Note: Descriptions are shown in the official language in which they were submitted.
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PLAS.MA. FROM'VITII.IGO PATIENTS FOR TREATMENT OF MELANOMA
CROSS REFERENCE TO RELATED APPLICATIONS
[o0o1] This application claims priority from US Provisional Application Number
60/773,335, filed
February 15, 2006, which is incorporated herein by reference in its entirety
FIELD OF INVENTION
[oo02] This invention is directed to compositions and methods of treating
melanoma in a subject.
Specifically, the invention is directed to the use of plasma containing
antibodies directed against
melanoma in the treatment of melanoma.
ts BACKGROUND OF THE INVENTION
[0003] Melanomas are aggressive, frequently metastatic turnors derived from
either melanocytes or
ritelanocyte related nevus cells ("Cellular and Molecular Immunology" (1991)
(eds) Abbas A. K.,
Lechtman, A. H., Pober, J. S.; W. B. Saunders Company, Philadelphia: pages 340-
341). Melanomas
2o make up approximately three percent of all skin cancers and the worldwide
increase in melanorna is
unsurpassed by any other neoplasm with the exception of lung cancer in women
("Cellular and
Molecular Immunology" (1991) (eds) Abbas, A. K., Lechtiman, A. H., Pober, J.
S.; W. B. Saunders
Company Philadelphia pages; 340-342; Kirkwood and Agarwala (1993) Principles
and Practice of
Oncology 7:1-16). Even when melanorna is apparently localized to the skin, up
to 30% of the patients
25 will develop systemic metastasis and the majority will die (Kirkwood and
Agarwala (1993) Principles
and Practice of Oncology 7:1-16). Classic modalities of treating melanoma
include surgery, radiation
and chemotherapy. In the past decade immunotherapy and gene therapy have
emerged as new and
promising methods for treating melanoma.
30 [0004] Strong evidence that an immune response to cancer exists in humans
is provided by the existence
of lymphocytes within melanoma deposits. These lymphocytes, when isolated, are
capable of
recognizing specifia tumor antigens on autologous and allogeneic melanomas in
an MHC restricted
fashion. (Itoh, K. et al. (1986), Cancer Res. 46: 3011-3017; Muul, L. M., et
al. (1987), J. Immunol.
1
CA 02642572 2008-08-15
WO 2007/095293 PCT/US2007/003919
138:989-995); Topalian, S. L., et al., (1989) J. Immunot. 142: 3714-3725;
Darrow, T. L., et al., (1989) J.
Immunol. 142: 3329-3335; Hom, S. S., et al., (1991) J Immunother. 10:153-164;
Kawakami, Y., et al.,
(1992) J. Immunol. 148: 638-643; Hom, S. S., et al., (1993) J Immunother.
13:18-30; ONeil, B. H., et
al., (1993) J. Imrnunol. 151: 1410-1418). TIL from patients with metastatic
melanoma recognize shared
antigens including melanocyte-melanoma lineage specific tissue antigens in
vitro (Kawakami, Y., et al.,
(1993) J. Immunother. 14: 88-93; Anichini, A. et al., (1993) et al., J. Exp.
Med 177: 989-998). Anti-
melanoma T cells appear to be enriched in TIL probably as a consequence of
clonal expansion and
accumulation at the tumor site in vivo (Sensi, M., et al., (1993) T. Exp. Med
178:1231-1246). The fact
that many melanoma patients mount cellular and humoral responses against these
tumors and that
to melanomas express both MHC antigens and tumor associated antigens (TAA)
indicate that
identification, characterization and use of additional melanoma antigens will
be important for
immunotherapy of patients with melanoma.
SUMMARY OF THE INVENTION
[0005] In one embodiment, the invention provides a composition for the
treatment of melanoma in a
subject, comprising: plasina isolated from a vitiligo patient.
[0006] In another embodiment, the invention provides a composition for the
treatment of melanoma in a
2o subject, comprising: plasma isolated from a melanoma patient.
[0007] In one embodiment, the invention provides a method for treating
melanoma in a subject,
comprising the step of administering to the subject a preparation comprising
plasma isolated from a
vitiligo patient.
[ooos] In another embodiment, the invention provides a method for treating
melanoma in a subject,
comprising the step of administering to the subject a preparation comprising
plasma isolated from a
melanoma patient.
l00091 In one embodiment, the invention provides an article of manufacture
comprising a suitable
packaging material and plasma isolated from a vitiligo patient.
2
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[o001o] In another embodiment, the invention provides an article of
manufacture comprising a suitable
packaging material and plasma isolated from a melanoma patient. _
[oooii] Other features and advantages of the present invention will become
apparent from the following
s detailed description examples and figures. It should be uinderstood,
however, that the detailed
description and the specific examples while indicating preferred embodiments
of the invention are given
by way of illustration only, since various changes and modifications within
the spirit and scope of the
invention will become apparent to those skilled in the art from this detailed
description
DETAILED DESCRIPTION OF THE INVENTION
[oa012] This invention relates in one embodiment to the use of plasma
comprising antibodies to
melanoma antigens in the treatment of melanoma. In another embodiment his
invention relates to
plasma from a group of vitiligo patients or pooled from a group of vitiligo
patients.In one embodiment,
1s the invention provides a composition for the treatment of inelanoma in a
subject, comprising: plasma
isolated from a vitiligo patient, or in anotli.er embodiment, the plasma used
in the compositions and
methods described herein is isolated from a melanoma patient. In another
embodiment his invention
relates to plasma from a group of melanoma patients or pooled from a group of
inelanoma, patients.
[00013] In another embodiment, the term "vitiligo" may be referred to as
leucoderma. In one
embodiment, the depigmented areas are lacking in the skin pigment melanin, and
in another
embodiment, the disease is the result of the destruction or inhibition of the
melanin secreting
melanocytes in the affected areas. In yet other embodiments, there may be some
Iiereditary component
to the disease, since approximately 30% of the cases have a familial
correlation. In one embodirnent, the
disease may be the result of an autoimmune or a specific metabolic defect may
be involved, or,
environmental factors to play a role in the aetiology of the disease in
another embodiment.
