PSP94: USE FOR TREATMENT OF HYPERCALCEMIA AND BONE METASTASIS

PSP94 : UTILISATION DANS LE TRAITEMENT DE L'HYPERCALCEMIE ET DE LA METASTASE OSSEUSE

English Abstract

The present invention discloses the use of PSP-94 for treating a patient
suffering from
hypercalcemia of malignancy. Prostate secretory protein of 94 amino acids (PSP-
94) is
known to serve as prognostic marker in hormone-dependent prostate. In the
current study the
effect of PSP-94 on prostate cancer growth, metastases to the skeleton and
hypercalcemia of
malignancy was examined. A marked dose-dependent decrease in primary tumor
volume was
seen in experimental animals receiving PSP-94. Furthermore, while control
animals routinely
developed hypercalcemia due to PTHrP production, treatment with PSP-94 led to
a near
normalization of plasma calcium and a marked reduction in PTHrP production as
determined
by radioimmunoassay and immunohistochemistry. Results obtained demonstrate the
ability of
PSP-94 to be an effective treatment modality for prostate cancer. Furthermore,
decrease in
plasma PTHrP and calcium levels can serve as useful biochemical markers for
monitoring the
efficacy of this novel anti-tumor agent.

Claims

We claim:
1. The use of PSP-94 for treating a patient suffering from hypercalcemia of
malignancy.
2. The use as defined in claim 1, wherein said hypercalcemia of malignancy
arise from
cancer selected from the group consisting of prostate cancer, breast cancer
and
carcinomas.
3. The use of PSP-94 for reducing PTHrP levels in a patient in need thereof.
4. The use of PSP-94 to reduce the development of skeletal metastasis.
5. The use of PSP-94 in the manufacture of a pharmaceutical composition for
the treatment
of hypercalcemia of malignancy.
6. The use of PSP-94 in the manufacture of a pharmaceutical composition for
the treatment
of skeletal metastasis.
7. A method for evaluating, the efficacy of PSP-94 tumor treatment in a
patient having a
tumor, said method comprising measuring plasma calcium levels in said patient.
8. A method for evaluating, in a patient, the efficacy of PSP-94 treatment of
hypercalcemia
of malignancy, said method comprising measuring plasma calcium levels in said
patient.
9. A method for evaluating, in a patient, the efficacy of PSP-94 treatment,
said method
comprising;
a) measuring plasma calcium from a patient with a tumor or with hypercalcemia
of
malignancy before the patient's treatment with PSP-94
b) measuring plasma calcium from a patient with a tumor or with hypercalcemia
of
malignancy after the patient's treatment with PSP-94; and
c) comparing values obtained in step a) with values obtained in step b).
39

10. A method for evaluating, the efficacy of PSP-94 tumor treatment in a
patient having a
tumor, said method comprising measuring plasma PTHrP levels in said patient.
11. A method for evaluating, in a patient, the efficacy of PSP-94 treatment of
hypercalcemia
of malignancy, said method comprising measuring plasma PTHrP levels in said
patient.
12. A method for evaluating, in a patient, the efficacy of PSP-94 treatment,
said method
comprising;
a) measuring plasma PTHrP from a patient with a tumor or with hypercalcemia of
malignancy before the patient's treatment with PSP-94
b) measuring plasma PTHrP from a patient with a tumor or with hypercalcemia of
malignancy after the patient's treatment with PSP-94; and
c) comparing values obtained in step a) with values obtained in step b).
40

Description

CA 02407548 2002-11-08
hl'h~.~:
PSP-94: Use for Treatment of Hyperca~ICemia and Done Metastasis
BACK(~R()llNl) Oli"'fHl~; INVh:N'fION
Tl~e prostate gland, which is bound exclusively in male mammals, produces
several
cumponent5 of semen and blood and several regulatory peptides 1'he prostate
gland
cc~mlorises stroma and epithelium cells, the latter group consisting ol~
columnar secretory cells
and basal nonsecretorv cells. A proliherutic:~n of theses basal cells as
v~:ell as stromu cells gives
rise to benign prostrrtic hyperplasia (hPH), which is ~~ne cornmon prostate
disease. Another
common prostate disease is prostatic adenocrurinoma (Cal'), which is the most
common of
tl7e tatrrl pathophysiological prostate ~~ancer~, rind involves rr malignant
transformation of
I S epithelial cells in the peripheral region of th;c prostate gland.
Prostatic adenocarcinoma and
benign prostatic hyperplasia are two common prostate diseases. Lvhich have a
high rate of
incidence in the aging human rxrale p~>pulation. Approximately one c>ut of
every four males
above the age of SS sutf'ers from a prostate disease of some (~orrr or
another. Prostate cancer
is the second most common cause of ::ancer related death in elderly merr, with
approximately
9t~,()()(> cases diagnosed and about ?6.0U0 dt~rrthas reported annually in the
United States.
A distinct feature of prostate cancer i~ its al-~ilit:y to cause osteol~lastic
skeletal metastases
which contributes to the hi~~h rate oi' mc>rbicOy and mortality associated
with this hormone
dependent malignancy. Additionrrlly_ a significant number ol~ patients with
prostate cancer
?5 exhibit an increase in their plasma caiciurn levels due to the prculuction
of PTHrP by tumor
cells. tlypercalcemia has been necogni~.ed ;rs a eo~mplic;ution of
r7~alignaney since 1920 and
occurs in at least IS-?0'% of patients harbouring a vro-iety of caru:crs
including prostate
cane:er. Although no single agent has been ~i~own to he uniduely responsible
for the
hypercalcemia of malignancy (I-IM), increased prcrdu~:aion of parathyroid
hormone related
_~0 peptide (PTHrP) by tumor cells has Icd to its estrrh~lishment as the:
major pathogenetic factor
responsible for fIM. This is of particular siynilicance in prostate and breast
cancer which are
oaten associated with skeletal metastasis where osteolytic effects of P~I'HrP
results in
_~

CA 02407548 2002-11-08
irrcrcased bone rescarption and hypercalcen~ia.
( linical prostate; cancer can he treated succ-essl~ully trt its early stage
when the cancer is well
confined within the prostate glr.rnd. l-fowevcr, increased productron of many
i~actors including
:~ ~,~~owth factors, sex steroids, angiogcnic I~ac~W rs and ;ioroteases such
as urokinase (uPA) and
matrix metalloproteinases (1~9N1Ps) by tum,~r fells and their sus-rounding
stroma is associated
with high mortality. Despite recent advances in the therapeutic modalities for
organ confined
pre:ostate cancer including surgery anal r°aclic:tthe;rapy, limited
success lras been obtained in
tmutin~ hormone-independent rnetastatic prostate cancer.
Prostate specific antigen (PSA) and I~rostat~ secrettory protein ~~1 94 amino
acids (herein
referred to PSP-9=I or PSP) are known to serve as prognostic markers for
disease progression.
Lil<c I'SA, PSP-94 levels in scrum, urine, and prostate tissue of loatients
with prostate cancer
arc inversely related to tumor grade. In prc:~,~icura work, described in
United Stales Patent No.
1 i 5.~?8.01 1 (the entire content of which ys inuorloorrrtcd herein by
reference), pharmaceutical
preparations (i.e., compositions) ol~ native Inurnan seminal plasma PSP-~~
were: provided for
irrhrbiting in-vitrc:r and in-viva cancerous prtrstute, gastrointestin;rl and
breast tumors. In
additional work disclosed in Canadian patent application No: 2,:x_59,0>0 (the
entire content of
wick is incorporated herein by reference), t;re ohility of PSI.~-94
t~r~agm~nts, such as PCK314S,
tc; be used in the inhibition of tur7ror ~_lrowtf-~ and more particularly, in
the inhibition of
prostate cancer tumor growth was illustrated.
In the present study, the effect of PSP-~)4 on prostate cancer turncor growth
and especially bone
meaastases was evaluated. her these studies 5yngeneic i-n viva madcl of rut
prostate cancer using
?5 the rat prostate cancer cell line Inunnirrg R3~'?7 Mat Ly Lu trarnslected
with the full length cDNA
er7coding rat PTHrP was used (Rabbani, S..4. et ai., Int. J. Cancer, ~i0: ?57-
?C4. Ir)99). Following
suh~-cutaneous (S.C.) or intracurdiac tLr'~.) irroculaticrn of Mat Ly Lu-
P~I"I~rP cells, the ability of
different doses of PSP-O4 or PCK314' to redtrcc tumor ~r~owth, metastases,
tumoral PTHrP
production, plasma calcium and plasma PTI IrP was evaluated- ~~rnino acid
sequence homology
between human and rat PSP and similarity ir7 their tertiary structures as
determined by highly
conserved cysteine residues has ~.rllowid the rrsc: of human PSP-94 for these
studies (Fernlund, F.
et al., Arch. Biochem.Biophys. :334:7 3-8?, 39~)(~). Due to the hy~,I levels
of PTHrP production

CA 02407548 2002-11-08
tk~eae animals routinely develop hypevrcaic:emia, a common complication in
many patients
snrflering from prostate cancer (Iwarraura, IVI., ct al., l:.irology, 43: (a75-
(x'79, 1994; Iwamura, M. et
al., lium. Pathol. 26: 797-801, 1995), lJse of thls homologous rrrodel for
prostate cancer al lows
for Iuil interaction between the host cnvirorrmcnt an~a growth facaors (EGF,
TGF-(3) (Helawelt,
('i.C). et al., BJLJ Int. 89:23()-240, 20()?) and proteases (ul'A, MMl's)
(Rabbani, S.A., et al., Int. J.
C"anccr, 87: 27O-282, 2000, Ral7bani S.A., ut al., In viva, 1'?: l ; ,...14?,
1998) secreted by tumor
cells. These prostate cancer cells arc hormone-independent allowing I~or the
evaluation of the
effect of PSP-94 on late stage prostate carver.
SUMN1ANY OH 1'I-IE INVI:N"1'ION
The invention disclosed herein provides pharmaceutical cc>rnpositions and
method for Creating
laaticnts with hypercalcemia of rnalit~nanc;y and skeletal metasU.~sis. PSP-94
(native PSP-94
(nPSP-94) (SEQ ID NO. l )) and rHuPSI'94 ~ recombinant human PSP-94 (SEQ ID
No.?)) as well
15 as derivatives (fragments) such us for example the decapeptide as set forth
rn SEQ ID NO: 3, the
polypeptidc as set forth in SEQ II) N(>: ~~ (hcalypcptidc 7-21 ). the
polypeptidc as seC forth in SEQ
11) NO: 5 (PCK314S), the polypepticle as set fnrth in SEQ lD NO: O
(polypeptide 7G-94), and
polypeptide analogs arc used herein to trc;rt conditions related to
hypercalcemia and skeletal
metastasis. Calcium tray also oc used herein as a surrogate marker ol~ the
efficacy of PSP-94
?(.l tumor treatment.
In a first aspect, the present invention relate; the use of PSP-94 and analog
thereof for treating a
p,~tient (with a malignancy) suffering from hyperc~_rlcernia of rrralignancy
(i.e., for treating
hvpercalcemia of malignancy). More t:rarti~ularly, the present irmenticm
relates to the use ofa
?5 pulypeptide selected 1i-om the group consisting of PS.f'-94, PCK3145, the
polypeptide 7-?1, the
dccapeptide, the polypeptide '-7(:i-94, and analog thereof tc~ reduce (treat a
patient with)
hypcrcalcemia (related to) of malignancy. 'l lais aspect of the invcuution
also encompass methods
fear treating a patient suffering from hypcrcalcemia of malignancy which
comprise administering
to the patient a pharmaceutical composition comprising PSP-94, the
holypeptides mentioned
i0 herein <rnd analogs thereof. This aspect of the invention also encompass
the use of PSP-94 for
reducing (lowering) calcium levels in a patient suffering 1'rono
ttypercalcemia of malignancy.

CA 02407548 2002-11-08
In accordance with the present invention. the malignancy rnay he an hormone-
independent
malignancy. It is to he understood herein than hypercalcemia of malignancy may
arise from
various source including prostate canc:cr, breast cancer, lung carcinoma,
hepatocellular
carcin oma, etc. rfherefare. treatment of hypercalcemia of malignancy with PSP-
94, the
polvpeptide described herein and analogs thereof may be necessary in patient
with prostate
cower, breast cancer, lung ~arcinc.~ma, hcpatovellular ~ar~inoma, etc.
Treatment of
hypercalcemia cof malignancy i,, not a~cstri~cd to any type; of malignancy.
In another aspect, the present invention relates to the use of PSP-94 to
prevent occurrence of
l0 hypercaleemia of malignancy and to ;,ontroi the: indu~.aion (onsc ) of
Irypercaleemia in a patient.
lri yet another aspect, the present invention relates to the use: of PSP-94 to
prevent (control)
P'hftrP increase in a patient.
Irr an additional aspect, the present invention relates t~-~ the use oi~;.~
polypeptide selected from the
group consisting of PSP-9:~, PrK31~~t5, the polypeptide 7-2l, th~.
dectrpeptide, the polypeptide
7f~-t)~, and analog thereof to reduce ( i~or rcdrrcinl;/lov~%ering) the Ivet
(biosynthesis, expression,
transcription, translation, production, score lion 1 or activity of PTE-irP in
a patient in need thereof
?0 lr~ a further aspect, the present invention relates to the use of' a
polypeptide selected from the
group consisting of PSP-9~E, E'(::'K31=t5, the polypept:ide 7-? 1, the
decapeptide, the polypeptide
7G-9=~, and analog thereof tc> recJuce tl7e prc7c:u~tion o1~ager7ts
resp~:>nsik~le f'or the development crf
(an hypercalcemie condition) h~dperculcemia including E''l'HrI'.
?s In a further aspect, the present invention sr totes to the use of PSP-94 to
reduce (delay] the
dmclopment of skeletal metastasis. Mlore particularly. the presen; invention
relates to the use of
a polypeptide selected from the ~~roup consisting of PSP-~)~, PC:K31~S, the
polypeptide 7-?1, the
decapeptide, the polypeptide 7(':~-94, and analog thereof to black (reduce,
impair, delay) the
development (progression) of skeletn! met~r:,tttsis. This rtspeci. of the
invention also encompass
3O methods for treating a patient with skeletal metastasis comprising
administering to the patient a
pharr~~aceutical composition comprising PSl'_9~I, the polypeptide:; described
herein and analogs
th~~rcot .

