U.S. patent application number 12/755001 was filed with the patent office on 2010-09-23 for methods and compositions for optimizing blood and tissue stability of camptothecin and other albumin-binding therapeutic compounds.
Invention is credited to Thomas G. Burke, Daniel C. Carter.
Application Number | 20100240602 12/755001 |
Document ID | / |
Family ID | 23058589 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240602 |
Kind Code |
A1 |
Burke; Thomas G. ; et
al. |
September 23, 2010 |
METHODS AND COMPOSITIONS FOR OPTIMIZING BLOOD AND TISSUE STABILITY
OF CAMPTOTHECIN AND OTHER ALBUMIN-BINDING THERAPEUTIC COMPOUNDS
Abstract
The present invention provides methods and formulations for
optimizing the anti-cancer and anti-HIV activities of a
camptothecin drug, including camptothecin and its related analogs
including 9-aminocamptothecin and 9-nitrocamptothecin. The
invention involves methodologies and formulations that limit human
serum albumin-mediated reduction of the anti-cancer and anti-HIV
effects of the camptothecins, and the methods and formulations
provide combination therapies in which binding of the camptothecin
agent to human serum albumin can be modulated by the administration
of a competing agent such as ibuprofen, clofibrate or clofibric
acid that also binds human serum albumin. Reduced camptothecin drug
binding to human serum albumin can result in elevated camptothecin
free drug levels and thus improve the effectiveness of treatment
regimens involving these drugs. Further agents such as methotrexate
and AZT can also be used in cancer and HIV-positive patients
employing camptothecin drugs.
Inventors: |
Burke; Thomas G.;
(Lexington, KY) ; Carter; Daniel C.; (Huntsville,
AL) |
Correspondence
Address: |
STITES & HARBISON PLLC
1199 NORTH FAIRFAX STREET, SUITE 900
ALEXANDRIA
VA
22314
US
|
Family ID: |
23058589 |
Appl. No.: |
12/755001 |
Filed: |
April 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10101513 |
Mar 20, 2002 |
7691872 |
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12755001 |
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60276908 |
Mar 20, 2001 |
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Current U.S.
Class: |
514/26 ; 514/161;
514/167; 514/171; 514/196; 514/221; 514/283; 514/52 |
Current CPC
Class: |
A61K 31/655 20130101;
A61K 31/4745 20130101; A61K 31/59 20130101; A61K 31/00 20130101;
A61K 31/407 20130101; A61K 31/57 20130101; A61K 31/59 20130101;
A61K 45/06 20130101; A61K 31/655 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/555
20130101; A61P 35/00 20180101; A61K 31/57 20130101; A61K 31/4745
20130101; A61P 31/18 20180101; A61K 31/407 20130101; A61K 31/555
20130101 |
Class at
Publication: |
514/26 ; 514/283;
514/171; 514/167; 514/196; 514/221; 514/52; 514/161 |
International
Class: |
A61K 31/7048 20060101
A61K031/7048; A61K 31/4375 20060101 A61K031/4375; A61K 31/56
20060101 A61K031/56; A61K 31/59 20060101 A61K031/59; A61K 31/545
20060101 A61K031/545; A61K 31/55 20060101 A61K031/55; A61K 31/714
20060101 A61K031/714; A61P 31/18 20060101 A61P031/18; A61P 35/00
20060101 A61P035/00; A61K 31/60 20060101 A61K031/60 |
Claims
1. A method for increasing the free drug levels of a camptothecin
drug that binds human serum albumin (HSA) during anti-topoisomerase
I-based therapy in humans, said method comprising administering, to
a human or animal patient in need of said therapy, at least one
HSA-binding compound so as to block the camptothecin binding site
on HSA and thus reduce the binding of the camptothecin drug to HSA
in human blood or plasma so that the free drug levels of the
camptothecin drug will be increased and so that greater levels of
the camptothecin will reach the drug target at the treatment site,
wherein the HSA-binding compound is administered separately from
the camptothecin drug.
2. The method according to claim 1, wherein the HSA-binding
compound is selected from the group consisting: of long chain fatty
acids (C.sub.16-C.sub.20; including oleic, palmitic, linoleic,
stearic, arachidonic, and palmitoleic); medium chain fatty acids
(C.sub.6-C.sub.14; including caprylate or octanoate); phospholipids
(lysolecithins, oleoyllysophosphatidic acid, phosphatidylcholine,
phosphatidylethanolamine); eicosanoid derivatives (Leukotrienes,
thromboxanes, prostaglandins A, E, F, and I); steroid hormones
(cholesterol, testosterone, pregnenolone, cortisol, androsterone,
indol, progesterone, estrogen); vitamin D (both monohydroxyvitamin
D and dihydroxyvitamin D); bile salts (lithocholate,
chenodeoxycholate, deoxycholate, ursodeoxycholate, cholate,
glycolitocholate, glycochenodeoxycholate, taurochenodoxycholate,
glycodeoxycholate, glycocholate, taurocholate); bilirubins
(bilirubin, biliverdin, xanthobilirubin, EZ-cyclobilirubin,
.delta.-bilirubin); porphyrins (hematin, protoporphyrin); warfarin;
salicylates, ibuprofen; prednisone; iophenoxate; sulfisoxazole;
phenylbutazone; oxphenylbutazone; digitoxin; indomethacin;
tolbutamide; furosemide; phenyloin; chlorpropamide; chlorthiazide;
the penicillins (including oxacillin, benzylpenicillin);
acetotrizoate; isulfobromophthalein; deacetylcolchicine;
dansylamide; dansylglutamine; dansylsarcosine; indomethacin;
phenylpropazone; azobenzene derivatives; sulfobromophthalein;
triiodobenzoate; benzodiazepine (including diazepam); flufenamate;
iopanoate; ethacrynate; panproxen; clofibrate; L-tryptophan;
N-acetyl-L-tryptophan; 6-methyltryptophan; thyroxine;
3,5,3'-L-triiodothyronine; indole propionate; kynurenine;
ethacrynate; panproxen; chlorophenoxyisobutyrate;
3'azido-3'-deoxythymidine; non-steroidal anti-inflammatory agents
containing ionized carboxyl groups; gossypol;
meso-2,3-dimercaptosuccinic acid; captopril;
N-2-mercaptoethyl-1,2-diaminopropane; disulfuramacetaminophen,
dis-dichlorodiamineplatinum 9II; pyridoxal 5'-phosphate;
aquocobalamin form of vitamin B12; folate; ascorbate (and its
oxidation product dehydroascorbate); melatonin;
.alpha.-melanotropin; gastrin; corticotropin and methotrexate, the
camptothecins (both .alpha.-hydroxy-.delta.-lactone congeners and
.beta.-hydroxy-.delta.-lactone congeners), and combinations of the
above.
3. The method according to claim 1, wherein the HSA-binding
compound is ibuprofen.
4. The method according to claim 1, wherein the HSA-binding
compound is selected from the group consisting of clofibrate and
clofibric acid.
5. The method according to claim 1, wherein the HSA-binding
compound is administered intravenously or orally.
6. The method according to claim 1, wherein the binding of the
HSA-binding compound to HSA results in the displacement of the
camptothecin drug from its HSA binding site.
7. The method according to claim 1 wherein the camptothecin drug is
selected from the group consisting of camptothecins that contain
either an E-ring .alpha.-hydroxy lactone pharmacophore or an E-ring
.beta.-hydroxy lactone pharmacophore, homocamptothecins,
homosilatecans, 9-aminocamptothecin, 10-hydroxycamptothecin,
10,11-methylenedioxycamptothecin,
9-nitro-10,11-methylenedioxycamptothecin,
9-chloro-10,11-methylenedioxycamptothecin,
9-amino-10,11-methylenedioxycamptothe-cin, 9-nitrocamptothecin,
topotecan, and combinations of the above.
8. A method for increasing the free drug levels of a camptothecin
drug that binds human serum albumin (HSA) during anti-topoisomerase
I-based therapy in humans, said method comprising administering, to
a human or animal patient in need of said therapy, at least one
HSA-binding compound selected from the group consisting of
clofibrate and clofibric acid so as to block the camptothecin
binding site on HSA and thus reduce the binding of the camptothecin
drug to HSA in human blood or plasma so that the free drug levels
of the camptothecin drug will be increased and so that greater
levels of the camptothecin will reach the drug target at the
treatment site, wherein the HSA-binding compound is administered
separately from the camptothecin drug.