[000141 In one embodiment, vitiligo is subdivided into two clinical types:
vitiligo non segmentalis (type
A) and vitiligo segmentalis (type B). Type A is more common, has a potential
lifelong evolution and is
associated with ICoebner phenomenon and frequently with autoimmune diseases,
such as Sutton nevus,
thyroid disorders, juvenile diabetes mellitus, pernicious anemia and Addison's
disease. Type B is rarer
and has a dermatomal distribution , wherein following a rapid onset and
evolution it usually exhibits a
stable course. The natural course of the disease is unpredictable in one
embodiment, but it is progressive
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in other embodiments. In one .embodiment some degree of spontaneous
repigmentation occurs in 10-
20 10 of patients, occurring in a perifollicular'pattem.
[00015] In one embodiment, there is a relationship between vitiligo and
melanoma [.Flarning R, Cui J,
Bystryn JC. -Relation between the incidence and level ofpigment cell
antibodies and disease actfvity in
vitiligo. J Invest Derrnatol 97: 1078-80, 1991; Fishman P, Azizi E. Shoenfeld
Y, et al. Vitiligo
autoantibodies are effective against melanoma. Cancer 72: 2365-2369, 1993. ;
Gilhar A, Zelickson B,
Ulman Y, Etzioni A_ In vivo destruction of melanocytes by the IgG fraction of
serum from patients with
vitiligo. J Invest Dermatol 105: 683-686, 1995.; Rigel DS, Rogers GS, Friedman
RJ. Prognosis in
lo malignant melanoma. Dermatol Clin North Am 3:309-14, 1985. Byslryn JC,
Rigel D, Friedman RJ,
Kopf A. Prognostic sign fcance of hyperpigmentation in malignant melanoma.
Arch Dermatol 123:
1053-5, 1987; D'aelio R, Frati C, Fattarossi A, Aiuti F. Peripheral T cell
subset imbalance in patients
with vitiligo and in their apparently healthy first degree relatives. Ann
Allergy 65: 143-5, 1990.; Bystryn
JC, Naughton GK. Immunity to pigrnented cells in vitiligo and melanoma. Fed
Proc 43:1664-5, 1984.;
ts Lerner AB, Nordlund J.1: Should vitiligo.be induced in patients after
resection of primary melanoma?
Editorial. Arch Dermatol 113: 421, 1977; Donaldson RC, Canaan SA Jr, McLean
RB, Ackerman LV.
Uveitis and vitiligo associated with BCG treatment for malignant melanoma.
Surgery 76:771-8, 1974;
Naughton GK, Eising M, Bystryn JC. Detection of autoantibodies to melanocytes
in vitiligo by specific
immunoprecipitation Arch Dermatol 81:540-2, 1983; Norris DA, Kissinger RM,
Naughton GK
20 Bystr,yn JC. Evidence for immunologic mechanisms in human vitiligo:
patients' sera induce damage to
human melanocytes in vitro by 'complement-mediated damage and antibody
dependent' cellular
cytotoxicity (ADCC). J Invest Dermatol 90: 783-9, 1988] and in another
embodiment, in patients with
malignant melanoma, circulating antibodies against melanoma cells are present.
Because these
antibodies also destroy normal, non-malignant melanocytes, vitiligo develops
in other embodiments, in
25 malignant, melanoma patients. In one embodiment, the prognosis in melanoma
patients is better when
hypo-pigmentation develops [Koh HK, Sober AJ, Nakagawa H, et al. Malignanat
melanonza and
vitiligo-like leukoderma: An electron nzicroscopic study. J Am Acad Dermatol
9:696-708, 1983,
Nordlund JJ, Kirkwood JM, Forget BM, et aL Vitiligo in patients with
metastatic melanoma: A good
prognostic sign. J Anz Acad Dermatol 9:689-96, 1983]. Therefore in one
embodiment, the selective
3o destruction of pigmented cells occurring in vitiligo is natural
immunotherapy for melanoma, and plasma
used in the composition described herein, which is isolated from melanoma
patients in which vitiligo
developed, or in another etnbodiment, from vitiligo patients, is useful in the
treatment of inelanoma
according to the methods described herein. = -
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(00016) In one embodiment, the plasma used in the compositions and methods of
the invention, is
isolated from vitiligo patients in which the vitiligo is characterized as
diffuse vitiligo. The term "diffuse'
vitiligo ' refers'in one embodiment to "generalized vitiiigo", or "vitiligo
non segmentalis (type Aj",
wherein the patchy white' areas are often symmetrical, flat, have definite
borders, and may affect'or
spread-to any part of the body.
[00017] In one embodiment, the plasma used in the methods and compositions
described herein, '
developed in a subject suffering from melanoma, in which the vitiligo
developed as a response to the
io melanoma treatment.
[00018] In one embodiment - melanoma, or in another embodiment, metastatic
melanoma responds to a
variety of immunotherapies, including in one embodiment, the administration of
IL-23 and the adoptive
transfer of T cells along with IL-2. In another embodiment, some melanoma
patients develop vitiligo
during or after immunotberapy, and T cells that are reactive'to both melanoma
cells and cu[tured
melanocytes have been detected in these patients, indicating that in one
embodiment, T cells inay be
involved in the destruction of melanoma and melanocytes. In one embodiment, T
cells specific for
melanocyte proteiins play an important role in melanoma rejection and -in the
development of vitiligo in
vivo. In one embodiment, the vitiligo developed in melanoma or metastatic
melanoma might not be
2o caused by autoimmunity, or in another embodiinent, the immune responses
might not be strong enough
to detect the antibodies specific to the melanoma cell phenotype or the
metastatic melanoma cell
phenotype, which, are different from on another. In one embodiinent, the
plasma used for the treatment
of melanoma in a subject in the compositions and methods described herein, is
isolated from a patient,
or pool of patients, in which vitiligo is shown to have been autoimmune in
nature.
[00019] In one embodiment, plasma refers to the liquid medium in which blood
cells are suspended and
. which contains salts, proteins and other organic compounds. In one
embodirnent, "fresh" blood plasma,
or in another embodiment, frozen (stored) and subsequently thawed plasma,
recovered plasma, or source
plasma is used for purposes of the compositions and methods described herein.