CA 02407548 2002-11-08
In an additional aspect, the present invention relates to the use of a
polypeptide selected from the
«roup consisting of PSP-94, P('K31 ~5, the polypeptide 7-~.'l, they
decupeptide, the polypeptide
i6 ~)4, and analog thereof to contrc:,l the level of n.olecules involved in
calcium production,
v-herein said molecules are selected from :he group consisting of vitamine B,
calcitonine and
biological equivalents thereof.
In yet an additional aspect, the present ir~ver~ticn~ relates to the use of a
polypeptide selected from
tlne Croup consisting of PSP-d4, PCKB 145, the polypeptide 7-? I . the
decapeptide, the polypeptide
7<~-~4, and analog thereof conjugated w°ith °~isphosphonates,
RGD peptides (Arginine-Glycine-
Aspartic acid peptides), ostex~blast, and osteocla.rst specific: proteins to
improve their
hmavailibility to the skeleton.
In another aspect, the present invention relates to a pharmaceutical
composition comprising;
I S a) a polypeptide sclectect from the r,rc~up consisting of PSP-94, PCK3145,
the
pcolypeptidc 7-? I , the decapc ptide, the polypeptidc '76-r)4, and analog
thereof; and
b) a pharmaceutically acceptable carrier.
for the treatment of hypercalc.ernia ot-n~rali~nancy or for the treatment of
skeletal metastasis.
?0 In a further aspect, the present invention relates to the use: of 1';sP-f)4
in the manufacture of a
pharmaceutical composition fur the treatment of hylocrcalccmia of malignancy.
More
particularly, the present invention relates t~~ the use ~~f a polypelUide
selected from the group
consisting of PSP-94, I'CKB 145, the p olypeptide ?-? t , the decapeptide, the
polypeptide 76-94,
and analog thereof for the manul~actr~re ol~ ~r l7htrr;nu~,:cutical
composition for the treatment c~f
?5 hvpercalcemia of malignancy.
In yet a further aspect, the present invention relates to the use of PSP-~4 in
the manufacture of a
pharmaceutical composition for treatment o~ skeletal metastasis. Mare
particularly, the present
invention relates to the use ol~ a polypeptide selectod from the grc:mp
consisting of PSP-~)4,
s0 P('K3145, the polypeptide 7-? 1. the decapeptide, the polypeptidc 76-94,
and analog thereof for
the manufacture of a pharmaceutical compositrim for the treatment of skeletal
metastasis.

CA 02407548 2002-11-08
lr~ an additional aspect, the present invention relates to a method of
treating a patient with a
condition related to hypercalcctnia of malignancy comprising ~tdmiraistering
to the patient a
pharmaceutical compcosition comprising a polypeptide selected ir-onn the
groulo consisting of PSP-
O4, I'CK3145, the polypeptide 7.-21, the clcvapc:l?tidc, the polypeptide 7(i-
94, and analog thereof
arid a pharmaceutically acceptable carrier.
In yet an additional aspect, the present invention relates to a mr~thod of
treating a patient with
si<eleta) metastasis comprising administering to the patient a pharmaceutical
composition
comprising a polypcptidc selected from the ~~rou.p consisting ol~ PSP-94,
PCK3I45, the
polvpeptide 7-'?I, the decapeptidc, the polypeptide 7~-~7=t. and analog
thereof and a
pharmaceutically acceptable- carrier.
Irr a further aspect, the present invention rcl;dt-es to the use of
pc.~lypcptid~ selected from tl7e group
consisting of PSP-J4, PCK314:i, the polylo°ptide 7-2I, the
dec~rt~eptid~~, the polypeptide 76-94,
and analog thereof in combination with hormone therapy, chemcUherapy or
radiation therapy.
In accordance with the present invention, PSP-9~ may be selected from the
group consisting of
native PSP-94 (nPSP-~~4) and rI-~uPSP94.
?U In accordance with the present invention. the pofypeptidc: may Lie used
with an antibody, an
hormone or an anticancer drug. including for cxarnple, (without being
restricted to) mitomycin,
idarubicin, cisplatin, 5-l~luoro~-uracil, methotrcxatc, adriumycin,
daunomycin, taxal (i.e.,
p,rclitaxel), and taxol der-iv~rtiv~ (e.g..docctnxel, taxanc).
?5 In another aspect, the present invcntic~n relate to a method forcvaluating,
the efficacy of PSP-94
tumor treatment in (of) a lortient h~rving a arnoor, said method comprising
measuring plasma
c~rleiurn levels of said patient.
In a further aspect the present application .-elates to a method fear
evaluating, in a patient the
30 efficacy of PSP-94 treatment of hvpercalccmia ol~ nmli~;nane:y, said method
comprising
measuring plasma calcium levels (in) of~ said patient.
7

CA 02407548 2002-11-08
In vet a further aspect, the present ir7vention revlates i.o a method for
evaluating, in a patient, the
etticacy of PSP-94 treatment, said method comprising;
a j measuring plasma calcium tiwm a patient with a tumor or with hypercalcemia
of
malignancy bctore the patient's trcatrnent with PSP-94,
b) treasuring plasrna calcium i ron~ a patient with a iurnor or with
trypercaleemia of
malignancy after the putien~'s treatment with PS f'-94; and
c) comparing values obtained m step a,) with values obtained in step b).
In another aspect, the present invention relates to ~i method for evaluating,
the efficacy of PSP-~4
tumor treatment in (a1~) a patient having a tumor, said met.hocl comprising
measuring plasma
P~rEIrP levels of said patient.
In a further aspect the present application i~elutes to a method for
evaluating, in a patient, the
cificacy of PSP->4 treatment of loyperculcemia of muli~r~uncy, said method
comprising
nrcasuong plasma PTHrP levels (in) of said patient.
In yet a further aspect. the. present inventic>r~ relates t~~ a method for
evaluating, in a patient, the
ei ticacy of PSP-94 treatment, said method curnprrsing;
a) measuring plasma PrHrP (levels) from a patient with a tumor or with
?() hypercalcemia of malignancy bc:-fore his (the patient's) treatment with
PSP-94,
b) treasuring plasma P rrlrl' (levels) from a patient with a tumor or With
hypercalccrnia of malignan~~~ at't:er his (the patient's) treatment with PSP-
~4; rind
c) cornparir~D values oht,rined in step a) ~'vith values abtained in step b).
?i As used herein, "polypeptidcs" refers to anv peptide or protein comprising
two or more amino
acids 'joined to each other by peptide bonds or modifcd peptide: bonds (i.e.,
peptide isosteres).
"Pulypeptide" refers to both short chains, cemomonly referred as peptides,
uligupeptides or
oligomers, and to longer chains generally rcier~r~ed to as proteins. As
described above,
pc5lylat;ptides miry cc~nt~rin amino acids other than the ?0 gene-encoded
amino acids.
A;; used herein, the term "tumor" relates to solid or non-solid turnc:>rs,
mctastasic or non-
metastasis tumors, tumors of dii~fer~,nt tissr_t~~ origin including, but not
limited to, tumors

CA 02407548 2002-11-08
c»iginating in the liver, lung, bruin, lymph nude, bone marrow, adrenal gland,
breast, colon,
pancreas, prostate, stomach, or reprcrducti~. a trtrcl (cervix, ovaries,
endometriurn ete.). 'the
term "tumor" as used herein. refers also to all ncopl:rstic cell gr-~,rwtf~
and proliferation,
whether malignant or benign, and all pre-canc:.~rous and c~rncerous cells and
tissues.
:ls used herein, "pharmaceutical composition" means therapeutically effective
amounts of the
sr~~ent together with pharmaceuticallw ac~el~tablt diluents, preservatives,
solubilizers,
emulsifiers, adjuvant and/or carriers. A. "therapeutic,ally effective amount"
as used herein
rcfm.s to that amount which prcrvidca a thcr~rpcutic effect for a gmen
condition and
1t) administration regimen. Such compcrsiticon; arc liquids or lyophilized or
otherwise dried
formulations and include diluents ul various buffer content (e.~~., Tris-HCI.,
acetate,
phosphate), pH and ionic strength, additives such as albumin car gelatin to
prevent absorption
tc~ surfaces, detergents (e.g.,'I'u~een '~0,'I'ween 80, Platonic F68. bile
acid salts). Solubilizing
a~~errts (e.g., glycercol, polyethylene glycerol ), anti-oxidants (c;.g..
ascorbic acid., sodium
1 ~ n-~etabisulfice), preservatives (e.g., thimeno:~al, benzyl alcohol,
p;rrabens), bulking substances
or tc>nicity modifiers (e.g., lactcnse, nnannitol), covalent attachment of
hc>lymers such as
polyethylene glycol to the protein, cr~mpletation with metal ions,, or
incorporation of the
material into or onto particulate preparatic»rs of polymeric compounds such as
polylactic acid,
pt~lyglycolic acid, hydrogels, etc, or unto liposomes, microemulsions,
micelles, unilamellar or
20 multilamellar vesicles, erythrocyte glro:;ts, err splleroplasts. Such
compositions will influence
the lohysical state, solubility, stability, rate of in viva release, arid rate
c>1' in viva clearance.
C'~_mtrulled or sustained release cuml7ositions include torrnul~rtiun in
lipophilic depots (e.g.,
fatty acids, waxen, coils). Also ce.>mprehended by the invention arc
particulate compositions
ccsatcd with polymers (e.g., polcrxamcrs or poloxamines). Other embodiments of
the
?5 cc>rnpositions of the invention incorporate particulate f~cwms protective
coatings, protease
inhif~itor-s or permeation enhancers f~c3r various routes of administration,
including parenteral,
pulmonary, nasal and oral routes. In mne en rbcadirrreni. the pharmaceutical
composition is
administered parentera(ly, paracancerally, transmuco,ally, transdcrmally,
intramuscularly,
intravenously, intradermally, subcutanec>uslv, intrapcritonealy,
~r~traventricularly,
30 intracraniallv and intratum<orallv, etc.
Further, as used herein "pharmaceutic°ally acceptable carrier"' or
"pharmaceutical carrier" are
c;.

CA 02407548 2002-11-08
known in the art and include, hut arc not limited to, 0.01-0.1 M and
preferably 0.05 M
phosphate buffer or 0.8 ~~. saline. Additionally, such pharrnaccutically
acceptable can~iers may
he aqueous or non-aqueous solutions, suspensions, ;.end emulsions. Examples at
non-aqueous
solvents are propylene glycol, polyethylene glycc>i, ~;eget.rhle mils such as
olive oil, and
in,jcctablc organic esters such as ethyl olearc:. Aqueous carriers include
water,
alcoholic/aqueous solutions, emulsions or suspcnsic:~ns, including saline and
buffered media.
I-';u~cnteral vehicles include sodium chloride: solution, Ringer's dextrose,
dextrose and sodium
chlcoride, lactated Ringer's orl'ixed oils. Ir~trrrveno~us wehiclcs include
fluid and nutrient
rcplenishers, electrolyte replenishers such as those based on Rrn~;cr's
dextrose, and the like.
t() Preservatives and other additives may also kic present, such as. tear
example, arttimicrobials,
antioxidants, collating agents, inert uses and the: like.
Mutant (variant. analog, derivative) loalypc:ltides encompassed by the present
invention includes
mutant that will passers one or more mutati~ms. which are deletrons (e.~.,
truncations), insertions
1 i (c~.g., additions), or substitutions of amino acid residues. l~lutants can
be either naturally
o~:cur-ring (that is to say, purificcl or isolated lrcom a natural source)
c:rr synthetic (for example, by
pvr(orming site-directed mutagenesis an the enc:odinl; DNA or mode by other
synthetic methods
such as chemical synthesis). It is thus apparent thal the pcrlypelotides of
the invention can be
either naturally occurring or recombinant (that is to say prepared from the
recombinant DN,4
?0 techniques). Mutant palypeptide derived trom PSP'-94 (nativ=e PSP-O4 (nPSP-
94) ; SEQ ID
N().r I or rHuPSPO4 (r-rcombinant human I'SP-94) : SEQ ID NO.:2) as well as
derived from the
p~Uypeptide described herein (PCK.iI-~5 rSF;~ lD NO.:S), decapeptidc (SEQ ID
NO.: 3),
pc>lypeptidc 7-? I (SEQ ID NO.4), polypeptide 76-c)4 (SEQ ID N0.6)) having the
biological
activity described herein (effect on hypercalcemia ;.md bane rnctastasis) are
included in the
?5 present application.
As may be appreciated, a number of rnc~dii~i~ations may be rnade to the
palypeptides and
fragments of the present invention wrthout d.leleteriously atf~ectin~; the
biological activity of
the holypepddes or fragrner~ts. t'olypehtides of the present invc=rrtion
comprises for example,
30 those containing amino acid sequences mociiPied either by natural
processes, such as
pcrsttranslational processing, or by chi:rrrical modification tcchnic.lues
which are known in the
art. Modifications may occur aroywhc;re in ar polypeptidc including the
polypeptide backbone,

