U.S. patent application number 10/788649 was filed with the patent office on 2004-09-02 for liposomal antineoplastic drugs and uses thereof.
This patent application is currently assigned to Inex Pharmaceuticals Corporation. Invention is credited to Ahkong, Quet F., Madden, Thomas D., Semple, Sean C..
Application Number | 20040170678 10/788649 |
Document ID | / |
Family ID | 26910152 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040170678 |
Kind Code |
A1 |
Madden, Thomas D. ; et
al. |
September 2, 2004 |
Liposomal antineoplastic drugs and uses thereof
Abstract
This invention relates to liposomal antineoplastic agents (e.g.,
camptothecin) compositions and methods of using such compositions
for treating neoplasia and for inhibiting angiogenesis. The
compositions and methods are useful for modulating the plasma
circulation half-life of an active agent.
Inventors: |
Madden, Thomas D.;
(Vancouver, CA) ; Semple, Sean C.; (Vancouver,
CA) ; Ahkong, Quet F.; (Surrey, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Inex Pharmaceuticals
Corporation
Burnaby
CA
|
Family ID: |
26910152 |
Appl. No.: |
10/788649 |
Filed: |
February 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10788649 |
Feb 27, 2004 |
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09896812 |
Jun 29, 2001 |
|
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60264616 |
Jan 26, 2001 |
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60215556 |
Jun 30, 2000 |
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Current U.S.
Class: |
424/450 ;
514/283 |
Current CPC
Class: |
A61K 9/1278 20130101;
A61P 35/00 20180101; A61K 31/4745 20130101; A61K 31/4745 20130101;
A61K 45/06 20130101; A61K 9/127 20130101; A61K 2300/00 20130101;
A61P 7/02 20180101; A61K 9/1272 20130101 |
Class at
Publication: |
424/450 ;
514/283 |
International
Class: |
A61K 031/4745; A61K
009/127 |
Claims
What is claimed is:
1. A liposomal formulation comprising: a) a liposome having an
active agent encapsulated therein; and b) an empty liposome.
2. The liposomal formulation of claim 1, wherein the ratio of
liposomes containing said active agent to said empty liposomes is
from about 1:0.5 to 1:1000.
3. The liposomal formulation of claim 2, wherein the ratio of
liposomes containing said active agent to said empty liposomes is
from about 1:1 to 1:100.
4. The liposomal formulation of claim 3, wherein the ratio of
liposomes containing said active agent to said empty liposomes is
from about 1:2 to 1:10.
5. The liposomal formulation of claim 4, wherein the ratio of
liposomes containing said active agent to said empty liposomes is
from about 1:3 to 1:5.
6. The liposomal formulation of claim 1, wherein said active agent
is an antineoplastic drug.
7. The liposomal formulation of claim 6, wherein said
antineoplastic drug is a camptothecin.
8. The liposomal formulation of claim 7, wherein said camptothecin
is a member selected from the group consisting of irinotecan,
topotecan, 9-amino camptothecin, 10,11-methylenedioxy camptothecin,
9-nitro camptothecin, TAS
103,7-(4-methyl-piperazino-methylene)-10,11-ethylenedio-
xy-20(S)-camptothecin and
7-(2-N-isopropylamino)ethyl)-20(S)-camptothecin.
9. The liposomal formulation of claim 8, wherein said camptothecin
is topotecan.
10. The liposomal formulation of claim 9, wherein said
antineoplastic drug is a vinca alkaloid.
11. The liposomal formulation of claim 10, wherein said vinca
alkaloid is a member selected from the group consisting of
vincristine, vinblastine, vinorelbine and vindesine.
12. The liposomal formulation of claim 11, wherein said vinca
alkaloid is vincristine.
13. The liposomal formulation of claim 11, wherein said vinca
alkaloid is vinorelbine.
14. The liposomal formulation of claim 1, wherein the ratio of said
active agent to lipid is about 0.005-1:1 (w/w).
15. The liposomal formulation of claim 14, wherein the ratio of
said active agent to lipid is about 0.05-0.9:1 (w/w).
16. The liposomal formulation of claim 15, wherein the ratio of
said active agent to lipid is about 0.1-0.5:1 (w/w).
17. The liposomal formulation of claim 1, wherein said active agent
comprises free active agent and precipitated active agent.
18. The liposomal formulation of claim 17, wherein at least 50% of
said active agent is precipitated active agent.
19. The liposomal formulation of claim 1, wherein said liposomes
containing said active agent comprise sphingomyelin.
20. The liposomal formulation of claim 19, further comprising
cholesterol.
21. The liposomal formulation of claim 20, wherein the
sphingomyelin and cholesterol are present at a molar ratio from
75/25 mol %/mol % sphingomyelin/cholesterol to 30/50 mol %/mol %
sphingomyelin/cholesterol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/896,812, filed Jun. 29, 2001, now pending; which is
related to U.S. Provisional Application No. 60/215,556, filed Jun.
30, 2000, entitled "Liposomal Camptothecins and Uses Thereof," and
U.S. Provisional Application No. 60/264,616, filed Jan. 26, 2001,
entitled "Liposomal Antineoplastic Drugs and Uses Thereof," all of
which are incorporated herein by reference in their entireties for
all purposes.
BACKGROUND OF THE INVENTION
[0002] This invention relates to liposomal compositions and methods
of using such compositions for treating neoplasia and for
inhibiting angiogenesis.
[0003] Many anticancer or antineoplastic drugs have been
encapsulated in liposomes. These include alkylating agents,
nitrosoureas, cisplatin, antimetabolites, and anthracyclines.
Studies with liposomes containing anthracycline antibiotics have
clearly shown reduction of cardiotoxicity and dermal toxicity and
prolonged survival of tumor bearing animals compared to controls
receiving free drug.
[0004] Liposomal anticancer drugs modify drug pharmacokinetics as
compared to their free drug counterpart. For a liposomal drug
formulation, drug pharmacokinetics will be largely determined by
the rate at which the carrier is cleared from the blood and the
rate at which the drug is released from the carrier. Considerable
efforts have been made to identify liposomal carrier compositions
that show slow clearance from the blood and long-circulating
carriers have been described in numerous scientific publications
and patents. Efforts have also been made to control drug leakage
rates from liposomal carriers, using for example, transmembrane
potential to control release.
[0005] Therapeutic camptothecins, such as Topotecan
(9-dimethylaminomethyl-10-hydroxy-camptothecin; Hycamtin.TM.), and
Irinotecan, are a semi-synthetic, water soluble derivative of
camptothecin, an alkaloid extracted from the stem wood of the
Chinese tree Camptotheca acuminata (Wall, et al., J. Am. Chem. Soc.
88:3888-3890 (1966)). Camptothecins belong to the topoisomerase
inhibitor class of antineoplastic agents, specifically inhibiting
the action of the nuclear enzyme topoisomerase I which is involved
in DNA replication (Hsiang, et al., Cancer Res. 48:1722-1726
(1988)). As such, topotecan exhibits a cell cycle-specific
mechanism of action, acting during S-phase (DNA replication) to
cause irreversible double strand breaks in DNA that ultimately lead
to G2 cell cycle arrest and apoptosis. In the free form, the drug
has a broad spectrum of activity against a range of tumor cell
lines and murine allograft and human xenograft tumor models
(McCabe, F. L. et al., Cancer Invest 12:308-313 (1994); Emerson, et
al., Cancer Res. 55:603-609 (1995); Thompson, Biochim. Biophys.
Acta 1400:301-319 (1998); Ormrod, et al., Drugs 58:533-551 (1999);
Hardman, et al., Anticancer Res. 19:2269-2274 (1999)). More
recently, evidence has emerged that topotecan has strong
anti-angiogenic properties that may contribute to its anti-tumor
mechanism of action (O'Leary, et al., Clin. Cancer Res. 5:181-187
(1999); Clements, et al., Cancer Chemother. Pharmacol. 44:411-416
(1999)). All these treatments are associated with dose-limiting
toxicity such as non-cumulative myelosuppression leading to
anaemia, neutropenia and thrombocytopenia, and
gastrointestinal-related toxicity, including mucositis and
diarrhea. Clinically, topotecan has been approved for second-line
therapy in ovarian and small cell lung cancer (SCLC) and is
currently the focus of extensive clinical evaluation.
[0006] Lipid formulations of camptothecins have been proposed as
therapeutic agents (see, U.S. Pat. No. 5,552,156 and PCT
Publication No. WO 95/08986. However, not all lipid formulations
are equal for drug delivery purposes and extensive research
continues into formulations which demonstrate preferred
characteristics for drug loading and storage, drug administration,
pharmacokinetics, biodistribution, leakage rates, tumor
accumulation, toxicity profile, and the like. With camptothecins,
the field is further complicated because dose limiting toxicities
in humans may be 10-fold lower than in mice (Erickson-Miller, et
al., Cancer Chemother. Pharmacol. 39:467-472 (1997)).
[0007] Improved liposomal formulations of antineoplastic agents
could prove very useful. It is an object of the instant invention
to provide lipid formulated antineoplastic agents having novel
clinical utility.
SUMMARY OF THE INVENTION
[0008] The present invention provides compositions and methods
useful for modulating the plasma circulation half-life of an active
agent (e.g., topotecan). The liposomal formulations have increased
clinical efficacy and decreased collateral toxicity. In addition,
the present invention provides methods and liposomal compositions
for treating neoplasia and inhibiting angiogenesis.