"Source plasma" is
3o defined in one embodiment, as plasma collected by plasmapheresis and
intended for further use such as
fractionation into other materials in certain embodiments. In one embodiment,
source plasma is used as
a transfusion product in the compositions and methods described herein. Frozen
(stored) plasma is
maintained in one embodiment at sub-freezing temperatures, such as at storage
conditions of -20 to -80
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degrees centigrade until thawed and used. "Fresh" plasma is refrigerated in
one embodiment or
maintained on ice until transfusion in another embodiment. In one embodiment,
the plasma used for
= transfusion according to the. methods described herein, is fresh-frozen
plasma, recovered plasma, source
+
plasma or combination thereof.
[0oo2o] In one embodiment, blood from a vitiligo patient, or in another
embodiment, from a melanoma
patient in which antibodies to melanoma antigens are identified, is drawn by
standard methods into a
collection bag, or a plastic.collection bottle for example, sodium citrate,
CPD, CPD Al, ACD, heparin,
or similar anticoagulants for preparation of plasma. In one embodiment, the
plasma is separated from
to whoie blood prior to being frozen. In one embodiment, fresh plasma is
separated from whole blood by
centrifugation, or fractionated by other standard methods in other
embodiments. In another embodiment
the plasma is collected by plasmapheresis wherein the plasma is separated
during or the collection
procedure by a variety of techniques. In one' embodimerit, the term
"plasmapheresis" refers to the
separation of blood into plasma fraction and cellular component fraction
providing for the collection of
plasma alone, with the cellular components being returned to the donor with a
suitable portion of
replacement fluid
[00021] In one embodiment, the compositions described herein, or the
preparations used in the methods
described herein comprise plasma that is isolated from vitiligo patients and
further comprise a
pharmaceutically. acceptable carrier, or excipient, flow agent, processing
aid, diluent or a combination
thereof in other embodiments. rn one embodiment, the carrier, excipient,
lubricant, flow aid, processing
aid or diluent is a gum, or a starch, a sugar, a cellulosic material, an
acrylate, calcium carbonate,
magnesium oxide, tale, lactose monohydrate, magnesium stearate, colloidal
silicone dioxide or mixtures
thereof in other embodiments.
[00022] The formulation may in one embodiment, be in the form of a bolus, or
in the form of aqueous or
non-aqueous isotonic sterile injection solutions or suspensions. These
solutions and suspensions may be
prepared from sterile powders or granules having one or more pharmaceutically-
acceptable carriers or
diluents, or a binder such as gelatin or hydroxypropyl-methyl cellulose,
together with one or more of a
so lubricant, anticoagulant, preservative, surface-active or dispersing agent
or a combination thereof.
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[00023] In another embodiment, the composition further comprises a binder, a
disintegrant, a buffer, a
protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an
emulsifier, a stabilizing agent, a
viscosity increasing agent, a sweetner, a film forming agent, or any
combination thereof.
s [00024] In one embodiment, the composition is a particulate composition
coated with a polymer (e.g.,
poloxamers or poloxamines). Other embodiments * of the compositions of the
invention incorporate
particulate forms protective coatings, protease inhibitors or permeation
enhancers for various routes of
administration, including parenteral, pulmonary, nasal and oral. In one
embodiment the pharmaceutical
composition is administered topically, parenterally, paracancerally,
transmucosally, transdermally,
intramuscularly, intravenously, intradermally, subcutaneously,
intraperitonealy, intraventricularly,
intratumorioally, or intracranially.
[00025] In one embodiment, the compositions of this invention may be in the
form of a pellet, a tablet, a
capsule, a solution, a suspension, a dispersion, an emulsion, an elixir, a
gel, an ointment, a cream, or a
suppository.
[00026] In another einbodiment, the composition is in a form suitable-for
oral, intravenous, intraaorterial,
intramuscular, subcutaneous, parenteral, transmucosal, transdermal, or topical
administration. In one
embodiment the composition is a controlled release composition. In another
embodiment, the
composition is an immediate release composition. In one embodiment, the
composition is a liquid
dosage forin. In another embodiment, the composition is a solid dosage form.
[00027] The compounds utilized in the methods and compositions of the present
invention may be
present in the form of free bases in one embodiment or pharmaceutically
acceptable acid addition salts
thereof in another embodiment. In one embodiment, the term "pharmaceutically-
acceptable salts"
embraces salts commonly used to form alkali metal'salts and to form addition
salts of free acids or free
bases. The nature of the salt is not critical, provided that it is
phannaceutically-acceptable. Suitable
pharmaceutically-acceptable acid addition salts of cornpounds of Formula I are
prepared in another
embodiment, from an inorganic acid or from an organic acid. Examples of such
inorganic acids are
3o hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and
phosphoric acid. Appropriate
organic acids may be selected from aliphatic, cycloaliphatic, aromatic,
araliphatic, heterocyclic,
carboxylic and sulfonic classes of organic acids, example of which are formic,
acetic, propionic,
succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,
glucuronic, maleic, fumaric, pyruvic,
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aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic,
phenylacetic, mandelic, embonic
(pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-
hydroxyethanesulfonic,
toluenesulfonic, sulfanilic, cyclohexylaminosulfonic,' stearic, algenic, b-
hydroxybutyric, salicylic,
galactaric and galacturonic acid. Suitable pharmaceutically-acceptable base
addition salts include
s metallic salts made from aluminum, calcium, lithium, magnesium, potassium,
sodium and zinc or
organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine,
ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these
salts may be prepared by
conventional means from the corresponding compound by reacting, in another
embodiment, the
appropriate acid or base with the compound.
(oo028] In one embodiment, the term "pharmaceutically acceptable carriers"
includes, but is not limited
to, may refer to 0.01-0.1M and preferably 0.05M phosphate buffer, or in
another embodiment 0.8%
saline. Additionally, such pharmaceutically acceptable carriers may be in
another embodiinent aqueous
or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable
organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions
or suspensions, including
saline and buffered rnedia.