CA 02407548 2002-11-08
the amino acid side-chains and the amino 'n-carboxy termini. It will be
appreciated that the
same type of modification may be present in the same or varying degrees at
several sites in a
given polypeptide. Also, a given polypeptide rnay contain many types of
modifications.
Polypeptides may he hranched as a result c~f uhiquitination, ctr~d they rnay
be cyclic, with or
:~ without branching. C:'yclic, hranchecl and branched cyclic loolypcptides
may result from
posttranslational natural processes or may ire made by synthetic methods.
Modifications
comprise for example, without limitation, .rcetylation, acylation, addition of
acetomidomethyl
(~'~cm) group, AI)P-ribosylaticrn, ~rmrd~rtiorr, covalent attachment to
fiavin, covalent
attachment to a heme moiety,.,ovalcnt attachment of a nucleotide or nucleotide
derivative,
i0 cr>valent attachment of a lipid or lipid cleri ~ativc:, covaleru attachment
of phosphatidylinositol,
cross-linking, cyclization, disulfide bond f~~~rmation, demethylation,
fcomation of covalent
cross-links, formation of cystinc, formation of pyro~.lutarnate, formylation,
garnma-
carhoxylation, glycosylation, Gt ( anchor furrnution, hydroxylation,
iodination, methylation,
r~ryristoylation, oxidation, loroteolytic proc;c°ssing,
phusphorylution, prmylation, racemization,
15 sclenoylation, sulfation, transfer-RNA mediated addition of amino acids to
proteins such as
arginylation and ubiquitination (for reference see, Prutcin-structure and
molecular proterties,
?"'r Icl., T.F. Creighton. W.H. E~reeman and Company, New-fork, 1993).
t)ther type of polypeptide modification may c.ornC»-ise, for example, amino
acid insertion (i.e.,
?(l addition), deletion and substitution (i.c., replacement ), either
conservative or non-
conservative (e.g., D-amino aci~~ls, dcsamin'a acid:;) in the polypeptide
sequence where such
cl7ange;s do not substantially altar the overall biological activity of the
polypeptidc.
f'olypeptides of the present inve~ntior~ comprise for example, laiologically
active mutants,
variants, fragments, chimeras, and analogs; Irugmcnts c:r~compass aminc,7 acid
sequences
2_5 having truncations of one cor more anoino acids, wherein the truncation
may originate from the
amino terminus (N-ter~rninus), carboxy terminus tt.'.-t~~rrninus), ~»- from
the interior of the
protein. Analogs of the invention involve ao insertion or a substitution of
one or more amino
acids. Variants, mutants, fragments, chimeras ,.rnd analogs may have the
biological property
01 polypepCides of the present invenUon which is to inhibit growth of
prostatic
30 aclenocurcinomu, stomach cancer, breast cancer, endc>metrial, ov,crian cn~
other cancers of
epithelial secretion, or benign prostate hyperplasia (RI H).

CA 02407548 2002-11-08
I:;xample of substitutions may be these, which ttrc ccmservtrtivc (i.e.,
wherein a residue is
replaced by another o1 the same general type). As i<; understood, naturally
occwr-ning amino
acids may be sub-classified as acidic, basic, neutral and polar, or neutral
and non-polar.
i~urthermore, three of the encoded arnir~o aids are aromatic. It rnay be of
use that encoded
polypeptides differing from the determined polypcptide of the present
invention contain
si.rhstitutcd colons for amino a~~ids, which arc from the same gr~;nrp as that
of the amino acid
he replaced. Thus, in some cases, the basic amino u;:ids 1_,ys, Arg and His
may be
interchangeable; the ~rcidic amino acrd;, Asp and r:ilu may be
interchangeable; the neutral
polar amino acids Ser, Thr, Cys, Calm arnd :lsn may he interchangeable; the
non-polar
t() aliphatic amino acids Gly, Ala, Val, lle, and Lc~u are interchangeable but
because of size Gly
and Ala are more closely related and V'al, IIe and Leu are more closely
related to each other,
and the aromatic amino acids Pl7e, Tr-p and 'hyr rnay be interchangeable.
It should be further noted that if the I~olypeptides are made synthetically,
substitutions by
15 amino acids, which are not naturally encoded by DN.A rnay also be made. For
example,
alternative residues include the omeyr ~tmirrco acids cif the formula
NH~(CH~)"("OOH wherein
n is ?-6. These are neutral nonlolar amino acids, as are sarcosinc, t-butyl
alanine, t-butyl
glycinc, N-methyl isoleucine. and norlcucine. Phenylglycine may substitute for
Trp, Tyr or
Phc: citrulline and rnethionine <.;ulfoaidc ar~~ neutral nonpolar, cy-steic
..rcid is acidic, and
?0 ornithinc is basic. Praline may be substituted with hvdroxyprolir~e and
retain the
ccmformation confer7iing properties.
It is known in the art chat mutants or variants may be generated by
substitutional mutagenesis
and retain the. hiological activity of the pol~peptides of the present
invention. These variants
?5 have at least one amino acid residue rn the ~rotcin molecule removed and a
different residue
inserted in its place. For example, orae Site of interest for substitutional
mutagenesis may
include but are not restricted to sites rdcvntified as the active sitc(s), or
irnmunological site(s).
Other sites of interest may be thaw, for example, in which particular residues
obtained from
various species arc identical. These positions rnav be important 1'c~r
biological activity.
30 Exarnplcs of substitutions identified us "corrscrvative substitutions" arc
shown in table 1. If
such substituti<ms result in a ch4rngc not desired, then other type of
substitutions, denominated
"exemplary substitutions" in table l, or as further described herein in
reference to amino acid
l'

CA 02407548 2002-11-08
classes, are introduced and the products screened.
In some cases it may lae of interest to rnod~fy the biological activity oi~ a
polypeptide by amino
acid substitution, insertion, or deletion. F~~r e.xarnple, modification of a
polypeptide may
result in an increase in the polvpeptide's brologic:al activity, may modulate
its toxicity, may
result in changes in bioavailubility c>r in stability, or may rTrodulate its
immunulogic~rl activity
~~r rmmunological identity. Substantial rru>difv~i;atiorts in function or
immunological identity
are accomplished by selecting substrtutions ttt~rt differ significantly in
their effect on
r~larrltalnlng (a) the structure of the l>olypelttide backbone in the area of
the substitution, for
example, as a sheet or helical conformation. (b) the char~~c or
ly~drophobicity of the molecule
at the target site, or (c) the bull,; of'the aside chain. Naturally occurring
residues are divided
intcs groups based on common side chu.in properties:
( 1 ) hydrophobic: norleucine, methionine (Met). Alanine (Ala), Valine (Val),
Leucine
1 '~ (Lea), Isoleucine (Ile )
(2) neutral hydrolohilic;: C'.vsteine (C:ys), Serine (Ser'), rfhrec>nine (Thr)
(3) acidic: Aspartie acid (Asp), Glutuanic acrd (Glu)
basic: Asparagine (Asn), Glutamine (Gln), I~istidine (rlis), Lysine (Lys),
Arginine
(Arg)
30 (S,) residues that influence chain orientation: Glycine (Gly), Praline
(Pro); and
(6) aromatic: ~hryptophan ;Trp). Tyrosine (Tyr), Phenyfulanine (Phe)
Mon-conservative substitutions will cmail exchanging a rnenrber of one of
these classes for
anot her.
l3

CA 02407548 2002-11-08
'hABLE 1.
Preferred
amino acid
suhstituti~>n
(>riginal Exemplary sab:;titucic~nConservative substitution
residue
alla (Aj Val, Leu, II~.-_-_--_Vul _~___~_
_. . _.___- - --_.__. _--_ __-__.___ _ ___._._
_ ..-_ ._ ____-_
\r<~ (Rj Lys, Glro, Asn ' Lys
.~'~sn (N) - Gln, Hi,, Lyc, --rCiln -. -__ _ _
- Arg _-
j ;\af5 (D) - ~lo ________.. _____C'lu_._______~-_
__._..___
___S e,' _ _- __ S e'. _
___-__-_.______
__.. ~~ .____-.. _--___-_. ~Sp ____.__._--
(iln (Q) .__.- _
_. .__ __._--_._---__j-__ _.__.___ ___~ _.-__ ____ _
-~_ __- _.-__- _
Clu (E) I Asp Asp
-~________ _.___~-_____ _. _-__
~ ( ~ I ~ ______ _
(G ) ~____ __ Pro
___
_____.
Pro
l lis (H I ,~lyt _.,~.At.g ______'_
--_ -_
Asn,
Ciln.
Lys
,
lle I f j l.eu, Phe Leu ___-__-_ _
__ Val,
IVlet,
Ala,
i
norleuc ,
i
ne
- _ _
~ ,__ __. _ _
~_ __ t
_
i ~ Mc~
I eu (Lj Norleucine, Ile
lle,
Vul,
'
Ala,
Phe
I 1 y~ (K) A~,g _-~_~L~_____.__-_
__ __ Clo, _
As,~
-~
__-
___-(_________..____-_
_ _-._ -_
____ _..
:~-le~ (M) y I I~u
Lcu
Phi
,
Ile
_ - __ __.__._____ _
P i-_ ~
-__________
_____
le (F) Lea Leu
~ V~u,
Ilc.
Ala
_ _.._____ __~.__.___ _.___ ___.__.____.~_.__-_
__- ________ Gly
i Prc, (P) ___
_
_._
i
CTIv
S a n i S ~', __.__ =r h [, __ _
) - .T ____ _ _ . _
1-___.__
--__
.____
;- ___- _.__ _ -
___-_ ___- _ _~- _-..-_
;'l for ('h) --_ ____~ Ser
-_
i
Ser
___.-__ __j____________ _____ -___~ _ _-___
___~_ _
.____._
T'rp (W) i l'yr
Tyr
_ ._- _ -_1._-___ _ __ _ ___ _ __-.__
Tyr (Yj __-_ -_
-__ ( I'he
.
___
~
Trp,
Phe,
'Thr,
Ser
'~Val (V) _~ AI~IJ~y_._____ ~___
- I _
,
I,cu,
Met,
f'I~c.
norleucine j
!_.. _.. _____._______-__i_-_______-_ ___.~___-__.__._________
._____ _
Example of analogs of PCK314~ (SE:Q ID NO: 51 ea;emplified by amino acid
substitutions has
been illustrated bclcow.
14

CA 02407548 2002-11-08
Position I 5 10 l5
P(_'K314S I-? w' Q T D N (~' l'h C T C Y E T
?;, w' (~) X~ D X, (' X, X~ C' X r C XX, X
For example, X, could be glutarnic acid (i.e.. ';lutarrmt-e) ((ilu),
asp;.rrtic acid (a.rspartate) (Asp),
car asparagine (Asn), X~ could he thrconinc (~fhr) or serine (Ser) ~rnd X~
could be tyrosine
i ( l'yr) or phenylalanine (Phe).
Polypeptides that are polypeptide analogs of PSI'-941 (nPSF'-94 (SEQ 1D NO.:
t) or rHuPSP94
(SEQ ID NO.:'?)) and/or analogs of PCK3145 (SEQ ID NO.:S), the decapeptide
(SEQ ID
NO.: 3), the polypeptide 7-21 (SEQ ID N0.4), the polypeptide 76-94 (SEQ IL>
N0.6))
1() include, for example, the t~ollc~w ~inv~:
a polypeptide analog of at least five contiguous ;.rmino acids of SEQ ID NO:
2, of SEQ ID
NO: 3, of SEQ II~> NO: 4, c>f SEQ 1D NO: 5, or of SEQ ID 1'~O: (i;
15 a polypeptide analog of at least two co~~tiguous ;amino ,.rcids ol~ SEQ ID
NO: 2, of SEQ ID
NO:3,ofSEQIDN0:4,c>fSE(~IDIx:():5.orofSEQIDNO:C~;
a polypeptide analog consisting of the amino acid sequence X, W Q X~ D X, C X,
X~ C XZ
C X~ X, X, , wherein X, is either- gluta:nic acid (Glu), asparagine (Asn) or
aspartic acid
?0 (Asp), X, is either threonine ('fhr) or swine (Ser), and X3 is either
tyrosine (Tyr) or
phenylalanine (Phe);
a polypeptide analog comprising, SEQ ID NO: 5 and having an addition of at
least one
amino acid to its amino-terminus;
~5
a polypeptide analog comprisin~T SEQ ID :NO: 5 and having an addition of at
least one
amino acid to its carboxy-terminus;
a polypeptide analog comprising two to ten units of SE;Q ID NO: 5;
I5

CA 02407548 2002-11-08
a polypeptide analog comprising tvvo W fifty units of SEQ ID NO: 5;
~u loolypepticle analog consisting vrf a sequence: of from two t<a 1'ourtecn
amino acid units
wherein the amino acid units arc sele<:l~d ti'om the group of amino acid units
of SEQ ID
NO: 5 c:onsistin~ of glutamic acid (Glu), trylntophan (.~fr'p), glutamine
(Gln), threonine
(Thr), aspartic acid (Asp), arsparagine (.~sr~), c:ysteine (C'ys), or tyrosine
(T,yr);
a polypeptide analog having at Icast 9() ~l of its amino acid sequence
identical to the
amino acid sequence set forth in SEQ 1D NC>: 5:
a polypeptide analog having at least 7Ci ~~. of its amino acid sequence
identical to the
amino acid sequence set forth in SEQ tD NO: 5:.
and a polypeptide analog having at Ica.,t S(:) ~i~~ of its aminco acid
sequence identical Co the
amino acid sequence set forth in SEQ ID NO: 5,
or any polypeptide analog of PSP-94 (nPSP-9~l (SEQ 1D NO.:1 ) or rHuPSP94 (SEQ
ID
NO.:?)) as well as the derivative descnhed herein (PCKa 14a (SEQ ID N0.:5),
decapeptide
?0 (SEQ ID NO.: ~), polypeptide ;'-? 1 (~EQ ID NO.~), polypeptide 7(i-~~ (SEQ
ID NO.fi))
having the biological activity described herein (effect on ttypcrcalcemia and
bone metastasis).
:amino acids sequence inser-tiorts (e.g., additions) include amino and/or
carboxyl-terminal fusions
ranging in length from one residues to polypeptides containin~~ a hundred or
more residues, as
?5 wvell as intrasequence insertions of single or multil>Ie amino arid
residues. Other insertional
uar~ants include the fusion c>f the 1'~- c~r C-terminus of tire prate-in to a
homologous or
hetcrologous polypeptide forming a ~hime~~a. Chimcric pcrlypeptides (i.e.,
chimeras, polypeptide
analog) comprise sequence of the polypeptides of the present invention fused
to homologous or'
lreterologous sequence. Said homolof;ous or lreterc~logcrrrs sequence
encompass those which,
31) when formed into a chimera w=ith tire polypeptides of the present
invention retain one or more
biological or immunological properties.
1 fi

