systemic activity of a low dose oral natural alpha interferon (alfa-n3) as measured by cdna...
TRANSCRIPT
Systemic Activity of a Low Dose Oral Natural Alpha Interferon (alfa-n3) as
Measured by cDNA Microarray Analysis
W. Carter1, D. Strayer1, W. Mitchell2, P. Faber3, H. Wu4, K. Mounzer5, T. Tong6;
1Hemispherx Biopharma, Inc., Philadelphia, PA, 2Vanderbilt University, Nashville, TN,
3Cleveland Clinic Foundations, Cleveland, OH, 4Drexel University, Philadelphia, PA,
5Philadelphia Fight, Philadelphia, PA, 6Princess Margaret Hospital, Hong Kong
Ba
ck
gro
un
d: E
xpo
sure
of th
e o
rom
uco
sa to
low
do
ses o
f alp
ha
in
terfe
ron
(IFN
) ha
s be
en
rep
orte
d to
lea
d to
bio
log
ical e
ffects in
a
nim
als a
nd
hu
ma
ns. H
ow
eve
r, the
op
tima
l do
se/sch
ed
ule
of lo
w
do
se o
ral (L
DO
) IFN
to a
chie
ve a
system
ic an
tiviral e
ffect h
as n
ot
be
en
de
term
ine
d. A
na
tura
lly de
rived
alp
ha
IFN
(Alfe
ron
N
Inje
ction
®) h
as b
ee
n a
pp
rove
d fo
r trea
tme
nt o
f con
dylo
ma
ta
acu
min
ata
. It is active
at d
ose
s sign
ifican
tly low
er th
an
tho
se u
sed
for
reco
mb
ina
nt a
lph
a IF
N.
Me
tho
ds
: Stu
dy A
: asym
pto
ma
tic HIV
infe
cted
sub
jects w
ith C
D4
le
vels >
40
0 w
ere
trea
ted
with
50
0 IU
Alfe
ron
® L
DO
da
ily for 1
0 d
ays.
RN
A fro
m p
erip
he
ral b
loo
d le
uko
cytes w
as iso
late
d fro
m b
loo
d
colle
cted
be
fore
, du
ring
an
d p
ost-th
era
py u
sing
Pa
xge
ne
tech
no
log
y fo
r RN
A iso
latio
n. cD
NA
micro
arra
y an
alysis w
as u
tilized
to id
en
tify g
en
es w
hich
we
re m
od
ula
ted
as a
resu
lt of th
e A
lfero
n®
LD
O d
osin
g.
Stu
dy B
: no
rma
l he
alth
y volu
nte
ers b
ein
g stu
die
d in
a sim
ilar m
an
ne
r in
Ho
ng
Ko
ng
.
Re
su
lts: In
itial re
sults in
Stu
dy A
ind
icate
a te
mp
ora
l ind
uctio
n o
f IFN
re
late
d g
en
e a
ctivity in p
erip
he
ral b
loo
d le
uko
cytes fo
llow
ing
the
ora
l a
dm
inistra
tion
of 5
00
IU o
f a m
ulti-sp
ecie
s na
tura
l leu
kocyte
IFN
. F
urth
er stu
die
s will cla
rify the
ran
ge
an
d m
ag
nitu
de
of th
e e
ffect
rela
tive to
a p
are
nte
rally a
dm
iniste
red
do
se.
Co
nc
lus
ion
: Exp
erim
en
ts to d
ate
ind
icate
tha
t a b
iolo
gica
l cockta
il of
na
tura
l hu
ma
n in
terfe
ron
spe
cies a
dm
iniste
red
ora
lly ha
s system
ic b
iolo
gica
l activity b
ase
d o
n u
pre
gu
latio
n o
f IFN
rela
ted
ge
ne
s in
pe
riph
era
l blo
od
leu
kocyte
s. Be
cau
se a
lph
a IF
Ns a
re b
roa
d sp
ectru
m
an
tiviral/im
mu
no
mo
du
lato
ry mo
lecu
les, p
ote
ntia
l ap
plica
tion
s in
nu
me
rou
s IFN
-sen
sitive d
isea
ses e
xist, inclu
din
g a
pp
licatio
n to
re
spira
tory in
fectio
ns su
ch a
s avia
n in
flue
nza
.