[00029] In one embodiment, the compositions described herein, may include
compounds modified by the
covalent attachment of water-soluble polymers such as polyethylene glycol,
copolymers of polyethylene
glycol and polypropylene glycol, oarboxymethyl cellulose, dextran, polyvinyl
alcohol,
polyvinylpyrrolidone or polyproline are known to exhibit substantially longer
half-lives in blood
following intravenous injection than do the corresponding unmodified compounds
(Abuchowski et al.,
1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications tnay
also increase the
2s compound's solubility in aqueous solution, eliminate aggregation, enhance
the physical and chemical
stability of the compound, and greatly reduce the immunogenicity and
reactivity of the compound. As a
result, the desired in vivo biological activity may be achieved by the
administration of such polymer-
compound abducts less frequently or in lower doses than with the unrnodified
compound.
[00030] The compositions described herein, which may be used in the
preparations used in the methods
described herein, can be prepared by known dissolving, mixing, granulating, or
tablet-forming
processes. For oral administration, the aative ingredients, or their
physiologically tolerated derivatives in
another embodiment, such as salts, esters, N-oxides, and the like are mixed
with additives customary for
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this purpose, such as vehicles, stabilizers, or inert diluents, and converted
by customary methods into
suitable forms for administration, sucb as tablets, coated tablets, hard or
soft gelatin capsules, aqueous,
alcoholic or oily solutions. Examples of suitable inert vehicles are
conventional tablet bases such as
lactose, sucrose, or cornstarch in combination with binders such as acacia,
cornstarch, gelatin, with
disintegrating agents such as cornstarch, potato starch, alginic acid, or with
a lubricant such as stearic
acid or magnesium stearate.
[ooo31] Examples of suitable oily vehicles or solvents are vegetable or animal
oils such as sunflower oil
or fish-liver oil. Preparations can be effected both as dry and as wet
granules. For parenteral
to administration (subcutaneous, intravenous, intraarterial, or intramuscular
injection), the active
ingredients or their physiologically tolerated derivatives such as salts,
esters, N-oxides, and the like are
converted into a solution, suspension, or emulsion, if desired with the
substances customary and suitable
for this purpose, for example, solubilizers or other auxiliaries. Examples are
sterile liquids such as water
and oils, with or without the addition of a surfactant and other
pharmaceutically acceptable adjuvants.
Illustrative oils are those of petroleum, animal, vegetable, or synthetic
origin, for example, peanut oil,
soybean oil, or mineral oil. In general,,water, saline, aqueous dextrose and
related sugar solutions, and
glycols such as propylene glycols or polyethylene glycol are preferred liquid
carriers, particularly for
injectable solutions.
[00032] In addition, the composition can contain minor amounts of auxiliary
substances such as wetting
or emulsifying agents, pH buffering agents which enhance the effectiveness of
the active ingredient.
[00033] An active component which, in one embodiment is the plasma isolated
from a vitiligo patient, or
in another embodiment, from a melanoma patient in which antibodies to melanoma
antigens are
identified, can be formulated into the composition as neutralized
pharmaceutically acceptable salt forms_
Pharmaceutically acceptable salts include the acid addition salts (formed with
the free amino groups of
the polypeptide or antibody molecule), which are formed with inorganic acids
such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like.
Salts formed from the free carboxyl groups can also be derived from inorganic
bases such as, for
example, sodium, potassium, ammoniuni, calcium, or ferric hydroxides, and such
organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and
the like.
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[00o34] In one embodiment, the plasma isolated from a vitiligo patient, or in
another embodiment, from a
melanoma patient in which antibodies to melanoma =antigens = are identified,
is administered in a
therapeutically effective amount. The 'actual amount administered, and the
rate and time-course of
administration, will depend in one embodiment, on the nature and severity of
the condition being
s treated. Prescription of treatment, e.g. decisions on dosage, timing, etc.,
is within the responsibility of
general practitioners or specialists, and typically takes account of the
disorder to be treated, the
condition of the individual patient, the site of delivery, the method of
administration and other factors
known to practitioners. Examples of techniques and protocols can be found in
Remington's
Pharmaceutical Sciences.
[ooo35] Alternatively, targeting therapies may be used in another embodiment,
to deliver the plasma
compositions described herein, more specifically to certain types of cell, by
the use of targeting systems
such as antibodies or cell specific ligands. Targeting may be desirable in one
embodiment, for a variety
of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise
require too high a dosage, or if
it would not otherwise be able to enter the target cells.
[00036i The compositions described herein, are formulated in one embodiment
for oral delivery, wherein
the active compounds which in one embodiment is plasma isolated from a
vitiligo patient, or in another
embodiment, from a melanoma patient in which antibodies to melanoina antigens
are identified, may be
incorporated with excipients and used in the form of ingestible tablets,
buccal tables, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. The tablets, troches,
pills, capsules and the like may
also contain the following: a binder, as gum tragacanth, acacia, cornstarch,
or gelatin; excipients, such as
dicalcium phosphate; a disintegrating agent, such as corn starch, potato
starch, alginie acid and the like;
a lubricant, such as magnesium stearate; an anticoagulant, such as sodium
citrate or any of the whole
blood anticoagulant-preservative solution such as ACD, CPD, CPD-Al, Adsol and
the like; and a
sweetening agent, such as sucrose, lactose or saccharin may be added or a
flavoring agent, such as
peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form
is a capsule, it may
contain, in addition to materials of the above type, a liquid carrier. Various
other materials may be
present as coatings or to otherwise modify the physical form of the dosage
unit. For instance, tablets,
pills, or capsules may be coated with shellac, sugar, or both. Syrup of elixir
may contain the active
compound sucrose as a sweetening agent methyl and propylparabens as
preservatives, a dye and
flavoring, such as cheiry or orange flavor. In addition, the active compounds
inay'be incorporated into
sustained-release, pulsed release, controlled release or postponed release
preparations and formulations
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(00037] Controlled or sustained release compositions include formulation in
lipophilic.depots (e.g. fatty
acids, waxes, oils). Also ooinprehended by the invention are particulate
compositions coated with
polymers (e.g. poloxamers or poloxamines) and the compou.nd coupled to
antibodies directed against
tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-
specific receptors.
[00038] In one embodiment, the composition described herein, or the
preparation used in the methods
described herein, can be delivered in a controlled release system. For
example, the plasma isolated from
a vitiligo patient, or.in another embodiment, from a melanoma patient in which
antibodies to melanoma
antigens are identified, may be administered using intravenous infusion, an
implantable osmotic pump, a
transdermal patch, liposomes, colloidosomes, polymerosomes or other modes of
administration. In one
embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref.