CA 02407548 2002-11-08
A protein at least 50 ~i« identical, as deterrrrined by rnethods known to
those skilled in the art
(Ior example, the methods described by S>b~ith, 'h.E. and Waterrnan M.S.
(1981) Ad.
ippl.Math., ?:48?-489, or Needleman, S.ti. and Wunsch, C.D. ( 1970)
J.MoI.Biol., 48: 443-
ts3), to those polypcptides crl the present invention are include) in the
invention, us are
t»-oteins at least 70 ~:c: or 80'%~~ and more preferably at least 90 ~%'r
identical to the protein of the
present invention. This will generally be aver tt region c7f' at least 5,
preferably at least 20
ccmti~uous amino acids.
It is to be understood herein, that if a "range" or "group" of substaraces
(e.g, amino acids),
substituents" or the like is mentioned or il~ other types of a particular
characteristic (e.g.
temperature, pressure, chemical stru:aure, tune, c.tc. ) is mentioned, the
present invention
relates to and explicitly incorpor-ate~ hcrcio each and every specific member
and combination
ryt' sub-ranges or sub-groups therein whatsoever. Thins, any spec-ified range
or group is to be
understood us a shorthand way of rcl'errin~ to each and very member of a range
or group
1 ~ individually as well as each an d every possible sub-ranges or sub-groups
encompassed
therein; and similarly with respect tn any sub-ranges or sub-groups therein.
Thus, fco
example,
- with respect to a temperature greater Chart lU0" C', this is to be
understood as
?() specifically incorpc»-ating herein each and every individual temperature
state, as
well as sub-range, ahove 100" ~ ', such a, For exampac; 1 () t" C, 105" C and
up, 1 10"
C and up, I I S" C and up. t 10 t=_~ l B5'' C, 115" c to 1 ~5" (', 102" C to t
50" C, up to
,,
210 C, etc.;
?S - with respect to reaction tine, a time cnf 1 minute. or rnorc is to be
understood as
specifically incorpc>r<rtin'~ herein each and every individual time, as well
as sub-
range, above 1 minute, such as for example I minute, 3 to i5 minutes, 1 minute
to
?0 hours, 1 to 3 hot.trs, ll hour>, 3 hours to ?() hours etc.;
31) - with respect to polypeptides, a polypeptide analog consisting of at
least two
contiguous amino acids of a ptrrticular snquencc is t<, be understood as
specifically
incorporating each and every individual possibility, such as for example, a
17

CA 02407548 2002-11-08
polypeptide analog. consisting elf amino acid I and ?, a polypeptide analog
consisting of amino acids 2 and B, a poiypeptidc analog consisting of amino
acids
3 and 4, a holypeptide analog consisting of amino acids O and 7, a polypeptide
analog consisting oi~ amino acids 9 and lU, a polypeptide analog consisting of
s amino acids 3(i and 37, a pulypeptide analog consisting of amino acids g3
and 9~,
etc.
and similarly with respect to other parameters such as, concentrations,
elements, etc...
l() It is also to be understood herein that "a" ctr "~Tm" is a reference to
the gram weight unit; that
"C." is a reference to the Celsius ten~rperature unit.
BRIEI! DESCRIY7'ION OF THE ;uRA W1NGS
In drawings which illustrate examplar;y embodin rents of the present
invention:
1~i~~ure 1 illustrates the ef~iect ofPSP-J<~ on Mat l.y L,u-PTHrPccll grc>wth.
Each point represents
the mean of 3 different experiments. Signil icant differences from control
cells (MatLyLu-C.MV)
?0 .rnd PTHrP transfectcd cells (MatLvLu-E''fHrP) in the abscencc of PSP-9~
are represented by
a.stcrisks (p<().()5 ).
ligurc 2A illustrates the effect of PSP-94 can Mat Ly Lu-P~TNrI'tumc.~r volume
(in cm3). Results
ncl~rescnt the mean ~= SEM of _5 animals ire each group in ~3 different
experiments. Significant
2~ difference from control tumor-bearing animals receiving vehicle alone
(C'I'L) are represented by
asterisks (p<0.t~5).
Figure 2B also illustrates the effect of PSl'-9~ on Mat Ly Lu - F''rI-trP
tumor volume (in cm')
fti_sulls r°eprescnt ~ SEM o1 six animals irr e~r~:h group. Significant
difference in tumor
30 v~uurne is shown by asterisks (p<O.t)_5).
lvi'rure 3 illustrates the effect of 1'SP-9~~ on animal weight. f2esults
represent the meirn ~ SEM of
l8

CA 02407548 2002-11-08
~ animals in each group in 3 different f;xperimenl.s.
Figtu-c 4 illustrates the effect of PSF'-94 orr M;~t I_y Lu-PTI-irP tumor
weight. Results represent
the mean ~ SEM of ~ animals irt each group in ~i different experiments.
Significant difference
from control tumor-bearing animals receivrn~; vehicle alone (C'A'L) are
represented by asterisks
( p<~0.().5).
Figure SA illustrates the effect of PSP~-94 ~.>n spinal metastases resulting
in the development of
luind limb paralysis. Results represent the mean -~~ SI:M of ~ anitruals in
each group in 3 different
experiments. Signilic:ant difference rn ~>> of non-paralyzed animals
f°rom control tumor-bearing
;rnimals receiving vehicle alone (CrI L~ are represented by asterisks
(p<O.OS).
hi~,ure SB alsc»Ilustrates the effect of PSF'-94 on spinal metastases
resulting in the
development of hind limh paralysis
Figure GA illustrates the effect of PSF'-~4 on plasma PT'>EIrP in tumor
bearing animals. Results
represent the mean ~ SEM of 5 animals in each group in 3 different
experiments. Significant
~lili~erence from control tumor°-bearing animals receiving vehicle:
alone (CTL) are represented by
asterisks (p<U.~S). Results obtained Yor nc~n-tumor bearing animals (N) are
also illustrated.
?0
E~i~~ure 6B illustrates the. effect of PSP-94 on plasrnr calcium in armor
bearing animals. Results
ehresent the rncan ~ SEM of 5 anrmals rrr eacla group in ~i dii~ferent
experiments. Significant
dif i~erence from control tumor--hearing animals rc.cerving vehicle alone
(CTL) are represented by
asterisks (p<0.05). Results ot,t<tinecl for rron-tumor bearing animals (N) are
also illustrated.
'?.S
Figure 7A also illustrates the effect of PSP-c)4 on F~lusma.t PTHrP in tumor
bearing animals.
Results represent ~ SEM of 6 di fferent ani rnals in each gecnaj7. Significant
difference from control
(C'TL) is marked by asterisks r,p<O.t)5 ). Results obtained for r~on-tumor
bearing animals (N) are
al so i I I ustratecf
:~i0
Figure 7B also illustrates the effect of PSP-94 on plasma calcium of tumor
bear7ng animals.
Results represent ~ SEM of C~ different animals i n each group. Signif icant
difference from control
19

CA 02407548 2002-11-08
(~,"I,L) is marked by asterisks (p<0.0>). Results <obtained 1~c» ~ non-tumor
bearing: .animals (N) are
also illustrated.
higure 8 illustrates the effect of PSP-94 ors PTI-1rP production by Mat l.y Lu-
PTHrP tumors. A
representative photomicrograph of three such experiments is shown at a
magnification 200X.
Figure 9A illustrates the effect of PSP--~)4 un DNA fragmentation of Mat Ly Lu-
PTHrP cells in
nitr~u. A representative photograph of three suc:.h experiments is shown.
It) Figure 9B illustrates the effect of PSP-~)4 on DNA fragmentation of Mat Ly
Lu-PTHrP cells in
s~ior>. All animals were sacrificed art the end of the study and their primary
tumors removed,
laaraffin embedded, sectioned and processed by TUN EL assay a s described
herein (upper panel)
or counterstained with Hoescht reagent (lo~~~er panel ). 'Three animals were
present in each group
and three sections were analyzed for each animal. At least ten random fields
of observation were
evaluated. A representative photomrcrograph for three such experiments in each
group is shown.
'Magnification 2()0X
1=figure IOA illustrates the effect of PCK-3 t 4S on plasma PTHrP levels in
tumor bearing animals
using a radioimmunoassay. Results rcpre,cnt the mean t SEM of ~ animals in
each group in ~i
dif~fercnt experiments. Significant differences frorrr control tumor-bearing
animals receiving
vehicle alcme (CTL) are represented by asterisks (p<0.05). Results cobtained
for non-tumor
hearing animals (N) are also illustrated.
Fi Sure IOB illustrates the effect of PCK-~ 9 ~5 on plasma c;.rlcium levels in
tumor bearing animals.
Results represent the mean :~ SEM of '~ animals in each gr-nup in 3
diffeuf°nt experiments.
Si~~nificant differences I~r~om control turrrcn~ hearing, animals receiving
vehicle alone (C'T'L) are
represented by asterisks lp<'0.()~). CZesirlt:; ~obtuined for non--trrrnor
bearing animals (N) are also
r I I r.rstrated.
1~igure I I . Ef feet of PCK-31~~5 on i~xperimental skeletal metastases
resulting in the development
of hind limb paralysis. Results reprcsem the mean ~- SEM of 5 animals in each
group in 3
different experiments. Significant differences in percentage of non-paraly?ed
animals, from

CA 02407548 2002-11-08
control tumor-bearing anitrrals receiving vehicle alone (C'Tl_;J are
represented by asterisks
( g~<t).()5).
DETAIL~IJ1D DESCRIP'f ION Oli rI'HE INVENTION
In the current study we have examined the effect ol~ PSP-94 or PC:K3145 on
prostate cancer
v~rcowth and metastasis to the skeleton. I-~or these studies, MatC.yl,u rut
prostate c:ancercells were
transfected with full-length c.DNA encoding parathyroid hor-nrone related
protein (PTl4rP).
MatC.yLu-PTHrP cells were inoculated ~sub~ut~rneousfy (S.C.) into the right
dank or- via
intrac;ardiac route (-I.C.'.) into the lent ventrrclc of syngcnic male
Copenhagen rats. lntrucardiac
inoculation of l~l~rtLyLr,r cells routinely r~:sults in tumor metastasis to
the lumbar vertebrae
resulting in hind limb paralysis. 'lime of hind limb paralysis and tumor
volume, was measured
~rnd comparison was made between PSP-O4- c.~r 1'C.K3145-treated animals and
control animals
receiving vehicle alone. At the end of tine study, animals were sacrificed and
serum Ca+'
1 > icalcium, Ca~',1 and P7'HrP levels in control and experimental animals
were determined. Primary
tumors and skeletal metastasis to lumbar mrtebrae were also cxa-rmined for
PTHrP production by
imrnunohistochemistry. At the end oi' this study, affected lumbar vertebra
were removed for
radiological and iistological analysis. Tvidence of tumor cell apoptosis was
monitored by
subjecting histological specimens m Hoechst staining and rl,liNEL assays.
1'SP-94 (native) was generated as described in U.S. patent No.: 5,428,011. The
PCk;3145
pe3lypeptide was generated as described, for example, in <'anadian patent
application No.:
?, 359,6.50.
Animal Protocols. Inbred male (.'openlragen rats weighing 200 - 250g were
obtained. from
Harlan Sprague-Dawley (Indianapolis, IN ). Before inoculation, Mat l.y Lu-F''f
HrP tumor cells
grwowing in serum-ccmtaining medium wea~e washed with Hanks buffer,
trypsinired, and collected
by centrifugation at 1500 rl~m for 5 ruin. ('~chbarou, A. et al., Cancer Res.,
54:2372-2377, 19)4;
Rubbani, S.A. et al., (nt. J. <'zmcei~, 80: 'S7-2fi4, 1909: Rabbani, S.A. et
af., Endocrinology,
( 36:5416-5422, 1995 ). Cell pellets ( 10 x 10' cells) were. resuspended in
1U01t1 saline andinjected
using 1 ml insulin syringes into the: left v-cntricle of rats anaesthetised
with ketamine/xylazine
cocktail. Animals were divided into e~ontrol groups which received vehicle
alone (PBS:
21

CA 02407548 2002-11-08
phosphate buffered saline) and experimental groups which were infused LP. with
different doses
(0.1 -- 10.0 Ltg/kg/day) of 1'SP-~)4 starting :rt the time of tumczr cell
inoculation (day 0) until the
day of skeletal metastases development. The time. after tumor cell inoculation
which was
required to develop hind limb paralysis (an index of spinal cord compression
due to lumbar
s vcr-tebrae metastases) was deterrnincd and percentage of starting number of
animals developing
hind-limb paralysis was plotted.
.~yltcrnatively, cell pellets (~ x l0' cells) were resuspended in 1001,11
saline and injected using; Iml
insulin syr7nges into the r-ig,ht flank of rats as desc-gibed herein. From the
tune of tumow cell
1() inoculation, experimental animals were treated with different doses 10, l,
1.0 or 10.0I,tg/kg/day)
of PSP-94 via S.C. injections for 1 ~ consecutive days. Conirc~l animals
received PBS alone as
vehicle control. All animals were numbered, kefnt separately and monitored
daily for the
c.levelopment of tumors. The tumor mass vv~as measured in ? dimensions by
calipers and tumor
volume was calculated according to the equation (I x ,1~~)/2 (l=length,
v~=width) (Rabbani, S.A. et
I ~ ul., Int. J. Cancer, 80: ?57-214, 1999; Rabbani, S..A. ct al.,
Endocrinology, 136:5416-5422, 195).
,~If control and experimental animals were wet shed every alternate day to
deterrrzine any adverse
effect of PSP-94. Both control and exper7nzental animals were sacrificed at
day 16 post tumor cell
rnoculation and their tumors were removed and weighed. Additionally, these
tumors were; used
for histological analysis as described herein. 131ood frcam alt control and
experimental animals
20 was collected on clay l6 for dc~term~naticna ~:>f pl<rsn ua C,',ar'+ and
f'THrI' levels.
Results presented herein are usually expa~essed as the morn ~: SE (standard
~c.rror) of a~ least
triplicate determinations, and statistical comparisons are based on the
Student's t test or ar:alysis
of variance. A probability value of <().05 was considered to be significant
(Glantz, S.A., Primer
2_5 c~f biostatistics, McGraw-Hill, new-York. 1981).