9. The method according to claim 8, wherein the HSA-binding
compound is administered intravenously or orally.
10. A method for improving the free lactone levels of at least one
camptothecin drug that binds in the carboxylate form to HSA during
anti-topoisomerase I-based therapy, said method comprising
administering, to a human or animal patient in need of said
therapy, at least one HSA-binding compound selected from the group
consisting of clofibrate and clofibric acid so as to block the
camptothecin binding site on HSA and thus reduce the binding of the
camptothecin drug to HSA in human blood or plasma so that the free
lactone levels of the camptothecin drug will be increased in human
blood or plasma and so that greater levels of the camptothecin will
reach the drug target at the treatment site, wherein the
HSA-binding compound is administered separately from the
camptothecin drug.
11. The method according to claim 10, wherein the HSA-binding
compound is administered intravenously or orally.
12. The method according to claim 10, wherein the binding of the
HSA-binding compound to HSA occurs by covalent or non-covalent
means.
13. The method according to claim 10, wherein the binding of the
HSA-binding agent to HSA results in the displacement of the
camptothecin drug from its HSA binding site.
14. A method for enhancing the cellular uptake and cellular
concentration of the camptothecin drug that binds to HSA during
anti-topoisomerase I-based therapy, said method comprising
administering, to a human or animal patient receiving said therapy,
at least one HSA-binding compound, wherein the compound is selected
from the group consisting of clofibrate and clofibric acid, so as
to block the camptothecin binding site on HSA and thus reduce the
binding of the camptothecin drug to HSA in human blood or plasma so
that the cellular uptake and cellular concentration of the
camptothecin drug will be enhanced in human blood or plasma and so
that greater levels of the camptothecin drug will reach the drug
target at the treatment site, wherein the HSA-binding compound is
administered separately from the camptothecin drug.
15. The method according to claim 14, wherein the HSA-binding
compound is administered intravenously or orally.
16. A therapeutic composition, comprising an effective therapeutic
amount of at least one camptothecin drug that binds HSA during
anti-topoisomerase I-based therapy in humans and at least one HSA
binding compound selected from the group consisting of clofibrate
and clofibric acid.
17. The composition of claim 16, further comprising one or more
additional agents selected from the group consisting of
antineoplastic agents and anthracyclines.
18. A method for improving the effectiveness of a therapeutic
treatment regimen using a camptothecin drug that binds to HSA
during anti-topoisomerase I-based therapy comprising administering
to a human or animal patient in need of said therapy a HSA-binding
compound, wherein said HSA-binding compound is selected from the
group consisting of clofibrate and clofibric acid, so as to block
the camptothecin binding site on HSA and thus reduce the binding of
the camptothecin drug to HSA in human blood or plasma so that the
effectiveness of the therapeutic treatment regimen of the
camptothecin drug will be improved and so that greater levels of
the camptothecin will reach the drug target at the treatment site,
wherein the HSA binding compound is administered separately from
the camptothecin drug.
19. The method according to claim 18 wherein said therapeutic
treatment regimen comprises therapeutic treatment for AIDS.
20. The method according to claim 18 wherein said therapeutic
treatment regimen comprises therapeutic treatment for cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 10/101,513, filed Mar. 20, 2002, and
the application claims the benefit of U.S. Provisional Application
Ser. No. 60/276,908, filed Mar. 20, 2001, all of said applications
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates in general to methods of optimizing
camptothecin and other albumin-binding compounds for therapeutic
use, and in particular to a method of using human serum albumin
binding compounds to increase the stability and effectiveness in
humans of camptothecin compounds and other albumin-binding
compounds which have been shown to possess important therapeutic
attributes, such as anti-cancer activity, in murine cells or other
in vitro studies, but which have been far less successful in humans
due to rapid lack of stability in human plasma. In addition, the
invention relates to the use of human serum albumin binding
compounds in conjunction with camptothecin compounds and other
therapeutic agents that bind to human serum albumin in methods of
treating or enhancing treatments against diseases such as cancer
and/or HIV.
BACKGROUND OF THE INVENTION
[0003] Camptothecin (CPT) has been shown to inhibit the growth of a
variety of animal and human tumors. Camptothecin and its related
congeners display a unique mechanism of action: they stabilize the
covalent binding of the enzyme topoisomerase I (topo I), an
intranuclear enzyme that is overexpressed in a variety of tumor
lines, to DNA. This drug/enzyme/DNA complex leads to reversible,
single strand nicks that, according to the fork collision model,
are converted to irreversible and lethal double strand DNA breaks
during replication. Therefore, due to the mechanism of its
cytotoxicity, CPT is S-phase specific, indicating that it is only
toxic to cells that are undergoing DNA synthesis. Rapidly
replicating cells like cancerous cells spend more time in the
S-phase relative to healthy tissues. Thus, the overexpression of
topo I combined with the faster rate of cell replication provide a
limited basis for selectivity via which camptothecins can effect
cytoxicity on cancerous cells rather than healthy host tissues. It
is important to note that due to the S-phase specificity of the
camptothecins, optimal inhibition of topo I requires continuous
exposure to the camptothecin agent.
[0004] A closed alpha-hydroxy lactone (E) ring of CPT is an
essential structural feature. An intact ring is necessary for the
diffusion of the electroneutral form of the drug across membrane
barriers and into cells by passive transport and, directly relevant
to its in vivo anti-tumor potency, is required for the successful
interaction of CPT with the topoisomerase I target. This essential
lactone pharmacophore hydrolyzes under physiological conditions (pH
7 or above) and, therefore, the drug can exist in two distinct
forms: 1) the biologically active, ring-closed lactone form; and 2)
the biologically-inactive, ring-open carboxylate form of the parent
drug. Unfortunately, under physiological conditions the drug
equilibrium favors hydrolysis and, accordingly, the carboxylate
form of the camptothecin drug persists. The labile nature of this
alpha-hydroxy lactone pharmacophore has significantly compromised
the clinical utility of the camptothecins, as continuous exposures
to the active lactone form are requisite for efficacy purposes.
[0005] In human blood and tissues, the camptothecins exist in a
equilibrium of active lactone form vs. inactive carboxylate form
and the directionality of this equilibrium can be greatly affected
by the presence of human serum albumin (HSA). Time-resolved
fluorescence spectroscopic measurements taken on the intensely
fluorescent camptothecin lactone and camptothecin carboxylate
species have provided direct information on the differential nature
of these interactions with HSA. The lactone form of camptothecin
binds to HSA with moderate affinity yet the carboxylate form of
camptothecin binds tightly to HSA, displaying a 150-fold
enhancement in its affinity for this highly abundant serum protein.
Thus, when the lactone form of camptothecin is added to a solution
containing HSA, the preferential binding of the carboxylate form to
HSA drives the chemical equilibrium to the right, resulting in the
lactone ring hydrolyzing more rapidly and completely than when
camptothecin is in an aqueous solution without HSA. In turn, this
effect has negatively impacted the topoisomerase I inhibitory
activity of many camptothecins and, by extension, negatively
affects their clinical utility.
[0006] The important role that HSA plays in the stability of the
camptothecins varies relative to drug structure. For drugs such as
camptothecin and 9-aminocamptothecin, HSA functions as a biological
sink for the carboxylate form. As a result, in whole human blood,
5.3% of camptothecin and only 0.5% of 9-aminocamptothecin remain in
the lactone form at equilibrium. In contrast, A, B-ring
substitutions of CPT, specifically at the 7- and 10-positions, can
inhibit the preferential binding interactions between the
camptothecin carboxylate and HSA. Accordingly, camptothecin
congeners such as topotecan and SN-38, the biologically active form
of the prodrug CPT-11, display lactone levels at equilibrium of
11.9% and 19.5%, respectively. Ultimately, by modulating the
circulatory and tissue levels of free and active camptothecin drug,
HSA can negatively impact the anti-cancer efficacy of the
camptothecin agent.
[0007] The effect of serum albumins on camptothecins also differs
markedly between lower vertebrates and humans and this variance has
obscured the judicious selection of analogs for advancement to
clinical trials. These interspecies difference have lead to
significant anomalies when the data from animal models and clinical
studies are compared. In particular, 9-aminocamptothecin has
displayed striking activity in murine models bearing brain tumors.
