Note: Descriptions are shown in the official language in which they were submitted.
O 94/26250 ~ ~ ~ PCT/US93/04645
-1-
METHOD FOR TREATING NEUROLOGICAL DISORDERS
BACKGR UND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for treating a neurological disorder
using a slow-release vehicle for delivery of a therapeutic agent to the
cerebrospinal (CSF) of a human.
2. Description of the Relafed Art
Neurological disorders are among the most difficult diseases to treat. A major
complicating factor in treating such disorders is the inability of many drugs
to
penetrate the blood-brain barrier when the agent is administered systemically.
This ineffectiveness of classical drug delivery to address this need is
particularly
problematic with respect to chronic neurological disorders, such as those
caused by benign or malignant cell proliferation or various viral etiologic
agents.
Among the most difficult chronic neurological disorders to treat are those
derived from metastatic infiltration, such as neoplastic meningitis.
Neoplastic
meningitis results from the metastatic infiltration of the leptomeninges by
cancer, and is most commonly a complication of acute leukemia, lymphoma,
or carcinoma of the breast and lung. Autopsy studies indicate that 5 to 8
~ percent of solid tumor patients develop metastasis to the leptomeninges
during
the course of disease. Evidence suggests that the incidence of neoplastic
meningitis may be increasing, in part due to increased survival from effective
systemic therapies. (Bleyer, Curr. Probl. Cancer, 12:184, 1988).
WO 94!26250 PCT/US93/04645~
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2-
Standard treatment for neoplastic meningitis includes single agent or
combination intrathecal chemotherapy and radiation therapy. Radiotherapy to
the entire neuraxis often produces severe marrow depression and has not
been satisfactory in controlling active leptomeningeal disease except in
leukemic meningitis. (Kogan, in Principle and Practice of Radiation Oncology,
Perez, et al. eds., Lippincott, Philadelphia, PA, pp. 1280-1281, 1987).
Systemic
chemotherapy likewise is not generally effective in active meningeal
malignancy
because of poor drug penetration through the blood-brain barrier. (Blasberg,
et al., Can. Treat. Rep., 01:633, 1977; Shapiro, et al., New Eng. J. Med.,
29$:161, 1975). Cytarabine, one of the three chemotherapeutic agents most
commonly used for intrathecal therapy of neoplastic meningitis, is a cell-
cycle
phase specific agent that kills cells only when DNA is being synthesized.
Consequently, optimal tumor kill with agents such as cytarabine requires
constant infusion or frequent daily injections to maintain therapeutic
concentrations for extended periods in CSF. This procedure is uncomfortable
for patients, time consuming for physicians, and associated with an increased
risk of infectious meningitis. Therefore, there is a need for a slow-releasing
depot formulation which can allow a therapeutic agent to persist in contact
with
a neurological disorder in order to achieve an ameliorative effect. The
present
invention addresses this need.
O 94/26250 ~ ~ ~ ~ ~ PCTIUS93/04645
-3-
SUMMARY OF THE INVENTION
The present invention arose from the seminal discovery that the clinical
effectiveness of therapeutic agents in the treatment of neurological disorders
in humans could be greatly enhanced if the therapeutic agent was
administered as part of a dispersion system. This therapeutic approach allows
effective dose levels of the agent to be maintained over a relatively long
period
of time such that the neurological disorder is continuously exposed to the
agent. Surprisingly, the dispersion system containing the therapeutic agent
can be effectively administered intralumbar even though the primary foci of
the
neurological disorder are centered in the cranium region, such as the
ventricles.
WO 94/26250 ~ ~ ~ ~ ~ ~ ~. . PCTIlJS93104645~
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE shows the ventricular CSF pharmokinetics of intraventricularly
administered DTC 101 as a function of dose from 12.5 to 125 mg.
FIGURE 2 shows the maximum CSF cytarabine concentration [Panel A] and
AUC [Panel B] as functions of dose.
FIGURE 3 shows a comparison of the ventricular (closed circles) and lumbar
(open circles) cytarabine concentrations [total and free, Panels A and B,
respectively] and DTC 101 particle count [Panel C] as functions of time
following intraventricular administration of DTC 101.
1 o FIGURE 4 shows cytarabine concentration in the ventricular CSF as a
function
of time (solid lines), and lumbar CSF cytarabine concentration at 3 minutes
and
14 days (broken lines), following intralumbar administration of DTC 101.
~O 94/26250 PCT/US93/04645
-5-
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention is directed to a method for ameliorating neurological
disorders which comprises administering a therapeutic agent to the cerebral
spinal fluid (CSF). The surprising ability of the therapeutic agent to
ameliorate
the neurological disorder is due to the presentation of the therapeutic agent
in
a dispersion system which allows the agent to persist in the cerebro-
ventricular
space. The ability of the method of the invention to allow the therapeutic
agent
to persist in the region of the neurological disorder provides a particularly
effective means for treating those disorders which are chronic and, thereby,
are
1 o particularly difficult to achieve a clinical effect.