CA 02407548 2002-11-08
EXAMPI,>N:~ 1
>H;ffect of PSP-94 on MatLylJu-PTIirP t',ell Growth, Morphology and Invasion.
S (.:ells and cell culture. The Dunnin~T R33~27 Mat L.y Lu cell line
(available, for example, under
,~'~me,r-ican Type Culture Collection Ilo.: JIJU-S) was transfected with full
length c:DNA encoding
rat P'CHrP as previously described (Ralobar~i~ S.A. et al_, Int. J. Cancer,
80: 257-2.64, 1999). One
caf the three well character-i-red monc~clcmal e;ell lines Mat Ly Lu-P'THrP-8
was used throuF;hout
t'he course of these studies. Cells were maintained in vitn« in RI'MI 1640
supplemented with 2
1() rnM L-glutamine (Life Technologies, lnc. Grand Island, N."f'.),
10°l fetal bovine serum (F~BS),
100 units/ml penicillin-strelatomycin sulphate iLife Technologies, Inc.l> and
2~~OnM
c.luxamethasonu and 6418 (600rng/ml) acc~~rdir~g to previously established
methods of culture of
E.hese experimental cells (Rabbani, ~i.A. et al., Int. J. C.'.ancer, 8(~: 2S7-
264, 199!7).
I S Cell nu>rphology. Morphological analysis of control and experimental Mat
Ly .Lu-PTHrF' cells
treated with PSP-94 was carried out by plating Sx 10~ cells/ well in 6-well
plates (;Falcon Plarsties,
Oxnard, CA) in the presence of 10'% FBS. C.'ells were examined daily for any
change in: their
morphology and photographed (Rabbani, S.A. et al., Endocrinology, 136:5416-
5422, 1995).
"20 Invasion. Effect of PSP-94 on Mat Ly Lu-PTHrP tumor cull invasive capacity
was examined by
?-compartment Boyden (.'hamber ('1'rans~~z ll, Costar, Cambrid~;e, MA) and
basement membrane
Matr-igel (Becton Dickinson l.trbware;, Budi'orcl M,A) as previously described
(Lie, D.F. et al.,
Prostate, 27:269-276, 1995).
2~ Growth curve. For growth curves. Mat L.,,y Lu-PTFIrP cells were plated in 6-
well plates (Falcon
Plastics, Oxnard, CA) at seeding densities of 5x 10' cells/wcll. Mat Ly Lu-
F'THrP cell; were
grown in the presence ol~ (). I , 1.0 8: 10.0 ~tg/ml of 1'SP-94 or vehicle
alone for up to 3 days and
the ability c:~t PSP-94 to altar cell de:~ublin~~, time was evaluated daily.
Medium eras changed every
tavo days. The number of cells was counted in a model Z C__"oulter counter
(Coulter Electronics,
30 Beds, UK). Comloarison was also made with doubt ing time o1' wild type
untransfected Mat Ly Lu
cells.
23

CA 02407548 2002-11-08
'Those results presented in Fligurc; 1 shows Mat Ly Lu cells trunsfccted with
vector alone (C-MV)
or vector expressing PTHrP v~ere seeded at a density of Sx 10' eel Is/wel I in
6-we-11 plates. Mat Ly
!.u-P'THrP cells were treated with >'SP-9~ and were trypsinized and counted
using a coulter
counter as described herein. Change ~n cell number l~c~llowin~, treatment with
f 0.0 ~g/ml of PSP-
u4 for 72 hrs is shown. 'rransfectic>n of :Flat l~y l_u with P'l'Hrl' cDNA
resulted in reduced
doubling time and increase in tumor cell gri~wth due to the growth promoting
effects of PTHrP.
'k~hus, Mat Ly Lu-PTHrP cells had a highea tutu of cell prolifer~rtion as
compared to control. Mat
I.y Lu cells transfected with vector alone. A significant dec:ruase in Mat Ly
Lu-PTHrF' cell
«rcwth was seen following treatment with 10.0 I,tg/ml of PSP-~~4 for 72 hrs
(Figure 1). Lower
doses of PSP-~)4 (0.1 and l .() Ftg/ml) failed tc> exhibit any signifrcant
change on iumorcell growth
tdata not shown). Treatment of Mat Ly l.cr-PTHrP ~;calls with 11).0 ~tg/ml of
PSP-94 for 3 days
resulted in a noticeable change in tumor cell morphology whore tumorcells were
found to change
their normal spindle-like shape to a more rc7undud and condcnsc:d appearance
~;ciata not shc.~wn).
I~Jsing a Boyden Chamber IV1atrigel invasion assay, trll doses of PSP-
~)4failed tu;.tlterthe invasive
~:apacity of Mat Ly Lu-P'f'I-tr!' cells (data out shown).
>IxaMP><.E z
Effect of PSI'-9a on Ntat t.y Lu-t''t'HrY tumor growth.
Male Copenhagen rats were inocul,rted with Mat Lv Lu-!'THrP cells via S.C.
route of injection
into the right flank as describu.d above-. Stardn~ t'rom the d~,ry o1 tunu~r
cell inoculation animals
were infused S.C., below the tumor cell inoculation site. witkt different
doses of PSP-94 (0.!-10.0
l,tg/kg/day) for up to l _S days. Effect of PSP-~)4 On ru.ducrng tumor growth
was evaluated b;y daily
determination of tumor volume with comparison being made to control tumor-
bearing animals
?S receiving vehicle alone.
Results presented in Figure ?A show Malc C_'openhagen rats injected S.C. into
uhe right flank with
IxlOr' Mat Lv Lu-I~THrP culls. Star-tin~~ on the time of tumor cull
inoculation animals were
infused daily with different doses of PSt'-94 for tiftu.en consecutive days as
described herein.
:30 Tumor volume was measured at timed intervals and comparrson was made
v.vith that of tumor-
hcaring animals receiving vehicle alone as control (C'fL). Results of Figure
~:Ei also show Male
?4

CA 02407548 2002-11-08
(:.'openhagen rats inoculated s.c with 10'' >Llat Ly Lu-PTHrP cells. After 3
days of tumor cell
inoculation, animals were injected with vehicle alone (Ctl) ordifi~erer~t
doses (0. t, l, l0l,tg/kg) of
PSP-~)4 (nPSP) at the cite of turner call injection. 'Tumor volume (expressed
in cubic centimeter
(~rrr')) was determined at tuned intervals. Control animals showed a
progressive increa;;e in
t~anu>r volume throughout the duration of the study. In contrast to this,
experimental animals
receiving PSP-94 showed a marked dose-dependent reduction in tumor volume
throughout the
course of this study (Figure ?A, ?B ~.
During this study. hoth control and uxperi~nental animals were monitored for
any noticeable
side effects and cachexia resulting in weight loss. Results presented in
Figure 3 shows Male
(:'openhagen rats injected S.(',. into the rigsn. flank with I x l0" Mlat Ly
l.u-PTHrP cells.
Starting on the time of tumor cell inoculation animals were infused with
different doses of
1'SP-)4 for fif~tecn consecutive days as dc;~.~r-ibed herein. All animals were
weighed at tinned
intervals and comparison was made with that of tunu~r hearing animals
receiving vehicle
1 S alone as contrcol (CTL). i~Jo significant change in the weight of control
and experimental
groups of animals that can be attributed tea any potential vide effect of PSP-
94 treatment
tF'igure 3) was observed.
h:~;AMPI,E 3
Effect of I'SP-9:I on Mat Ly l.u-P'1'HrP tumor weight.
In order to determine the effect of 1'SP-9~t on tumor weight, animals
inoculated with Mat Ly
Lu-PTHrP via S.C. route of injection were sacrificed at. the end of the study
(day 16) anti their
?5 tumors excised and weighed.
Results presented in Figure 4 shows Male Copenhagen rats inoculated with 1 x
lOc' Mat Ly Lu-
P'~HrP cells via subcutaneous injection into the right flank. Starting from
the day of tumor cell
inoculation animals were adrrrinistered with different doses of PSP-94 t'or
fifteen consecutive
3(1 days as described herein. At the end of tHc study tumors from control
(CTL,), vehicle treated
animals and PSP-94 treated itnirr~als w~~rc cxcisevd and weighed. Control
animals receiving
vehicle alone exhibited large tumz3rs while treatment with different doses of
PSP-94 (0.1-10.0
'' S

CA 02407548 2002-11-08
ftg/kg/day) resulted in a signifiicant close-dependent decrease in tumor
weight (Figure 4).
Inoculation of made Copenhar~~n rats with Nlat Ly Lu-PTIIrP cells into the
right flank via S.C.
injections resulted in the development of primary tumors. Whereas control,
vehicle treated
animals developed large primary tumors, t:- .atmcnt with different doses of
PSP-94 resulted in
a dose-dependent decrease in their tumors mass. These anti-turrror effects
were not associated
with ~tny noCiccahle side effects or vcight: loss of experimenter! animals.
~;XAMPLF: ~1
Effect of PSP-94 and 1'CK3145 on the development ot'skeletal metastases.
Since the major cause of prostate cancer related mortality is the development
of metastases,
evaluation of the effect of PSP-94 on delaying the development of skeletal
metastases was
1 ~ c:arried out by inoculating ntalc C:opcnhagen rats with Mat l.y Lu-P'I'HrP
cells via LC. route
:nt<> the left ventricle. Routine. injection o!~ Mat Ly Lu cells into the
Ici~t ventricle results in the
clevclopment of skeletal metastases cuusirr~~ compression of the spinal coed
leading to hind-
limb paralysis .
?0 Mett I~y Lu-P'I'HrP cells were thus inoculated in male Copenhagen rats via
L(.. injection into
the: left ventricle. Starting from the day of tumor cell inoculation (day 0),
aniunals were
a dministered with different doses <af I'SI'--94 (0.1-10.0 ~g/kg/ctay) via LP.
(intraperitoneal)
route. The effect of PSP-c)4 on deluyinL~ the development of' skeletal
metastases was evaluated
by doily monitoring of the animals for the development of hind-limb paralysis.
~>
Results presented in Figure SA show Malc Cc>penhagcn rats inc>culated via I.C.
route into 'the left
ventricle with IOx 10~ Mat L.y L.u-P'rHrP cells. Starting on the time of tumor
cell inoculation (day
O) animals were infused with different doses of PSP-94 (0.1 - I0.0
l,tg/kg/day) until the day of
development of hind-lir~ot~ paralysis as described he:rcin. Animals receiving
vehicle alone as
30 control (CTL) or PSI'-94 were morritorcd daily for the deve(opmc;nt of hind-
limb paralysis. and %
animals not paralyzed at different tirrre points in each group was calculated.
All ( 100%) control
animals inoculated with Mat L,y Lu-PTI-IrP ells and receiving vehicle alone
developed hind-limb
?(~

CA 02407548 2002-11-08
paralysis by day 1 ~. Whi le 0.1 and I .() Itg/kg/day PSP-J4 had no
significant effect on the time of
hind limb paralysis (data not shown), treatment with I().0 Ixg/kg/day PSP-94
resulted in a
statistically significant delay in the number° of anirrrals developing
bind limb paralysis. Percentage
01' total number of animals receiving 1'SP-9-I nat developing mind limb
paralysi~~ .at different days
S is shown in Figures SA and s13.
A second set of expcr-imentation was performed. Results presented in Figure SB
also show
Male Copenhagen rats inoculated via the intracardiac (i.c) route with 5 x 10~
>'vlat Ly Lu-
PTHrP cells. After ~i days of tumor cell inoe;ulation, animals were injected
by intraperitoneal
I() route with vehicle alone (Ctl) ur~ different doses of PSP-94 (n1'SP).
'rime to the development
mf hind limb paralysis in Ctl and animals receiving 10 ftg/kg/day of PSP-94 is
shown.
Percentage of total number of animals recd vin'; PCK314S not developing hin<.I
limb paralysis at
different days is shown in Frgure 1 1. Results presented in Figure l l show
Male Copenhagen rats
IS inoculated via I.C. injection with t(P x l0' Mat t.y Lu-P~I'Hrl' cells.
Starting on the say of tumor
:.ell inoculation (day ()), zrnimals w-ere infr.rsed with different doses or
PCK-3145 (i.0-100.0
,ug/kg/dayj until the day c>f hind-limb paralysis development as discussed
herein. Animals
recei ving vehicle alone as control (('TL) or PC.K-3145 were monitored daily
for the development
of hind-limb paralysis and percentage of animals not paralyzed at different
tune points in each
?0 group was calculated.
While all control, vehicle treated annmals developed hind-limb paralysis by
day 13,
a.rdministration of the hif;hest dose of PS I'-9~f starting from the time of
tumor cell inoculation
resulted in modest delay in skeletal metastases. Such results suggest low
bioavailability of PSP-
?5 ~)=~ to the skeleton, a common drawbaok associated with developing
effective therapeutic agents
for skeletal metastases (Rabbani, S.A. et al., <:_'anct-r res., 58:3~=I(,l-
B4b5, 198;1.
27