AB
ST
RA
CT
Study A Design
• Prospective, Open-label
• Dose-Ranging
• 1:1:1 Randomization to 3 dosage groups• 500 IU/day• 1000 IU/day• 2000 IU/day
• 60 Total Subjects (20 in each group)
• Treatment Duration 10 days
Alferon LDO® Study Medication
• Supplied as a liquid solution
• Packaged in sealed polypropylene lined foil pouches
• Each pouch contains 1.0 ml of Alferon® LDO or Placebo
• Taken orally each day for 10 days
• No food or water is taken 30 minutes prior to through 30 minutes after administration
Alferon LDO® Dosing and Blood Sampling Schedule
Study Day Number and Event
Day Number
0* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
LDO Dosing
X X X X X X X X X X
Blood Samples Drawn
￪ B1 +
B2
￪ T1
￪ T2
￪ T3
￪ T4
￪ T5
*Day 0 = Baseline period in which two separate samples (B1 and B2) are drawn.
Goal: Compare gene expression of T1-T5 Samples to two Baseline Samples Combined (i.e. B1+B2)
Meth
od
of cD
NA
M
icroarray A
nalysis
Array co
nstru
ction
. The array used in this study com
prised a subset of sequence verified cDN
A clones from
the R
esearch Genetics Inc. 40,000 clone set representing 950 genes containing adenylate/uridylate rich
elements and 18 genes potentially involved in A
U-directed m
RN
A decay, 855 IS
Gs representing an
expansion of a previously described clone set containing confirmed and candidate genes stim
ulated by IFN
s in diverse cell types, 288 genes responsive to the viral analog poly(I).poly(C
), and 85 housekeeping genes.
Targ
et RN
A p
reparatio
n. T
arget RN
A w
as generated in a T7 polym
erase based linear amplification reaction.
Tw
o μg total R
NA
and 5 pmol of T
7-(dT)24 prim
er (5’GG
CC
AG
TG
AA
TT
GT
AA
TA
CG
AC
TC
AC
TA
TA
GG
G
AG
GC
GG
-(dT)24-3’ in a total volum
e of 5.5 μl w
as incubated at 70°C for 10 m
in and chilled on ice. For first
strand cDN
A synthesis, the annealed R
NA
template w
as incubated for 1 h at 42°C in a 10 μ
l reaction containing first strand buffer (Invitrogen), 10 m
M D
TT
, 1U per μ
l anti-RN
ase (Am
bion), 500 μM
dNT
Ps and 2
U per μ
l Superscript II, (Invitrogen). S
econd strand synthesis was for 2 h at 16°C
in a total reaction volume of
50 μl containing first strand reaction products, second strand buffer (Invitrogen), 250 μ
M dN
TP
s, 0.06 U per
μl D
NA
ligase (Am
bion), 0.26 U per μ
l DN
A polym
erase I, (NE
B) and 0.012 U
per μl R
Nase H
(Am
bion) follow
ed by the addition of 3.3 U of T
4 DN
A polym
erase (3 U per μ
l; New
England B
iolabs) and a further 15 m
in incubation at 16°C. S
econd strand reaction products were purified by phenol:chloroform
:isoamyl alcohol
extraction in Phaselock m
icrocentrifuge tubes (Eppendorf) according to m
anufacturer’s instructions and ethanol precipitated. In vitro transcription w
as performed using the T
7 megascript kit (A
mbion) according to a
modified protocol in w
hich purified cDN
A w
as combined w
ith 1μl each 10X
AT
P, G
TP
, CT
P and U
TP
and 1 μl
T7 enzym
e mix in a 10 μ
l reaction volume and incubated for 9 h at 37°C
. Am
plified RN
A w
as purified using the R
neasy RN
A purification kit (A
mbion).