Biomed. Eng. 14:201
(1987); Buehwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J.
Med. 321:574 (1989). In
another embodiment, polymeric materials can be used. In another embodiment, a
controlled release
system can be placed in proximity to the therapeutic target, i.e., the
melanoma site, thus requiring only a
fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of
Controlled Release, supra,
vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in
the review by Langer
(Science 249:1527-1533 (1990).
[00039] Such compositions are in one embodiment liquids or lyophilized or
otherwise dried formulations
and include diluents of various buffer content (e.g., Tris-HCI:, acetate,
phosphate), pH and ionic
strength, additives such as albumin or gelatin to prevent absorption to
surfaces, detergents (e.g., Tween
20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g.,
glycerol, polyethylene glycerol),
anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives
(e.g., Thimerosal, benzyl alcohol,
parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol),
covalent attachment of
polymers such as polyethylene glycol to the protein, complexation with metal
ions, or incorporation of
the material into or onto particulate preparations of polymeric compounds such
as polylactic acid,
polglycolic acid, hydrogels, etc., or onto liposomes, niicroemulsions,
micelles, unilamellar or
multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions
will influence the
physical state, solubility, stability, rate of in vivo release, and rate of in
vivo clearance. Controlled or
sustained release compositions include forinulation in lipophilic depots
(e.g., fatty acids, waxes, oils).
Also comprehended by the invention are particulate cbmpositions coated with
polymers (e.g.,
poloxamers or poloxamines). Other embodiments of the compositions of the
invention incorporate
11
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WO 2007/095293 PCT/US2007/003919
particulate forms, protective coatings, protease inhibitors, or permeation
enhancers for various routes of
administration, including parenteral, pulrrionary, nasal, and oral.
(00040] In another embodiment, the compositions of this invention comprise one
= or more,
s pharmaceutically acceptable carrier materials.
[00041] In one embodiment, the carriers for use within such compositions are
biocompatible, and in
another embodiment, biodegradable. In other embodiments, the formulation may
provide a relatively
constant level of release of one active component. In other embodiments,
however, a more rapid rate of
io release immediately upon administration may be desired. In other
embodiments, release of the plasma
isolated from a vitiligo patient, or in another embodiment, from a melanoma
patient in which antibodies
to melanoma antigens are identified, may be event-triggered. The events
triggering the release of , the
plasma isolated from a vitiligo patient, or in another embodiment, from a
melanoma patient in which
antibodies to melanoma antigens are identified, may be the same in one
embodiment, or different in
3s another embodiment. Events triggering the release of the active components
may be exposure to
moisture in one embodiment, lower pH in another embodiment, or temperature
threshold in another
embodiment. The formulation of such compositions is well within the level of
ordinary skill in the art
using known techniques. Illustrative carriers useful in this regard include
microparticles of poly(lactide-
co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like.
Other illustrative postponed-
20 release carriers include suprainolecular biovectors, which comprise a non-
liquid hydrophilic core (e.g., a
cross-linked polysaccharide or oligosaccharide) and, optionally, an external
layer comprising an
amphiphilic compound, such as phospholipids. The amount of active compound
contained in one
embodiment, within a sustained release formulation depends upon the site of
administration, the rate and
expected duration of release and the nature of the condition to be treated
suppressed or inhibited.
[000.42] In one embodiment, the plasma isolated from a vitiligo patient, or in
another embodiment,
plasma from a melanoma patient is isolated from a single patient, or from a
pool of patients. In one
embodiment, blood is collected into a container that already has an
appropriate anticoagulant, or
anticoagulant-preservative solution in it and stored for later separation. The
separated plasma is then
frozen for later use as an active agent in the compositions and methods as
described herein. In one
einbodiment, the isolated plasma is frozen at a rate that ensures the
viability of the components. In
another embodiment, following the freezing of the plasma, the frozen plasma is
further lyophilized.
12
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[00043] In one embodiment, the plasma isolated from a melanoma patient in
which antibodies to
melanoma antigens such as HMW-MAA or tyrosinase are identified, is used in the
methods and
compositions of the invention. In one embodiment, melanoma is immunogenic and
can activate host
immune responses capable of controlling the disease and= causing tumor
regression. -
[ooo44] In one embodiment, the term. "antibody" inelude complete antibodies
(e.g., bivalent IgG,
pentavalent IgM) or fragments of antibodies in other embodiments, which
contain an antigen binding
site. Such fragment include in one embodiment Fab, F(ab')2, Fv and single
chain Fv (scFv) fragments. In
one embodiment, such fragments may or may not include antibody constant
domains. In another
io embodiment, F(ab)'s lack constant domains which are required for complement
fixation. scFvs are
composed of an antibody variable light chain (VL) linked to a variable heavy
chain (VH) by a flexible
linker. scFvs are able to bind antigen and can be rapidly produced in
bacteria. The invention includes
antibodies and antibody fragments which are produced in bacteria and in
mamrnalian cell culture. An
antibody obtained from a bacteriophage library can be a complete antibody or
an. antibody fragment. In
ts one embodiment, the domains present in such a library are heavy chain
variable domains ('Vn) and light
eliain variable domains (VL) which together comprise Fv or scFv, with the
addition, in another
einbodiment, of a heavy chain constant domain (CHI) and a light chain constant
domain (CL). The four
domains (i.e., VH - Cu1 and VL - CL) comprise an Fab. Complete antibodies are
obtained in one
embodiment, from such a library by replacing missing constant domains once a
desired VH - VL
2o combination has been identified.
[00045] In one embodiment, the plasma is isolated from melanoma patients
wherein the melanoma
tumour remains localized for long periods with no detectable spread (stage I
melanoma) and comprises
antibodies able to cause the death of autologous tumour -cells. In one
embodiment, the plasma isolated
25 from a melanoma patient in which antibodies to melanoma antigens are
identified, are taken from a
melanoma patient or pool of patients where the disease is at an early stage of
the disease or from a
patient or pool of patients in which the disease is at an advanced stage. A
person skilled in the art will
recognize that at each stage of the melanoma, different melanoma-associated
antigens (MAA's) may be
present, which will create a different profile of circulating antibodies in
the isolated plasma. In one
3o embodiment, the isolated plasma is taken from a patient or pool of,
patients where the tumour is a
primary tumor at stage I. In one embodiment, plasma is isolated from melanoma
patients whose lesions
are excised and are presumed cured.