CA 02407548 2002-11-08
hx I~IfTPLh. 5
Effect of I'SP-94 and NCK31~5 on plasma P1'tirY and calcium levels and tumoral
(''fHrP production.
i
In order to determine the effect of PSP-94 and PCK;i 145 on plasma PTHrP and
calcium lc;vels
animals inoculated with Mat Ly Lu-PTNrP cells via S.C, route were sacrificed
at the end of the
study, plasma was collected and l~'TI-LrP levels were analyzed using a
radioimmunoassay.
(.'omparison was made between pl,rsma ccollcctcd from normal, non-tumor
bearing animals,
I() v°ontrol tumor hearing animals recd ving vuhiclc: alone ~.rnd
plasma collected from experimental
animals receiving different doses of PSP-~)~t (().l-1(l.U lug/k~ day). Plasma
calcium levels were
determined by atomic absorption sp~ctropi~otorrretry (model 7t):B, Perkin-
Elmer, Norwalk, CT).
For plasma PTHrP, all sarnlrlcs were tested in two dilutions in P'ITHrP R.I.A.
kit (Nichols Institute
Diagnostics, San Juan Cap~strano, C'A.) u~cording t.c.~ manuia.rcturers
instructions.
ormal non-tumor bearing animals showed halal levels of plasma PTHrP whereas
animals
incacutated with Mat L.y l.r.r-P'rHrP cells and receiving vehicle alone showed
marked elevated
levels of immunoreactive plasma PTHrP levels. Treatment ol~turnor hearing
anirnals with PSP-94
resulted in a dose-dependent decrease in l>I4rsma PTHrP levels (Figure 6A).
Analysis of plasma
?0 collected from normal non-tumor hearing animals ,and tumor bearing animals
receiving vehicle
alone revealed a marked increase in plasma calcium of control tumor fearing
animals at the time
ol' sac:rificc on day 1 (i past tumor cell inoculation. In contr<~st,
experimental groups of animals
receiving dil~fer-ent doses of PSP-94 resulted in significant reduction in
their plasma calcium
levels. The highest dose of PSP-94 ( t().(? ftg/kg/dayj resulted in near
normalization of plasma
calcium of these experimental group of :rnirnals (Figure 6 B). See also
results presented in
Figures 7A and 7B.
Results cof Figure f A and 6I3 show Male; C~openhsrgen rats inoculated S.C.
with lxlOc' Mat Ly
l..u-PTHrP cells. Starting on the time of tumor cell inoculation animals were
administered with
different doses of PSP-94 f'cn~ fifteen ccansecutive days as described herein.
All animals were
sacrificed at the end of the study (clay 16) and plasma was collected from
control (CTL) vehicle
treated animals rrnd PSP-94 treated animals and analyzed for immunoreactive
plasma P"T'HrP

CA 02407548 2002-11-08
(iP'f1-1rP) levels using radioimmunoussay as described herein (Figure 6A) or
for plasma calcium
levels as described herein (Figure OB). Plasma PTI-IrP levels in normal non-
tumor bearing
animals (N) (Figure 6A) and plasma calcium from normal, non-tumor bearing
animals are also
shown (N) (Figure 6B).
;'~ second set of experiment was perlormeci. Similar results arc presented in
Figures 7A and
?B. Results of Figure 7A shows Mule Copenhagen rats inoculated s.c with 10c'
Mat Ly Lu-
I'"I'IIrP cells. Following 3 days of tumor et°II inoculation, anim~rls
wc,re treated with vehicle
~rlc>ne (Ctl) or different doses ( I .0, or 10.0 ftg/kg/day) of PSP-9~~t for
18 days. Animals were
s~rcr-ificed on day ? 1 and plasm~.r P'THrP was determined. P'I'HrP levels
(expressed in
picornole equivalents/liter) of non-tumor i»aring arrirt~als is also shown
(N). Results of
hi~~ure 7B shows Male Copenhagen rats inoculated s.c with 10'' Mat Ly Lu-
PT'HrP cells.
Following 3 days of tumor cell inoculation, animals were treated with vehicle'
alone (Ctl) or
different doses (1.0, cor 10.() f,tg/k<r :lay) of 1'SP-~)4 for 18 days.
Animals were sacrificed on
1.5 day ?1 and plasma calcium (expresved in millimolar (mM)) was determined.
Plasma calcium
of non-tumor bearing animatls is also shown (N).
All animals were sacrificed at the end oi' the study. Tumors f~r-om control
group treated with
vehicle alone and experimental groups treated with different doses of PSP-94
(0.1- 10.0
pg/kg/day) were excised, paraffin embedded, and sectioned and analyzed for
tumoral PTHrP
production by immunohistochemi~al reaction specrfic: icrr P~I-lirP using
method described
herein. Results of higure 8 show hrstologic sections of tumors from animals
receiving v~°hicle
alone (CTL) or different dosc;s of PSP-9~1 and stained with an antibody
specific for PTHrP as
described herein. Three animals were: present in each group and three tumor
sections were
?5 analyzed for each animal by evaluating ttt least ten random fields of
observation.
Histolo~ic Analysis. Fur imrnunohistological analysis, pr,irrtary tumor
samples were dewaxed by
hi:ating at 60°C and rehydrated in ~r graded alcohol series (10()x%7-
70°/<>). Anti-u~,.tt antibody against
P'fHrP was used as the larirnary antibody. ~fum<.rr sections were incubated
overnight at ~°C
followed by further incubatio n with hiotinylated universal antibody (Vector
Laboratories,
Burlingame, CA) for 4S-6() minutes. SeCt10r1S were rinsed with 'I'BST (Tris
buffered saline-
t~ween) followed by incubation with Vectastain ABC-AP Reagent (Vecaor
Laboratories,
.?c)

CA 02407548 2002-11-08
Burlingame, CA) for 30 minutes. These sections were ag~rin washed with TBS'T'
and incubated
witlo a Napthul AS-Mix PhosphatelFast Re:d solution (Sigma-Aldr-iche,
Oahville, ON). The
sections were finally cuunterstained with Methyl Green (Vector Laboratories,
Eurlingame, CA)
and mounted.
Intense color staining of tumors from c;ontrul groups of anirrrals receiving
vehicle alone was
observed. In contrast a dose dependent, decrease in P'rHrP imrnunostaining
w;.is observed in
experimental tumors from animals receiving dit~ferent doses of PSP->4 (Figure
8).
1() Similar results obtained in tumor bearing animals inoculated with PCK3145
are illustrated in
l~i~~ure l0A and B.
Kcsults presented in 1~igure l0A show Male Copenhagen rats inoculated S.C.
with 1 x 10G Mat
Ly I_.u-PTHrP cells. Starting can the time c~F tumor cell inoculation, animals
were administered
1.S ~.vith different doses of PCK-;~ 145 I~or fifteen ~::onserutive days as
described herein. All animals
were sacriticed at the end ol~ the study (day I(i) and plasrnu was collected
from control (G.'TL),
vehicle treated animals and PSP-O-1 treated ~rnirnals and analyzed for
immunureactive F'THrP
(iP'1'HrP) levels using a radiuimmunoassay its described herein. Plasma
P'rHrP' levels in normal
non-tumor bearing animals is a.rlsu -;hewn (N >.
?0
Results presented in Figure lOB show Ma.rle Copenhagen rats inoculated S.C.
with 1 x 10c' Mat
Lv Lu-PT I-1rP cells. Starting on the time of tun uor Lell inoculation,
animals ~,~'ere administered
with different doses of PCK-3145 fur fifteen consecutive days as described
herein. All animals
were sacrificed at the end of the study (clay IO) and plasma was collected
frurn vehicle treated
control animals (CTI..) and P(:'K-314i treated animals and analyzed for plasma
calcium levels as
described herein. Plasm.t calcium from nun-tumor bearing animals is also shown
(N).
As discussed above, upon sac-ritic.c of animals, plasma.t was collected and
analyzed for 1'THrP
and calcium levels. Norr~~al, non-tumor gearing animals have undetectable
levels of plasma
30 1?T'HrP whereas inoculation of aramals ~,vith Mat L.y L.u-P'I'lirP cells
resulted in marked
increase in their plasma PTHrP levels. lrr contrast te.~ this, treatment with
the different doses of
l'SP-)4 resulted in a dose-dependent de~r'ease in plasma P~'HrP leve-ls. In
addition, the same

CA 02407548 2002-11-08
dose-dependent decrease was observed in tumoral P~l'Hrl' production when tumor
samples
from control, vehicle treated and PS I'-94 tr~~.rted animals were subjected to
immunohistochemical analysis
I~eing the major pathogenctic factor cof hyI>ercalcemia of malignancy, plasma
calcium levels
cor7-elate with that of plasma P'rHrP levels (I~~amura, M., et al., Urology
43:675-679, 1994;
Iwamura, M., et al., Hum. Pathol. 2f~:797-X01. 1995; Suva, L.J., ct al.,
Science, 237:893-89fi,
1987). Inoculation of Mat Lv L.u-P'I~HrP cells into the animals resulted in a
m,.rrked increase
io their plasma calcium levels as compared to scrum from nc~rrnal, non-tumor
bearing
animals. Administration of different doses of PSP-94 rew.rlt~;d in a dose-
dependent decrease in
plasma calcium levels with the: highest dom, of PSP-94 leading to a near
normalization of
plasma calcium levels.
Using this model we were not only ~rble to demonstrate the anti-tumor effects
of PSP-94 by
reduction in tumor volume and weight but also biorhemie:al parameters like
plasma calcium
;.rnd P'fHrP levels also showed a marked decrease following therapy. A
significant finding in
then: studies was that while decrea se in tur-nor volume was dose-dependent,
10.0 l,tg/kg/day
PSP-94 did not show a marked decrease in tumor volume as comparod to 1.0
~g/k~T day PSP-
94. In contrast, the ahility of I'SP-~'4 to reduce plasma calcium, plasma
PTHrI' and tumoral
?0 PlfHrP continued to show a dose-dependent effect with 10.0 pg/kg/day PSP-94
causing near
normalization of plasma curium and P'TI-IrP levels. 'These findings allow us
to speculate that
PSP-94 may also have additional effects including its ability to regulate PTI-
IrP production by
tumor cells or alter calcium homec~~stasis. Indeed PSP-94 has hec;n shown to
suppress follicle
stimulating hormone (FSI-I) which is knc~~arn to regulate intracellular
calciurr~ (Touyz, R.M. et
al., Biol. Reprod. 62:1067-1074. 2000). 5upprcssion of FSH by PSP-94 may serve
as an
additional mechanism to cause anti-turner cfic<as due to the growth-promoting
effects of FSH
iro prostate cancer (Poser, A.'1'. et 4r1., lJrol. tJncol., 6:131-1 38, 2001).
Furthermore, although
cloning and characterization of a Iwtativc PSP-94 receptor has not been per-
l~ormed several
Studies have provided evidence for the e~:istence of PSP-94 binding proteins
on prostate
caneer cells which could allow PSP-94 Finding to initiate a signaling cascade
that results in
the observed anti-tumor effects observec.l (Yang, J.P. et al., .i.Urol.
160:2240-2244, 1998;
Yang, J.P. et al., Prostate, 35:11-17, 19~~8).
:31

CA 02407548 2002-11-08
EXA~iV11'LE 6
Effect of PSP-94 Mat L,y Lu-P'fHrP tumor cell apoptosis irt vitro arid irt
vivo.
Iro'fUNEL assay, tissue sections vvera dewaxed and rehydrated by heating at
60°C followed by
washing in xylene and rehyclration ti~rouglr a graded series of er.hanol and
water. Tissues were
incubated with proteinase K for 30 ruin at 37''C and fixed, blocked and per-
meabilized. Apoptotie
cells were detected by 'fL!NEL assay ira .s,~trt cell death detection kii
(Ruche Mole,;.ular
l3iochemicals, Laval, QC) according to the manufacturers instru~ ion.
Positive'1'UNEL staining
was visualised by fluorescence microscopy.
In other experiments following TLINlJI. assay. tissue sections were
counterstained with Hoechst
X3258 (Sigma-Aldrich, Oakvilie, ('anada). Hoechst staining was added to
tissues at a final
concentration of 24pg/ml in PBS and incubated fc;r IS minutes at room
temperature. Tissue
Sections were washed and visualized by fluorescence microscopy using a blue
screen (Rabbani,
5.:~1., et al., Int. J.C'.ancer, 8'7:276-282, 20()0). All results of
imrnunohistochemistry and TLJNEL
.essay were evaluated and interpreted by two independent examiners.
'?() In order to investigate the underlying rrrulecular n-echanisrn of action
of PSP-94 in reducing
tumor growth Mat Ly Lu-P'CHrP cells wore cultured in the presence of PSP-94 (
10.0 ~tg/mI) or
vehicle alone for different time intervals. ~aenornic DNA was collected from
cells cultured in the
presence of vehicle alone or PSP-~)~J. Briefly, for DNA fragmentation, Mat Ly
Lu-PTHRP cells
were plated in 6 well plans (Falcon Plastics, Oxnard, CA). C'.ells were
treated with PSP-94 ( 10.0
Ixg/ml) for up tea 72 hours. DNA from treated cells incubated with PSP-94 and
cells treated with
vehicle alone was collected usin4~ a PhenoI:Choloroform:lsoamyl arlcohol
solution (50:48:2).
Equal amounts of DNA were subjected to gel clectrolohore;sis on a 2% agarose
gel. DNA
fragmentation was visualised by C1V lig.lm using a transilluminator. More
particularly, results
presented in Figure c)A show Mat l.y Lu-1'THrP cells cultured in the presence
of vehicle alone or
:~() PSP-94 (10.0 ltg/ml) fur up to S)6 hours. DNA was isolated and run in a,n
agarose gel, as
described above. These results show that control Mat l.y l.u-PTHrP cells
cultured with vehicle
alone exhibited no signs ol~ DNA i ragmentation. I-Iowever, experimental Mat
f.y Lu-PTHrP cells
~2