The term "neurological disorder" denotes any disorder which is present in the
brain, spinal column, and related tissues, such as the meninges, which are
responsive to an appropriate therapeutic agent. Among the various
neurological disorders for which the method of the invention is effective are
those which relate to a cell proliferative disease. The term "cell
proliferative
disease" embraces malignant as well as non-malignant cell populations which
often appear morphologically to differ from the surrounding tissue. Thus, the
cell proliferative disease may be due to a benign or a malignant tumor. In the
latter instance, malignant tumors may be further characterized as being
primary
tumors or metastatic tumors, that is, tumors which have spread from systemic
sites. Primary tumors can arise from glial cells (astrocytoma,
oligodendroglioma, glioblastoma), ependymal cells (ependymoma) and
supporting tissue (meningioma, schwannoma, papilloma of the choroid plexus).
In children, tumors typically arise from more primitive cells
(medulloblastoma,
neuroblastoma, chordoma), whereas in adults astrocytoma and glioblastoma
are the most common. However, the most common CNS tumors in general
WO 94126250 PCT/US93/04645,""
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are metastatic, particularly those which infiltrate the leptomeninges. Tumors
that commonly metastatically invade the meninges include non-Hodgkin's
lymphoma, leukemia, melanoma, and adenocarcinoma of breast, lung, or
gastrointestinal origin.
The method of the invention is also useful in ameliorating neurological
disorders which arise as a result of an infectious disease. Aseptic meningitis
and encephalitis are CNS diseases which are caused by a virus. Among the
viral infections which may benefit most from the ability of the method of the
invention to allow the therapeutic agent to persist are those viral diseases
1 o caused by a slow virus or a retrovirus. Of particular interest among the
retroviruses are the Lentivirus, which include HTLV-I, HTLV-II, HIV-1 and HIV-
2.
Neurological disorders which arise due to an infectious disease caused by a
prokaryote can also be treated according to the method of the invention.
Typically, the procaryotic etiologic agent is a bacteria such as Hemophilus
influenzae, Neisseria meningitides, Streptococcus pneumonia, Pseudomonas
aeruginosa, Escherichia coli, Klebsiella-Enterobacter, Proteus, Mycobacterium
tuberculosis, Staphylococcus aureus, and Listeria monocytogenes.
Alternatively, the infectious disease can be caused by a eukaryote, such as a
fungus. Important fungi which can be treated according to the method
invention include Cryptococcus, Coccidioides immitis, Histoplasma, Candida,
Nocardia, and Blastomyces.
The therapeutic agents used according to the method of the invention are
administered to the CSF in a delivery system such as synthetic or natural '
polymers in the form of macromolecular complexes, nanocapsules,
microspheres, or beads, and lipid-based systems including oil-in-water
~O 94/26250 .~ PCTIUS93/04645
emulsions, micelles, mixed micelles, synthetic membrane vesicles, and resealed
erythrocytes. These systems are known collectively as dispersion systems.
Typically, the particles comprising the system are about 20 nm - 50 ~m in
diameter. The size of the particles allows them to be suspended in a
pharmaceutical buffer and introduced to the CSF using a syringe. The
administration may be intraventricularly or, more preferably, intrathecally.
Most
preferred is injection of the particles by intralumbar puncture
Materials used in the preparation of dispersion systems are typically
sterilizable
via filter sterilization, nontoxic, and biodegradable, for example, albumin,
ethylcellulose, casein, gelatin, lecithin, phospholipids, and soybean oil can
be
used in this manner. Polymeric dispersion systems can be prepared by a
process similar to the coacervation of microencapsulation. If desired, the
density of the dispersion system can be modified by altering the specific
gravity to make the dispersion hyperbaric or hypobaric. For example, the
dispersion material can be made more hyperbaric by the addition of iohexol,
iodixanol, metrizamide, sucrose, trehalose, glucose, or other biocompatible
molecules with high specific gravity.
One type of dispersion system which can be used according to the invention
consists of a dispersion of the therapeutic agent in a polymer matrix. The
2o therapeutic agent is released as the polymeric matrix decomposes, or
biodegrades, into soluble products which are excreted from the body. Several
classes of synthetic polymers, including polyesters (Pitt, et al., in
Controlled
Release of Bioactive Materials, R. Baker, Ed., Academic Press, New York,
1980); polyamides (Sidman, et al., Journal of Membrane Science, 7:227, 1979);
polyurethanes (Maser, et al., Journal of Polymer Science, Polymer Symposium,
...
CA 02162854 2002-07-12
X6:259, 1979); polyorthoesters {Heller, et al., Polymer Engineering Science,
X1:727, 1981 ); and polyanhydrides {Leong, et aL, Biomaterials, _1:364, 1986)
have been studied for this purpose. Considerable research has been done on
the polyesters of PLA and PLA/PGA. Undoubtedly, this is a consequence of
convenience_and safety considerations. These polymers are readily available,
since they have been used as biodegradable sutures, and they decompose
into non-toxic lack and giyoofic aads (see, U.S. 4,578,384; U.S. 4,765,973;
incorporated by reference).