[0009] As such, in one embodiment, the present invention provides a
method for modulating the plasma circulation half-life of an active
agent, comprising: (a) providing a liposome having free active
agent and precipitated active agent encapsulated therein; and (b)
varying the amount of the active agent that is precipitated in the
liposome. Surprisingly, by varying the amount of active agent that
is precipitated in the liposome, it is possible to modulate the
release kinetics of the active agent into the plasma. Preferred
active agents are antineoplastic drugs, such as a camptothecin
(e.g., topotecan).
[0010] In another embodiment, the present invention provides a
liposomal formulation, comprising: a) an antineoplastic drug; and
b) a liposome having free antineoplastic drug and precipitated
antineoplastic drug, wherein the precipitated antineoplastic drug
in the liposome is at least 50% of the total antineoplastic drug.
By tailoring the amount of precipitated antineoplastic drug in the
liposome, it is possible to control the release of the drug, both
in vitro and in vivo. In certain preferred embodiments, high
intraliposomal concentrations of the active agent (e.g., topotecan)
results in a high amount of precipitated form. In this aspect,
subsequent release rates of the drug in vivo are slow. In certain
aspects, a slow release rate is preferable and more efficacious
compared to a fast release rate.
[0011] In yet another embodiment, the present invention provides a
liposomal formulation, comprising: a) an active agent; b) a
liposome having free active agent and precipitated active agent
encapsulated therein; and c) an empty liposome.
[0012] In this aspect, the serum half-life of the liposome is
prolonged by including empty liposomes in the formulation. It will
be readily apparent to those of skill in the art that any of a
variety of lipids can be used to form the liposomal compositions of
the present invention. In a presently preferred embodiment, the
lipid comprises a mixture of sphingomyelin and cholesterol,
preferably at a spingomyelin:cholesterol ratio (molar ratio) of
about 30:70 to about 60:40. In one preferred embodiment, the
liposome comprises sphingomyelin and cholesterol in a 55:45
ratio.
[0013] In still another aspect, the present invention provides a
method of treating a solid tumor in a human afflicted therewith,
the method comprising administering to the human an effective
amount of a liposomal formulation of the present invention in a
pharmaceutically acceptable carrier. A variety of solid tumors can
be treated using the compositions of the present invention. In a
preferred embodiment, the solid tumor to be treated is selected
from the group consisting of solid tumors of the lung, mammary,
colon and prostate. In another preferred embodiment, the method
further comprises co-administration of a treatment or active agent
suitable for treating neutropenia or platelet deficiency.
[0014] In a preferred embodiment, a liposomal topotecan is used to
treat the solid tumors. In addition, it will be readily apparent to
those of skill in the art that any of a variety of lipids can be
used to form the liposomal compositions of the present
invention.
[0015] Other features, objects and advantages of the invention and
its preferred embodiments will become apparent from the detailed
description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A-C shows the pharmacokinetic behavior of a liposomal
formulation of vinorelbine. Panel A shows the rates of drug leakage
from circulating carriers for three formulations of differing
drug:lipid ratio (0.1:1, 0.2:1, 0.3:1). Drug release is dependent
upon drug:lipid ratio with the slowest rate of release seen for the
highest ratio (0.3:1). Panel B shows lipid recovery in the blood.
Panel C shows that modulation in drug release rates from the
carrier results in changes to the blood clearance half-life for
vinorelbine.
[0017] FIGS. 2A-C shows a corresponding behavior when plasma drug
levels are used to follow pharmacokinetics. Panel A shows drug
retention versus time. Panel B shows lipid recovery versus time.
Panel C shows drug recovery versus time.
[0018] FIGS. 3A-C shows the pharmacokinetic behavior of
formulations of liposomal vinblastine as a function of drug:lipid
ratio (blood PK). Drug leakage from the liposomal carrier is
determined by the initial drug:lipid ratio with slower release for
formulations of higher drug ratio. Panel A shows drug retention
versus time. Panel B shows lipid recovery versus time. Panel C
shows drug release rates correlate with changes to drug clearance
half-life from the blood.
[0019] FIGS. 4 A-C shows the pharmacokinetic behavior of
formulations of liposomal vinblastine as a function of drug:lipid
ratio (plasma PK). Panel A shows drug retention versus time. Panel
B shows lipid recovery versus time. Panel C shows drug release
rates correlate with changes to drug clearance half-life from the
plasma.
[0020] FIGS. 5A-C shows the influence of lipid dose on PK behavior
(blood PK). As illustrated therein, similar rates of drug release
(A), lipid clearance (B) and drug clearance (C) are seen for a
liposomal vinblastine formulation of drug:lipid ratio 0.3:1 over a
lipid dose range of 16.6 mg/kg to 50 mg/kg.
[0021] FIGS. 6A-C shows the influence of lipid dose on PK behavior
(plasma PK). As illustrated therein, similar rates of drug release
(A), lipid clearance (B) and drug clearance (C) are seen for a
liposomal vinblastine formulation of drug:lipid ratio 0.3:1 over a
lipid dose range of 16.6 mg/kg to 50 mg/kg.
[0022] FIGS. 7A-B shows the pharmacokinetic behavior of two
formulations of liposomal topotecan of differing drug:lipid ratios.
Panel A shows that when topotecan is loaded to a drug:lipid ratio
of 0.11:1, a much slower drug release rate is seen resulting in a
much longer plasma clearance rate compared to Panel B having a
formulation of lower drug:lipid ratio of 0.02:1.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0023] The activity of many anticancer drugs is dependent on their
pharmacokinetic behavior. This pharmacokinetic behavior defines the
drug concentrations and period of time over which cancer cells are
exposed to the drug. In the case of most anticancer drugs, longer
exposure times are preferred as this results in increased killing
of the cancer cells. In general, several parameters are used to
describe drug pharmacokinetics. Plasma clearance half-time and area
under the curve (AUC) are examples. The plasma clearance half-time
is the time required for half of the administered drug to be
removed from the plasma. The AUC is a measure of plasma drug levels
over time and provides an indication of the total drug exposure.
Generally, increased plasma clearance half-life and plasma AUC for
an anticancer drug correlate with increased therapeutic
efficacy.
I. Modulating Active Agent Release
[0024] The present invention describes methods and formulations for
modulating drug release from liposomes. In one embodiment, the
present invention provides a method for modulating the plasma
circulation half-life of an active agent, comprising: (a) providing
a liposome having free active agent and precipitated active agent
encapsulated therein; and (b) varying the amount of the active
agent that is precipitated in the liposome. Preferably, the "free
active agent" and the "precipitate active agent" are the same
active agent, however the present invention is not so limited. As
used herein, the term "modulating" can mean either increasing or
decreasing the release rate of the active agent from the liposomal
carrier. For antineoplastic active agents, modulating is preferably
decreasing or slowing the release rate of the active agent.
[0025] In preferred aspects, the liposomes of the present invention
contain both encapsulated free active agent and precipitated active
agent. The amount of active agent that is precipitated within the
liposome can be varied using a variety of mechanisms. For example,
by varying the active agent to lipid ratio the amount of active
agent that is precipitated can be increased or decreased. Drug
loading at low drug:lipid ratios, results in low concentrations of
active agent (e.g., topotecan) in the liposome interior and hence
most, if not all of the entire drug is in solution i.e., not
precipitated or free. Low precipitation amounts result in a fast
release rate of the drug from the liposome. Conversely, a high
drug:lipid ratio results in high intraliposomal concentrations and
high precipitation amounts. When the drug is in a precipitated
form, subsequent release rates in vivo or in vitro are slow. For
antineoplastic drugs (e.g., topotecan), slow release rates are
preferable.
[0026] Without being bound by any particular theory, it is believed
that the liposomes of the present invention undergo a
"precipitation-dissoluti- on mechanism" (PDM), which dictates drug
release. In the PDM mechanism of the present invention, the
dissolution rate of precipitated active agent (e.g., topotecan)
within the lipsomome's interior into the internal solution of the
liposome is slow, compared to the rate of release of active agent
out of the liposome to the exterior and is thus rate determining.
That is, the rate of dissolution of the precipitated drug to free
drug in the liposome's interior determines how fast the drug will
be released into the plasma.
[0027] In certain embodiments, the active agent to lipid ratio can
be varied by the addition of empty liposomes. In general, liposomes
whether empty or those having active agents contained therein are
cleared by cells of the reticuloendothelial system (RES).
Typically, the RES will remove 80-95% of a dose of injected
liposomes within one hour, effectively out-competing the selected
target site for uptake of the liposomes. A variety of factors which
influence the rate of RES uptake of liposomes have been reported
including, liposome size, charge, degree of lipid saturation, and
surface moieties. By including empty liposome vesicles, it is
possible to shield the liposomes containing active agent from the
RES. Thus, empty liposome vesicles actually extend the blood
circulation lifetime of the liposomes by acting as "decoys". An
extended circulation time is often needed for liposomes to reach
the target region, cell or site from the site of injection. The
empty liposomal vesicles keep the RES busy and as a result, the
serum half-life of the liposomes having active agent contained
therein is increased.
[0028] In certain other aspects, a component(s) is added to the
liposome that will enhance the precipitation of the active agent.
In this aspect, a variety of charged ions can be used to increase
the amount of precipitated active agent in the vesicle's interior.
In preferred aspects, divalent, trivalent or polyvalent anions are
used. Suitable anions include, but are not limited to, carboxylate
(--CO.sub.2.sup.-), sulfonate (SO.sub.3.sup.-), sulfate
(SO.sub.4.sup.-2), hydroxide (--OH), alkoxides, phosphate
(--PO.sub.4.sup.-2), and phosphonate (--PO.sub.3.sup.-2). Those of
skill in the art will know of other components, which will enhance
the amount of precipitated active agent in the liposome's
interior.