However, the pharmacokinetics of 9-aminocamptothecin in mice are
quite different from those in humans; notably, 9-aminocamptothecin
lactone levels are approximately 100-fold higher in murine blood
relative to human blood. This discrepancy is due to the reduced
binding of the carboxylate form of 9-aminocamptothecin to murine
albumin. The logical extension of this finding is that
approximately 100-fold more free lactone, which is able to cross
cell membranes or the blood-brain barrier, is present in the mouse
than it is in humans. The clinical relevance of this interspecies
variation is underscored by a recent trial: 99 brain cancer
patients were treated intravenously with 9-aminocamptothecin; the
therapy was grossly ineffective (one partial responder) due to the
likelihood that 99.5% of the drug was in the carboxylate form,
bound to HSA and unable to transverse the blood-brain barrier.
[0008] The inherent blood instability of camptothecin has resulted
in an extensive research effort to surmount the problem. Efforts to
realize a blood stable camptothecin agent with potent anti-tumor
activity have been primarily focused on formulation, such as
liposomal preparations of the drug, and rational drug design, such
as the development of the class of beta-hydroxy lactone
camptothecins known as the homocamptothecins. The work described
herein describes a third approach to maintaining a potent and more
blood stable camptothecin congener: the modulation of camptothecin
drug binding to HSA by implementing competing molecules that also
bind HSA.
[0009] The camptothecins are not unique in their ability to bind
albumin, as a variety of small molecules interact with this
protein. A relatively large protein, 67 kD, albumin is distributed
both in the plasma and in the interstitial fluid. Being one of the
most abundant plasma proteins, its circulatory level ranges from 35
to 50 mg/ml (approximately 0.6 mM). The principal biological
function of HSA is to maintain colloid osmotic pressure in the
vascular system and to transport fatty acids and bilirubin.
However, by hydrophobic and/or ionic interactions, a variety of
small molecules bind tightly to albumin. Electroneutral and basic
drugs may bind to albumin by hydrophobic binding interactions, and,
as albumin has a net cationic charge, anionic drugs bind avidly to
albumin via electrostatic interactions. Albumin possesses two
well-characterized binding pockets, as well as other general
binding sites. Site I is known as the warfarin binding site, which
also binds drugs such as phenylbutazone, sulfonamides, phenyloin,
and valproic acid. Site II is referred to as the diazepam site,
which is also the binding site for benzodiazepines, tryptophan,
ibuprofen, naproxen, octanoic acid, clofibric, iopanice,
probenecid, semi-synthetic penicillins and medium chain fatty
acids. Other general binding sites include sites for bilirubin,
digitoxin and a variety of fatty acids. Recent x-ray
crystallography and competition data obtained by the present
inventors reveal that camptothecin carboxylate preferentially
associates with a characterized drug binding site in subdomain IB,
which overlaps with one of the main long-chain fatty acid binding
sites, protoporphyrin and other drugs and compounds, although it
possesses secondary affinity to binding sites I and II.
Interestingly, in vivo small molecule binding to albumin is
saturable at therapeutically relevant drug levels.
[0010] The ability of human serum albumin to avidly bind to a
variety of small molecules offers the possibility of competitively
attenuating the negative effects human serum albumin on the in vivo
anti-cancer and/or anti-HIV activity of camptothecin compounds and
numerous other compounds such as camptothecin that have extremely
high binding affinity for human serum albumin.
[0011] However, no prior methods have recognized or attempted to
deal with the problem caused by the human serum albumin binding
activity, and thus methods and compositions are needed which can
attenuate the negative effects of human serum albumin on the
stability of compounds such as camptothecin compounds, e.g.,
camptothecin or 9-aminocamptothecin, and other compounds or drugs,
such as protease inhibitors, which have a high affinity for human
serum albumin.
SUMMARY OF THE INVENTION
[0012] It is thus an object of the present invention to utilize
human serum albumin binding molecules in a method of achieving
increased stability of compounds, such as camptothecin compounds,
which have a high affinity for human serum albumin and which are
thus generally less effective than optimal when administered in the
human bloodstream.
[0013] It is also an object of the present invention to provide
therapeutic methods of administering compounds such as camptothecin
that have a high affinity for albumin in humans by adding a human
serum albumin binding compound with the ability to bind to one or
more binding sites on human serum albumin so that the compounds
having high affinity for albumin can become more stable when
administered and thus are far more effective than therapeutic drugs
administered without such additive binding compounds.
[0014] It is still further an object of the present invention to
provide a method of treating cancer wherein a camptothecin compound
is administered in conjunction with an appropriate human serum
albumin binding agent.
[0015] It is still further an object of the present invention to
provide a method of treating HIV infection wherein a protease
inhibitor is administered in conjunction with an appropriate human
serum albumin binding agent.
[0016] It is even further an object of the present invention to
provide a wide range of compounds which can effectively be used to
increase and optimize the stability of camptothecin compounds when
administered to humans.
[0017] It is even further an object of the present invention to
provide a method of utilizing an agent which can bind to one or
more sites on human serum albumin and yet which also has anti-tumor
or tumoricidal effects and which can thus be administered in
conjunction with camptothecin compounds so as to even further
enhance the cancer-fighting properties of camptothecin
compounds.
[0018] It is even further an object of the present invention to
provide a method of utilizing an agent which can bind to one or
more sites on human serum albumin and yet which also has anti-HIV
abilities and which can thus be administered in conjunction with
protease inhibitors so as to even further enhance the HIV-fighting
properties of protease inhibitors.
[0019] It is even further an object of the present invention to
provide a method of utilizing an agent which can bind to one or
more sites on human serum albumin and yet which also has anti-HIV
effects and which can thus be administered in conjunction with
camptothecin compounds so as to even further enhance the
HIV-fighting properties of camptothecin compounds.
[0020] These and other objects are achieved via the present
invention which implements combination therapy consisting of
competitor molecules that can bind human serum albumin (HSA) and
thereby inhibit albumin binding of drugs which have a high binding
affinity for human serum albumin, such as camptothecin compounds
and protease inhibitors, and thus increase the effectiveness and
safety of these drugs when administered to humans. This invention
overcomes multiple obstacles that have been associated with
therapies based on drugs such as camptothecin which have high
binding affinity for human albumin. First, as a result of this
binding interaction, the competitor effects elevated free
camptothecin drug levels in human blood and tissues. Secondly, this
invention also overcomes the negative effects of human serum
albumin on the in vivo stability of some camptothecin drugs, such
as camptothecin, 9-aminocamptothecin, and 9-nitrocamptothecin. For
camptothecin, 9-aminocamptothecin, and 9-nitrocamptothecin, it has
been demonstrated that the inactive, carboxylate form of the drug
binds tightly to human serum albumin. This binding promotes a shift
in the lactone/carboxylate equilibrium to favor the formation of
the carboxylate form of the drug. A competitor molecule that
reduces the binding of camptothecin carboxylate to human serum
albumin can shift the drug equilibrium to favor re-lactonization of
the camptothecin agent. As the equilibrium shifts to favor the
formation of the active, lactone form of the camptothecin agent,
the anti-tumor activity of the drug is preserved. Third,
preservation of the electroneutral, lactone form of the
camptothecin agent should enhance the cellular uptake and cellular
concentration of the agent, as on the electroneutral drug species
may transverse the plasma membrane. Thus, the present invention
provides a method for improving camptothecin-based anti-cancer
and/or anti-HIV therapies.
[0021] The competitive displacement of the camptothecin drug can
occur by allosteric inhibition or by direct binding of the small
molecule to the camptothecin binding pocket(s). The camptothecin
agents herein can include camptothecin, 9-nitrocamptothecin,
9-aminocamptothecin, SN-38, the .beta.-hydroxy-.delta.-lactone
camptothecins, such as the homocamptothecins and homosilatecans,
and any other camptothecin agent that physically interacts with
human serum albumin either in its lactone or carboxylate form. The
competing small molecule can include a diverse array of molecular
entities that exhibit a binding affinity for human serum albumin.
Examples include aspirin, ibuprofen, AZT, methotrexate, warfarin,
and the medium chain triglycerides, such as caprylate. The patient
may be administered a single competitor or a series of distinct
competitors, which can be administered individually or as a
mixture. The camptothecin agent and the competitor(s) can be
co-administered or administered separately in order to enhance the
desired therapeutic effect. The camptothecin agent and the
competitor(s) can be administered orally and/or intravenously in
order to enhance the desired therapeutic effect.