Solid polymeric dispersion systems can be synthesized using such
1 o polymerization methods as bulk polymerization, inttrrfaaal poiymerizatiai;
solution polymer~at~n, ar>d ring opening polymerization (Odian, G., Principles
of Polymerizaffon, 2nd ed., John Whey & Sons, New York, 1981). Using any
of these methods, a variety of different syr>thetic polymers having a broad
range of mechanical, chemical, and biodegradable properties are obtained; the
differences in properties and characteristics are controlled by varying the
parameters of reaction temperatures, reactant ~ntrations, types of solvent,
and reac~on time. If desired, the solid polyrrisric dispersion system can be
produced iniGatfy as a larger mass which is then ground, or otherwise
processed, into particles small enough to maintain a dispersion in the
2o appropriate physiologic buffer (see, for example, U.S. 4,452,025; U.S.
4,389,330; and U.S. 4,696,258).
The mechanism of release of therapeutic agent from biodegradable slabs,
cylinders, and spheres has been described by Hopfenberg {in Controlled
Release Polymeric Formulaflons, pp. 2&32, Paul, D.R. and .Harris, F.W., Eds.,
American Chemical Society, Washington, D.C., 1976). A simple expression
O 94126250 ~ ~ ~ ~~ PCT/US93/04645
-9-
describing additive release from these devices where release is controlled
primarily by matrix degradation is:
Mt~M~ = 1 - (1 _ kot/Coa~n
where n = 3 for a sphere, n = 2 for a cylinder, and n = 1 for a slab. The
symbol a represents the radius of a sphere or cylinder or the half-thickness
of
a slab. Mt and M~ are the masses of drug release at time t and at infinity,
respectively.
Most preferred as a dispersion system according to the invention are synthetic
membrane vesicles. The term "synthetic membrane vesicles" denotes
1 o structures having one or more concentric chambers, commonly known as
liposomes, as well as structures having multiple non-concentric chambers
bounded by a single bilayer membrane.
When phospholipids are dispersed in aqueous media, they swell, hydrate, and
spontaneously form multiiamellar concentric bilayer vesicles with layers of
aqueous media separating the lipid bilayer. Such systems are usually referred
to as multilamellar liposomes or multilamellar vesicles (MLVs) and have
diameters ranging from about 100nm to about 4~m. When MLV's are
sonicated, small unilamellar vesicles (SUVs) with diameters in the range of
from
about 20nm to about 50 nm are formed, which contain an aqueous solution in
2o the core of the SUV.
The composition of the synthetic membrane vesicle is usually a combination
of phospholipids, particularly high-phase-transition-temperature
phospholipids,
usually in combination with steroids, especially cholesterol. Other
phospholipids or other lipids may also be used.
WO 94/26250 PCT/LTS93104645"",
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Examples of lipids useful in synthetic membrane vesicle production include
phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and
. .,
gangliosides. Particularly useful are diacylphosphatidylglycerols, where the
lipid
moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon
atoms, and are saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine, and
distearoylphosphatidyl-
choline.
in preparing vesicles containing a therapeutic agent, such variables as the
efficiency of drug encapsulation, lability of the drug, homogeneity and size
of
the resulting population of vesicles, drug-to-lipid ratio, permeability
instability of
the preparation, and pharmaceutical acceptability of the formulation should be
considered. (Szoka, et al., Annuai Reviews of Biophysics and Bioengineering,
,x:467, 1980; Deamer, et aL, in Liposomes, Marcel Dekker, New York, 1983, 27;
Hope, et al., Chem. Phys. Lipids, 4~( :89, 1986).
If desired, it is possible to produce synthetic membrane vesicles with various
degrees of target specificity. The targeting of vesicles has been classified
based on anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific, cell-specific,
and
organelle-specific. Mechanistic targeting can be further distinguished based
upon whether it is passive or active. Passive targeting utilizes the natural
tendency of vesicles to distribute to cells of the reticulo-endothelial system
,
(RES) in organs which contain sinusoidal capillaries. Active targeting, on the
other hand, involves the alteration of the vesicle by coupling the vesicle to
a
specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein,
or
by changing the composition or size of the vesicles in order to achieve
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targeting to organs and cell types other than the naturally occurring sites of
localization. Alternatively, vesicles may physically localize in capillary
beds.
Another dispersion system which can be used according to the invention is
resealed erythrocytes. When erythrocytes are suspended in a hypotonic
medium, swelling occurs and the cell membrane ruptures. As a consequence,
pores are formed with diameters of approximately 200-500 A which allow
equilibration of the intracellular and extracellular environment. If the ionic
strength of this surrounding media is then adjusted to isotonic conditions and
the cells incubated at 37 ° C, the pores will close such that the
erythrocyte
1 o reseals. This technique can be utilized to entrap the therapeutic agent
inside
the resealed erythrocyte.
The surface of the dispersion system may be modified in a variety of ways.
Non-lipid material may be conjugated via a linking group to one or more hydro-
phobic groups, for example, alkyl chains from about 12-20 carbon atoms. In
the case of a synthetic membrane vesicle delivery system, lipid groups can be
incorporated into the lipid bilayer in order to maintain the compound in
stabile
association with the membrane bilayer. Various linking groups can then be
used for joining the lipid chains to the compound.
