbc d10/11 plt base plt d6 plt d10/11
mean 7.9 0.5 4.8 817 101 1232** +/-sd +/-2.1 +/-0.2 +/-0.7 +/-142
+/-48 +/-167 number 5 4 3 5 4 3
[0066] The data in table 2 show additional results. In the NAC
animals, NAC was infused by the left carotid aortic infusion method
30 min prior to chemo, at a concentration of 1200 mb/m2. White
blood cells (wbc) and platelets (pit) were counted at baseline
before the experiment and at 6 and 9 days after treatment with the
chemotherapeutic agents. The data in Table 1 show that NAC
treatment decreased the nadir blood count for both white cells and
platelets, and blood counts recovered from chemotherapy faster.
TABLE-US-00002 TABLE 2 wbc base wbc d6 wbc d9 plt base plt d6 plt
d9 chemo alone mean 6.4 1.6 5.2 878 187 599 +/-sd +/-0.3 +/-0.4
+/-0.7 +/-46 +/-70 +/-187 number 6 6 6 6 6 6 chem + NAC mean 5.1
3.3 6.2 721 388 1155 +/-sd +/-0.4 +/-0.7 +/-1.6 +/-59 +/-95 +/-154
number 6 6 6 6 6 6
[0067] These data show that with NAC the platelets recovered from
chemotherapy faster.
[0068] In addition, the experiment with the same chemotherapy
agents at the same doses with and without BSO (buthionine
sulfoximine) shows improved white blood cell recovery and higher
nadirs with the chemoprotectant (NAC alone or with STS at the doses
listed above) in FIG. 2A. Similar data are shown with platelets in
FIG. 2B, and with granulocytes in FIG. 2C.
EXAMPLE 2
[0069] This example shows the results of NAC delivery by different
means of catheter-based administration. In this experiment,
radiolabeled NAC was administered to rats by three different
routes. Liver and kidney tissue, in addition to the ipsilateral and
contralateral hemispheres of the brain, were analyzed for
radioactivity. Results are shown as the percent of the injected
radioactive dose per gram of tissue. Route indicates intravenous
(i.v.), intra-arterial into the right carotid artery (i.a.), or
left carotid with internal artery occlusion and aortic infusion
(left carotid aortic infusion). The left carotid aortic infusion
intra-arterial method uses descending aorta placement of the
catheter tip and NAC administration. TABLE-US-00003 route left hem
right hem liver kidney number i.v. 0.02 0.03 0.59 0.72 2 i.a. 0.03
0.41 0.57 0.70 3 Left carotid 0.04 0.04 0.29 1.42 2 aortic
infusion
[0070] These data show that i.a. delivery provided much brain
delivery in the infused hemisphere. When the NAC was administered
i.v., negligible amounts were found in brain (0.03 % of the
injected dose, n=2). When radiolabeled NAC was administered
intraarterially into the right carotid artery of the rat, high
levels of radiolabel were found throughout the right cerebral
hemisphere. Delivery was 0.41% of the injected dose (n=3),
comparable to the levels found in liver (0.57 % of the injected
dose) or kidney (0.70 % of the injected dose). In contrast, the
left carotid aortic infusion method prevented brain delivery. The
Left carotid aortic infusion method also changed bio-distribution
in peripheral tissues in that liver delivery was decreased and
kidney delivery was increased. The change in tissue delivery with
different modes of administration is likely due to NAC being
related to the amino acid cysteine that is rapidly bound by tissues
via the amino acid transporters. In summary, the method of
administration of NAC markedly affected its biodistribution.
EXAMPLE 3
[0071] This example tested whether the inventive method for
intra-arterial infusion (via the left carotid artery, with left
internal artery occlusion) could reduce brain delivery of NAC and
increase systemic delivery. Brain delivery was 0.04 % of the
injected dose with the inventive method (n=2).
[0072] In conjunction with other pharmacological and physiological
data, these results show that NAC is protective when
N-acetylcysteine is administered prior to, (preferably 30 minutes
prior to), the cytotoxic agent at a dose which provides a serum
concentration of NAC of between 0.5 mM to 15.0 mM, preferably 5 mM
to 12.5 mM. Generally, a dose of between 40.0 mg/kg to 1000 mg/Kg
of N-acetylcysteine will provide an appropriate serum concentration
in humans and other mammals.