[0022] Another important aspect of the present invention is the use
of this type of drug displacement therapy utilizing binding
compounds for albumin for any drug or other beneficial compound
which will be amenable to improvements in safety (by lowering the
effective dose through displacement) or efficacy by allowing a
higher concentration of the active principle during therapeutic
treatment. Accordingly, the present invention also is directed to
the addition of an albumin-binding compound to improve the
effectiveness and/or safety of drugs or therapeutic compounds
which, other than camptothecin, also show binding affinity for
human serum albumin. Of the top 200 pharmaceuticals as of 1999, a
substantial number have high binding affinity for albumin and in
most cases become at least 97% bound to albumin in the human
patient. As a result, the effectiveness of these drugs can be
severely limited in some cases, or far greater doses are necessary
to achieve a desired result, and these inordinately higher doses
almost invariably lead to greater drug side-effects which can often
negate the therapeutic benefit of the drugs.
[0023] The high binding affinity in many drugs for albumin also has
created problems in developing effective new drugs because many
drugs are tested first in vitro or in environments outside the
human body wherein the presence of human serum albumin is not
provided for. As a result, many newly developed drugs work
extremely well in these initial tests, but then are rendered less
effective or entirely useless when administered to human patients
because of their high affinity to human serum albumin which has not
been accounted for. In addition to the camptothecin compounds set
forth in detail herein, numerous other drugs will also be improved
through introduction of albumin binding compounds in accordance
with the present invention, including drugs such as protease
inhibitors which have shown some initial effectiveness in anti-HIV
treatment. In accordance with the present invention, the anti-HIV
treatments that employ protease inhibitors with a high binding
affinity for albumin will be greatly enhance when such treatments
will be administered in conjunction with administration of an
effective amount of the albumin-binding compounds in accordance
with the present invention.
[0024] The present invention thus provides a method of utilizing
the ability of human serum albumin to avidly bind to a variety of
small molecules so as to competitively attenuate or eliminate
negative effects of human serum albumin on the in vivo anti-cancer
and/or anti-HIV capabilities of camptothecin compounds and other
therapeutic compounds such as protease inhibitors which have high
affinity for human serum albumin via one or more binding sites on
serum albumin. Because the human serum albumin binding sites and
their affinity for many small molecules have been well
characterized, many of these small molecules are ideal for in vivo
administration and will be useful in the present invention and can
be utilized when it is necessary to target one or more particular
binding sites. A number of suitable small molecules can thus be
employed as human serum albumin binding competitors to effect the
displacement of camptothecin drugs and compounds, either in the
lactone or carboxylate form, and of other therapeutic compounds,
such as protease inhibitors, which also have high binding affinity
to human serum albumin. Generally, it is contemplated that
treatment with albumin binding compounds in accordance with the
present invention will be particularly effective with those drugs
or other therapeutic compounds that exhibit about 90% or greater
binding with HSA.
[0025] X-ray crystallographic experiments performed using
apparatuses and methods previously described in patents such as
U.S. Pat. No. 4,833,233, U.S. Pat. No. 4,886,646 and U.S. Pat. No.
5,585,466, incorporated herein by reference, have revealed the
camptothecin binding sites to be overlapping with long-chain fatty
acids and ibuprofen consistent with the solution chemistry.
[0026] Inhibiting the binding of the camptothecin agent to human
serum albumin, or other therapeutic compounds to human serum
albumin, will thus enhance free drug levels of that therapeutic
compound in the blood and tissue. Given that a diverse assortment
of small molecules binds to HSA, these small molecules may be
administered singly or as a mixture with the camptothecin agent or
other therapeutic compound to enhance their free drug levels.
Moreover, via inhibiting the binding of the carboxylate form of a
camptothecin drug, a shift in the equilibria occurs that favors the
formation of the biologically active and electroneutral lactone
species. Lastly, as many of these small molecules exhibit
pharmacological activity, they may be utilized dually for their
competitive binding to human serum albumin and for their desired in
vivo effect. Thus, agents such as methotrexate, AZT, and a number
of additional small molecules as set forth below may be used to
enhance the free drug levels of camptothecin or other therapeutic
drugs, such as protease inhibitors, and substantially enhance their
respective biological effects in humans.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1-6 reflect test results with regard to camptothecin
compounds (CPT) and competitor binding agents.
[0028] FIG. 1 is a graphic representation of competitor binding
tests between CPT and ibuprofen.
[0029] FIG. 2 is a graphic representation of competitor binding
tests between hCPT and ibuprofen.
[0030] FIG. 3 is a graphic representation of competitor binding
tests between SN38 and Caprylic Acid.
[0031] FIG. 4 is a graphic representation of competitor binding
tests between CPT and Caprylic Acid.
[0032] FIG. 5 is a graphic representation of competitor binding
tests between SN38 and HSA.
[0033] FIG. 6 is a graphic representation of competitor binding
tests between hCPT and Caprylic Acid.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0034] For the purpose of clarity in the detailed description of
the invention, the following definitions and detailed description
of the invention are provided below.
Hydrolysis of the Camptothecins
[0035] The .beta.-hydroxy-.delta.-lactone members of the
camptothecin class of anti-cancer drugs exhibit the following
chemical equilibrium at pH 7 and above:
##STR00001##
[0036] The electroneutral lactone species, as depicted on the left,
represents the biologically active form of the camptothecin agent.
The carboxylate species, as depicted on the right, represents the
biologically inactive form of the agent. The
.beta.-hydroxy-.delta.-lactone camptothecins, also known as the
homocamptothecins and homosilatecans, also undergo hydrolysis,
however, there is no chemical equilibrium as the reaction is not
reversible under normal physiological conditions. The hydrolysis of
the .beta.-hydroxy-.delta.-lactone camptothecins is detailed
above.
HSA Binding of the Carbon/Late Species
[0037] The carboxylate species of the camptothecin agent may bind
HSA at specific, defined sites, as detailed by crystallographic and
displacement studies, and may also bind directly to HSA at
non-specific sites that have yet to be clearly defined. Binding may
occur by hydrophobic and/or ionic interactions between HSA and the
camptothecin carboxylate form.
HSA Binding of the Lactone Species
[0038] The lactone species of the camptothecin agent may bind HSA
at specific, defined sites, as detailed by crystallographic and
displacement studies, and may also bind directly to HSA at
non-specific sites that have yet to be clearly defined. Binding
between HSA and the camptothecin lactone form may occur by
non-covalent means.
HSA Binding of the Competitor
[0039] The competitor may bind to HSA at specific, defined sites,
as detailed by crystallographic and displacement studies, and may
also bind HSA at non-specific sites that have yet to be clearly
defined. Binding between the competitor and HSA may occur by
covalent or non-covalent mechanisms.
GENERAL DEFINITIONS
[0040] Before the present compositions and methods are disclosed
and described, it is to be understood that this invention is not
limited to specific drugs, human serum albumin selective ligands,
pharmaceutical carriers, or administration regimens, as such may,
of course, vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only and is not intended to be limiting.
[0041] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reverence to a "a pharmacologically active
agent" includes mixtures of two or more such ligands, and the
like.
[0042] By the term "pharmacologically active agent" or "drug" as
used herein is meant any chemical material or compound suitable for
administration to a mammalian, preferably human, individual, which
induces a desired local or systemic effect. In general, this
includes: anorexics; anti-infectives such as antibiotics and
antiviral agents, including many penicillins and cephalosporins;
analgesics and analgesic combinations, antiarrythmics;
antiarthritics; antiasthmatic agents; anticholinergics;
anticonvulsants; antidiabetic agents; antidiarrheals;
antihelminthics, antihistamines; anti-inflammatory agents;
anti-migraine preparations; antinasuseants; antineoplastics;
antiparkinsonism drugs; antipruritics; antipsychotics;
antipyretics; antisense agents; antispasmodics; cardiovascular
preparations including calcium channel blockers and beta-blockers
such as pindolol; antihypertensives; central nervous system
stimulants; cough and cold preparations, including decongestants;
diuretics; gastrointestinal drugs; sympathomimetics; hormones such
as estradiol and steroids; hypnotics; immunosuppressives; muscle
relaxants; parasympatholytics; psychostimulatants; sedatives;
tranquilizers; vasodilators including general coronary, peripheral
and cerebral; xanthine derivatives.