Whether a ligand or a receptor, the number of molecules bound to a synthetic
membrane vesicle will vary with the size of the vesicle, as well as the size
of
~ the molecule to be bound, the binding affinity of the molecule to the target
cell
receptor or ligand, as the case may be, and the like. In most instances, the
bound molecules will be present on the vesicle from about 0.05 to about 2
mol%, preferably from about 0.1 to about 1 mol%, based on the percent of
,.i~~~,,~ P i i ~ i
CA 02162854 2002-07-12
-t 2-
bound molecules to the total number of molecules in the outer membrane
bilayer of the vesicle.
In general, the compounds to be bound to the surface of the targeted delivery
system will be Ggands and receptors which will allow the dispersion system to
actively "home in" on the desired tissue. A ligand may be any compound of
interest which will specifically bind to another compound, referred to as a
receptor, such that the ligand and receptor form a homologous pair. The
compounds bound to the service of the dispersion system may vary from small
haptens from about 125-200 molecular weight to much larger antigens with
' 10 molecular weights of at least about 6000, but generall~r of less than 1
million
molecular weight. Proteinaceous ligands and receptors are of partia~la~r
interest. In general, the surface membrane proteins which bind to speafic
effedor molecules are referred to as receptors. As preserdly used, however,
most receptors will be antibodies. These antibodies may be monodonal or
1 s polydonal and may be fragments thereof such as Fab, F(ab~~, and Fw which
are capable of tending to an epitopic determinant. Ted~nfor binding of
proteins, such as antibodies, to synthetic membrane vesides are well known
(see, for example, U.S. 4,806,466 and U.S. 4,857,735 .
2o The term "therapeutic agent" as used herein for the compositions of the
'' invention includes, without limitation, drugs, radioisotopes, and immun-
ornodulators. Similar substances are known or can be readily ascertained by
one of skill in the art There may be certain combinations of therapeutic agent
with a given type of dispersion system which are more compatible than others.
25 For example, the method for producing a solid polymeric dispersion may not
be compatible with the continued biological activity of a proteinaceous
~O 94/26250 PCT/US93/04645
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therapeutic agent. However, since conditions which would produce an
uncompatible pairing of a particular therapeutic agent with a particular
dispersion system are well known, or easily ascertained, it is a matter of
routine
to avoid such potential problems.
The drugs which can be incorporated in the dispersion system include non-
proteinaceous as well as proteinaceous drugs. The term "non-proteinaceous
drugs" encompasses compounds which are classically referred to as drugs
such as, for example, mitomycin C, daunorubicin, vinblastine, AZT, and
hormones. Of particular interest are anti-tumor cell-cycle specific drugs such
1 o as cytarabine, methotrexate, 5-fluorouracil (5-FU), floxuridine (FUDR),
bleomycin, 6-mercapto-purine, 6-thioguanine, fludarabine phosphate,
vincristine,
and vinblastine. Similar substances which can also be used according to the
invention are within the skill of the art.
The proteinaceous drugs which can be incorporated in the dispersion system
include immunomodulators and other biological response modifiers as well as
antibodies. The term "biological response modifiers" encompasses substances
which are involved in modifying the immune response in such manner as to
enhance the particular desired therapeutic effect, for example, the
destruction
of tumor cells. Examples of immune response modifiers include such
2o compounds as lymphokines. Examples of lymphokines include tumor necrosis
factor, the interleukins, lymphotoxin, macrophage activating factor, migration
inhibition factor, colony stimulating factors and the interferons. Interferons
which can be incorporated into the dispersion systems include a-interferon,
(3-interferon, and y-interteron and their subtypes. In addition, peptide or
polysaccharide fragments derived from these proteinaceous drugs, or
WO 94/26250 , , PCT/US93/04645~
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independently, can also be incorporated. Those of skill in the art will know,
or
can readily ascertain, other substances which can act as proteinaceous drugs.
In using radioisotopes to treat cell proliferative disorders, such as tumors,
certain radioisotopes may be more preferable than others depending on such
factors, for example, as tumor distribution and mass, as well as isotope
stability
and emission. Depending on the type of malignancy present some emitters
may be preferable to others. In general, a and ~3 particle-emitting
radioisotopes
are preferred in immunotherapy. For example, if a patient has solid tumor foci
a high energy ~3 emitter capable of penetrating several millimeters of tissue,
1 o such as 9°Y, may be preferable. On the other hand, if the
malignancy consists
of single target cells, as in the case of leukemia, a short range, high energy
a
emitter such as 2'2Bi may be preferred. Examples of radioisotopes which can
be incorporated in the dispersion system for therapeutic purposes
are'251,'3'I,
90~,~ s7Cu~ 212Bi, 2»At, 2'2Pb, 4'Sc,'°9Pd and'~Re. Other radioisotopes
which
can be incorporated are within the skill in the art.
When an antibody is incorporated into the dispersion system, the antibody,
whether monoclonal or polyclonal, may be unlabeled or labeled with a
therapeutic agent. The term "antibody" or "immunoglobulin" as used herein,
includes intact molecules as well as fragments thereof, such as Fab, F(ab~2,
2o and F~, which are capable of binding to an epitopic determinant on a cell
proliferative or infectious neurological disorder etiologic agent. When
coupled
to an antibody, the therapeutic agent can be coupled either directly or
indirectly. One example of indirect coupling is by use of a spacer moiety.