EXAMPLE 4
[0073] This example shows the inventive process being used to
compare bone marrow toxicity of a chemotherapeutic agent (alone or
in combination) to administration of NAC alone (1000-1200
mg/m.sup.2 alone or in combination) and NAC plus STS. The
chemotherapy agents were carboplatin (200 mg/m.sup.2) melphalan (10
mg/m.sup.2) and etoposide phosphate (100 mg/m.sup.2). The dose of
STS in the rat was 8 g/m.sup.2 that is the equivalent to 20
g/m.sup.2 in humans. The method of administration was left carotid
aortic infusion method. These data are expressed according to
lineages of bone marrow cell in Table 3a and 3b. TABLE-US-00004
TABLE 3a wbc wbc Platelet Plt plt wbc chemo mean 9.2 0.7 3.2 759.0
63.3 164.0 sd 3.6 0.2 1.6 260.5 59.0 91.0 n 7 4 3 7 4 3 Wbc chemo +
mean 7.1 1.4 5.2 772.3 132.0 1559.0 sd 2.7 1.1 1.7 171.5 36.8 567.1
n 4 2 2 4 2 2 wbc chemo + NAC + mean 9.5 2.8 8.6 936.5 554.0 1950.0
sd 6.0 0.4 3.6 186.0 147.1 134.4 n 2 2 2 2 2 2
[0074] TABLE-US-00005 TABLE 3b wbc base wbc d6 wbc d9 plt base plt
d6 plt d9 chemo + BSO mean 6.8 0.8 5.4 930 92 387 +/-sd +?-0.8
+/-0.2 +/-1.1 +/-63 +/-28 +/-139 number 11 11 6 11 11 6 BSO + chem
+ NAC mean 8.4 2.2 7.6 818 341 1560 +/-sd +/-1.5 +/-0.3 +/-1.2
+/-99 +/-79 +/-194 number 9 9 6 9 9 6
[0075] These data show the synergistic effect of a combination of
STS and NAC as the combined thiol-based chemoprotectant agents
administered according to the inventive process.
EXAMPLE 5
[0076] This example provides the results of an in vitro experiment
using a combination of a taxane (paclitaxel) with a
paclitaxel-cytotoxic enhancing agent BSO and a glutathione-reviving
10 agent NAC in cultured tumor cells. Paclitaxel is cytostatic in
cultured cells at concentrations from 1 to 10 micromolar, that is,
the tumor cells do not grow but they are not killed either. At 20
uM, paclitaxel begins to be cytotoxic and at 30 uM it is completely
toxic in cultured tumor cells. When the tumor cells were pretreated
cells with BSO, paclitaxel addition was completely cytotoxic in
vitro at doses as low as 5 uM.
[0077] NAC did not change the dose response for paclitaxel alone.
However, NAC completely reversed the enhanced toxicity from the BSO
treatment, returning paclitaxel concentration effects to the
non-BSO level. This experiment was repeated twice with the data
provided in Table 4. TABLE-US-00006 TABLE 4 Treatment paclitaxel
dose live cells (WST fluorescence) paclitaxel alone 5 1.274 +/-
.071 20 0.771 +/- .056 paclitaxel + NAC 5 1.369 +/- .061 20 0.823
+/- .094 BSO + paclitaxel 5 0.056 +/- .004** 20 0.045 +/- .004**
BSO + paclitaxel + NAC 5 1.419 +/- .095* 20 0.732 +/- .100*
*significantly different from BSO and paclitaxel **significantly
different from paclitaxel alone
EXAMPLE 6
Chemoprotection Against Cytotoxicity
[0078] The dose/response for rescue from chemotherapy cytotoxicity
was evaluated for four different small molecular weight sulfur
containing chemoprotectants. Each chemotherapeutic agent was used
at a concentration affording approximately 90% lethality in the
absence of BSO (20 .mu.g/ml melphalan, 200 .mu.g/ml carboplatin, 15
.mu.g/ml cisplatin). Over all, N-acetylcysteine was the most
effective of the thiol agents tested, on a .mu.g/ml basis. The
concentration dependence for protection with N-acetylcysteine in
comparison to D-methionine is shown in FIGS. 3A and 3B, and Table 5
shows the EC50 for protection afforded by each protective agent. As
shown in FIG. 3A AND Table 5, the cytotoxicity of each alkylator
was reduced by 75-90% by concurrent administration of
N-acetylcysteine, but N-acetylcysteine was more active against
melphalan (EC50=74.+-.18 .mu.g/ml) than the platinum agents. In
contrast, as shown in FIG. 3B and Table 5, D-methionine did not
protect against melphalan toxicity at the doses tested (50 to 1000
.mu.g/mI), although it was highly protective against cisplatin
toxicity, with a half-maximal concentration of 140.+-.41 .mu.g/ml.