[0029] Moreover, the drug: lipid ratios can be varied using the
size of the liposome. The larger the liposome vesicle used, the
smaller the drug:lipid ratio. In certain aspects, both the active
agent to lipid ratio and the size of the liposome are varied to
optimize the efficacy of the active agent.
[0030] The amount of encapsulated active agent that is precipitated
in vesicle will vary and is somewhat dependent on the active agent
itself. In certain embodiments, the amount of precipitated active
agent is at least about 25% to about 95% (such as about 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95%)
of total active agent. For topotecan, the amount of the
precipitated active agent encapsulated in the liposome is at least
50% of the total active agent.
[0031] In preferred aspects, when the active agent is an
antineoplastic drug, using higher drug:lipid ratios results in
higher amounts of encapsulated precipitated drug. As a result, drug
release from the liposomes in vivo is slower than for similar
compositions prepared at lower drug:lipid ratio. These higher
drug:lipid ratio liposomes exhibit extended plasma half-life and
increased plasma AUC values. Advantageously, these formulations
exhibit improved antitumor efficacy.
[0032] In certain embodiments, the ratio of active agent: lipid is
about 0.005-1:1 (w/w).
[0033] Preferably, the ratio of active agent:lipid is about
0.05-0.9:1 (w/w) and more preferably, the ratio of active
agent:lipid is about 0.1-0.5:1 (w/w). By modulating the plasma
circulation half-life of the active agent, it is thus possible to
maximize or optimize efficacy of the active agent.
II. Compositions and Methods of Making Liposomal Formulations
[0034] Liposome, vesicle and liposome vesicle will be understood to
indicate structures having lipid-containing membranes enclosing an
aqueous interior. The structures can have one or more lipid
membranes unless otherwise indicated, although generally the
liposomes will have only one membrane. Such single-layered
liposomes are referred to herein as "unilamellar." Multilayer
liposomes are referred to herein as "multilamellar."
[0035] The liposomes that are used in the present invention are
preferably formed from lipids which when combined form relatively
stable vesicles. An enormous variety of lipids are known in the
art, which can be used to generate such liposomes. Preferred lipids
include, but are not limited to, neutral and negatively charged
phospholipids or sphingolipids and sterols, such as cholesterol.
The selection of lipids is generally guided by consideration of,
e.g., liposome size and stability of the liposomes in the
bloodstream.
[0036] Preferred liposome compositions for use in the present
invention include those comprising sphingomyelin and cholesterol.
The ratio of sphingomyelin to cholesterol in the liposome
composition can vary, but generally is in the range of from about
75/25 mol %/mol % sphingomyelin/cholesterol to about 30/50 mol
%/mol % sphingomyelin/cholesterol, more preferably about 70/30 mol
%/mol % sphingomyelin/cholesterol to about 40/45 mol %/mol %
sphingomyelin/cholesterol, and even more preferably about 55/45 mol
%/mol % sphingomyelin/cholesterol. Other lipids can be included in
the liposome compositions of the present invention as may be
necessary, such as to prevent lipid oxidation or to attach ligands
onto the liposome surface. Generally, if lipids are included, the
other inclusion of such lipids will result in a decrease in the
sphingomyelin/cholesterol ratio. Liposomes of this type are known
as sphingosomes and are more fully described in U.S. Pat. No.
5,814,335, the teachings of which are incorporated herein by
reference.
[0037] A variety of methods are available for preparing liposomes
as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng.
9:467 (1980); U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028, the
text Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York,
1983, Chapter 1; and Hope, et al., Chem. Phys. Lip. 40:89 (1986),
all of which are incorporated herein by reference. The protocol for
generating liposomes generally includes: mixing of lipid components
in an organic solvent; drying and reconstituting liposomes in
aqueous solvent; and sizing of liposomes (such as by extrusion),
all of which are well known in the art.
[0038] Alternative methods of preparing liposomes are also
available. For instance, a method involving detergent dialysis
based self-assembly of lipid particles is disclosed and claimed in
U.S. Pat. No. 5,976,567 issued to Wheeler, et al., which avoids the
time-consuming and difficult to-scale drying and reconstitution
steps. Further methods of preparing liposomes using continuous flow
hydration are under development and can often provide the most
effective large scale manufacturing process.
[0039] Preparation of liposomal formulations having active agents
(e.g., camptothecins) requires loading of the drug into the
liposomes. Loading can be either passive or active. Passive loading
generally requires addition of the drug to the buffer at the time
of the reconstitution step. This allows the drug to be trapped
within the liposome interior, where it will remain if it is not
lipid soluble, and if the vesicle remains intact (such methods are
employed, for example, in PCT Publication No. WO 95/08986, the
teachings of which are incorporated herein by reference).
[0040] Active loading is in many ways preferable, and a wide
variety of therapeutic agents can be loaded into liposomes with
encapsulation efficiencies approaching 100% by using a
transmembrane pH or ion gradient (see, Mayer, et al., Biochim.
Biophys. Acta 1025:143-151 (1990) and Madden, et al., Chem. Phys.
Lipids 53:37-46 (1990)). Numerous ways of active loading are known
to those of skill in the art. All such methods involve the
establishment of some form of gradient that draws lipophilic
compounds into the interior of liposomes where they can reside for
as long as the gradient is maintained. Very high quantities of the
desired drug can be obtained in the interior, so much that the drug
may precipitate out on the interior and generate a continuing
uptake gradient.
[0041] Particularly preferred for use with the instant invention is
ionophore-mediated loading as disclosed and claimed in U.S. Pat.
No. 5,837,282, the teachings of which are incorporated by reference
herein. The ionophore-mediated loading is an electroneutral process
and does not result in formation of a transmembrane potential. With
hydrogen ion transport into the vesicle there is concomitant
magnesium ion transport out of the vesicle in a 2:1 ratio (i.e. no
net charge transfer). In the case of topotecan, it is thought that
the agent crosses the membrane in a neutral state (no charge). Upon
entry into the vesicle, topotecan becomes positively charged. As
ionophore-mediated loading is an electroneutral process, there is
no transmembrane potential generated.
[0042] An important characteristic of liposomal camptothecins for
pharmaceutical purposes is the drug to lipid ratio of the final
formulation. As discussed earlier, drug:lipid ratios can be
established in two ways: 1) using homogenous liposomes each
containing the same drug:lipid ratio; or 2) by mixing empty
liposomes with liposomes having a high drug:lipid ratio to provide
a suitable average drug:lipid ratio. For different applications,
different drug:lipid ratios may be desired. Techniques for
generating specific drug:lipid ratios are well known in the art.
Drug:lipid ratios can be measured on a weight to weight basis, a
mole to mole basis or any other designated basis. Preferred
drug:lipid ratios range from about 0.005:1 drug:lipid (by weight)
to about 0.2:1 drug:lipid (by weight) and, more preferably, from
about 0.1:1 drug:lipid (by weight) to about 0.3:1 drug:lipid (by
weight).
[0043] A further important characteristic is the size of the
liposome particles. For use in the present inventions, liposomes
having a size of from about 0.05 microns to about 0.15 microns are
preferred.
[0044] The present invention also provides liposomal compositions
(e.g., camptothecin) in kit form. The kit can comprise a ready-made
formulation, or a formulation, which requires mixing of the
medicament before administration. The kit will typically comprise a
container that is compartmentalized for holding the various
elements of the kit. The kit will contain the liposomal
compositions of the present invention or the components thereof,
possibly in dehydrated form, with instructions for their
rehydration and administration
[0045] The liposome compositions prepared, for example, by the
methods described herein can be administered either alone or in a
mixture with a physiologically acceptable carrier (such as
physiological saline or phosphate buffer) selected in accordance
with the route of administration and standard pharmaceutical
practice. Generally, normal saline will be employed as the
pharmaceutically acceptable carrier. Other suitable carriers
include, e.g., water, buffered water, 0.4% saline, 0.3% glycine,
and the like, including glycoproteins for enhanced stability, such
as albumin, lipoprotein, globulin, etc. These compositions may be
sterilized by conventional, well-known sterilization techniques.
The resulting aqueous solutions may be packaged for use or filtered
under aseptic conditions and lyophilized, the lyophilized
preparation being combined with a sterile aqueous solution prior to
administration. The compositions may also contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents and the like, for example, sodium
acetate, sodium lactate, sodium chloride, potassium chloride,
calcium chloride, etc. Additionally, the composition may include
lipid-protective agents, which protect lipids against free-radical
and lipid-peroxidative damages on storage. Lipophilic free-radical
quenchers, such as .alpha..-tocopherol and water-soluble
iron-specific chelators, such as ferrioxamine, are suitable.
[0046] A wide variety of active agents are suitable for the
liposomal compositions and methods of the present invention. In a
preferred aspect, the active agents are antineoplastic drugs.