CA 02407548 2002-11-08
cultured in the presence of PSP-94 ( 10.0 ~;~lmt ) exhibited marked DNA
fragmentation after 72
hours of treatment (Figure 9A),
'hhc degree of DNA fragmentation was also analyzed in viva using '1'L1NEL
assay which can
serve as a marker for apoptusis. More particularly, results presented in
Figure 9B are derived
i rorn tissue collected from Male Copenhagen ra.rts inc>culated mth t x t Or'
Mat Lv Lu-PTHrP cells
and infused with different doses of PSP-9~ for fifteen consecutive days as
descry bed herein. All
animals were sacrificed ;~t the end of the study and their prsmary tumors
re"moved, paraffin
enobc;dded, sectioned and processed by TIJNIL assay {upper p~mel) as described
herein.
Following TUNEL assay, tPrey were cc~unt~rst~uned with I ~oes~ht reagent
(lower panel). Tumor
,ectians treated with PSF'-~~4 {10.(i ftg/l:~T'day) were significantly more
TUI'~tEL positive as
:compared to vehicle treated control tumors (Figure OB). Countc:rstairring
with Hoechst reagent
revealed the presence of apoptotic bodies in tissue sections from animals
treated with PSP-94.
Furthermore, condensed chromatin, whicia is c:haractcristic of apaptc>tic
cells, was observed in
t 5 PSP-94 treated tumors. C'ontrul, vehicle treated tumors exhibited normal
DNA staining patterns
(Figure 9B). rhhese ire vr'tru and in vir~c~ lindlngs demonstrate that indeed
reduction in tumor
volume following treatment with PSF'-94 is clue to its ability tea prorrrote
tumor cell apoptosis.
As indicated above, TUNI<L. analysis carried c>ut on tumor4rl sections from
control and
?0 experimental animals revealed that PSP-~)~f treated tumors are more TUNEL
positive as
compared tea control turners, indicdrting a higher degree of apolotosis in PSP-
94 treated
animals. In addition to this, cuunterstaining with I-Ioechst reagent revealed
condensed,
apoptotic chromatin in PSP-~)4 treated tumors whereas control tumors exhibited
normal DNA
staining.
~5
Collectively, the results of this study demonstrate PSP-94 and PCK3145 to be
effective
inhibitors of hormone-independent, late ~;tage prostate cancer growth and its
associated
I~ypercalcemia of malignancy witl-rout manifesting, any noticeable cytotoxic
effects. The use
_~iC) of polypeptides, their peptidumimetic analogues alone ur rn combination
with currently
available chemutherapcutic agents will provide unique opportunities to block
prostate cancer
progression with highly effective nun-tu.xic biotherapcutic; agents which can
be deliver°d over
33

CA 02407548 2002-11-08
a long period of time without any drug asst;elated side effects. 'These
approaches will go a
lon~.a way in reducing prostate cancer assocrated morbidity and mortality.
All publications and patent arpplicati~ms citv.d in this specification are
herein incorporated by
r~:f~ere:nce as if each individual publication <>r patent application were
specifically and
individually indicated to be incorporated by reference. 'Chc citation of any
publication is for
its disclosure prior to the tiling date and should not be construed as an
adnrissicm that the
present invention is not entitled to antedate such publication by virtue of
prior invention.
1() Although the foregoing invention has been described in some detail by way
of illustration and
example for purposes of clarity of understanding, it is readily apparent to
those of ordinary
>kill in the art in light of the teachings of itois invention that certain
changes and modifications
may be made thereto withi>ut departing frc>rn the spirit or scope of the
appended claims.
l5
3~

CA 02407548 2002-11-08
S_'~;QC1ENCE LISTING
( ':. ) GENERAL. LrJFORMATIC>N:
( :, ) APPL,ICAN'I' : PROCYOIV B ~rO?HARM;'?. LN(.' .
( :ii. ) TITL~I;I OF' II'~i :NTIO ~: PSP- 94 : i1S E FOR 'PREATMEN i' OF
IC) IIYE'ERCALCIIMT~ AND BONE METr,STF;SIS
( i i i ) NUMBER OF SE(~LJI?NC.'ES : 6
tv) C.'ORRI~SPONDENCEADDRE:>S:
IS (A) ADF?ESSF;E: BR:)ULLETTE; K,OSIE
(B) ;STREET: 1200 RENE-LE;SVE~UE
IiL~7D 6VEST
(C) uRC)V/STATE: QUEBEC
(D) ~r'C,LJT~1TR'r : CANrIDF,
(E,'~ PC :~'rALi ZIP C.-;=)DL;: H3E3
5C'9
( v 1 COMPUTER READABi~E E'ORC'
( A ) MEIJIUM T~'PE~ : F LOPPY DISK
(B) COI~IPLJTI:R: I:,M I'C COMPATIBLE
(C) P'IRAT.,'NC7 S':'S'I'EM: P(.'_-DOSIMS
_1)OS
?1 (D) SCWTWAII:E:Aat'II (TEX'C)
( vi ) CURRENT hPPLICr2;T LON :.)A'CA:
( A ) r~,PPL ICn.TION NUZrIBER
( F3 ) _ I I~ ID1G D:4TE
(C) ~4LA.SSI"I~'AC." ON:
;vii) PI~.IOR APPL:LCF~i'ION D:'1TA:
(A) T~PPLTC,'~TTON NU~IBI~R: ('A '<?, 361, 736
(B; b'IL:ING DATF_: ?0('7-11-08
~> (Ci C;~AS STi.;ICA'I'CON:
(viii)A'I'TORiVEY'/PA'rI~:NT AGF;NT I~II'ORMFSrIC;N:
(A) NAME: BROULt.~ETTE~ KCSIE
(L3) RE.',GISTRATICCd NO.: 3939
(C') ~f'FER.F;I'dCE/L;::)CKE~~ NC:. : 0650f;,- C)52-C:'A-02
( D ) '1' f ,7:_' . f~.0 . : i :> 1 9 ) :5 9'7 8 5 0 0
(I~:) E'r:.~ NC:.: (w:L4) 397 8515
-i5
(?) INFORI~LATION FOR SE;(~ ID NC): 7 :
( i ) SEQUEN('E CI .ARAC'I i~,RISTICS :
(A) L~ENG'rl!: 94 ~a~MINO A~"ID~,
( ?.) 'I'~CPE: AMI1~1( r'1C'TDS
SU ( C ) S'TRANI~GDNE; ""~~ : SINGLE;
LL~) 7:'~;)I'OLtoG'C: '~INEAR
(ii)MOLL;CULE TYPE: F'F.'C>'11~L:Dd
( i i i ) HYPOTHE"'I Cr';L :
( iv) T~d'rI-SENSE:
>S (Z-) FRAGML;2(IT TYPE:
(vi')ORIC~IDdAL, SO!JRCE:
(A) ORGANCSM:
( Vi i i IMSZErDI A,'rE; ~~oURC ~;
(vi:i.) POSiTIOlv IN GEIV:)ME
(A) CHROMOSOME,-' IECIM;~',':I'P:
(B) MAF~ F'Cr~::l:'P-'OV:
((:) UNI'1:3:
( i~~ ) FE;A~fUI'E
~5

CA 02407548 2002-11-08
(A) NP.ME/KE'v.':
(B) I_C~CATIO"1:
(C) IDENTII~'ICATIO..I METFfOD:
i D) ~~THER INI~'ORMA'I'IOIV
S ( x ) PUBLICF:rION LNFORM: iTIr:7N
( A ) F:CITI~OKS -
( B ) 'I'ITL~E
( C ) JC7UR NAh
( D ) VOL(JME
IC) (E) IS~IIE:
(F) PAn~F,S:
i G ) IeITF'
( H ) I70C UMEN-1' NO .
(I) I~'TL'YNG DATE:
IS (J) :''1JF~I,ICATION i)ATE:
(K) T~.EL~I~VAI~I'I' R.ES-DUES IL; SE;~~
ID I'.0:1-:
(x:i.) SEQIIENCF~; DEF;~CRIP'I-CON: SEI~ ID
NO: J
;:er Cys '~yr Ile Pr:~ Asn (::=lu G:i 7 Va 1. hro Gl.y
Phe A:~y Ser 'Thr Arg
5 10 I5
~1
L.,ys Cys Met. Asp Leu Lys c ~ly l,sn L,; :. H.i s Pro I ~ a A:>r Ser (.:~lu
Trp
20 2' 30
i;lt Thr Asp Asrz Cys Glu Thr c::ys Tt~r Cys T~rr Gl.oz T:z:. Glu Ile Ser
35 ~a0 4p
''ys Cys Thr I~eu Val Sec. Thx- i'ro V<;1 C:l.y 'Py: A:,;p Ly ; Asp Asn Cy
30 50 p5 ~:~()
I1.:_ Ar<7 Ilee Phe Lys I~y; Glu ;fist ('_~~s T.'y:; 'I'yr I l.e Vat Val Gltz
Ly:
?0 '75 80
35 ~~ys A.sp Pro T_~ys Lys Thr Cys :>er 'J ;: l '~e.r C'lu Trp I W> Ile
f35 90
( 2 ) INP'ORMA'I'ION FOR SEQ ID NO : 2 :
(i ) SEQUENCE CI-IAR.ACTI?RTS'I'IC;::
(A) LENGTH: 102 AMINO t~,CLDS
(F3) T~'PE: ANIINC ACIDS
(C' ) S"'RANL;:EDNE:":; : S-INGI~E
( M ) T()POL~C:~GY' : L (NEAR
( ii ) MOLECUL.~P~ TyFE: FE~c''TEIN
SO (vz.)ORTGIrdA~ SOIIRC:E:
(A) ORGANISM:
(xi) SEQUE1'T;"E Di°;SCRII''1'ION: SEA) :I:D N0: ?.:
~5
C~)~_, Ala Al.aTyrVal Pne;.~er~ PhE: ProAsn
Glu Glu C'ys 'Pyr I)e G:Lu
5 t0 15
Cily Val, Gl.yAsp:Ier Arg-.,ys C'ysAsp LysGly
Pro Thr Me:t Leu Asn
z0 '': pCr
L:y:~ Hi.s IleAsr:Ser Trplin 'T'hr Asn GluThr
Pro Giu Asp Cys (:ys
35 40

CA 02407548 2002-11-08
'I'hr C'ys Tyr Glu Thr Glu Ile Se'r Cy: CIi:~ 'rh:c i,o~u ~raL Ser 'rhr Pro
~~0 '_i5 60
V,:al Gly Tyr A,sp Lys Asx~ Asn Cyr~s G ~n Arg I12 Phe hys Lys Glu AsLn
~>'a 70 75 80
C'y~s Lys Tyr Ile Val V<iI Glu I_,ys Ly,: Asp Pro Ly~~ L,ys Thr Cys Ser
85 9() 95
IU '~'-xSer Glu Trp Ile I Le
100
('~? ) INF ORMATION FOR S:.=::Q ID FvO: 3
(i) SEQUEIVC.I? CHFaE~.CTEKLS'IICS:
(A; ~EIJGTi3: 1 0 I?".~IINO ACa DS
(F3) 'Yi'-_:~: z~fdINO ~oC.'IDS
(C:~ S'lItANDI:1)rIES-: SINGI~~,:
~~) (D) PO?OLOi;Y: L~l!eE:AR
( i.i ) MOL,E:C'UL,E: r'YPIv.: PI2(:'I'F,~ fv
f vi) ORIGINAL SGURCI:
(A) ~'~R~:~P.NI:;in:
~5
(xi ) SEQUENCE D1~SCRI:~"I':ION: S~Q TD N0: 3 :
f'yr 'Phr Cys Spr Val Se.r Glu ero <;..y 11<:
':> ~ (i
3l)
( 2 ) INFORMhTION FOR. :~I~~k~ ID VO : 4
~S (i) SEQUF?NC'E CHARAC'I'n,RISTICS:
( A ) TWNG'I'I-? : 15 "~1'I'INO AC:I DS
( E i 'I"~'I?E : AMINO ACIDS
( C.' ) STRANI-EDNES S : S INGI,E
(I)) TOPOi~(:C~Y: I.INEAH
( 1. L ) MOLfCI_':i.uF TYI''f: PR;)TEI:N
ivi)ORIGI:;VA.L SOL RCE:
( is ) GIt~~AN I SL~I
(xi) SEQUENCE DI~:SCRII'I'I<)N: SEQ ID N0: 4:
Asr. Glu Gly Val Pro Gly Asp Ser ''hr Arg L~ys <.:ys I~I~-~t Asp Leu
1 ~ !.0 7.5
(<y) INFORMATION FOR SEp ID N0: ~:
SS ( i ) SEQf.IE,;PdCEI CH,ARAC'P ~:RIS'I'ICS :
(i'? ) LE;NGT'-I : 1'p AMLNO Ai: CDS
( I3 ) 'TYPE : AMIN i AC' I:IOS
f (." :7.'RAI'7EDNE;:->; : (=ITNCIL:
(D:~ TOPOL~JGY: ,INIAR
OO (-ii ) MOLI~C'UI,fTYPE: P?.c:~TFIN
(vi ) ORIc;INAL SOLtRCE
sA) ORGAPIIS~M:
.37