The maximum magnitude of protection was variable between
experiments, ranging from about 70% to 100% protection, and
protection was consistently less 5 for carboplatin than for
cisplatin or melphalan. All agents tested required a significantly
higher dose to protect against carboplatin than against cisplatin
or melphalan. On a .mu.g/ml basis, glutathione ethyl ester was the
least effective protective agent.
[0079] Chemotherapy agents were fixed at the 90% lethal dose for
each agent. Each EC50 measurement comprised 6 concentrations with 4
wells per concentration, and each dose response 10 was performed
twice for each protectant. EC50 concentrations, in .mu.g/ml are
reported as the average .+-. pooled standard deviation for two
independent experiments. For the combination of D-methionine with
melphalan, protection was not detected. For each chemoprotectant, t
test comparisons were done for melphalan versus cisplatin,
cisplatin versus carboplatin, and carboplatin versus melphalan, and
significant differences are indicated (**=P<0.01;
***=P<0.001). TABLE-US-00007 TABLE 5 Protection against
chemotherapy cytotoxity Chemoprotective agent EC50 (.mu.g/ml)
Glutathione ethyl chemotherapy N-acetylcysteine Sodium thiosulfate
D-methionine ester Melphalan 74 .+-. 18*** 110 .+-. 78 None*** 303
.+-. 124*** Cisplatin 151 .+-. 15** 86 .+-. 76** 140 .+-. 41*** 530
.+-. 30*** Carboplatin 200 .+-. 15*** 442 .+-. 203** 379 .+-.
123*** 995 .+-. 48***
EXAMPLE 7
Cytoenhancement and Chemoprotection in Combination
[0080] The effects of BSO cytoenhancement and thiol chemoprotection
on the dose/response relationships for cytotoxicity of the
alkylating chemotherapeutics were evaluated in the B.5 LX-1 cells.
BSO cytoenhancement consisted of preincubation with 100 .mu.M BSO
for about 18-24 hours prior to addition of chemotherapy, and rescue
consisted of 1000-2000 .mu.g/ml of thiol chemoprotectant added
immediately after chemotherapy. As shown in FIG. 4A and Table 6,
BSO consistently decreased the EC50 for cytotoxicity and increased
the maximum degree of toxicity. The specific case of carboplatin
and N-acetylcysteine is shown in FIG. 4A. Glutathione depletion
with BSO increased carboplatin cytotoxicity, reducing the EC50 by
48% (P<0.01). As detailed in Table 6, similar BSO
cytoenhancement was found with melphalan (53% reduction of EC50,
P<0.001), while the EC50 for cisplatin was reduced only 29%
(P<0.05). Chemoprotection with N-acetylcysteine blocked
carboplatin toxicity as well as BSO-enhanced cytotoxicity. Similar
chemoprotection was found with additional thiol agents, sodium
thiosulfate and glutathione-ethyl ester, but D-methionine was only
effective against the platinum agents.
[0081] Cytoenhancement and chemoprotection against non-alkylating
chemotherapeutic agents was also evaluated. As shown in FIG. 4B,
Glutathione depletion with BSO did not increase the cytotoxicity of
etoposide phosphate, nor did N-acetylcysteine decrease the
cytotoxicity of etoposide phosphate. Similarly, no enhancement or
protection was found with methotrexate or doxorubicin in the B.5
LX-1 cells, although carcinoma cells of gastric origin showed some
enhancement with BSO (data not shown). Interestingly, although the
growth inhibitory dose of taxol (approximately 10 nM) was not
altered by BSO, glutathione depletion did shift the cytotoxic dose
of taxol from 15 ,.mu.M to 2 .mu.M, and this enhanced cytotoxicity
was completely reversed with N-acetylcysteine (not shown).