[0043] As used herein, the term "competitor" refers to a chemical
material or pharmacologically active agent suitable for
administration to a mammalian, preferably human. The competitor
exhibits binding affinity for serum albumin and, in general,
includes: long chain fatty acids (C.sub.16-C.sub.20; including
oleic, palmitic, linoleic, stearic, arachidonic, and palmitoleic);
medium chain fatty acids (C.sub.6-C.sub.14; including caprylate or
octanoate); phospholipids (lysolecithins, oleoyllysophosphatidic
acid, phosphatidylcholine, phosphatidylethanolamine); eicosanoid
derivatives (leukotrienes, thromboxanes, prostaglandins A, E, F,
and I); steroid hormones (cholesterol, testosterone, pregnenolone,
cortisol, androsterone, indol, progesterone, estrogen); vitamin D
(both monohydroxyvitamin D and dihydroxyvitamin D); bile salts
(lithocholate, chenodeoxycholate, deoxycholate, ursodeoxycholate,
cholate, glycolitocholate, glycochenodeoxycholate,
taurochenodoxycholate, glycodeoxycholate, glycocholate,
taurocholate); bilirubins (bilirubin, biliverdin, xanthobilirubin,
EZ-cyclobilirubin, .delta.-bilirubin); porphyrins (hematin,
protoporphyrin); warfarin; salicylates, ibuprofen; prednisone;
iophenoxate; sulfisoxazole; phenylbutazone; oxphenylbutazone;
digitoxin; indomethacin; tolbutamide; furosemide; phenyloin;
chlorpropamide; chlorthiazide; the penicillins (including
oxacillin, benzylpenicillin); acetotrizoate; isulfobromophthalein;
deacetylcolchicine; dansylamide; dansylglutamine; dansylsarcosine;
indomethacin; phenylpropazone; azobenzene derivatives;
sulfobromophthalein; triiodobenzoate; benzodiazepine (including
diazepam); flufenamate; iopanoate; ethacrynate; panproxen;
clofibrate; L-tryptophan; N-acetyl-L-tryptophan;
6-methyltryptophan; thyroxine; 3,5,3'-L-triiodothyronine; indole
propionate; kynurenine; ethacrynate; panproxen;
chlorophenoxyisobutyrate; 3' azido-3'-deoxythymidine; non-steroidal
anti-inflammatory agents containing ionized carboxyl groups;
gossypol; meso-2,3-dimercaptosuccinic acid; captopril;
N2-mercaptoethyl-1,2-diaminopropane; disulfuramacetaminophen,
dis-dichlorodiamineplatinum 9II; pyridoxal 5'-phosphate;
aquocobalamin form of vitamin B12; folate; ascorbate (and its
oxidation product dehydroascorbate); melatonin;
.alpha.-melanotropin; gastrin; corticotropin and methotrexate.
[0044] An "effective amount" of a pharmacologically active agent is
intended to mean a nontoxic but sufficient amount of the agent,
such that the desired prophylactic or therapeutic effect is
produced. As will be pointed out below, the exact amount of a
particular agent that is required will vary from subject to
subject, depending on the species, age, and general condition of
the subject, the severity of the condition being treated, the
particular drug used and its mode of administration, and the like.
In addition, other factors, such as an assay of patient albumin
levels prior to administering the therapy and adjusting the drug
levels accordingly is often utilized to properly set a treatment
regiment for a particular patient. Thus, it is not possible to
specify an exact "effective amount" of any particular
pharmacologically active agent. However, an appropriate effective
amount may be determined for any particular drug by one of ordinary
skill in the art using only routine experimentation.
[0045] By the term "pharmaceutically acceptable" to describe a
carrier or excipient is meant a material that is not biologically
or otherwise undesirable, i.e., the material may be administered
along with the selected pharmacologically active agent without
causing any desirable biological effects or interacting in a
deleterious manner with any of the other components of the
pharmaceutical composition in which it is contained.
[0046] The term "camptothecin drug" or "camptothecin compound" is
inclusive of camptothecins that contain either an E-ring
.alpha.-hydroxy lactone pharmacophore or an E-ring .beta.-hydroxy
lactone pharmacophore, which includes the homocamptothecins and
homosilatecans. As used herein, the camptothecin analogs
9-aminocamptothecin, 10-hydroxycamptothecin,
10,11-methylenedioxy-camptothecin,
9-nitro-10,11-methylenedioxycamptothecin,
9-chloro-10,11-methylenedioxycamptothecin,
9-amino-10,11-methylenedioxycamptothecin, 9-nitrocamptothecin,
topotecan, and other analogs of camptothecin, are collectively
referred to as camptothecin drugs or compounds.
DESCRIPTION OF THE INVENTION
[0047] The present invention accomplishes multiple tasks. First,
administration of a HSA binding competitor elevates free
camptothecin levels in blood and human tissues by inhibiting
camptothecin drugs from binding to human serum albumin. Secondly,
for those camptothecin drugs that bind human serum albumin in the
carboxylate form, this invention induces a shift in the lactone
carboxylate equilibrium that enhances in vivo drug lactone levels.
Third, Enhanced free drug levels and elevated lactone levels in
vivo result in greater cellular uptake and enhanced activity.
[0048] Under physiological conditions, the camptothecin drug exists
in a equilibrium of the active lactone and inactive carboxylate
forms. In human blood and tissues, binding of the camptothecin drug
to human serum albumin can occur when said drug is either in the
biologically inactive, carboxylate form or in the biologically
active, lactone form. Camptothecin, 9-aminocamptothecin, and
9-nitrocamptothecin bind human serum albumin predominantly in the
carboxylate form. In contrast, SN-38, the biologically active agent
of the camptothecin prodrug CPT-11, binds human serum albumin in
the lactone form. Binding of the camptothecin drug, whether in the
carboxylate or lactone form, reduces the levels of free drug in the
blood and tissue.
[0049] As described above, the present invention relates to the use
of human serum albumin binding molecules which are administered in
conjunction with camptothecin compounds in order to achieve greater
stability in the human bloodstream and thus allow for the
camptothecin compounds to be more effective when administered in
human treatment regimens. The invention thus contemplates
therapeutic methods, such as methods to treat diseases such as
cancer or HIV, wherein camptothecin compounds are administered in
humans in conjunction with a suitable human serum albumin binding
compound. Even further, it is contemplated that the human serum
albumin binding compound selected for use in accordance with the
present invention will ideally be one that additionally enhances
the effect of the free camptothecin compounds. In this regard,
agents such as methotrexate, AZT, and a number of additional small
molecules as set forth below may be used to enhance free
camptothecin drug levels and substantially enhance their respective
biological effects in humans.
[0050] The following is a list of molecules that bind human serum
albumin and are thus contemplated for use in accordance with the
present invention:
Long Chain Fatty Acids (C.sub.16-C.sub.20):
[0051] Oleic, palmitic, linoleic, stearic, arachidonic, and
palmitoleic Note for fatty acids, at pH 7 they exist as salts, and
thus may more accurately be defined not as palmitic acid but as
palmitate.
Medium Chain Fatty Acids (C.sub.6-C.sub.14):
Phospholipids:
[0052] Lysolecithins, oleoyllysophosphatidic acid,
phosphatidylcholine, phosphatidylethanolamine
Eicosanoid Derivatives:
[0053] Leukotrienes, thromboxanes, prostaglandins A, E, F, and
I
Steroid Hormones:
[0054] Cholesterol, testosterone, pregnenolone, cortisol,
androsterone, indol, progesterone, estrogen
Vitamin D: Both Monohydroxyvitamin D and Dihydroxyvitamin D.