Currently, there are approximately twenty recognized classes of
approved antineoplastic drugs. The classifications are
generalizations based on either a common structure shared by
particular drugs, or are based on a common mechanism of action by
the drugs. A partial listing of some of the commonly known
commercially approved (or in active development) antineoplastic
agents by classification is as follows:
[0047] Structure-Based Classes:
[0048] 1. Fluoropyrimidines-5-FU, Fluorodeoxyuridine, Ftorafur,
5'-deoxyfluorouridine, UFT, S-1 Capecitabine;
[0049] 2. Pyrimidine Nucleosides-Deoxycytidine, Cytosine
Arabinoside, 5-Azacytosine, Gemcitabine,
5-Azacytosine-Arabinoside;
[0050] 3. Purines-6-Mercaptopurine, Thioguanine, Azathioprine,
Allopurinol, Cladribine, Fludarabine, Pentostatin, 2-Chloro
Adenosine;
[0051] 4. Platinum Analogues-Cisplatin, Carboplatin, Oxaliplatin,
Tetraplatin, Platinum-DACH, Ormaplatin, CI-973, JM-216;
[0052] 5. Anthracyclines/Anthracenediones-Doxorubicin,
Daunorubicin, Epirubicin, Idarubicin, Mitoxantrone;
[0053] 6. Epipodophyllotoxins-Etoposide, Teniposide;
[0054] 7. Camptothecins-Irinotecan, Topotecan, 9-Amino
Camptothecin, 10,11-Methylenedioxy Camptothecin, 9-Nitro
Camptothecin, TAS
103,7-(4-methyl-piperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothe-
cin, 7-(2-N-isopropylamino)ethyl)-20(S)-camptothecin;
[0055] 8. Hormones and Hormonal Analogues-Diethylstilbestrol,
Tamoxifen, Toremefine, Tolmudex, Thymitaq, Flutamide, Bicalutamide,
Finasteride, Estradiol, Trioxifene, Droloxifene,
Medroxyprogesterone Acetate, Megesterol Acetate, Aminoglutethimide,
Testolactone and others;
[0056] 9. Enzymes, Proteins and Antibodies-Asparaginase,
Interleukins, Interferons, Leuprolide, Pegaspargase, and
others;
[0057] 10. Vinca Alkaloids-Vincristine, Vinblastine, Vinorelbine,
Vindesine;
[0058] 11. Taxanes-Paclitaxel, Docetaxel.
[0059] Mechanism-Based Classes:
[0060] 1. Antihormonals-See classification for Hormones and
Hormonal Analogues, Anastrozole;
[0061] 2. Antifolates-Methotrexate, Aminopterin, Trimetrexate,
Trimethoprim, Pyritrexim, Pyrimethamine, Edatrexate, MDAM;
[0062] 3. Antimicrotubule Agents-Taxanes and Vinca Alkaloids;
[0063] 4. Alkylating Agents (Classical and Non-Classical)-Nitrogen
Mustards (Mechlorethamine, Chlorambucil, Melphalan, Uracil
Mustard), Oxazaphosphorines (Ifosfamide, Cyclophosphamide,
Perfosfamide, Trophospharnide), Alkylsulfonates (Busulfan),
Nitrosoureas (Carmustine, Lomustine, Streptozocin), Thiotepa,
Dacarbazine and others;
[0064] 5. Antimetabolites-Purines, pyrimidines and nucleosides,
listed above;
[0065] 6. Antibiotics-Anthracyclines/Anthracenediones, Bleomycin,
Dactinomycin, Mitomycin, Plicamycin, Pentostatin, Streptozocin;
[0066] 7. Topoisomerase Inhibitors-Camptothecins (Topo I),
Epipodophyllotoxins, m-AMSA, Ellipticines (Topo II);
[0067] 8. Antivirals-AZT, Zalcitabine, Gemcitabine, Didanosine, and
others;
[0068] 9. Miscellaneous Cytotoxic Agents-Hydroxyurea, Mitotane,
Fusion Toxins, PZA, Bryostatin, Retinoids, Butyric Acid and
derivatives, Pentosan, Fumagillin, and others.
[0069] The objective of all antineoplastic drugs is to eliminate
(cure) or to retard the growth and spread (remission) of the cancer
cells. The majority of the above listed antineoplastic agents
pursue this objective by possessing primary cytotoxic activity,
effecting a direct kill on the cancer cells. Other antineoplastic
drugs stimulate the body's natural immunity to effect cancer cell
kill. The literature is replete with discussions on the activity
and mechanisms of all of the above drugs, and many others.
[0070] Exemplary methods of making specific formulations of
liposomal camptothecins and, in particular, liposomal topotecan are
set out in the examples below.
III. Methods of Using Liposomal Camptothecins
[0071] The liposomal compositions (e.g., camptothecins) of the
present invention are used, in the treatment of solid tumors in an
animal, such as a human. The examples below set out key parameters
of the drug:lipid ratios, dosages of active agent and lipid to be
administered, and preferred dose scheduling to treat different
tumor types.
[0072] Preferably, the pharmaceutical compositions are administered
parenterally, i.e., intraarticularly, intravenously,
intraperitoneally, subcutaneously or intramuscularly. More
preferably, the pharmaceutical compositions are administered by
intravenous drip or intraperitoneally by a bolus injection. The
concentration of liposomes in the pharmaceutical formulations can
vary widely, i.e., from less than about 0.05%, usually at or at
least about 2-5% to as much as 10 to 30% by weight and will be
selected primarily by fluid volumes, viscosities, etc., in
accordance with the particular mode of administration selected. For
example, the concentration can be increased to lower the fluid load
associated with treatment. Alternatively, liposomes composed of
irritating lipids can be diluted to low concentrations to lessen
inflammation at the site of administration. The amount of liposomes
administered will depend upon the particular camptothecin used, the
disease state being treated and the judgement of the clinician, but
will generally, in a human, be between about 0.01 and about 50 mg
per kilogram of body weight, preferably between about 5 and about
40 mg/kg of body weight. Higher lipid doses are suitable for mice,
for example, 50-120 mg/kg.
[0073] Dosage for the active agent (e.g., camptothecin) will depend
on the administrating physician's opinion based on age, weight, and
condition of the patient, and the treatment schedule. A recommended
dose for free topotecan in Small Cell Lung Cancer is 1.5 mg/M.sup.2
per dose, every day for 5 days, repeated every three weeks. Because
of the improvements in treatment now demonstrated in the examples,
below, doses of active agent (e.g., topotecan) in humans will be
effective at ranges as low as from 0.015 mg/M.sup.2/dose and will
still be tolerable at doses as high as 15 to 75 mg/M.sup.2/dose,
depending on dose scheduling. Doses may be single doses or they may
be administered repeatedly every 4 h, 6 h, or 12 h or every 1 d, 2
d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d, 9 d, 10 d or combination thereof.
Preferred scheduling may employ a cycle of treatment that is
repeated every week, 2 weeks, three weeks, four weeks, five weeks
or six weeks or combination thereof. In a presently preferred
embodiment, treatment is given once a week, with the dose typically
being less than 1.5 mg/M.sup.2.
[0074] Particularly preferred topotecan dosages and scheduling are
as follows:
1 Dosage (mg/M.sup.2/dose) Period Repeat Cycle every: 0.15 1d
.times. 5d 3 weeks 0.5 1d 1 week 1.5 1d 1 week 15 1d 3 weeks 50 1d
3 weeks
[0075] The invention will be described in greater detail by way of
specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of non-critical parameters, which can be changed or
modified to yield essentially the same results.
IV. EXAMPLES
A. Materials and Methods
[0076] 1. Materials. Topotecan (Hycamtin.TM., SmithKline Beecham)
was purchased from the pharmacy at the British Columbia Cancer
Agency. Sphingomyelin (SM) was purchased from Avanti Polar Lipids.
Sphingomyelin from Northern Lipids was used in an early study, but
was less soluble in ethanol than the Avanti version. Cholesterol
(CH) and the divalent cation ionophore A23187 were purchased from
Sigma. [.sup.3H]-cholesterylhexadecy- lether (Dupont) was used as a
lipid marker.
[0077] 2. Mice. Female, ICR, BDF-I or athymic nu/nu (6-8 weeks)
were purchased from Harlan-Sprague Dawley (Indianapolis, Ind.). All
animals were quarantined for one week prior to use. All studies
were conducted in accordance with the guidelines established by the
Canadian Council on Animal Care (CCAC) and the Institutional Animal
Care and User Committee (IACUC).
[0078] 3. Formulation of topotecan by the Mg-A23187 method.
Topotecan was encapsulated in SM:CH (55:45, mol/mol) liposomes
using the Mg-A23187 ionophore method according to U.S. Pat. No.
5,837,282. The initial drug-to-lipid ratio was 0.10 (w/w) and drug
loading was typically 95-100%. The external buffer consisted of 10
mM PBS, pH 7.5 and 300 mM sucrose. All formulations were analyzed
with respect to particle size, drug loading efficiency, pH, and
drug and lipid concentration.
[0079] 4. Drug preparation and dosing. Each vial of topotecan
(Hycamtin.TM.) was hydrated in 1.0 ml of sterile water, giving a
topotecan concentration of 4.0 mg/ml. Subsequent dilutions were
.mu.l made in 0.9% sterile saline to maintain the low pH required
for the lactone species of the drug. Unused drug in the water stock
solution (4.0 mg/ml) was stored at 4.degree. C. in the absence of
light. Liposome encapsulated topotecan was diluted in 0.9% saline
to the required concentration for administration. All drug
administrations were at 10 ml/kg (200 .mu.l/20 g mouse) via the
lateral tail vein.
[0080] 5. Pharmacokinetic and in vivo leakage studies. The
pharmacokinetics and drug leakage of free and liposome encapsulated
topotecan were evaluated in ICR mice over 24 h following i.v.
administration via the lateral tail vein. Two different
drug-to-lipid ratios, i.e., 0.10 (w/w) and 0.02 (w/w), were used to
examine the influence of drug-to-lipid ratio and lipid dose on drug
leakage and PK behavior. Encapsulated topotecan was administered at
1 mg/kg (10 or 50 mg/kg lipid) and 5 mg/kg topotecan (50 mg/kg
lipid). Correspondingly, the PK behavior of free topotecan was
evaluated at and 1 and 5 mg/kg. Total topotecan in blood was
determined by a fluorescence assay preceded by precipitation of
plasma proteins. Topotecan was quantified by spectrofluorimetry at
an excitation (2.5 nm slit width) and emission wavelength (2.5 nm
slit width) of 380 and 518 nm, respectively. Lipid levels in plasma
were determined by liquid scintillation counting of the
[.sup.3H]-CHE label.