[0082] Whether the cytoenhancement and chemoprotection seen in the
B.5 LX-1 SCLC cells was a generalized phenomenon was evaluated by
testing similar experimental conditions in the GM294 human
fibroblast cell strain. Cells were treated with or without
chemotherapy at the approximately half maximal dose found in the
B.5 LX-1 cells, with or without pretreatment with BSO. As shown in
FIGS. 3A and 3B, although melphalan, cisplatin and carboplatin were
all somewhat more cytotoxic in the fibroblasts as compared to the
tumor cells, nevertheless BSO enhanced the toxicity of all three
alkylators. In fibroblasts, N-acetylcysteine was partially to
completely chemoprotective against the cytotoxicity induced by
melphalan, cisplatin and carboplatin, independent of BSO treatment.
As shown in FIG. 5, neither BSO cytoenhancement nor
N-acetylcysteine chemoprotection affected the cytotoxicity of
etoposide phosphate in fibroblasts.
[0083] Half-maximal cytotoxic concentrations (EC50) are expressed
as .mu.g/ml. Each EC50 measurement comprised 6 concentrations with
4 wells per concentration, and each dose response was performed in
triplicate for each chemotherapeutic agent. Data are reported as
the mean .+-.pooled standard deviation for three independent
experiments. P values are shown for the reduction in EC50 by BSO
treatment (*=P<0.05, **=P<0.01, *** =P<0.001).
TABLE-US-00008 TABLE 6 Effect of BSO on the cytotoxicity of
alkylating chemotherapeutics Chemotherapy EC50 (.mu.g/ml) Melphalan
Cisplatin Carboplatin Without BSO 13.8 .+-. 1.8 8.9 .+-. 2.4 103
.+-. 21 +BSO 6.4 .+-. 0.6*** 6.4 .+-. 0.9* 55 .+-. 15**
EXAMPLE 8
Time Dependence for Chemoprotectant Rescue From Chemotherapy
Cytotoxicity
[0084] How long the addition of chemoprotectant could be delayed
after treatment with chemotherapy and remain effective was
evaluated. Cells were treated with doses of chemotherapy providing
approximately 90% lethality, for melphalan (20 .mu.g/ml),
carboplatin (200 .mu.g/ml), or cisplatin (15 .mu.g/ml). The thiol
chemoprotectants were added either concurrently with chemotherapy
or up to 8 hours after chemotherapy. For melphalan, chemoprotection
was reduced if administration of STS was delayed for 2 hours,
whereas sodium thiosulfate was still protective for the platinum
chemotherapeutics if delayed up to 4 hours after treatment as shown
in FIG. 6. Similarly, delayed administration of N-acetylcysteine
and glutathione ethyl ester reduced their protective activity
against melphalan cytotoxicity, whereas both agents maintained
protective activity against platinum cytotoxicity. Separately, all
three agents were completely protective if added within 1 hour of
melphalan, rather than 2 hours as shown in FIG. 6. Chemoprotection
was not effective against etoposide phosphate cytotoxicity at any
time point.
[0085] The time dependence of D-methionine rescue of cisplatin
cytotoxicity was also evaluated. Unlike chemoprotection with
thiosulfate, N-acetylcysteine, or glutathione ethyl ester, the
protection afforded by D-methionine was significantly reduced by
delayed administration. If delayed for 2 hours after cisplatin,
D-methionine protection was reduced by 41.2.+-.10.2 % compared to
the maximal protection seen with simultaneous addition, while
delaying D-methionine to 4 hours reduced protection by 66.1.+-.4.5
% compared with simultaneous addition. Pretreatment with
D-methionine for 30 min prior to addition of cisplatin did not
increase the amount of protection compared to simultaneous
addition.
EXAMPLE 9
Effects of Cytoenhancement and Chemoprotection on Apoptosis
[0086] Apoptosis was evaluated by measuring Caspase-2 enzymatic
activity and by in situ TUNEL staining. Treatment of B.5 LX-1 cells
with melphalan resulted in an increase in Caspase-2 activity that
was amplified by BSO pretreatment at low melphalan concentrations
as shown in FIG. 7A. The increase in caspase activity was variable
between experiments and ranged from 50-100% at 7-8 h to 250-600% at
20-24 h after treatment with melphalan. TUNEL staining also
demonstrated melphalan-induced apoptosis. In the experiment shown
in FIG. 7B, TUNEL staining after melphalan treatment was positive
in 29 of 3643 cells, compared to 7 of 4395 cells in the untreated
control, and BSO treatment prior to melphalan increased the
positive staining to 800 of 1699 cells. In both the caspase-2 assay
as shown in FIG. 7A and the TUNEL staining assay as shown in FIG.