These spacer moieties, in turn, can be either insoluble or soluble (Diener, et
al.,
Science, x:148, 1986) and can be selected to enable drug release from the
antibody molecule at the target site. Examples of therapeutic agents which can
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be coupled to the antibody for immunotherapy are drugs and radioisotopes,
as described above, as well as lectins and toxins.
Lectins are proteins, usually isolated from plant material, which bind to
specific
sugar moieties. Many lectins are also able to agglutinate cells and stimulate
s lymphocytes. However, ricin is a toxic lectin which has been used
immunotherapeutically. This is preferably accomplished by binding the alpha-
peptide chain of ricin, which is responsible for toxicity, to the antibody
molecule
to enable site specific delivery of the toxic effect.
Toxins are poisonous substances produced by plants, animals, or microorgan-
isms that, in sufficient dose, are often lethal. Diphtheria toxin is a
substance
produced by Corynebacterium diphtheria which can be used therapeutically.
This toxin consists of an a and (3 subunit which under proper conditions can
be separated. The toxic a component can be bound to an antibody and used
for site specific delivery to a target cell for which the antibodies are
specific.
Other therapeutic agents which can be coupled to the monoclonal antibodies
are known, or can be easily ascertained, by those of ordinary skill in the
art.
The labeled or unlabeled antibodies can also be used in combination with
other therapeutic agents such as those described above. Especially preferred
are therapeutic combinations comprising a monoclonal antibody and an
2o immunomodulator or other biological response modifier. Thus, for example, a
monoclonal antibody can be used in combination with a-interferon. This
treatment modality enhances monoclonal antibody targeting of carcinomas by
increasing the expression of monoclonal antibody reactive antigen by the
carcinoma cells (Greiner, et al., Science, x:895, 1987). A dispersion system
based on the use of a synthetic membrane vesicle with multiple non-concentric
WO 94/26250 PCT/US93/04645~
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chambers is particularly useful with combination therapy, since the non-
concentric chambers can be loaded with different therapeutic agents. Those
of skill in the art will be able to select from the various biological
response
modifiers to create a desired effector function which enhances the efficacy of
the monoclonal antibody or other therapeutic agent used in combination.
When the monoclonal antibody of the invention is used in combination with
various therapeutic agents, such as those described herein, the administration
of the monoclonal antibody and the therapeutic agent usually occurs
substantially contemporaneously. The term "substantially contemporaneously"
1 o means that the monoclonal antibody and the therapeutic agent are adminis-
tered reasonably close together with respect to time. Usually, it is preferred
to administer the therapeutic agent before the monoclonal antibody. For
example, the therapeutic agent can be administered 1 to 6 days before the
monoclonal antibody. The administration of the therapeutic agent can be daily,
or at any other interval, depending upon such factors, for example, as the
nature of the neurological disorder, the condition of the patient and half-
life of
the agent.
The term "therapeutically effective" as it pertains to the compositions of the
invention means that the therapeutic agent is present at a concentration
2o sufficient to achieve a particular medical effect for which the therapeutic
agent
is intended. Examples, without limitation, of desirable medical effects which
can be attained are chemotherapy, antibiotic therapy, and regulation of ,
metabolism. Exact dosages will vary depending upon such factors as the
particular therapeutic agent and desirsable medical effect, as well as patient
factors such as age, sex, general condition and the like. Those of skill in
the
~O 94/26250 PCTIUS93104645
-17-
art can readily take these factors into account and use them to establish
effective therapeutic concentrations without resort to undue experimentation.
The foregoing is meant to illustrate, but not to limit, the scope of the
invention.
Indeed, those of ordinary skill in the art can readily envision and produce
6 further embodiments, based on the teachings herein, without undue
experimentation.
WO 94!26250 PCTIUS93/04645~
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EXAMPLE 1
PRODUCTION OF DEPO/ARA-C yDTC1011 ,
This example describes the production of a synthetic membrane vesicle having
multiple non-concentric chambers containing ara-C which are bounded by a
single bilayer membrane.
Dioleoyl lecithin (8.3 g), dipalmitoylphosphatidyl glycerol (1.66 g),
cholesterol
(6.15 g), and triolein (1.73 g) were mixed with 800 ml of chloroform (the
lipid
phase) in a 13-liter glass homogenizes vessel fitted with a 2-inch mixing
blade.
Next, Cytosine arabinoside (33 mg/ml) was dissolved in 0.151 N HCI (at a final
volume of 1.2 liters) and added to the homogenizes vessel. To form a water-in-
oil emulsin, the mixing blade was rotated at 8000 rpm for 10 minutes.
Low ionic strength aqueous component comprising free base lysine (40 mM)
and glucose (3.2%) was added to the homogenizes to form the chloroform
spherules and the mixing blade was rotated at 3500 rpm for 90 seconds. To
remove the chloroform, nitrogen gas was bubbled through the mixture at 62
liters per minute for 30 minutes while the vessel was heated to 35 ° C.