[0081] 6. MTD studies. MTD studies were performed in the host mouse
strain corresponding to each tumor model. Single dose and multidose
MTD were determined by monitoring weight loss over time. The MTD
was defined as the dose that resulted in 20% weight loss.
[0082] 7. Myelosuppression and neutropenia studies. Alteration in
peripheral blood cell levels as a consequence of topotecan
administration was assessed over 4-6 weeks in ICR mice. Blood was
collected into EDTA microtainer tubes at Day 1, 3, 5, 7, 14, and 21
following i.v. administration of free or liposome encapsulated
topotecan at 10 mg/kg. Empty vesicles were administered as a
control. CBC and differential analysis was performed at Central
Labs for Veterinarians (Langley, BC) to quantify cellular levels,
ratios and morphology.
[0083] 8. Tumor Models. The L1210 murine leukemia model and the
CT-26 murine colon metastases model were employed as in standard
protocols. Human MX-1 and LX-1 cell lines were obtained from the
DCTD Tumor Repository in Frederick, Md. These cell lines were
received as tumor fragments and were propagated in NCr nude mice by
serial transplantation of 3.times.3 mm fragments. Experiments were
not initiated until the cell lines had been through 3 passages in
nude mice and the tumor lines were restarted when the passage
number reached 10.
[0084] 9. Efficacy Studies. All dosing of free and liposomal
topotecan was administered by the intravenous route at 10 ml/kg via
the lateral tail vein. In the L1210 and CT-26 models, dosing
occurred on day 1 (tumor cell injection=day 0). For the MX-1 and
LX-1 tumor models, tumor volume was determined by repeated
perpendicular measurements of tumor dimensions and using the
formula:
Volume (mm.sup.3)=(L.times.W.sup.2)/2
[0085] Dosing was initiated in the MX-1 and LX-1 models when tumors
had clearly demonstrated growth and were in the range 100-300
mm.sup.3.
[0086] Since most drugs exhibit a balance between a biological
effect and toxicity, it is useful to examine a parameter that
incorporates both of these attributes. The most commonly employed
parameter is therapeutic index (TI). Traditionally, therapeutic
index is defined as:
TI=LD.sub.50/ED.sub.50
[0087] However, since it is no longer permissible to perform LD50
studies, therapeutic index for these studies has been defined as
follows:
TI=MTD/MED.
[0088] In the above formula, MTD is the maximum tolerated dose,
defined as that dose that causes a mean weight loss of 20% in a
group of animals; and MED is the minimal effective dose, defined as
the dose that produces an optimal % T/C value of .ltoreq.40 in the
solid tumor models or an % ILS of 50.+-.10% in the survival
models.
B. Results
[0089] 1. Pharmacokinetics and drug leakage. The influence of
liposome encapsulation and drug-to-lipid ratio on plasma
pharmacokinetics and drug leakage of topotecan was examined over 24
h in ICR mice. Liposome encapsulation of topotecan (drug-to-lipid
ratio, 0.11, wt/wt) had a dramatic influence on the
pharmacokinetics parameters of the drug (see, FIG. 1, top; and
Table 1). At a 5 mg/kg dose of topotecan, a 164-fold increase in
plasma AUC, a 24-fold increase in C.sub.max and a 24-fold increase
in the plasma a half-life were observed for the liposomal drug
relative to the free drug (see, Table 1). Historically, large
improvements in AUC and plasma half-lives of liposomal drugs have
resulted in enhanced delivery of the drug to disease-sites (such as
tumors), a process known as "disease-site targeting".
[0090] The formulations used in this study were prepared by the
Mg-A23187 ionophore method. There was an initial rapid release of
drug in the first 10-30 minutes after iv administration (see, FIG.
1, bottom), followed by a more gradual release phase. The
t.sub.1/2release for the Mn-A23187 and Mg-A23187 formulations were
.about.3 h and .about.5-7 h, respectively; however, very little
drug was present in either formulation at 24 h.
[0091] For most liposomal drug formulations, the pharmacokinetic
properties of the encapsulated drug are controlled by the lipid
composition and dose. Liposomal topotecan has been shown to exhibit
exceptional anti-tumor activity, even at very low drug doses (0.5
mg/kg; drug-to-lipid ratio, 0.10, wt/wt). At these drug doses and
drug-to-lipid ratio, liposome elimination from the plasma is
expected to be rapid. Therefore, to determine whether the
pharmacokinetics of topotecan at low doses could be improved, a low
drug-to-lipid ratio (0.02, wt/wt) formulation of topotecan was
investigated. Interestingly, in this study, the low drug-to-lipid
ratio formulation released the drug much faster than the higher
drug-to-lipid ratio (0.11, wt/wt) formulation. This result was
unexpected.
2TABLE 1 Pharmacokinetic parameters of free and liposomal
topotecan. Dose AUC Cmax .alpha..sub.1/2 Formulation (mg/kg) (h
.multidot. .mu.g/ml) (.mu.g/ml) Cl (ml/h) (h) .beta..sub.1/2 (h)
Free 1 1.97 0.75 13.9 0.14 11.8 5 2.77 2.17 49.6 0.26 11.4 TCS 1
65.7 16.3 0.417 2.79 5 453 51.0 0.302 6.16
[0092] All parameters were derived from one or two-compartment
models using WINNONLIN PK modeling software.
[0093] 2. Maximum tolerated doses. Single and multidose MTD studies
were performed in tumor bearing Balb/c, BDF-1 and NCr nu/nu mice.
Body weights of individual mice were monitored throughout each
study to evaluate the general tolerability of free and liposomal
topotecan and, where possible, to establish an MTD (see, FIG. 2).
The maximum tolerated dose of liposomal topotecan was 10 mg/kg on a
single administration, 7.5 mg/kg on a q7dx3 schedule and 5 mg/kg on
a q3dx4 schedule. The reported LD.sub.10 of free topotecan
following a single intravenous infusion in mice is 75 mg/M.sup.2
(.about.25 mg/kg) [Hycamtin.TM. product monograph]; however, very
little weight loss was observed at doses up to 40 mg/kg, although
this was considered the MTD due to acute responses. Drug quantities
were limited so doses higher than 40 mg/kg (administered over 5-10
minutes) were not pursued. It has previously been indicated that
the LD.sub.10 of free topotecan on a qdx5 schedule is 14 mg/M2/dose
(.about.4.7 mg/kg/dose) (Grochow, et al., Drug Metab. Dispos.
20:706-713 (1992)).
[0094] 3. Toxicity. The major dose-limiting toxicity of free
topotecan administered daily in humans for 5 consecutive days (dx5)
at 1.5 mg/M.sup.2/dose, the MTD, is non-cumulative
myelosuppression. As mentioned earlier, humans are more sensitive
than mice to myelosuppression and can only tolerate 11% of the MTD
in mice (1.5 vs 14 mg/M.sup.2). In this regard, dogs have been
shown to be a much better predictor of topotecan myelosuppression
in humans (Burris, et al., J. Natl. Cancer Inst. 84:1816-1820
(1992)). However, mice should be suitable for comparing the
relative myelosuppressive effects of free and liposome encapsulated
topotecan.
[0095] In a study, the maximal reduction in peripheral WBC counts
occurred at day 3 post-injection following administration of
liposomal topotecan. A comparison of peripheral blood cell levels
and morphology was then made at day 3 following administration of
free or liposome encapsulated topotecan or empty vesicles (see,
Table 2). The dose used for this comparison was the MTD of
liposome-encapsulated topotecan (10 mg/kg). A significant reduction
in circulating neutrophils was observed for liposomal topotecan
relative to free topotecan (.about.10-fold), empty vesicles
(.about.10-fold) or control animals (.about.20-fold). Total WBC
levels and the lymphocyte sub-population were reduced approximately
2-fold for liposomal topotecan relative to control animals. No
significant differences were observed in these parameters for free
topotecan at the same dose. At day 21 post-injection total, WBC
levels for liposomal topotecan remained approximately 2.5-fold
lower than normal animals; however, neutrophils levels had
recovered from a 20-fold decrease to a 3-fold decrease relative to
normal mice. Lymphocyte levels remained .about.2-fold lower than
normal mice. No other significant differences were observed.
[0096] Analysis of serum chemistry parameters at day 3
post-injection revealed very few changes relative to untreated
animals (see, Table 3). The only change of note was a statistically
significant increase (.about.2-fold) in globulin levels and a
concomitant decrease in the albumin/globulin ratio for animals
treated with liposomal topotecan. No other significant changes were
observed.
3TABLE 2 Blood CBC and differential of ICR mice treated with a 10
mg/kg i.v. dose of free or liposome encapsulated topotecan. Day WBC
Differential Treat- Post- WBC Neutro Lympho Mono Eosino Baso RBC Hb
Hc PLT ment Injection (.times.10.sup.9/L) (.times.10.sup.9/L)
(.times.10.sup.9/L) (.times.10.sup.9/L) (.times.10.sup.9/L)
(.times.10.sup.9/L) (.times.10.sup.12/L) (g/L) (L/L)
(.times.10.sup.9/L) Control 6.47 .+-. 0.937 .+-. 5.23 .+-. 0.180
.+-. 0.059 .+-. 0.056 .+-. 8.67 .+-. 0.93 142 .+-. 12 0.438 .+-.