13
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[00046] The term disease stage refers in one embodiment to the different
stages of melanoma cancer.
Stage I is divided into stages IA and IB. In stage IA, the tumor is not more
than I millimeter thick, with
no ulceration_ The tumor is in the epiderrnis (outer layer of skin) and upper
layer of the dermis (inner
layer of skin). In stage IB, the tumor is either not more than I millimeter
thick, with ulceration, and may
have spread into the dermis or the tissue below the skin; or I to 2
millimeters thick, with no ulceration.
Stage II is divided into stages IIA, IIB, and IIC. In stage IIA, the tumor is
either I to 2 millimeters thick,
with ulceration; or. 2 to 4 millimeters thick, with no ulceration. In stage
IIB, the tumor is either 2 to 4
millimeters thick, with ulceration; or more than 4 millimeters thick, with no
ulceration. In stage IIC, the
tumor is more than 4 millimeters thick, with ulceration. The tumor may be of
any thickness, with or
without ulceration, and may have spread to I or more nearby lymph nodes. Stage
III is divided inta
stages IIIA, IIIB, and IIIC. In stage IIIA, the cancer may have spread to as
many as 3 nearby lymph
nodes, but can be seen only with a microscope. In stage IiIB, the cancer has
spread to as many as 3
lymph nodes and may not be visible without a microscope, or has satellite
tumors (additional tumor
growths within 1 inch of the original tumor) and has not spread to lymph
nodes. In stage IIIC, the cancer
either has spread to as many as 4 or more lymph nodes and can be seen without
a microscope, or has
lymph nodes that may not be moveable, or has satellite tumors and may have
spread to lymph nodes.
[00047] In one embodiment, the plasma used in the compositions and methods
described herein, is taken
from a patient or pool of patients wherein the Vitiligo developed prior to the
isolation of plasma.
[o0048] In one embodiment, the compositions described herein, are used to
treat melanoma in a subject.
In another embodiment, the invention provides a method for treating melanoma
in a subject, comprising
the step of administering to the subject a preparation comprising plasma
isolated from a vitiligo patient.
[00049] In one embodiment, the term "treat ' or "treating" refers to
suppressing, inhibiting, increasing lag
time before development of symptoms, reducing or alleviating symptoms
associated with melanoma,
extending the periods at a single disease stage, preventing or inhibiting
metastases, reducing number and
size of metastases loci and the like. In one embodiment, the plasma is
isolated from a melanoma patient
whose melanoma metastasized and as a response to treatment, developed
Vitiligo, at which point plasma
was isolated and used for the compositions described herein. In one
embodiment, that plasma may be
later used to suppress, inhibit or reduce the metastases.
14
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[,ooo5oi In one embodiment, the methods described herein are used with at
least orie other treatment '
modality, prior to, during or after the administration of the preparation of
plasma isolated from a vitiligo
patient, a melanoma patient where antimelanoma antibodies were identified, or
a melanoma patient
where vitiligo developed prior to isolation of plasriia. In one embodiment the
other treatment modality is
chemotherapy, immunotherapy, radiationtherapy, surgery or a combination
thereof. In one embodiment,
the other treatment modality is surgery, and the method comprises in one
embodiment further removing
surgically the melanoma prior to, during or after the administration of the
preparation plasma isolated=
from a vitiligo patient a melanoma patinet where antimelanoma antibodies were
identified, or. a
melanoma patient where vitiligo developed prior to isolation of plasma.
j000511 In one embodiment, the plasma donor for the compositions and methods
described herein is a
vitiligo patient, or a Vitiligo patient who has a diffuse vitiligo; a patient
in which the vitiligo developed
in response to melanoma; a melanoma patient, a melanoma patient where anti-
melanoma antibodies
were identified; or a combination thereof in other embodiments. In one
embodiment, the donor is a= pool
of donors.
[00052] The term "about" as used herein nzeans in=quantitative terms plus or
minus 5%, or in another
embodiment. plus or minus 10%, or in another embodiment plus or minus 15%, or
in another
embodiment plus or minus 20%.
[00053] The following examples are presented in order to more fully illustrate
the preferred embodiments
of the invention. They should in no way be construed, however, as limiting the
broad scope of the
invention.
EXAMPLES
Example 1: Vitiligo Plasma for the.Treatment of Melanoma in Mouse Models
[00054} These experiments are done to evaluate the anti tumor effect of plasma
obtained from individual .
and pooled vitiligo and controls subjects in the B16-F10 experimental
metastasis model in syngeneic
mice.
CA 02642572 2008-08-15
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Animals [00055] Femiale C57BL/6J mice are purchased at 6-8 wks of age at study
initiation. Administration of the
plasma takes place= following veterinary Inspection -and I-Wk acclimatization,
marking and
' randomization.
Experunental Procedures,
(00056) Over a 4-wk experimental duration, purchase, propagation and
preparation for injection of tumor.
cells takes place. Intravenous injection of tumor cells is carried out, while
daily morbidity and mortality
io are checked Body weight measurements are done twice weekly. Vitiligo
sourced plasma is injected-
intraperitoneally once daily (including weekends). Mice are euthanized, their
lungs excised, weighed
and fixated, followed by metastasis count.
[00057] The following table describes the treatment grouping done:
'I'reatment
_;,.:_ - =:~;=,; _ _ -1;;-.: - ~=s:;= '/,;:.:_ . ,w:~,r.=.:::;w.:;~;;~~:
;~::: `-~. v,, .~ {~=
Group n Model
~~}7v vS~Y'S~-:G:.l =L..~ y.a:::i _ !~:~~Ck J _ - .== }.Yi,`~.~:::~h=~
yj:ei ; : ar 't=: _ .=--?Ei;i .','=ki. s:R:~:+c:. - x.r ~;:5-:u;Ji. t3iic A
.:5;:
.C:+iS"c5: 'i4`.^=rl.: _ _ %?i:'1*' 2ft'o`:kskc=s_,:.T.::,_~
Induction
r........y.~a; ''=,>5....=k . ;;::, .r.~l.. _ ~... ~,t. Q.. :h.. :>:~.