The
resulting product was purified and concentrated by diafiltration using
polysulfone hollow-fiber with 0.1~C pore size and 8 ftz surface area.
~O 94/26250 PCT/LJS93/04645
21~2$~~
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EXAMPLE 2
INTRATHECAL AND INTRAVENTRICULAR TREATMENT WITH
ENCAPSULATED DEPO/ARA-C
A. PATIENTS AND METHODS
Twelve patients with a histologically proven diagnosis of cancer and radiologi-
cal or cytologic evidence of neoplastic meningitis were treated. This human
investigation was performed after approval by the UCSD Human Subjects
Committee. There was no pertormance status requirement and prior intra-CSF
chemotherapy was allowed. The patients were given 47 total doses of DTC
1 o 101. There were 4 patients with hematological malignancies and 8 with
solid
tumors (TABLE 1 ). Concurrent systemic chemotherapy was given to 5
patients.
WO 94/26250 PCT/US93/04645~
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TABLE '1
PATIENT CHARACTERISTICS
Total Number of Patients 12
Male
Female
saes i(range, ~ er ars) 6-73
Median 38
Diagnoses
Chronic myelogenous leukemia in blast crisis 1
AIDS-related non-Hodgkin's lymphoma 2
Multiple myeloma i
Breast cancer 2
Non-small cell lung cancer 1
Head and neck cancer 1
Renal cell tumor 1
Melanoma 1
Primitive neuroectodermal tumor 2
Prior Therap~r for Meningeal Disease
With prior therapy 9
2o Without prior therapy 4
T~rpes of Prior Therapy
Methotrexate 6
Cytarabine 3
ThioTEPA 3
interferon 1
~O 94/26250 PCT/LTS93/04645
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An Ommaya reservoir was placed in the right lateral ventricle in all but one
patient with chronic myelogenous leukemia in blast crisis. Therapy consisted
of DTC i 01 suspended in a preservative-free 0.9% NaCI solution administered
intraventricularly or by the lumbar intrathecal route as a single injection
once
every 2-3 weeks. The reservoir was flushed with autologous CSF after DTC
101 dosing and at each CSF sampling.
B. TREATMENT
The initial dose of 12.5 mg was escalated (25, 37.5, 50, 75, 125 mg) after at
least 3 cycles in 2 patients who were available for evaluation. Treatment was
continued until disease progression or to a maximum of 7 doses. Initial work-
up included history and physical examination and complete neurological
examination; CBC and platelet count; CSF sample for cytology; serum
chemistries; CT or MR brain scan with and without appropriate contrast agents
and Indium-DTPA CSF flow studies (Chamberlain, ef al., Neurol., 4_Q:435-438,
1990; Chamberlain, et al., Neurol., 41:1765-1769, 1991 ). Before each cycle of
DTC 101, complete neurological history and examination, blood counts, and
chemistries were done, and CSF samples were obtained for cytologic
examination. Complete cytologic response was defined as two consecutive
negative CSF cytology examinations at least one week apart; anything less
2o than a complete response was considered as no response. Progressive
disease was defined as conversion from negative to positive cytology.
Changes in parenchyma) CNS lesions or lesions outside the CNS were not
used as part of response determination since these were not expected to be
influenced by intra-CSR therapy. Treatment-induced toxicities were scored
using the "Common Toxicity Scale" of the National Cancer Institute.
WO 94!26250 ~ ~ ~' ~ ~ (PCT/US93/04645~
Figure 1 shows the CSF pharmacokinetics of cytarabine following intraventricu-
lar administrations of DTC 101 at various doses ranging from 12.5 to 125 mg,
where CSF samples were obtained from the same ventricle into which DTC 101 ,
had been injected. Panel A, total cytarabine concentration; panel B, free
cytarabine concentration. Each data point is an average from at least three
courses and the error bars represent standard errors of mean. Following
intraventricular administration of a maximum tolerated dose (75 mg), the
ventricular concentration of free cytarabine (cytarabine that had been
released
from DepoFoam particles into the CSF) decreased biexponentially with an
average initial (a) half-life of 9.4 ~ 1.6 hrs (SEM), and terminal (/3) half-
life of 141
~ 23 hrs (SEM). The total ventricular concentration (free plus encapsulated
cytarabine) decreased in a similar biexponential manner.
Pharmacokinetic Studies
Ventricular CSF and blood samples were obtained immediately before injection
and at 1 hr and then 1, 2, 4, 7, 14, and 21 days following injection. Lumbar
CSF samples were obtained in selected patients as a part of evaluation for
lumbar CSF cytology at one of these same time points. For intralumbar
injections, a sample from the lumbar sac was obtained 3 minutes after
injection
in lieu of the 1 hr sample. All CSF and blood samples were collected in tubes
2o containing tetrahydrouridine at a final concentration of 40 ~rM to prevent
in vitro
catabolism of cytarabine to uracil arabinoside (ara-U) by cytidine deaminase.