CA 02407548 2002-11-08
(xi) SEQUEIV1CE bESC'RTPI': JN: SEQ ID N0: p
G a M'x~p Gin '1'hr Asp Asn Cys G a The C~,rs 'I'hr Cy~~ 'I'yr Glu Thr
2~.) 15
(2.1 INFORMATION FOR :pI:Q I:D N~): 6:
IU l i ) SEQLENCE CHA'~ACTE:<:IS I' LCS
( A ) LENGTH : 1 J A.~ INO AC: I DS
(B) ~"YPI~I: AMINO ~,(.'II7~
(C) ;:;TF;A1VDEJNESS: SIN!~LF
(D) 1'OPOLOG'r': L~I;vdEAR
I S ( ii ) MOLECIJhL: i'YPE : PR01"ETN
~,~i ) ORIGLN:"~:i~ :~OUF''E;
(A) ~1RC~I~Nl~iM:
(xi) SEQLIEL3CI~; DEwCF;IP" IODI: SEy7 1D NO: E.:
Le:~ V al Val G 1 a Lys Lys Asp Ieru I_~;-s L.ys ';I':m <;y:; S~~t- Val Ser
Gl~.
~ 1 (i 15
? 5 ' r ~.> I:1 <~ I l a
:3b

CA 02407548 2002-11-08
20()2-11-l77 sE~q 1>_:~t_ (06508--051-C'.F~-(72)
SEQ>:.E;.IC~S LISTING
() GENERAL INFORMAT:LON:
~?PPL~ICANT: PROC'YON E3IOPHARMA I vC
.l t 'I'ITLE OF It'TVEZ'dTI(.)PJ : PS r --9~ : =_~,SE F'OR 'I'REA'I'MEN I OF'
IIYPERCt'~LC'EMIA ANL) BC>NE NFTAS'I ';STS
i '~ i ) NUMBER OF SE:QUENO:E:p : v
a<1 <~ORRESPONDENCE AD~)R?aS
(A) ADRES::r.E: I3FLOUILET'lE Kt_)S1.E',
(B) S'PRF:E~L': 1:.OCI RF:NE-L~SV-~:Qtu: BLVi) WEST
1(: ) F'RO'v'/;;'L'ATi,;: QL::'BIC:
( D ) C,'()UN'I'.~ Y : i ANADF-.
(F:? P!nS:'i"-~:,~!ZI_" (.'OL>H:: H3B 'i:::5
vi COMPUTER READABLE :~'ORM:
(A) P!LEDLIJM TYE'E: F!~Oh7?Y DI;~k:
(E3) COMi?LJ'I'E I~'.: I:3M ''C'. C'()D'IPA'I":F3_.~I:
(C ) OPE'i?A'I'INC SYS'J ~~,M : PC:-DOS 'M;J -DOS
ii)) Si>F,.I'r~JP,RFr ::°~5~:."I:C ('rI~'..~!':f')
;G) CLJRREN''I' AYPI'_ICA'1:'1C)1V i')AT:~:
(A) API?L:ICAT ION l.U':iMBF:R:
(3) 1~ l~IT,I(i I'-;'',TE.:
(C1 CL,~?S;;IFT.;'ATIO'3:
t v i i ) F'RIOR AP I'L:~ CAT Ic:~N DATh
fA) Ar''E'L:LCA'I'ICN NEJC4B.ER: C'A 2, 361 ,'736
B ) F, 1 L -., I,d G I F~'I' E : ;?, O C 1, - l-1 - O 8
(C) Cr:.~SSnIA~;cF'rlcrs:
(~,~i ii ) ATTORNEY% PATENT' AC;EN'T LPdFC:iFIf,ATlON:
(A) N.-'~M~:: BI-rOLJLL~I.'('~.,E K:OS:~F,
(B) R~~C;LSTR~..T.TOL) E~,TO.: :3939
(C) RF'FERENC'E!DC.)c'Tlri,'C DIt). : OE'~OY-=O!>7-C'A-02
(r)) r?L. ro : ('>:~':) :3~~~ ;~~:,oc)
( E ) F'::za NC: . ( 51.. ) '-> ;' f3 -; :; .'>
_' a: ) INF'ORMA'TION FOR SE;~~ LD N~.7 : 1
SEQUENCE C'HARA(:7.'ERI:S'I-'I.S:
(A) I,Ei~GTIi: 94 ~:-1:IN0 ACLD:I
( B ) '.~YPF : A:~1IN0 '-.C IDS
(C) :;TRANDF;17NESS: SD:(GLF:'
(D) ~i'!:)F'C7Li~t::~1': L,7:'IEAi?
~:~yi)MOLECLII_~F: 'L'YE'E: ?I=c)TEIN
i i i ) H Y PO'I'I~ET IC_A'
V ) ANTI _ >ETJSF::
. s~ ) FR.:~GMENT TYL'E
'.~i n ORTC>INAL S0L1RCF?:
( A ; C)R.GAN I ~:;M :
f v i. ) IMMEDIt'~TE SOURC I?
i i i ) POS IT ION .LN t:>E(JOIv'I F~;
oa~~~ 1

CA 02407548 2002-11-08
2002-:1.1--0 , s~ q li:~k: t 065C)~--051 -C:~:-02 )
(A) <'EiROMOSOMI/;~EC~t-IEN'I':
(B) MAP POSIT: ON:
(;r: UNi'1':~:
t J; ) F EATURE
(A) NAI'1E/f<;EIc':
(B) L,t)C".A'I'ION:
(~'v') IDENTIFICATION METHOD:
(D) 0'i'HEF: TN~''JRTrIA'I'L'ON:
v:,') PUBLICATION INFORM)'"L()'N:
( A ) AU''HCiR S
(B) '1'I":'~F.:
C ) ~~ O':1RNAL
( I> ) ~f'0 i , IJP~IF,
(~-',) ~S'-~UI';:
( F ) ~AGE;i
l,(~) _':~A'~E:
f !-I ) (nOCUMEN'I' a'VO .
( L ) ~ I'_~ I ;V(,1 Dw'W :
(J) ~Li3LIC.'.AT::ON Da.'I'E:
(F:) FI;;L~E'Ja'jI~1'i' R:SI:?oClES I:N ;:~E.c~ :D NJ:1:
(:.pSEQUENCE DESCRIP'?'I~~>IJ: Sr;y ID 1(:~: ..
SE - 'ys Tyr L'he ll.e Pro Asru C;7 ,a Gly Va7 Pro G1y Asp Seer Thr Arg
1. ', 10 15
~ < < I: ' y ~ 'l.~j A~~ri Ly:~ :;dis Prc; I le Asr.. :per C)lu '1rp
u~~ _> ~l,r_, Me c_ A.~ ~ ~eu L s c~
~~0 ~5 si)
G i z: 'Fhr Asp Asn Cys G7 a TYir Cys Phr C'_ys Ty ;Ilu 'I'hr Glu ~~le Ser
3 i 4 ( 4 _>
C,: s ~',:',~s Thr Leu Val Se.r 'I'Iur Pm) Va7 G1--y Tye A.sp Ly:: Asp Asn Cys
~~0 w5 E,0
C: ~i r?.rg I L r Phe Lys I~ys Glu A:-:p Cy: L,: ~; 'Py~ i :a ~'a ._ Va l Glu
Lys
6 _a ;' U 7 '~ 8 0
L:-.r s i-.r~p Pro Lys Lys '~'Yr <;ys S~_°r Va Sf°n G1 A
'i'rp 1 l~ I le
8 5 r) ~:)
(2,) INFC)RMATION FOR SF:Q 7:D N°J: 2:
( ~ ) SEQUENCE CHARAC~'i ERISTT:~.'S
(A) LENGTI-i: 102. =1MIN0 AC.'IOS
(B) TYFE: AMINU ~'~C'.IDS
(C) ;~'PRt~I:7H;i:)IVES:: SINGL.I:
(D) 'T'OPOLOCiy : I~I,.IEA.R
( :. i ) MOLFsCULE TYPE : 1'Re)TI~IN
( ~:;w ) ORIGINAL SOURCE-
(P,t.)RC:I.r'~NI:-~M:
(;:i) SEQUENCE DESCRIPTION: ;~Et~ ITv N0: ?.:
~i.i A1a Glm Ala Tyr Val Glu i'h~~ Sam ~~ys 'I'yr h'he Ll:~ Pro Asr. Glu
- 5 10 15
Page '>

CA 02407548 2002-11-08
2002-:11-07 seq list ( G6508- 0~1--CA~-02 )
t;~iy alai Pro Gl.y Asp Ser Trr Arg Lys ('y~; Met asp I~E.u Wfs Gly Asn
:' C.'~ 2'~ 3
Ly ~ H ;: Pro I l a Asn Ser G1u 'frp G Ln ''hr Asp Assn Cy~: 'Ci a Thr Cys
35 40 'I5
Thr C'ys Tyr Giu 'fhr Cl7.~s =-:~_e Se:v Cys ':fs 1't:r Leu 'Jai Sf~r 'f'hr
Pro
'il 5'~ 60
Vai tI7--f Tyr A.>p Ly:~ Asp f~:~rr Cy:GIrL =..rg -Ile P:~re Cay:~ L,vrs Ulu
Asp
t,5 70 ?5 8()
t;ly~ L,y; Tyr I7 a Val 'v'a ~. iiLi.~ Ly::: L,ys =jsp Pro Lys L~ys ~'hr t~ys
Ser
HJ '30 '~5
V_~ . Ser Clu Trp Il a Ile
100
( a' j INFORMATI<)N F'OR SF:4'~ ~L NO : 3
( S. ) SEQUENCE CHARAC'1.'E'i~ISTL;C;I
I,A) LE:;'dGTH: ~0 AP~I)~NO T~.C:IDS
(R) 'fYI'~ : AM.NO A~::CDS
(f:: ) :'7-RA.NDED~'~7ESS : SIDIf:II_.E
('.7) "'(:~1?~sh0t.i'i : L~IIJ'~:,'\R
r; . -; MOLECULE TYPE:: F'RCe'('E:IN
(v.~ i 1 ORIGINAL SOURCE:
iA) 0:~.~:;ANISh~:
(:; i ) SEQUENCE DESCRIP'I'IOIV: ~;EQ II NC): .3
T~;:x- 'rr~r Cys Ser Val S~Jr C~Lu PTO G1; Ile
I 5 10
~) INFORMATION FOR SE:Q II7 N~): 4:
i ; SEQUENCE CHARACTEI~ISTI~:S:
( ~ ) L,EI\'1G7.'H : 15 -~,MI NC> AC' IDS
(F~J TYPE: ?,MINiJ ACIL)S
(C1 :3'I'RANDI~:L)NES:>: SINGhE
( D ) 'fOF'OI O=:,s~.' : I: I :~IEAI~
i i ) MOLECULE TYPE: PI,cJTIrIN
t'~i ) ORTGINAL~ SOLJRCI;
(A) OIIt~AN:I SM:
}:..i ) ShQLIENCE DESCRII-'TLON: SF~'.~) v ::~ rI(>: 4
'a:i Glu Gly Val Pro ~;ly hsp ;>e=.° '~'ir fircr I.~~r:; (.'ys Ma: Asp
Leu
! i) 15
Pzg~ 3

CA 02407548 2002-11-08
2002-y1._.p~ s~~ li.t: ~OG5G8-0~,._..~A._p')
(2.: i:NFORMATTON FOR SF:Q :ID N0: -i :
i ; SEQUENCE CHARAC:TERI ~~'PIC:~
(A) 1,ENCI'I'H: ' 5 AbI' NU T~.CID::i
rB) TYPE: AM~NO A('I:DS
'C) :.i'I'RANDF',DhfF:~~S: iaJ~dGLE
(D) T'f_iPOL,OGY: ~~IT~?AR
i i ; MOLECUI-.F: T'i PE : PRCi'I';H I N
(«iicuRIGINAL SOURCE:
(A) ORGPNISl~~:
1 ~: - j SEQUEIJCE :7ESCRl P'I' I OTe~ : S T~Q II NC : '~
~~I . 'rn Gln 'Il_r Asp Asm i:ys Glu Thr ~:;y:~ 'rhr Cy~. 7'yr C;Lu rtr
1 5 D 1 '~
(:) I NFORivIATION F'OR Sc;Q ID N0: E~;
(:.) SEQUENCE CHARACTERISTIa:.S:
(A) LE:NG'IH: .'--9 AP4:.N0 11CID;~
(I3) '7"i-E'E: AMN~J lit:'-~D,:
( C' ) .I'7'RA:!VDED1IESS : :~.I=1;GLE
(I7) 'I'C;LanL,ClG'~ - ;,BEN=~°.ijR
r. ~ N:OLECUL;F; 'I":'PB;: F'ROTI_:TN
'~.'i )C>RIGIN:1L SOURCE:
( A ) ORGFiNI Slyf :
L=~i) SEQUENCE DESCR7:P'I'IC:)IQ: SEQ TL'- NO: 6:
I::.e Vr~l Val Glu Lys Lys Asp Pro Ly:: L;ys Th,~ Cys Sea Val Ser Glu
1 5 10 t'.i
Twla .Ile Z1e
Pale 4