0.045 717 .+-. 317 1.62 0.201 1.45 0.042 0.039 0.053 Free 3 6.70
.+-. 0.520 .+-. 5.90 .+-. 0.177 .+-. 0.031 .+-. 0.057 .+-. 8.47
.+-. 0.39 136 .+-. 05 0.444 .+-. 0.012 879 .+-. 145 1.95 0.200 1.70
0.072 0.021 0.040 21 5.16 .+-. 0.480 .+-. 4.33 .+-. 0.247 .+-.
0.034 .+-. 0.088 .+-. 9.81 .+-. 0.37 154 .+-. 04 0.493 .+-. 0.014
907 .+-. 059 1.18 0.122 0.93 0.180 .016 0.071 TCS 3 2.82 .+-. 0.048
.+-. 2.63 .+-. 0.109 .+-. 0.001 .+-. 0.034 .+-. 8.93 .+-. 0.76 141
.+-. 10 0.463 .+-. 0.033 564 .+-. 098 1.05 0.018 0.87 0.126 0.001
0.029 21 2.54 .+-. 0.282 .+-. 2.06 .+-. 0.133 .+-. 0.019 .+-. 0.064
.+-. 9.41 .+-. 0.83 154 .+-. 12 0.486 .+-. 0.035 1009 .+-. 161 1.43
0.167 1.36 0.142 0.011 0.060 Empty 3 4.68 .+-. 0.598 .+-. 3.66 .+-.
0.248 .+-. 0.081 .+-. 0.064 .+-. 7.77 .+-. 0.30 130 .+-. 05 0.416
.+-. 0.014 863 .+-. 143 1.13 0.238 0.93 0.168 0.044 0.055 21 5.05
.+-. 0.898 .+-. 3.78 .+-. 0.263 .+-. 0.038 .+-. 0.072 .+-. 9.36
.+-. 0.67 152 .+-. 08 0.483 .+-. 0.033 1366 .+-. 144 0.64 0.575
0.88 0.163 0.036 0.057
[0097]
4TABLE 3 Serum chemistry panel of ICR mice treated with a 10 mg/kg
i.v. dose of free or liposome encapsulated topotecan - day 3
post-injection. BUN Treat- (mmol/ Creatinine TP Albumin Globulin
Alb/Glob Bilirubin Alk Phos ALT AST CPK ment L) (.mu.mol/L) (g/L)
(g/L) (g/L) Ratio (.mu.mol/L) (IU/L) (IU/L) (IU/L) (IU/L) Control
11.3 .+-. 83 .+-. 6 46.7 .+-. 31.3 .+-. 1.5 15.3 .+-. 1.2 2.07 .+-.
0.15 4.7 .+-. 0.6 86 .+-. 12 27 .+-. 31 59 .+-. 22 87 .+-. 107 3.0
2.1 Free 9.4 .+-. 82 .+-. 18 48.0 .+-. 32.8 .+-. 1.3 15.2 .+-. 1.1
2.16 .+-. 0.15 3.8 .+-. 0.8 67 .+-. 35 13 .+-. 23 55 .+-. 10 56
.+-. 38 3.2 2.1 TCS 10.0 .+-. 96 .+-. 28 55.8 .+-. 28.8 .+-. 2.5
27.0 .+-. 10.1 1.18 .+-. 0.33 2.5 .+-. 0.6 73 .+-. 21 23 .+-. 17 77
.+-. 29 155 .+-. 54 3.9 11.8 Empty ND 68 .+-. 13 49.3 .+-. 33.0
.+-. 1.7 16.3 .+-. 0.6 2.00 .+-. 0.17 4.3 .+-. 0.6 70 .+-. 10 17
.+-. 15 53 .+-. 6 56 .+-. 26 1.2
C. Efficacy Studies in Murine and Human Tumor Models: Single Dose
Studies
[0098] 1. L1210 Murine Leukemia. The intravenous L1210 murine
leukemia model has been used extensively to evaluate differential
activity between free and liposome encapsulated chemotherapeutic
agents and was one of the original (1955-1975) models in the in
vivo NCI screen of novel chemotherapeutic agents (Plowman, et al.,
Human tumor xenograft models in NCI drug development. In
"Anticancer Drug Development Guide: Preclinical Screening, Clinical
Trials, and Approval" (B. Teicher, Ed.), Humana Press Inc., Totowa
(1997); Waud, Murine L1210 and P388 leukemias. In "Anticancer Drug
Development Guide: Preclinical Screening, Clinical Trials, and
Approval" (B. Teicher, Ed.), Humana Press Inc., Totowa (1997)). The
model is rapid--the mean survival of untreated animals is typically
.about.7-8 days--and the administered tumor cells seed in the liver
and bone marrow.
[0099] Administration of free topotecan as a single intravenous
dose had minimal effect on survival in the L1210 model (see, FIG.
3A). At the highest dose of free topotecan, a median survival of 13
days (44% ILS) was observed. There was one long-term survivor (day
60) in this group. In contrast, a single i.v. administration of
liposomal topotecan at either 5 or 10 mg/kg resulted in 100%
survival at day 60 (see, FIG. 3B). Median survival for a 1 mg/kg
dose was 13 days (44% ILS) and the survival curve was nearly
identical to that of the free topotecan administered at 30 mg/kg--a
30-fold improvement in potency. At higher doses (30 mg/kg) of the
liposomal topotecan, toxic deaths were observed. The MTD for
liposomal topotecan was 20 mg/kg in BDF-1 mice after a single i.v.
administration.
[0100] 2. CT-26 Murine Colon Carcinoma. The murine CT-26 colon cell
line is useful for drug screening since it readily grows as
subcutaneous solid tumors or can be administered intravenously and
used as a survival model. In addition, when the tumor cells are
administered by intrasplenic injection, followed by splenectomy,
the cells seed to the liver and give rise to an experimental
metastases model that more closely resembles the clinical
progression of colorectal cancer. The model has been used
extensively and is described, for example, in detail elsewhere.
[0101] In the CT-26 model, administration of a single dose of
topotecan had a modest impact on survival resulting in % ILS of
23-60% over the dose range 5-40 mg/kg (see, FIG. 4). Liposome
encapsulated topotecan, however, was highly active at doses greater
than 5 mg/kg, resulting in 100% survival (8/8) at day 90. At 10
mg/kg, 87.5% survival (7/8) was observed at day 90; however, the
tumor burden in dead animal was very low suggesting that this
animal may have died due to other factors, such as infection
related to myelosuppression. A dose response was observed for
liposomal topotecan, with the 2 mg/kg dose giving an % ILS of 54%.
This was determined to be the MED and was comparable to the % ILS
(58%) achieved using free topotecan at 40 mg/kg--a 20-fold increase
in potency.
[0102] 3. MX-1 Human Breast Carcinoma. MX-1 is an experimental
model of human breast cancer and has a reported doubling time of
3.9 days (NCI); in this study, the median doubling time was
consistently 3.6-3.7 days. The tumor cell line was derived from the
primary tumor of a 29-year-old female with no previous history of
chemotherapy and is provided by the DCTD (NCI) tumor repository as
a tumor fragment that is serially passaged in nude mice.
Histologically, MX-1 is a poorly differentiated mammary carcinoma
with no evidence of gland formation or mucin production. MX-1 was
one of 3 xenograft models (MX-1, LX-1, CX-1) that comprised the NCI
in vivo tumor panel and prescreen (1976-1986) for evaluating novel
chemotherapeutic agents (Plowman, et al., Human tumor xenograft
models in NCI drug development. In "Anticancer Drug Development
Guide: Preclinical Screening, Clinical Trials, and Approval" (B.
Teicher, Ed.), Humana Press Inc., Totowa (1997)). Since then, MX-1
has been incorporated into a larger panel of breast tumor models
(12 in total) to reflect a shift in NCI strategy from
"compound-oriented" discovery to "disease-oriented" discovery.
[0103] In staged (100-300 mm.sup.3) MX-1 tumors, free topotecan
exhibited dose-dependent inhibition of tumor growth (see, FIG. 5;
Table I). At the highest dose (40 mg/kg), an optimal % T/C of 24%
was obtained; while optimal % T/C values for 10 and 5 mg/kg were
66% and 78%, respectively. No drug-related deaths were observed and
all animals gained weight throughout the study. Liposome
encapsulation of topotecan had a marked impact on % T/C, with
optimal % T/C values of 8%, -49% and -62% following a single
administration of the drug at 2, 5 or 10 mg/kg, respectively. A
negative % T/C value is indicative of tumor volume regression from
the original staged tumor size (100-300 mm.sup.3). According to NCI
guidelines, an optimal % T/C<10% is considered significant
activity, while values <42% are the minimum acceptable limits
for advancing a drug further in development (Corbett, T. et al., In
vivo methods for screening and preclinical testing. In "Anticancer
Drug Development Guide: Preclinical Screening, Clinical Trials, and
Approval" (B. Teicher, Ed.), Humana Press Inc., Totowa (1997)).
Liposome encapsulation increased the toxicity of topotecan,
reducing the MTD to 10 mg/kg from >40 mg/kg for free
topotecan.