The heparinized blood samples were immediately placed on ice and plasma
was separated from blood cells by centrifugation. The CSF samples were
centrifuged at 600 X g for 5 minutes to separate DepoFoam particles from the '
free cytarabine fraction (the supernate). The DepoFoam pellet was lysed by
vortexing sequentially in 200 NI methanol and in distilled water. The free
~O 94/26250 PCTIL1S93104645
-23-
cytarabine fractions of CSF were analyzed without further processing. The
plasma was ultrafiltered (YMT membrane, No. 4104; Amicon Corp., Danvers,
MA). The CSF and plasma samples were stored frozen at -20 ° until
analysis
by a modification of previously described method (Kaplan, JG, et al., J Neuro-
Onc, x:225-229, 1990). The samples were analyzed on a high performance
liquid chromatography system (Waters Associates, Milford, MA) with 254 and
280 mm UV detectors, two Pecosphere C-18 reverse-phase columns (3X3C
Cartridge; Perkin-Elmer, Norwalk, CT) in tandem, and 6.7 mM potassium
phosphate/3.3 mM phosphoric acid mixture (pH 2.8) as an isocratic mobile
1 o phase at a flow rate of 1.0 ml/min. Retention time for cytarabine was 6
minutes
and that for the major metabolite, ara-U, was 7 minutes. There were no
intertering peaks.
The pharmacokinetic curves were fit to the biexponential function C(t) = Ae'~
+ BED, where C(t) is the concentration at time 5, A and B are constants and
a and (3 are the initial and terminal rate constants. The RSTRIP program
(MicroMath Scientific Software, Salt Lake City, UT) was used to perform the
curve fitting by iterative non-linear regression. The area under the concentra-
tion-versus-time curve (AUC) was determined by the linear trapezoidal rule up
to the last measured concentration and extrapolated to infinity. CSR clearance
of cytarabine was determined by dividing the dose of cytarabine by the AUC.
The initial volume of distribution of cytarabine in CSF (Vd) was calculated by
dividing the dose of cytarabine by the concentration measured at 1 hr.
TABLE 2 shows the detailed pharamcokinetic parameters as a function of dose.
The half-lives (T1~2), volumes of distribution (Vd), and clearances (CI) did
not
change significantly as the dose was escalated from 12.5 to 125 mg.
WO 94/26250 PCT/US93/04645"",
' _24-
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~O 94/26250 PCT/LTS93/04645
~~5~
25-
Figure 2 depicts the maximum ventricular cytarabine concentration (Panel A)
measured at 1 hr following DTC 101 administration, and the CSF drug
exposure (AUC, Panel B) as a function of dose administered intraventricularly.
Open and closed circles represent total and free cytarabine, respectively.
Each
s data point is an average from at least three courses nd the error bars
represent standard errors of mean. There was a linear relationship between
these pharmacokinetic parameters and dose, indicating that there was no
saturation of clearance process over the dose range examined. The total ara-U
AUC averaged 3.7 ~ 0.9% (SEM) of the total cytarabine AUC in the CSF. No
1 o cytarabine or ara-U was detected in the plasma (detection limit = 0.25
~g/ml
for both) at any time point.
Lumbar CSF samples were obtained during five courses in two patients
following intraventricular administration of DTC 101 at the maximum tolerated
dose (75 mg). Figure 3 compares the ventricular drug concentration and DTC
15 101 particle count with that in the lumbar subarachnoid space. Comparison
of the ventricular (closed circles) and lumbar (open circles) cytarabine
concentrations (total and free, Panels A and B, respectively) and DTC 101
particle count (Panel C) as functions of time following intraventricular
adminis-
tration of DTC 101. The initial ventricular free cytarabine concentration
20 decreased in an exponential fashion with a half-life of 6.8 hrs; cytarabine
became detectable in lumbar CSF at 1.25 hrs and then increased rapidly with
a doubling time of 0.53 hrs. Subsequently, the lumbar and ventricular
_ concentrations of free and total cytarabine decreased in parallel fashion,
with
the lumbar drug concentrations remaining comparable to those in the ventricle
25 throughout the terminal phase of the decay curve.
WO 94/26250 ~ ~ ~ ~ ~ ~ j~ PCT/CTS93/04645~
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Both ventricular and intralumbar CSF samples were obtained from four patients
given DTC 101 intrathecally by lumbar puncture. Figure 4 shows that a
therapeutic concentration of free cytarabine (>0.1 ~Cg/ml) was maintained for
3
to 6 days in ventricular CSF following intrathecal lumbar injection, and a
significant concentration of total cytarabine was found in the ventricular CSF
for
14 days following intralumbar administration. Cytarabine concentration in the
ventricular CSF as a function of time (solid lines), and lumbar CSF cytarabine
concentration at 3 minutes and 14-days (broken lines), following intralumbar
administration of DTC 101. Open squares and circles represent total
cytarabine concentrations and closed squares and circles represent free
cytarabine concentrations. A therapeutic concentration of free cytarabine was
maintained for more than 14 days in the lumbar subarachnoid space following
intralumbar injection.