7B, the effect of melphalan on apoptosis was reduced by the
chemoprotectant N-acetylcysteine. In both assays, activity was
maximal with low doses of melphalan, or with a 1 hour pulse
treatment with the doses used in the cytotoxicity assays.
Continuous treatment with the cytotoxic dose of melphalan actually
reduced caspase-2 activity and TUNEL staining.
[0087] Cisplatin and carboplatin were less effective than melphalan
at inducing caspase activity. Over a range of doses (100, 150, or
200 .mu.g/ml carboplatin, and 5, 10, or 15 .mu.g/ml cisplatin) and
times (8, 12, 16, 20, 24 h), each platinum agent increased
Caspase-2 activity by 50-100%. No significant amplification of
caspase activity was induced by BSO treatment. Additionally, no
reduction in caspase enzymatic activity could be detected after
addition of N-acetylcysteine, and in some experiments treatment
with N-acetylcysteine actually increased cisplatin-or
carboplatin-induced caspase activity. Samples of the cells used in
the caspase and TUNEL assays were also evaluated for membrane
permeability by trypan blue exclusion. In experiments producing
negative results with the caspase-2 or TUNEL assays, trypan blue
exclusion showed high numbers of non-viable cells after treatment
with carboplatin or cisplatin and this was increased by BSO
treatment.
EXAMPLE 10
Biodistribution of Radiolabeled NAC
[0088] The method of administration of NAC and its biodistribution
was tested. Intra-arterial infusion retrograde via the left
external carotid artery, with transient left internal artery
occlusion, was evaluated as a mechanism to essentially perfuse via
the descending aorta with limited delivery to the brain. When NAC
was administered i.v., negligible amounts were found in brain as
determined by the percent administered dose of 14C-NAC per gram of
tissue as shown in FIG. 8. Intra-arterial delivery in the right
internal carotid artery resulted in high levels of radiolabel in
the right cerebral hemisphere, and this was not increased by BBBD.
However, aortic infusion minimized brain delivery of NAC as shown
in FIG. 8. At the low dose of NAC (140 mg/kg), aortic infusion
decreased liver delivery and increased kidney delivery, where as
there was no change in liver and kidney delivery at high dose (1200
mg/kg) NAC (data not shown). The serum concentration of NAC 10 min
after administration of 1200 mg/kg was 2.4 0.6 mg/ml (n=6) as
determined by radiolabel remaining in the blood.
EXAMPLE 11
Toxicity of NAC
[0089] A dose escalation of NAC was performed in the rat. Initial
doses of 140-800 mg/kg NAC administered i.v. immediately after
chemotherapy (n=4), were well tolerated but provided no detectable
bone marrow protection. Doses of NAC above 1200 mg/kg (n=3) were
toxic whereas there was no toxicity at 1200 mg/kg. Therefore, 1200
mg/kg of NAC was used for the bone marrow protection studies,
except when administered in conjunction with STS where a dose of
1000 mg/kg was used due to volume considerations.
[0090] The toxicity of NAC was determined when infused for bone
marrow protection. As shown in FIG. 9. the mortality due to NAC was
significantly dependent on the route of administration, with i.v.
administration significantly more toxic than aortic infusion
(P=0.0014). Pretreatment with BSO markedly enhanced the toxicity of
NAC. Groups given i.v. NAC after BSO treatment were halted early
due to excessive mortality, with a stopping rule of 75% mortality
within n=4 animals. In selected animals (n=21) from all groups,
blood pressure monitoring indicated most animals that expired
experienced persistent acute hypotension, suggesting cardiac
toxicity. Of particular note, however, there was no mortality nor
any evidence of toxicity in 12 animals, without BSO, giving 1200
mg/kg NAC by aortic infusion 30 minutes prior to chemotherapy.