..-,..>....,... _ ..., ,..:. :::.;:'. . . .,.: . :...az~,
.,.:x.:.=:..:.. =:,-.<.._ ..... .. . ..... . . :.. ..-....,
1 10 Single IV Localized Vitiligo 50.ug Once
2 10 injection of Diffuse Vitili o per IP daily
3 10 2x105 BI6-Fi0 Melanoma mouse Days
4 10 melanoma cells Healthy 1-18
5 10 on day 0 Pooled Plasma
from 10 donors
with diffuse
Vitiligo
6 10 Pooled plasma
from
healthy donors
ts
[ooo58] Results show that pooled plasma froin donors exhibiting diffuse
vitiligo shows a marked
decrease in the number and size of melanoma metastases, as well as at least a
tumoristatic effect on the
metastasized melanoma tumors.
Example 2: Vitiligo Plasma for the Treatment of Human Melanoma in a Mouse
Model
[00o59] These experiments are done to evaluate the anti tumor effect of plasma
obtained from individual
and pooled vitiligo and controls subjects, in a human melanoma experimental
metastasis model in SCID
mice.
Anfinals
16
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WO 2007/095293 PCT/US2007/003919
[0006o7 Female CB17-SCID mice are purchased at 6-8 wks of age at study
initiation. Administration of
the plasma takes place following veterinary Inspection and 1-Wk
acclimatization, marking and
randomization.
Experfinental Procedures
[00061) Over a 4-wk experimental duration, purchase, propagation and
preparation for injection of tumor
cells takes place. Intravenous injection of human melanoma A375 tumor cells is
carried out, while daily
morbidity and mortality are checked. Body weigbt measurements are done twice
weekly. Vitiligo
i0 sourced plasma is injected intraperitoneally once daily (including
weekends). Mice are euthanized once
the first mouse dies, or for humane reasons, their lungs excised, weighed and
fixated in formalin or
Bulin Solution, followed by metastasis count.
[00062] The following table describes the Tumor cells dosing validation -
Induction
~,: : ;,<,,,,,k~:; ,_:,:: ar:,~ ;:<,;;x:i7<: ,;,;; =;`3~~:,:. ~,.
,e:.:i'F'~eii~:>
uriioir>Gi;lls:= <,; .2;z:Dose; ;;,r,,..R+b~~e;;o~~,:~;~; ,,R m.
Group n
,3..6:3t:?3-`=7 i?::X,.s;='' '=?r' - .:,.3=".xy~':$1' <ti:> .;~~;<
.,y .
. ... ~
iE~ %..='ti:i.F`:= ::5.: axe= : >aq ;~,; ;a=
. . ..
pioiS':: : 3.= T';' 1 .;-S~i~ : c~!
.....: -.::,.:.,::., .::..:..:.....
_.:..,.>~":;.:a..~,_>>.v_.:~;:.:.=::,C,E:_15,~......._.... ~:;=: ::._-...__--
..,.._.. _....... . ~ . -
1 6 Human 0
2 6 melanoma 10 IV Once
3 6 cells A375 2ae10
]5 '
[00063] The following table describes the Grouping Protocol
Treatment
.:.s...... ....: ..,;-~=. .;.. -., .: .., ,..:.. ~ - ,,:~ :,
Group n Model '+F=:Plasma:::Sburce:;:._ ::Dose=: ''.::r -butewo~:n<
_ ,,<.= _ ::ii;:o.. =::k.,~. -x,~ g3.: - ~:<:-.
:;.._ ..1...:z....:. ..
_ -as -- - r,=.9C`< =t':c=- -
:iEFl, . ~;<^~>o :.e:;=. - -
- - -
:;c...
...,:
~1? ,=}%.<:C.
,:.
:>= >
. . . , :..
- .. .....:,.:, .h= ............. .... .....: :.; ..::
InductXOn ,:
.:..:_....~;,....~. ~...~...::4~,.. :.:.ti= ..~~r,,,,._=:,.: =
,:,.:,_..:;.;:.>.:.
1 10 Single IV Localized Vitiligo 50Og Once'
2 10' injection of A Diffuse Vitiligo per IP daily
3 10 375 Huinan Melanoma mouse Days
4 10 melanoma cells ITealthy 1-18
5 10 on day 0 Pooled plasma
from 10 donors
with diffuse
Vitiligo
6 10 Pooled plasma
froin
healthy donors
17
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[po064] Results show that pooled plasma from donors exhibiting diffuse
vitiligo shows a marked
decmase in the number and size of melanoma metastases, as well as at least a
tumoristatic effect on the
metastasized melanoma tumors.
s
Example 3: Effect of Vitiligo Plasma on Mouse Melanoma cells
[00065] These experiments are done to evaluate the anti tumor metastases
effect of plasma obtained from
individual and pooled vitiligo and control subjects in the B16-F10
experimental metastasis model in
syngeneic mice.
Animals
[00066] Female C57BL/6J mice are, purchased at 6-8 wks of age at study
initiation. Administration of the
plasma takes place following veterinary Inspection and i-Wk acclimatization,
marking and
randomization.
Experirnet:tal Procedures .
[00067] Over a 4-wk experimental duration, purchase, propagation and
preparation for injection of tumor
cells takes place. Intravenous injection of tumor cells is carried out, while
daily morbidity and mortality
2o are checked. Body weight measurements are done twice weekly. Vitiligo
sourced plasma is injected 7
days following the injevtion of tujmor cells and is done intraperitoneally
once daily (including
weekends). Mice are euthanized, their lungs excised, weighed and fixated,
followed by metastasis count.
[00068] The following table describes the treatment grouping done:
Treatment
Group n Model Plasrria. SourCe Dose Route o~ Regmnen,;
Induction ~ k ' Adi tmstrat on ~<
........ . ..... . ~>. . ....,.. . . . ..:.. ... ....11.... .. ,. .. . a . .
., :. . .