Bile Salts: Lithocholate, Chenodeoxycholate, Deoxycholate,
Ursodeoxycholate, Cholate, Glycolitocholate,
Glycochenodeoxycholate, Taurochenodoxycholate, Glycodeoxycholate,
Glycocholate, Taurocholate
[0055] Bilirubins: bilirubin, biliverdin, xanthobilirubin,
EZ-cyclobilirubin, .delta.-bilirubin Gossypol (note high affinity
1.1 e-7, competes with bilirubin, antibiotic, promotes fertility)
Porphyrins: hematin, protoporphyrin Site I Ligands (domain IIA):
bilirubin, warfarin, salicylates, cyclic eicosanoids, hematin,
.omega.-dicarboxylic medium-chain fatty acids, long-chain fatty
acids, prednisone, iophenoxate (eliminated slowly due to extremely
high affinity, contrast agent), salicylates, sulfisoxazole,
warfarinS-, phenylbutazone, digitoxin, indomethacin, tolbutamide,
furosemide, phenyloin, chlorpropamide, chlorthiazide, oxacillin,
benzylpenicillin, acetotrizoate, phenol red, bromcresol green,
brophenol blue, isulfobromophthalein, methyl orange, methyl red,
evans blue, deacetylcolchicine, Phenol red, dansylalmide,
dansylglutamine, dansylsarcosine, indomethacin, phenylpropazone,
bromcresol purple, azobenzene derivatives, sulfobromophthalein,
triiodobenzoate, cibacron blue, various penicillins,
benzodiazepine, Site II Ligands (subdomain IIA): monocarboxylic
medium-chain fatty acids (C.sub.6-C.sub.14; in particular
octanoate), diazepam (the 2,3-benzodiazepines), flufenamate,
iopanoate, ethacrynate, panproxen, chlorophenoxyisobutyrate
(clofibrate), L-tryptophan, octanoate, thyroxine,
N-Acetyl-L-tryptophan, indole propionate, kynurenine,
6-methyltryptophan, 3, 5, 3'-L-triiodothyronine, triiodobenzoate,
ibuprofen, chloride ions, AZT (3' azido-3'-deoxythymidine,
non-steroidal anti-inflammatory agents containing ionized carboxyl
groups (Li et al., 1988; Wanwimolruk et al., 1991),
oxphenylbutazone Ligands at CySH-34: penicillamine,
meso-2,3-dimercaptosuccinic acid, captopril
(N-2-mercaptoethyl-1,2-diaminopropane, disulfuramacetaminophen,
cisdichlorodiammine-platinum9II) Miscellaneous: pyridoxal
5'-phosphate, Aquocobalamin form of vitamin B12, folate, ascorbate
and its oxidation product dehydroascorbate, melatonin,
a-melanotropin, gastrin, corticotropin, calcium, nickel, magnesium,
and copper
[0056] It is noted that the binding of some of these molecules to
human serum albumin may be readily followed by detection procedures
well known in the field. For example, binding of tryptophan may
easily be followed by fluorescence. In addition, different ligands
may either increase or decrease the affinity of a second ligand for
albumin to the extent multiple ligands are used.
[0057] In accordance with the invention, the above human serum
albumin binding compounds may be utilized in conjunction with human
therapies which can utilize camptothecins, and these albumin
binding compounds inhibit binding of camptothecin compounds to
human serum albumin present in human blood and plasma, which frees
the camptothecin drug for therapeutic purposes. In addition, it is
contemplated that the methods of the present invention may involve
administration of a cocktail on one or more of these binders, or a
single competing binding agent may be administered as needed.
[0058] It is also contemplated that these albumin binding compounds
may be administered before, during, or after administration of the
camptothecin agent. It is also contemplated that any camptothecin
agent that binds albumin, regardless of the effect albumin has on
the agent, will still be useful in accordance with the invention
since one goal of the therapy is to raise the vascular and tissue
levels of total free drug, and this goal will still be achieved
even if albumin has an effect on the agent
[0059] The present invention thus provides a method of utilizing
the ability of human serum albumin to avidly bind to a variety of
small molecules so as to competitively attenuate negative effects
of human serum albumin on the in vivo camptothecin compounds'
anti-cancer and anti-HIV activities. Because the human serum
albumin binding site and affinity for many small molecules have
been well characterized, many of these small molecules are ideal
for in vivo administration and will be useful in the present
invention and can be utilized when it is necessary to target
particular binding sites. A number of suitable small molecules such
as those described above can thus be employed as human serum
albumin binding competitors to effect the displacement of
camptothecin drugs and compounds, either in the lactone or
carboxylate form.
[0060] In accordance with the invention, the inhibition of the
binding of the camptothecin agent to human serum albumin will thus
enhance free drug levels in the blood and tissue. Given that a
diverse assortment of small molecules binds to HSA, these small
molecules may be administered singly or as a mixture with the
camptothecin agent or compound to enhance their free drug levels.
Additionally, as many of these small molecules exhibit
pharmacological activity, it is also contemplated they may be
utilized dually for their competitive binding to human serum
albumin and for their desired in vivo effect. Thus, agents such as
methotrexate, AZT, and a number of additional small molecules which
have therapeutic effects apart from their ability to bind human
serum albumin are preferably used in accordance with the invention
to even further enhance free camptothecin drug levels and
substantially improve their respective biological effects in
humans. These biological effects include their use as anti-cancer
and/or anti-HIV agents, as well as their use in any other
anti-topoisomerase I-based therapy.
[0061] The following examples are provided only to exemplify
various aspects of the preferred embodiments of the present
invention. It will thus be appreciated by those of skill in the art
that the techniques and compositions disclosed in the examples
which follow represent techniques and compositions discovered by
the inventors to function well in the practice of the invention,
and thus can be considered to constitute preferred modes for its
practice.
[0062] However, those of skill in the art will also appreciate that
the following examples are only exemplary aspects of the present
invention, the scope of which is defined by the claims appended
hereto, and thus many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
EXAMPLES
Example 1
HSA/Competition Experiments by Fluorescence Spectroscopic
Methods
Materials and Methods:
[0063] The camptothecin used in the experiments was obtained from
Boehringer Ingelhem (Lot#95-002). Dimethyl Sulfoxide (HPLC grade,
Aldrich, Milwaukee, Wis.) was used to prepare stock solutions of
camptothecin at various concentrations, which were stored in the
dark at -20.degree. C. Working solutions of 1.0.times.10.sup.-3 M
camptothecin carboxylate and camptothecin lactone were prepared by
diluting a stock solution of camptothecin in DMSO with PBS buffer
at pH values of 10.0 and 3.0, respectively. The Sigma Chemical Co.
(St. Louis, Mo.) supplied the human serum albumin (HSA) employed in
the binding experiments. A 2.5.times.10.sup.-3 M stock solution of
HSA was prepared in PBS buffer at a final pH of 7.40.+-.0.05. The
concentration of the HSA was determined on a weight-to-volume basis
(g/L). A Milli-Q UV PLUS purification system (Bedford, Mass.) was
used to acquire high-purity water.
[0064] For the competition binding experiments, 3.0.times.10.sup.-3
M camptothecin carboxylate and 1.0.times.10.sup.-3 M
homocamptothecin working solutions were prepared. Caprylic acid
obtained from Sigma Chemical Co. (Lot#72HO473) was one of the
competitive binders analyzed. Five different stock solutions of
varied caprylic acid concentration were made to satisfy
concentration specifications discussed below. Another competitive
binder studied was Ibuprofen obtained from Sigma Chemical Co.
(Lot#13HO7511). Four different stock solutions of varied ibuprofen
concentration were prepared. Both caprylic acid and ibuprofen stock
solutions were made-up in PBS buffer at a final pH of
7.40.+-.0.05.
Fluorescence Spectroscopy:
[0065] Steady-state fluorescence anisotropy measurements were
recorded using a SLM 9850 fluorometer interfaced with an IBM
computer. The samples were excited at an excitation wavelength of
370 nm by implementing an argon ion laser. The excitation
monochromator bandwidth was set to 4 nm. Fluorescence emission was
isolated from scattered light by utilizing 400 nm long band-pass
filters.
[0066] For the camptothecin and homocamptothecin binding
experiments with HSA, fourteen test tubes of varied HSA
concentration were prepared. Volumes of the 2.5.times.10.sup.-3M
HSA stock and PBS buffer pH of 7.40.+-.0.05 were combined in
fourteen test tubes to create different HSA concentrations ranging
from 0.5.times.10.sup.-6 M to 1.8.times.10.sup.-4 M. The test tubes
were placed in a VWR Scientific Waterbath (Model 1235) set at
37.degree. C. for approximately five minutes. Following this, the
first test tube was removed and a 5.0.times.10.sup.-6 M
concentration of the drug was prepared by adding an appropriate
volume of the 1.0.times.10.sup.-3M camptothecin or homocamptothecin
(37.degree. C.) working solution to the test tube. The drug and HSA
solution was immediately vortexed on a Vortex Genie 2.TM. from
Fisher Scientific for approximately three to five seconds.
Immediately after, the solution was transferred to a thermostatic
(37.degree. C.) sample cell and the anisotropy measurement was
recorded. The same procedure was followed for the remaining
thirteen test tubes. For each tube, the anisotropy measurement was
recorded within one minute upon the addition of the drug. This
short acquisition time secured that the anisotropy measurements
reflected the initial form of the drug added instead of a
lactone-carboxylate equilibrium form. The results of the
camptothecin and homocamptothecin HSA binding experiments can be
seen in the Figures.