EXAMPLE 12
Effect of NAC on Chemotherapy-induced Bone Marrow Toxicity
[0091] Chemoprotection against chemotherapy-induced bone marrow
toxicity was determined in BSO treated and untreated animals given
i.a. carboplatin, etoposide phosphate, and melphalan. NAC with or
without STS was administered either about 30 minutes prior to
chemotherapy or immediately after chemotherapy, and was
administered either i.v. or by aortic infusion. Chemoprotection was
found with NAC as shown by increased blood counts (white blood
cells, granulocytes, platelets) at the nadir, compared to no
chemoprotectant as shown in Table 7 and Table 8. Chemoprotection
was effective whether or not animals were pretreated with BSO as
shown in FIG. 10, and the magnitude of the chemotherapy-induced
bone marrow toxicity nadir was minimized and recovery to normal
platelet levels was improved. TABLE-US-00009 TABLE 7 Protection
against chemotherapy-induced myelosuppression White Cell
Granulocyte Treatment Rats Nadir Nadir Platelet Nadir No protectant
N = 6 24.5 .+-. 5.8 20.7 .+-. 11.0 22.7 .+-. 8.7 NAC i.v. post
chemo N = 6 46.0 .+-. 6.5* 57.9 .+-. 8.8* 36.8 .+-. 10.4 NAC i.v.
30 min prior N = 8 51.4 .+-. 9.4 93.5 .+-. 28.1 53.3 .+-. 12.7 NAC
aortic infusion post N = 6 25.9 .+-. 4.9 36.6 .+-. 17.0 26.8 .+-.
9.3 chemo NAC aortic infusion 30 min N = 6 69.7 .+-. 19.3 206.2
.+-. 125.1* 59.3 .+-. 17.9 prior NAC aortic infusion 30 min N = 6
53.8 .+-. 12.8* 83.4 .+-. 21.3* 47.0 .+-. 11.5 prior + STS post
chemo The mean and standard error are shown for nadir blood counts
as a percent of baseline. *indicates P < 0.05 compared to no
protectant, by Wilcoxon/Kruskal-Wallis rank sums tests.
[0092] TABLE-US-00010 TABLE 8 Protection against BSO-enhanced
chemotherapy-induced myelosuppression White Cell Treatment Rats
Nadir Granulocyte Nadir Platelet Nadir No protectant N = 11 12.9
.+-. 3.5 3.5 .+-. 1.3 9.1 .+-. 2.3 NAC aortic infusion post N = 7
13.9 .+-. 4.2 19.8 .+-. 14.7 11.7 .+-. 3.5 chemo NAC aortic
infusion 30 min N = 9 20.1 .+-. 2.7 115.4 .+-. 68.8* 23.1 .+-. 6.2
prior NAC aortic infusion 30 min N = 7 30.5 .+-. 6.5* 121.0 .+-.
40.2* 39.0 .+-. 7.4* prior + STS post chemo The mean and standard
error are shown for nadir blood counts as a percent of baseline.
*indicates P < 0.05 compared to no protectant, by
Wilcoxon/Kruskal-Wallis rank sums tests.
[0093] In animals that did not undergo BSO treatment, pretreatment
with NAC (1200 mg/kg) by aortic infusion, with or without follow-up
with STS, was the best treatment strategy as shown in FIGS. 10A and
10C. As shown in FIG. 10A, the chemotherapy-induced decrease in
granulocyte counts was completely blocked (p<0.05) and platelet
toxicity was reduced as shown in FIG. 10C by aortic infusion of NAC
30 minutes prior to chemotherapy.
[0094] In animals pretreated with BSO, good protection for
granulocytes was provided by aortic infusion of NAC about 30 min
prior to chemotherapy, but the best protection and the least
mortality was provided by NAC aortic infusion before and STS
immediately after chemotherapy as shown in FIGS. 10B and 10D. As
shown in FIG. 10B, the combination chemoprotection (NAC and STS)
regimen significantly blocked the toxicity for granulocytes
(p<0.01) and, as shown in FIG. I OD, platelets (p<0.01)
compared to animals that received no chemoprotection.
Chemoprotection also significantly enhanced the platelet recovery
from chemotherapy. STS alone did not give consistent bone marrow
protection (data not shown).
[0095] As noted, the discussion above is descriptive, illustrative
and exemplary and is not to be taken as limiting the scope defined
by any appended claims.
* * * * *