[0104] 4. LX-1 Human Lung Carcinoma. LX-1 is an experimental model
of human small cell lung cancer (SCLC). The tumor cell line was
derived from the surgical explant of a metastatic lesion found in a
48 year old male and is provided by the DCTD (NCI) tumor repository
as a tumor fragment that is serially passaged in nude mice. The
LX-1 model was part of the NCI in vivo tumor panel from 1976-1986
(Plowman, J. et al., Human tumor xenograft models in NCI drug
development. In "Anticancer Drug Development Guide. Preclinical
Screening, Clinical Trials, and Approval" (B. Teicher, Ed.), Humana
Press Inc., Totowa (1997)) and, although used less frequently now,
remains a useful xenograft model for comparative activity studies
between free and liposomal drugs because of its rapid growth
rate.
[0105] In general, the LX-1 model was less sensitive to the effects
of topotecan than the MX-1 model, for both free and
liposome-encapsulated drug (see, FIG. 6; Table I). Optimal % T/C
values for free topotecan were 43%, 55% and 67% for doses of 30, 10
or 5 mg/kg, respectively. Anti-tumor activity was improved through
encapsulation, resulting in % T/C values of 8%, 11% and 13% for
doses of 30, 10, or 5 mg/kg, respectively. Interestingly, all of
the liposomal topotecan doses exhibited similar activity. This was
an early study and subsequent studies in other models (see, FIGS.
4-6) indicate dose response beginning at doses<5 mg/kg. This is
consistent with the observation that camptothecin-class compounds
(and presumably other antineoplastic agents) can exhibit
"self-limiting" efficacy whereby, at doses above a critical
threshold dose, no further activity benefits are observed
(Thompson, Biochim. Biophys. Acta 1400:301-319 (1998)). This
situation could conceivably occur if the drug has limited tumor
cell access or if the drug is acting on, and destroying, the tumor
vasculature (i.e., has anti-angiogenic activity). In both
instances, a higher dose of drug would be expected to have
negligible benefit.
[0106] As observed in the L1210 study, encapsulation of topotecan
enhanced the toxicity of the drug and reduced the MTD. The MTD in
tumor-bearing nude mice was 10 mg/kg (.about.16% weight loss). At
30 mg/kg, 4/6 drug-related toxic deaths were observed and maximum
weight loss reached .about.29% (27-34% range).
D. Efficacy Studies in Murine and Human Tumor Models: Multiple Dose
Studies
[0107] 1. MX-1 Human Breast Carcinoma. To address the effectiveness
of multiple administration and prolonged exposure of the tumors to
drug, two multiple dose protocols were examined in MX-1
xenografts--q3dx4 and q7dx3 schedules. On the q4dx3 schedule, free
topotecan exhibited moderate activity at 2.5 and 10 mg/kg/dose and
minimal activity at 1.25 mg/kg/dose (see, FIG. 7; Table II).
Optimal % T/C values for free topotecan on this dosing schedule
were 55%, 30% and 27% for 1.25, 2.5 and 10 mg/kg/dose,
respectively. For the encapsulated topotecan administered on the
same dosing schedule, optimal % T/C values were--15%, -100%, -100%,
and -100% for 0.5, 1.25, 2.5 and 5 mg/kg/dose, respectively. All
regressed tumors were monitored for 60 days. At the end of this
period, all animals treated with .gtoreq.1.25 mg/kg/dose of
liposomal topotecan were considered tumor free.
[0108] On a q7dx3 dosing schedule, little activity was observed
with the free topotecan, either a 5 or 10 mg/kg/dose (see, FIG. 8;
Table II). At the same doses, liposomal topotecan induced complete
regression of the staged tumors. However, on this dosing schedule,
10 mg/kg/dose was too toxic and this portion of the study was
halted as 6/6 toxic deaths (or euthanasia's) were observed by day
24.
[0109] 2. LX-1 Human Lung Carcinoma. Initial studies (single dose)
in the LX-1 model indicated that free topotecan was inactive at
evaluated doses<30 mg/kg and liposomal topotecan inhibited tumor
growth, but did not induce regression. To improve this activity, a
multiple (q7dx3) schedule was examined for both free and liposomal
topotecan. In this instance, considerably greater activity was
observed for free topotecan compared to the single dose study and
optimal % T/C values of 5 and 40 were obtained for 30 and 10
mg/kg/dose, respectively. Liposomal topotecan also exhibited
significantly improved activity, resulting in complete regression
(with subsequent re-growth) at 5 mg/kg/dose. Optimal % T/C values
for liposomal topotecan in this model and dosing schedule were--55,
3 and 16 for 5, 2.5, 1.25 mg/kg/day, respectively.
[0110] 3. Therapeutic Index (TI) Comparisons. The therapeutic index
of free and liposomal topotecan was assessed in 4 different tumor
models on several different dosing schedules (see, Table 4). The
assumptions and definitions used to generate these numbers are
found in Table III. In some instances, a true MED or MTD was not
observed and was therefore estimated mathematically based on dose
response trends. For instance, an acute MTD of 40 mg/kg was
observed for free topotecan administered as a single bolus
injection, but the true MTD (based on weight loss) would likely be
closer to 60 mg/kg if the drug was infused over 5-10 minutes. Also,
complicating the analysis somewhat was the level of potency of the
liposomal formulation. Significant anti-tumor activity was achieved
at low drug doses and the MED had to be estimated in certain
studies. In these instances, a notation was made in Table 4.
[0111] In general, the increase in therapeutic index for liposomal
topotecan was relatively large for single dose administration (5,
10, 15 and 18-fold, depending on the model) and decreased with
increasing dosing frequency. This is illustrated in Table 4, where
the TI.sub.TCS/TI.sub.Free ratio was 4.7-7.5 and 3.3 for q7dx3 and
q3dx4 schedules, respectively. The decrease in the
TI.sub.TCS/TI.sub.Free ratio with more frequent dosing is
consistent with preclinical and clinical studies indicating that
the efficacy and toxicity of free topotecan is
schedule-dependent.
5TABLE 4 Relative Therapeutic Indices of Free and Liposomal
Topotecan in Murine and Human Tumor Models..sup.a Tumor Route of
Dosing Model Inoculation Schedule TI.sub.Free TI.sub.TCS
TI.sub.TCS/TI.sub.Free L1210 i.v. single 1.3 (2.0).sup.b 20 15.4
(10).sup.b (murine leukemia) CT-26 i.s. single 1.0 (1.5).sup.b 5.0
5 (3.3).sup.b (murine colon) MX-1 s.c. single 1.4 (2.1).sup.b 25
17.9 (11.9).sup.b (human q3dx4 15 50.sup.c 3.3 breast) q7dx3 2.0
15.0.sup.c 7.5 LX-1 s.c. single 1.3 (2.0).sup.b 13.3 10.2
(6.7).sup.b (human q7dx3 4.0 18.8 4.7 lung) .sup.abased on data in
Table II and III; formulas and definitions in Table IV.
.sup.bobtained using an acute MTD of 40 mg/kg; second value is
based on an estimated MTD (body weight) .sup.ca conservative
estimate that may be .about.2-fold greater; difficult to assess the
MED due to high activity at low doses.
E. Discussion
[0112] Topotecan is an excellent candidate for liposome
encapsulation. Briefly, topotecan is cell-cycle specific (S-phase)
and activity is greatly enhanced with prolonged exposure, topotecan
exhibits rapid plasma pharmacokinetics and the drug needs to be
maintained below pH 6.0 to retain biological activity. This is an
ideal scenario for using a relatively non-leaky liposome
formulation (such as SM:CH, 55:45) that has an acidic aqueous core.
The required acidic interior can be produced, for example, by
pH-loading or ionophore loading methodology. Here, it has been
demonstrated that encapsulation of topotecan in SM/CH liposomes by
the Mg-A23187 method results in dramatic enhancements in anti-tumor
efficacy. Modest enhancement of toxicity was also observed for
liposomal topotecan, but this was largely offset by substantial
dose reductions that achieved comparable and, in most instances,
superior efficacy relative to the free drug.
[0113] Therapeutic index (TI) is a useful parameter of drug
activity, as it is measure of the ratio of toxicity (MTD) to
biological activity (user defined endpoint, i.e., MED, ED.sub.50,
or ED.sub.80). In general, the lower the TI, the greater the risk
of toxicity since the dose of drug required to elicit a biological
effect approaches the MTD. Therapeutic index is particularly useful
for the evaluation of liposomal drugs since the relative change in
TI can be used to define the benefit (or lack thereof) of
encapsulation. As demonstrated herein, the TI improved from 3-18
fold depending on the model and dose schedule used. Therefore, the
improvement in biological activity observed following liposome
encapsulation of topotecan more than compensates for any increases
in toxicity.
[0114] Without intending to be bound by any theory, it is thought
that the significant improvements in anti-tumor activity and the
increased toxicity of the liposomal form of the drug result from
improved pharmacokinetics and the maintenance of the drug in the
active lactone form. In these studies, 84% of topotecan was present
in plasma as the lactone species after 24 h compared to 48% lactone
for free topotecan after only 5 minutes. Moreover, when the same
dose (10 mg/kg) of free and liposomal topotecan was administered
intravenously in mice, the concentration of lactone was
.about.40-fold higher at times<1 h. At 24 h, the lactone plasma
concentration for liposomal drug was 5.4 .mu.g/ml compared to 1.5
.mu.g/ml at 5 minutes for free drug--still 3.5-fold greater than
the peak lactone concentration for free topotecan.