[0067] The procedure followed for the competition binding
experiments was very similar to the description above. A
3.0.times.10.sup.-3M camptothecin carboxylate working solution was
prepared and kept at 37.degree. C. Only ten of the fourteen test
tubes described above were prepared. The HSA concentration varied
from 5.0.times.10.sup.-6 to 7.5.times.10.sup.-5 M. The maximum HSA
concentration was reduced due to background fluorescence present
from the HSA. Once the HSA/PBS solutions were prepared, an
appropriate volume of a competitor stock was added to each tube.
The competitor concentration was identical for all ten test tubes.
Caprylic acid competition concentrations of 1.0.times.10.sup.-4 M,
1.0.times.10.sup.-3 M, 5.0.times.10.sup.-3 M, 1.0.times.10.sup.-2 M
and 5.0.times.10.sup.-2 M were studied using the stock solutions
discussed earlier. The same competition concentrations for
Ibuprofen were studied excluding the 1.0.times.10.sup.-2M. Once the
competitor was added, the test tubes were placed in the waterbath,
like before, and the measurements were taken by employing the same
technique described for the HSA binding experiment. The results for
the caprylic acid and ibuprofen competition binding with
camptothecin carboxylate are shown on Figures CPT/HSA and CPT:
Caprylic Acid/HSA and CPT/HSA and CPT: Ibuprofen/HSA,
respectively.
[0068] The homocamptothecin carboxylate competition experiments
were carried out using a 1.0.times.10.sup.-3 M homocamptothecin
carboxylate working solution at 37.degree. C. Ten test tubes were
prepared using the same procedure described for the camptothecin
competition experiments. Caprylic acid and ibuprofen competition
concentrations of 1.0.times.10.sup.-4M, 1.0.times.10.sup.-3M,
5.0.times.10.sup.-3M, and 5.0.times.10.sup.-2M were studied.
Homocamptothecin carboxylate competition results are shown in
Figures hCPT/HSA and hCPT: Caprylic Acid/HSA and hCPT/HSA and hCPT:
Ibuprofen/HSA, respectively.
[0069] Background fluorescence from the HSA was detected in all of
the experiments. In the camptothecin carboxylate competition
experiments with caprylic acid and ibuprofen, the maximum scattered
light detected was 8% and 5%, respectively. The homocamptothecin
carboxylate competition experiments displayed higher values of
maximum scatter equal to 13% and 15% for the caprylic acid and
ibuprofen competition. In all cases, the percent of scattered light
decreased with increasing competition concentration.
Example 2
Procedure of Competition Binding and Stability of 9AC, DB172, DB67
and SN38 with the Presence of Various Drugs
1. Materials
[0070] Samples of 9AC, DB67, DB172 and SN38 were obtained from
various sources. Human serum albumin (HSA) was purchased from Sigma
Chemical (St. Louis, Mo.). Recovered human plasma was obtained from
Central Kentucky Blood Center (Lexington, Ky.) and stored at
-20.degree. C. Whole human blood was obtained from a healthy male
donor by drawing blood into sterile vacutainers containing heparin,
to prevent clot formation. Ultrafiltration tubes were purchased
from Millipore. (Centrifree; MW cutoff 30,000). Triethylamine and
HPLC-grade acetonitrile was purchased from Fisher Scientific (Fair
Lawn, N.J., USA). High purity water was provided by a Milli-Q UV
Plus purification system (Millipore, Bedford, Mass., USA). Stock
solutions of each drug were prepared in A.C.S. spectrophotometric
grade dimethylsulfoxide (DMSO; Aldrich, Milwaukee, Wis., USA) at a
concentration of 2.times.10.sup.-3M and stored in the dark at
-20.degree. C. until use. Phosphate buffered saline (PBS, pH 7.4)
refers to an aqueous solution of 8 mM dibasic sodium phosphate
(Na.sub.2HPO.sub.4), 1 mM potassium phosphate monobasic crystal
(KH.sub.2PO.sub.4), 137 mM sodium chloride (NaCl) and 3 mM
potassium chloride (KCl).
2. HPLC Apparatus:
[0071] All HPLC analyses were carried out on a Waters Alliance 2690
Separations Module equipped with a Waters.TM. 474 Scanning
fluorescence Detector, All separations were carried out on a Waters
Symmetry.RTM. C.sub.18 5 .mu.m 3.9.times.150 mm column with a
Waters Symmetry.RTM. C.sub.18 5 .mu.m 3.9.times.20 mm guard column.
For the separation of 9AC, which is higher fluorescence at low pH
and the acidification of mobile phase before separation will change
9AC carboxylate form to lactone form, the postcolumn acidification
was employed. The postcolumn acidification can separation
carboxylate and lactone before acidify and acidify the mobile phase
by pump 0.5 N HCl at a flow rate 0.5 ml/min before the drug goes
into the detector. A Xterra.TM. MS C.sub.18 5 .mu.m 3.9.times.150
mm column (stable at low and high pH) was used for postcolumn
acidifiation to stable the baseline. For the separation of 9AC,
mobile phase consisted of 20% acetonitrile and 80% of an aqueous
buffer containing triethylamine and acetate. The
triethylamine/acetate buffer (pH5.5) contained 2% triethylamine
added to distilled, deionized water with pH adjustment to 5.5 made
with concentrated acetic acid. Fluorescence excitation for 9AC was
set at 380 nm and emission at 450 nm. For DB172, the mobile phase
consisted of 57% acetonitrile and 43% triethylamine/acetate buffer.
Excitation and emission detectors settings of 371 nm and 428 nm,
respectively, were used. For DB67, the mobile phase consisted of
41% acetonitrile and 59% triethylamine/acetate buffer. Excitation
and emission detectors settings of 380 nm and 560 nm, respectively,
were used. For SN38, the mobile phase consisted of 25% acetonitrile
and 75% triethylamine/acetate buffer. Excitation and emission
detectors settings of 383 nm and 560 nm, respectively, were used.
Flow rates of 1 min/ml were used in all experiments. The mobile
phase was degassed by filter through a membrane filter (0.45 .mu.m,
Millipore). Fluorescence output signal was monitored and integrated
using Millennium.sup.32 Chromatography Manager software.
3. Protein Binding Studies:
Preparation of Standard Solution
[0072] A stock solution containing 2 mM of the drug of the interest
in DMSO was prepared and stored at -20.degree. C. For 9AC and DB67,
an aliquot of this stock was added to PBS pH 10.0 to form 100 .mu.M
carboxylate standard solutions.
Protein binding studies of 9-AC carboxylate
[0073] The present studies determined the protein binding to the
carboxylate form of 9-AC. Initial experiments were run to determine
the amount of 9-AC carboxylate lost during protein binding studies
due to adhesion to the ultrafiltration membrane. PBS (990 .mu.l) at
pH 7.4 was spiked with 9-AC carboxylate to form 1 .mu.M 9-AC
solution. After vortexing for 30 seconds, 500 .mu.l of the solution
was transferred to an ultrafiltration device and centrifuged for 15
minutes at 4500 rpm. A 100 .mu.l aliquot of the filtrate was added
to 600 .mu.l ice-cold methanol and vortexed. A 500 .mu.l aliquot of
the supernatant was removed and mixed with 25 .mu.l 12 N HCl. The
suspension was mixed with 1 ml of water, vortexed, and injected
(1000) onto the HPLC. The same protocol was repeated with 100 .mu.l
total (1 .mu.M 9-AC before ultrafiltration). The percentage
recovery was obtained: the filtrate concentration divided by the
total concentration.
Protein Binding of 9-AC Carboxylate to HSA (30 Mg/Ml), Human Plasma
with or without the Presence of Various Drugs.
[0074] Protein binding studies using HSA, human plasma with and
without the presence of various drugs were conducted in a similar
manner. HSA were prepared with PBS (pH 7.4). A mount of different
drug (phenylbutazone, ibuprofen, caprylic acid, aspirin,
warfarin-Na salt, L-tryptophan) was added to HSA or human plasma to
form different concentration of drug, In the test tube, 990 .mu.l
HSA solution or human plasma with or without various was spiked
with 9-AC carboxylate to form 1 .mu.M 9-AC solution. After
vortexing for 30 seconds, 500 .mu.l of the solution was transferred
to an ultrafiltration device and centrifuged for 15 minutes at 4500
rpm. A 100 .mu.l aliquot of the filtrate was added to a 600 .mu.l
ice-cold methanol, vortexed and centrifuged at 8000 rpm for 30
seconds. A 500 .mu.l aliquot of the supernatant was removed and
mixed with 25 .mu.l 12 N HCl. Subsequently, 1 ml of water was added
to the suspension and the mixture was vortexed and injected (100
.mu.l) onto the HPLC. The same protocol was repeated with 100 .mu.l
total (1 .mu.M 9-AC before ultrafiltration). The total drug
concentration was corrected for the apparent drug loss due to
adsorption of the drug to the ultrafiltration membrane using the
equation: Corrected total concentration=determined total
concentration.times.the percentage recovery.