TABLE 3 summarizes the toxicities of DTC 101 as a function of dose. The
toxicities were transient and in no instance did drug-related toxicity delay
therapy with a subsequent dose of DTC 101. There was one death due to the
occurrence of a toxic encephalopathy that developed 36 hours following
intraventricular administration of 125 mg of DTC 101. This patient was also
receiving concurrent whole brain irradiation (20 Gy in 5 fractions) for
partial
blockage of CSF flow at the base of brain. There were no hematological
toxicities attributable to DTC 101 except in one patient who had an autologous
bone-marrow transplant two months prior to DTC 101 administration. The
maximum tolerated dose of DTC 101 was 75 mg; dose limiting toxicity
occurred a dose of 125 mg, at which there was excessive vomiting and
encephalopathy (TABLE 3).
~O 94126250 ~ ~ ~ ~ ~ ~ PCTIUS93104645
-27-
TAB~.E 3
TOXICITY OF DTC 101 AS A FUNCTION OF DOSE
Dose (mg) 12.5 25 37.5 50 75 125
Patients 2 6 5 4 8 4
Courses 3 7 7 6 20 4
Fever 1 (1 )* 0 (4) 0 (1 0 0 0
) (3) (5) (1
)
Headache 0 (1 ) 0 (7) 0 (2) 2 0 1
(2) (4) (2)
Neck/back pain 0 (0) 0 (0) 0 (0) 0 0 0
(1 (3) (0)
)
Nausea/vomiting 0 (3) 1 (2) 1 (3) 0 2 3
(4) (4) (1
)
Cerebellar 0 (1 ) 0 (0) 0 (0) 0 0 0
(0) (0) (0)
Tinnitus 0 (0) 0 (1 0 (0) 0 0 0
) (0) (0) (0)
Encephalopathy 0 (0) 1 (1 0 (0) 1 0 1
) (0) (0) (1
)
Hyponatremia 0 (0) 0 (0) 0 (0) 0 0 0
(0) (1 (0)
)
* The numbers in the columns represent toxicities greater than grade 2.
The numbers in parenthesis represent toxicities of grade 1 or 2.
TABLE 4 shows that oral dexamethasone in doses of 2 to 4 mg twice per day
had a major effect on blunting toxicities associated with DTC 101. Fever,
headache, and nausea/vomiting were all reduced. There were three patients
who received the same doze of DTC 101 with and without oral dexamethasone.
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All three patients manifested toxicity without dexamethasone and in each
patient the toxicity was almost completely suppressed with concurrent oral
dexamethasone.
TABLE 4
EFFECT OF CONCURRENT
ORAL DEXAMETHASONE
ADMINISTRATION ON DTC XICITY
101 TO
~-1 Dexamethasone ~+~Dexamethas one
Number of courses 9 12
with toxicity grade 1-2 3-4 1-2 3 -4
Fever 44 0 8 0
Headache 44 0 8 0
Back/neck pain 33 0 0 0
Nausea/vomiting 22 22 17 8
Encephalopathy 0 0 0 0
Intraventricular and intralumbar routes were combined.
Four patients were treated with DTC 101 by the lumbar intrathecal route. The
toxicities were similar to those observed following intraventricular
administration
except 4 of 9 cycles were associated with grade 1 to 2 low back pain.
O 94/26250 ~ ~ ~ ~ ~ PCTIUS93104645
w
-29-
Nine of 12 patients had a positive CSF cytology immediately prior to
treatment.
Seven of these 9 cytologically evaluable patients cleared their CSF of
malignant
cells with DTC 101 treatment (TABLE 5). The duration of response ranged
from 2 to 26 weeks with a median of 16 weeks. One non-responding patient
had an AIDS-related non-Hodgkin's lymphoma and the other had a primary
brain tumor. Survival time for all patients on study ranged from 3 to 64 weeks
(median: 21 weeks).
WO 94/26250 PCT/US93/04645~
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TA_ BLE 5
SF CYTOLOGIC RESPONSE TO DTC 101
Number of Patients 12
Number with positive CSF cytology 9
Cleared CSF with DTC 101
Responders
Breast cancer 1
Non-small cell lung cancer 1
Multiple Myeloma 1
1 o CML in blast crisis 1
Melanoma 1
AIDS-related non-Hodgkin's lymphoma 1
Primitive neuroectodermal tumor 1
Non-Responders
AIDS-related NHL 1
Primitive neuroectodermal tumor 1
Three of twelve patients had evidence of neoplastic meningitis by CT or MRI
scan, but had negative CSF cytology prior to therapy and were not evaluable
for cytoiogic response. However, none of these three patients developed a
2o positive CSF cytology while on treatment.
~O 94/26250 PCTILTS93104645
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Surprisingly, responses were observed at all dose levels and were not limited
to the higher doses. One patient with multiple myeloma relapsed following an
initial cytologic response at the 25 mg dose level, then responded again to a
higher dose {37.5 mg) of DTC 101. Three of five patients presenting with
headache responded clinically to DTC 101 therapy. No clinical improvement
was observed in patients with focal (ophthalmoplegia or paraparesis) or
diffuse
(acute confusional state) neurologic deficits at the start of therapy.
A number of embodiments of the present invention have been described.
Nevertheless, it will be understood that various modifications may be made
1 o without departing from the spirit and scope of the invention. Accordingly,
it is
to be understood that the invention is not to be limited by the specific
illustrated embodiment, but only by the scope of the appended claims.