1 10 Single IV Localized Vitiligo 50 g Once
2 10 injection of Diffuse Vitiligo per iP daily
3 10 2x105 B16-F10 Melanonia mouse Days
4 10 melanoma cells Healthy 7-25
5 5 on day 0 Pooled plasma
from 10 donors
with diffuse
Vitiligo
18
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6 5 Pooled plasma
from
healthy donors
[00o69] Results show that pooled plasma from donors 'exhibiting diffuse
vitiligo shows a marked
decrease in the number and size of melanoma metastases, as well as at least a
tumoristatic effect on the
metastasized melanoma tumors.
5
Example 4: Vitiligo Plasma Effects on Human Melanoma Cells
[oo070] These experiments are done to evaluate the anti tumor effect of plasma
obtained from individual
and pooled vitiligo and controls subjects, in a human melanoma experimental
metastasis model in SCID =
10 mice.
Animals
[00071] Female CB17-SCID mice are purchase at 6-8 wks of age at study
initiation. Administration of
the plasma takes place following veterinary Inspection and I-Wk
acclimatization, marlcing and
randomization.
Experimental Procedures
[00072] Over a 4-wk experimental duration, purchase, propagation and
preparation for injeation of tumor
2o cells takes place. Intravenous injection of human melanoma A375 tumor cells
is carried out, while daily
morbidity and mortality are checked Body weight measurements are done twice
weekly. Vitiligo
sourced plasma is injected intraperitoneally once daily (including weekends)
starting 8 days after the
tumor cell injection, and are continued for 17 days thereafter. Mice are
euthanized at the 17`h day, their
lungs excised, weighed and fixated in formalin or Bulin Solution, followed by
metastasis count.
[00073] The following table describes the Grouping Protocol
Treatment
Group n Model Induction Plasma Souree Dose Ro~te of Re~urie;ri`;,
~ ..:.:.: ...... . ~. .. ... . . Ad~nmistra~i.o.n.. :. ;.
1 10 Single IV Localized Vitiligo 50 g Once
2 10 injection of Diffuse Vitiligo per IP daily
3 10 A375 human Melanoma mouse Days
4 10 melanoma cells Healthy 8-25
19
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5 -on day 0 Pooled plasma
from 10 donors
with diffuse
Vitiligo
6 5 Pooled plasma
from
healthy donors
[00074] Results show that pooled plasma from donors exhibiting diffuse
vitiligo shows a marked
decrease in the number and size of melanoma metastases, as well as at Ieast a
tumoristatic effect on the
metastasized melanoma tumors.
5
Example 5: Vitileo Plasma Administered for the Treatment of Patients with
Staee III or Stage IV
Inonerable Melanoma
[00075] The purpose of the example is to evaluate whether plasma obtained from
donors with Vitiligo,-
io can be administered for the treatment of patients with stage IIl or IV
inoperable melanoma. The phase I
part of the study is designed to determine whether the Vitiligo-derived plasma
can be safely
administered, and the phase 2 part of the study is designed to provide
additional safety data and to gain
an understanding of whether administrating Vitiligo plasma can improve the
clinical outcome for
melanoma patients versus the currently available data.
[00076] This is a randomized, multicenter, study where Vitiligo plasma is
administered as an intravenous
infusion on consecutives days followed by a rest period for a treatment cycles
of 28 days.
[00077] 1" cohort - 5 patients are randotnized to each of the 3 treatment
doses (100/ 200/ 400 mg/kg
bodyweight) - total of 15 patients. Patients are randomized to I of the 3
plasma dose levels. Each patient
is infused twice every 28 eight days and is followed for 28 days post the last
infusion to detect potential
safety issues. The 2 d cohort does not start accrual, prior to a satisfactory
safety analysis from the l'
cohort. Nevertheless, the 1t cohort patients continue treatment for up to the
copmetion of 6 cycles (each
cycle being 28 days) unless the patient develops progressive disease or
intolerable toxicity. When there
is evidence of clinical benefit (stable disease or tumor response defined as
partial or complete response),
patients may continue treatment beyond 6 cycles so long as toxicity remains
within acceptable limits;
these patients are taken off study after cycle 6, but receive treatment under
compassionate access while
following the same study schedule. Patients are replaced if they do not
complete at least treatment 2
CA 02642572 2008-08-15
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cycles and have at least one post-treatnnert disease assessment performed
(unless there is a clear
evidence of clinical progression after two (2) cycles).
[ooo78] 2 a cohort - Upon positive results from the safety analysis of the
first cohorl; 5 patients = are
randomized to each of the three above described treatment doses to a total of
15 patients. Patients'
treatments are administered similarly as to the 1" cohort. Patients are
replaced under the same
conditions as of the 1" cohort. Although recruitment is not suspended until
afler enrollment is completed
for this cohort, a safety analysis is done to include the first 30 patients,
including two infusions each and
a follow up period.
[00079] 374 cohort - Additional 9 patients are randomized to each of the 3
treatment doses (total of 27
patients). Patients' treatment is administered as described above. A Simon two-
stage designs is used to
evaluate the efficacy of the treatment, after 6 cycles at each of the above
described doses. Patients are
replaced if they do not complete at least 2 cycles of treatment and have at
least 1 post-treatment disease
assessment performed (unless there is clear evidence of clinical progression
after two (2) cycles).
[oooso] 4th cohort - Additional 21 patients (maximum of 63 patients) are
randomized to those treatment
arms (doses, as descibed above) which have shown at least one response (as
desciribed above) at the 3`d
cohort stage and are treated as described above. At the completion of the 4a'
cohort, if four or more
responses are observed for any dose level, then it is concluded that this dose
level is a candidate for
further clinical study. Patients are replaced in a similar method as in
previous cohorts.
[0008]) Results indicate that vitilogo plasma is a safe and effective compound
for the treatment of stage
III or IV inoperable melanoma.
[000822 The descriptions of the foregoing embodiments of the invention have
been presented for purpose
of illustration and description. They are not intended.to be exhaustive or to
limit the invention to the
precise forms disclosed herein, and obviously many modifications and
variations are possible-in light of
the.above teaching. The enibodiments were chosen and described in order to
best explain the principles
of the invention to thereby enable others skilled in the art to utilize the
invention in various
embodiments and with various modifications as are suited to the particular use
contemplated. It is
intended that the scope of the invention, be defined by the following claims
appended hereto.
21