[0075] The bound concentration was obtained by calculating
difference: corrected total concentration minus free concentration.
All experiments were run in triplicate.
Protein Binding Studies of Db67 Carboxylate
[0076] The present studies determined the protein binding to the
carboxylate form of DB67. Initial experiments were run to determine
the amount of DB67 carboxylate lost during protein binding studies
due to adhesion to the ultrafiltration membrane. PBS (990 .mu.l) at
pH 7.4 was spiked with DB67 carboxylate to form 1 .mu.M DB67
solution. After vortexing for 30 seconds, 500 .mu.l of the solution
was transferred to an ultrafiltration device and centrifuged for 15
minutes at 4500 rpm. A 100 .mu.l aliquot of the filtrate was added
to 600 .mu.l ice-cold methanol and vortexed. A 500 .mu.l aliquot of
the supernatant was removed diluted with 500 .mu.l PBS (pH 12) and
injected (10 .mu.l) onto the HPLC. The same protocol was repeated
with 100 .mu.l total (1 .mu.M DB67 before ultrafiltration). The
percentage recovery was obtained: the filtrate concentration
divided by the total concentration.
Protein Binding of Db67 Carboxylate to HSA (30 Mg/Ml), Human Plasma
with or without the Presence of Caprylic Acid
[0077] Protein binding studies using HSA, human plasma with and
without the presence of various drugs were conducted in a similar
manner. HSA were prepared with PBS (pH 7.4). A mount of caprylic
acid was added to HSA or human plasma to form different
concentration of drug, In the test tube, 990 .mu.l HSA solution or
human plasma with or without various was spiked with DB67
carboxylate to form 1 .mu.M DB67 solution. After vortexing for 30
seconds, 500 .mu.l of the solution was transferred to an
ultrafiltration device and centrifuged for 15 minutes at 4500 rpm.
A 100 .mu.l aliquot of the filtrate was added to a 600 .mu.l
ice-cold methanol, vortexed and centrifuged at 8000 rpm for 30
seconds. A 500 .mu.l aliquot of the supernatant was removed and
diluted with 500 .mu.l PBS (pH 12) and injected (10 .mu.l) onto the
HPLC. The same protocol was repeated with 100 .mu.l total (1 .mu.M
DB67 before ultrafiltration). The total drug concentration was
corrected for the apparent drug loss due to adsorption of the drug
to the ultrafiltration membrane using the equation:
Corrected total concentration=determined total
concentration.times.the percentage recovery.
[0078] The bound concentration was obtained by calculating
difference: corrected total concentration minus free concentration.
All experiments were run in triplicate.
Lactone Stability Studies
Lactone and Carboxylate Peak Area Ratio
[0079] A stock solution containing 2 mM of interested drug was
prepared and stored at -20.degree. C. The stock solution was
diluted 5-time with DMSO to form 0.4 mM stock. 2 .mu.l 0.4 mM stock
was added to 798 .mu.l DMSO to form 1 .mu.M Lactone form, or added
to 798 .mu.l PBS pH 10.0 to form 1 .mu.M carboxylate form, and
injected onto the column. The ratio of molar fluorescence
intensities of the lactone to carboxylate form (k) is calculated as
following:
Lactone/Carboxylate Ratio (k)=Average Peak Area of Lactone/Average
Peak Area of Carboxylate
Stability Study of 9AC, DB67, DB172 and SN38 in Human Whole Blood,
HSA or Human Plasma with or without Caprylic Acid
[0080] Weigh amount of caprylic acid and added to HSA, human plasma
and human whole blood to form a certain concentration of caprylic
acid (1 mM, 2 mM, 10 mM, 25 mM, 50 mM and 100 mM). For HSA and
human plasma, incubate the HSA or human plasma with or without
caprylic acid at 37.degree. C. and adjust pH to 7.4. For human
whole blood, it will form participate with caprylic acid when
adjust pH with HCl or NaOH. So, first adjust pH a little below 7.4
and then add caprylic acid to form pH 7.4 with caprylic acid in
whole blood. A 5 .mu.l 0.4 mM interest drug solution was added to
1995 .mu.l of HSA human plasma or human whole blood that had
previous been incubated at 37.degree. C. and adjusted to pH 7.4 to
form a 1 .mu.M solution. At each respective time interval, a 150
.mu.l volume was removed from the incubation tube and added to 600
.mu.l of ice-cold methanol (-20.degree. C.), vortex-mixed for 20s
and centrifuged at 4000 g for 1 min. The supernatant was directly
injected onto the HPLC column immediately. Aliquots were taken and
HPLC analyses was performed at times of 1, 10, 20, 30, 60, 120 and
180 minutes, respectively. The fraction of lactone form was
calculated as:
Fraction of lactone=lactone area/(lactone area+carboxylate area*k),
where k is the response factor defined as the ratio of molar
fluorescence intensities of the lactone to carboxylate form.
[0081] In the Tables appended hereto, competition binding and
stability of 9AC, DB172, DB67 and SN38 with the presence of various
drugs is shown, including Table 1.1 (Protein binding of 9AC
carboxylate (1 .mu.M) in HSA and human plasma) and Table 1.2
(Protein binding of DB67 carboxylate (1 .mu.M) in HSA and human
plasma).
Competition Binding and Stability of 9AC, DB172, DB67 and SN38 with
the Presence of Various Drugs
1. Competition Binding
1.1 Protein Binding of 9AC Carboxylate (1 .mu.M) in HSA and Human
Plasma
TABLE-US-00001 [0082] Matrix Compound added Percent 9AC bound HSA
(1 mg/ml) No 94.26 .+-. 0.25 HSA (1 mg/ml) Phenylbutazone (0.162
mM) 81.52 .+-. 1.16 HSA (1 mg/ml) Ibuprofen (480 mM) 0.00 .+-. 0.00
HSA (1 mg/ml) Caprylic acid (347 mM) 0.00 .+-. 0.00 HSA (30 mg/ml)
No 100.00 .+-. 0.00 HSA (30 mg/ml) Ibuprofen (10 mM) 24.82 .+-.
0.99 HSA (30 mg/ml) Caprylic acid (10 mM) 83.34 .+-. 0.88 Human
plasma No 100.00 .+-. 0.00 Human plasma Phenylbutazone (0.2 mM)
99.90 .+-. 0.04 Human plasma Aspirin (10 mM) 97.98 .+-. 0.12 Human
plasma Warfarin-Na salt (10 mM) 86.44 .+-. 0.67 Human plasma
L-Tryptophan (10 mM) 99.59 .+-. 0.03 Human plasma Ibuprofen (100
mM) 0.00 .+-. 0.00 Human plasma Ibuprofen (10 mM) 43.06 .+-. 0.76
Human plasma Ibuprofen (1 mM) 99.82 .+-. 0.04 Human plasma Caprylic
acid (100 mM) 0.00 .+-. 0.00 Human plasma Caprylic acid (80 mM)
15.05 .+-. 3.17 Human plasma Caprylic acid (60 mM) 5.14 .+-. 2.38
Human plasma Caprylic acid (40 mM) 22.73 .+-. 1.32 Human plasma
Caprylic acid (20 mM) 74.04 .+-. 1.82 Human plasma Caprylic acid
(10 mM) 89.62 .+-. 0.18 Human plasma Caprylic acid (1 mM) 99.86
.+-. 0.04
1.2 Protein Binding of DB67 Carboxylate (1 .mu.M) in HSA and Human
Plasma
TABLE-US-00002 [0083] Matrix Compound added Percent DB67 bound HSA
(30 mg/ml) No 99.22 .+-. 0.23 HSA (30 mg/ml) Caprylic acid (100 mM)
39.09 .+-. 0.88 HSA (30 mg/ml) Caprylic acid (10 mM) 75.52 .+-.
0.69 Human plasma No 98.58 .+-. 0.09 Human plasma Caprylic acid
(100 mM) 53.39 .+-. 1.64 Human plasma Caprylic acid (10 mM) 78.81
.+-. 0.31 Human plasma Caprylic acid (1 mM) 96.40 .+-. 0.06
* * * * *