6TABLE I Summary of Single Dose Anti-Tumor Activity and Toxicity
Parameters Anti-Tumor Activity T - Toxicity Model Dose % T/C.sup.a
C.sup.b % ILS.sup.c LCK.sup.d TF.sup.e DRD.sup.f MWL.sup.g L1210
Free 5 11 0/8 0/8 + (i.v.) Free 10 22 0/8 0/8 + NCTEF-005 Free 20
33 0/8 0/8 + Free 30 44 0/8 0/8 + Free 40 55 0/8 0/8 + TCS 1 44 0/8
0/8 + TCS 5 ** 8/8 0/8 + TCS 10 ** 8/8 0/8 -9.7 TCS 20 ** 7/7 1/8
-14.8 TCS 30 ** 3/3 5/8 -23.4 CT-26 Free 5 31 0/8 0/8 + (i.s.) Free
10 23 0/8 0/8 + NCTEF-005 Free 40 58 1/8 0/8 -0.4 TCS 2 54 0/8 0/8
+ TCS 5 ** 8/8 0/8 -6.8 TCS 10 ** 7/8 0/8 -19.1 MX-1 Free 5 78 0.2
0 0.02 0/6 0/6 + (s.c.) Free 10 66 1.4 13 0.12 0/6 0/6 + NCTEF-004
Free 40 24 4.2 35 0.35 0/6 0/6 + TCS 2 8 7.4 65 0.62 0/6 0/6 + TCS
5 -49 10.2 74 0.85 0/6 0/6 -0.4 TCS 10 -62 14.2 83 1.19 1/6 0/6
-18.3 LX-1 Free 5 67 1.4 0 0.13 0/6 0/6 + (s.c.) Free 10 55 1.9 0
0.18 0/6 0/6 + NCTEF-003 Free 30 43 2.9 7 0.27 0/6 0/6 -1.3 TCS 5
13 7.9 30 0.74 0/6 0/6 -1.7 TCS 10 11 8.7 22 0.82 0/6 0/6 -15.6 TCS
30 8 9.9 22 0.93 0/6 4/6 -29.0 .sup.aoptimal % T/C following final
treatment. Negative value indicates tumor regression. .sup.btumor
growth delay (difference in time for treated and control tumors to
reach 500 mm.sup.3). .sup.cincrease in lifespan relative to
untreated animals (expressed as %). .sup.dlog cell kill (gross).
.sup.etumor free animals at the end of study (i.e. no visible
tumors or long term survivors). .sup.fdrug related deaths.
.sup.gmaximum mean weight loss per treatment group. .sup.hpositive
weight change (i.e. at no time did weight decrease below
pre-treatment weight). ** long term survivors
[0115]
7TABLE II Summary of Single Dose Anti-Tumor Activity and Toxicity
Parameters Anti-Tumor Activity T - Toxicity Model Dose % T/C.sup.a
C.sup.b % ILS.sup.c LCK.sup.d TF.sup.e DRD.sup.f MWL.sup.g MX-1
Free 1.25 55 2.0 20 0.17 0/6 0/6 +.sup.h (q3dx4) Free 2.5 30 5.0 55
0.42 0/6 0/6 + NCTEF-006 Free 10 27 2.5 52 0.21 1/6 0/6 + TCS 0.5
-15 23.5 157 1.96 1.6 0/6 -0.3 TCS 1.25 -100 ** ** 6/6 0/6 -1.0 TCS
2.5 -100 ** ** 6/6 0/6 -11.5 TCS 5 -100 ** ** 6/6 0/6 -20.0 MX-1
Free 5 58 1.8 27 0.15 0/6 0/6 + (q7dx3) Free 10 61 2.0 ND.sup.i 0/6
0/6 -0.8 NCTEF-009 TCS 5 -100 ** ** 6/6 0/6 -7.6 TCS 10 -100
ND.sup.i ND.sup.i 6/6 6/6 -29.0 LX-1 Free 10 40 2.0 21 0.14 0/6 0/6
-6.2 (q7dx3) Free 30 5 20.9 58 1.53 0/6 0/6 -8.8 NCTEF-007 TCS 1.25
16 10.8 54 0.79 0/6 0/6 -7.7 TCS 2.5 3 23.2 79 1.70 0/6 0/6 -7.3
TCS 5 -55 30.2 100 2.22 0/6 0/6 -10.5 LX-1 Free 10 28 4.4 41 0/6
0/6 -3.6 (q7dx3) Free 30 9 25 72 0/6 2/6 -16.4 NCTEF-011 TCS 7.5
ND.sup.i ND.sup.i ND.sup.i 0/6 6/6 >-30 TCS 0.75 27 11.2 50 0/6
0/6 -1.3 .sup.aoptimal % T/C following final treatment. Negative
value indicates tumor regression. .sup.btumor growth delay
(difference in time for treated and control tumors to reach 500
mm3). .sup.cincrease in lifespan relative to untreated animals
(expressed as %). .sup.dlog cell kill (gross). .sup.etumor free
animals at the end of study (i.e. no visible tumors or long term
survivors). .sup.fdrug related deaths. .sup.gmaximum mean weight
loss per treatment group. .sup.hpositive weight change (i.e. at no
time did weight decrease below pre-treatment weight). .sup.inot
determined; toxic deaths in the liposome-encapsulated group. **
"cures"; no visible tumors by day 60.
[0116]
8TABLE III Definitions and Formulas for Toxicity and Anti-Tumor
Activity Parameters DRD Drug-related death. A death was considered
drug-related if the animal died or was euthanized within 15 days
following the final treatment with drug AND its tumor weight was
less than the lethal burden on control mice, or its weight loss was
greater than 20% that of the control animals. GI.sub.50 The
concentration of drug that causes 50% growth inhibition in a
population of cells in vitro. The NCI renamed the IC.sub.50
parameter to emphasize the correction for cell count at time zero.
Therefore, the formula is: GI.sub.50 = (T - T.sub.0)/(C - T.sub.0)
.times. 100 = 50 T and T.sub.0 are the optical densities at 48 and
0 h, respectively; C is the control (cell count) optical density at
0 h. % ILS Increase in lifespan (in percent). For survival models
this is calculated using the median survival times for the treated
(T.sub.treat) and control (T.sub.cont) animals, according to:
(T.sub.treat - T.sub.cont)/T.sub.cont .times. 100 For the solid
tumor models, the time for tumors to reach 2000 mm.sup.3
(.about.10% of body weight) was used as an ethical cutoff instead
of median survival. LCK Log cell kill (gross). This parameter
estimates the number of log.sub.10 units of cells killed at the end
of treatment, according to the formula: (T - C) .times.
0.301/median doubling time Net log cell kill can be calculated by
subtracting the duration of treatment from the tumor growth delay
(T - C) parameter as follows: [(T - C) - duration of treatment]
.times. 0.301/median doubling time A log cell kill of 0 indicates
that the cell population at the end of treatment is the same as it
was at the onset of treatment. However, a log cell kill of 4, for
example, indicates a 99.99% reduction in the initial cell
population. MBWL Maximum body weight loss (in percent). The animals
are weighed prior to the first administration of the drug (Wi) and
on various days during the study (Wd). The percent change in body
weight is calculated by: MBWL = (W.sub.d - W.sub.i)/W.sub.i .times.
100 MED Minimum effective dose. This is a somewhat arbitrary
parameter. For these studies we have defined the MED as the lowest
dose achieving an optimal % T/C .ltoreq. 40 (for solid tumor
models) or a % ILS of 40-60% (for survival models). MTD Maximum
tolerated dose. Dose of drug that results in a MBWL of .ltoreq.20%.
% T/C Optimal ratio of treated vs control tumors obtained following
the first course of treatment. These values are obtained by
subtracting the median tumor weight on the first day of treatments
(T.sub.i or C.sub.i) from the tumor weights on each observation day
according to the following formula: % T/C = (.DELTA. T/.DELTA. C)
.times. 100, where .DELTA. T > 0, or % T/C = (.DELTA. T/T.sub.i)
.times. 100, where .DELTA. T < 0 According to NCI activity
criteria, the following scoring system applies (Plowman, et al.,
Human tumor xenograft models in NCI drug development. In
"Anticancer Drug Development Guide: Preclinical Screening, Clinical
Trials, and Approval" (B. Teicher, Ed.), Humana Press Inc., Totowa
(1997)[22]: 0 = inactive, % T/C > 40 1 = tumor inhibition, % T/C
range 1-40 2 = tumor stasis, % T/C range 0 to -40 3 = tumor
regression, % T/C range -50 to -100 4 = % T/C range -50 to -100 and
>30% tumor-free mice TGD Tumor growth delay (also represented as
T - C). This parameter expresses the difference in time (in days)
for treated and control tumors to attain an arbitrary size
(typically 500 or 1000 mm.sup.3). TI Therapeutic index. Therapeutic
index is the ratio of a toxicity parameter (i.e. LD.sub.50,
LD.sub.10, MTD) and a biological activity parameter (i.e. ED.sub.50
- the dose that causes a defined biological response in 50% of the
treatment group). In general, TI describes the margin of safety for
a drug. For animal model studies this is traditionally described by
the formula: TI = LD.sub.50/ED.sub.50 However, since it is no
longer ethically permissible to perform LD.sub.50 studies, we have
defined therapeutic index for these studies as: TI = MTD/MED
[0117] It is to be understood that the above description is
intended to be illustrative and not restrictive. Many embodiments
will be apparent to those of skill in the art upon reading the
above description. The scope of the invention should, therefore, be
determined not with reference to the above description, but should
instead be determined with reference to the appended claims, along
with the full scope of equivalents to which such claims are
entitled. The disclosures of all articles and references, including
patent applications and publications, are incorporated herein by
reference for all purposes.
* * * * *