RN
A lab
eling
. Cy3 or C
y5 labeled cDN
A w
as prepared by indirect incorporation. Tw
o μg of am
plified RN
A, 1
μl dT
12-18 primer (1 μ
g per μl, Invitrogen), 2.6 μ
l random hexanucleotides (3 μ
g per μl, Invitrogen) and 1μ
l anti-R
NA
se (Am
bion) were com
bined in a reaction volume of 15.5 μ
l and incubated for 10 min at 70°C
. R
everse transcription was for 2 h at 42°C
in a 30 μl reaction containing annealed R
NA
template, first strand
buffer, 500 mM
each dAT
P, dC
TP
, dGT
P, 300 μ
M dT
TP
, 200 μM
aminoallyl-dU
TP
(Sigm
a), 10 mM
DT
T, 12.7
U per μ
l Superscript II. F
or template hydrolysis, 10 μ
l of 0.1M N
aOH
was added to the reverse transcription
reaction and the mixture w
as incubated for 10 min at 70°C
, allowed to cool at room
temperature for 5 m
in and neutralized by addition of 10 μ
l 0.1M H
Cl. cD
NA
was precipitated at –20°C
for 30 min after addition of 1 μ
l linear acrylam
ide (Am
bion), 4 μl 3M
NaA
c (pH 5.2) and 100 μ
l absolute ethanol then resuspended in 5 μl of
0.1M N
aHC
03. For dye-coupling the contents of 1 tube of N
HS
ester containing Cy3 or C
y5 dye (Am
ersham
Biosciences) w
as dissolved in 45 μl D
MS
O. F
ive μl dye solution w
as mixed w
ith the cDN
A and incubated for
1 h in darkness at room tem
perature. Labeled cDN
A w
as purified on a Qiaquick P
CR
purification column
(Qiagen) according to m
anufacturer’s instructions. Eluted cD
NA
was dried under vacuum
and resuspended in 30 μ
l of Slidehyb II hybridization buffer (A
mbion). A
fter 2 min denaturation at 95°C
the hybridization mixture
was applied to the m
icroarray slide under a coverslip. Hybridization proceeded overnight in a sealed m
oist cham
ber in a 55°C w
aterbath. Post-hybridization, slides w
ere washed successively for 5 m
in each in 2 X S
SC
0.1%
SD
S at 55°C
, then 2 X S
SC
at 55°C plus a final 5 m
in wash in 0.2 X
SS
C at room
temperature.
Data acq
uisitio
n an
d n
orm
alization
. Data w
ere acquired with a G
enePix 4000B
laser scanner and GeneP
ix P
ro 5.0 software. R
aw data w
ere imported into G
eneSpring 6.0 softw
are (Silicon G
enetics) and normalized
based on the distribution of all values with locally w
eighted linear regression (LOE
SS
) before further analysis.
ResultsInitial results in Study A indicate that Alferon LDO is well tolerated at the 500 and 1,000 IU/day dosage levels. cDNA microarray analysis identified 385 genes that were expressed > two fold over Baseline in two or more patients. Approximately a four fold increase in gene expression was seen at the 1,000 IU/day dosage level compared to 500 IU/day (p<0.0001). Seventeen genes were expressed > two fold over Baseline in over 35% of patient samples. PDZ and LIN domain 5 and 2’-5’ oligoadenylate synthetase-like were among the top five upregulated genes. 2’-5’ oligoadenylate synthetase is an important component of the interferon intracellular antiviral pathway.
Recent evidence shows that the virulence of influenza A including avian (H5N1) isolates correlates with the ability of the non-structural NS1 viral protein to bind to human PDZ domains and thereby abrogating the expression of antiviral genes in host cells including interferon pathways (Science xpress, 26 January 2006). Thus, the finding that Alferon LDO can upregulate PDZ domain expression raises the possibility that Alferon LDO could have an important role in abrogating the ability of influenza viruses including avian (H5N1) to evade human host defense mechanisms.
Alferon LDO® Dose Effect: Number of Genes with Expression Increased
> 2 Fold Over Baseline in Two or More Patients
Dose 500 IU 1,000 IUFold
Increase of MeanPatient # 1 2 3 Mean 4 5 6 Mean
Day 2 10 1 39 16.7 85 77 108 90 5.4
Day 5 14 4 23 13.7 54 72 35 54 3.9
Day 11 1 8 4 4.3 4 45 40 30 6.9
Day 12 3 15 - 9.0 19 44 28 30 3.4
Day 16 - 14 - 14 3 59 48 37 2.6
Mean 7.0 8.4 22 12.5 33 59 52 48 3.9
Student’s t-test, p-value <0.0001 (n=385)
Alferon LDO® Dose Effect: Number of Genes with Expression Increased > 2 Fold Over Baseline in Three or More Patients
Dose 500 IU 1,000 IUFold
Increase of MeanPatient # 1 2 3 Mean 4 5 6 Mean
Day 2 3 1 19 7.6 36 41 42 39.7 5.2
Day 5 2 2 6 3.3 16 17 16 16.3 4.9
Day 11 1 1 1 1.0 3 3 3 3.0 3.0
Day 12 1 2 - 1.5 7 8 8 7.7 5.1
Day 16 - 0 - 0 2 2 2 2.0 >5
Mean 1.8 1.2 8.7 3.9 12.8 14.2 14.2 13.7 3.5
Student’s t-test, p-value <0.0001 (n=252)
Alferon LDO® Dose Effect: Number of Genes with Expression Increased
> 3 Fold Over Baseline in Two or More Patients
Dose 500 IU 1,000 IUFold
Increase of MeanPatient # 1 2 3 Mean 4 5 6 Mean
Day 2 0 0 9 3.0 23 10 37 23.0 7.8
Day 5 5 1 5 3.7 16 19 4 13.0 3.5
Day 11 1 2 0 1.0 0 14 3 5.7 5.7
Day 12 1 3 - 2.0 3 10 2 5.0 2.5
Day 16 - 0 - 0.0 0 14 6 6.7 >5
Mean 1.8 1.2 4.7 2.6 8.4 13.4 10.4 10.7 4.1
Student’s t-test, p-value <0.0001 (n=69)
GE
NE
S E
XP
RE
SS
ED
> T
WO
F
OL
D O
VE
R B
AS
EL
INE
IN O
VE
R
35% O
F P
AT
IEN
T S
AM
PL
ES
Ex
pre
ss
ion
Fre
qu
en
cy
(%)
37
67
0In
terfe
ron
(alp
ha
, be
ta, a
nd
om
eg
a) re
ce
pto
r 11
7
37
67
0C
oa
gu
latio
n fa
cto
r III (thro
mb
op
las
tin, tis
su
e
fac
tor)
16
37
67
0F
GG
15
41
73
0G
luta
ma
te d
eh
yd
rog
en
as
e 1
14
41
73
0S
arc
og
lyc
an
, be
ta (4
3k
Da
dy
stro
ph
in-
as
so
cia
ted
gly
co
pro
tein
)1
3
41
47
33
Ma
jor h
isto
co
mp
atib
ility c
om
ple
x, c
las
s I, F
12
41
47
33
Inte
rfero
n in
du
ce
d tra
ns
me
mb
ran
e p
rote
in 2
11
41
73
0C
yto
ch
rom
e P
45
0, fa
mily
51
, su
bfa
mily
A,
po
lyp
ep
tide
10
41
73
0C
oa
gu
latio
n fa
cto
r II (thro
mb
in) re
ce
pto
r9
41
73
0P
rote
as
om
e (p
ros
om
e, m
ac
rop
ain
) 26
5
su
bu
nit, A
TP
as
e, 6
8
44
80
0N
-my
risto
yltra
ns
fera
se
27
48
87
0S
imila
r to K
IAA
01
60
ge
ne
pro
du
ct
6
52
67
33
2’-5
’ olig
oa
de
ny
late
sy
nth
eta
se
-like
5
52
93
0In
terle
uk
in 1
7 re
ce
pto
r4
52
93
0P
DZ
an
d L
IN d
om
ain
53
56
87
17
Ho
mo
sa
pie
ns
, clo
ne
ima
ge
:51
64
03
1, m
RN
A2
59
40
83
SF
RS
pro
tein
kin
as
e 1
1
Ov
era
ll1
,00
0 IU
50
0 IU
Ide
ntifie
d G
ene
Alferon LDO® Safety Summary
• No Serious Adverse Events Reported
• Only Mild Adverse Events Reported • Metallic taste in mouth• Flatulence/Bloating
• No Clinically Significant Changes in Laboratory Parameters
• No Changes in Karnofsky Performance Status (KPS)
CO
NC
LU
SIO
NS
Alfe
ron L
DO
® ma
y pro
ve to b
e an id
eal a
ntivira
l a
gen
t for pro
phylactic/th
era
peu
tic usag
e a
ga
inst
influen
za b
y virtue o
f the fo
llowin
g p
rop
erties:
•W
ell-tole
rated
, broad
-spe
ctrum
antivira
l, derived
fro
m an
alre
ady F
DA
ap
proved
prod
uct
•H
igh p
oten
cy na
tural in
terferon
prod
uct
•E
vide
nce o
f system
ic activa
tion
of inn
ate im
mu
ne
resp
onse
s by o
ral a
dmin
istratio
n in
hu
ma
ns
•E
ase
of oral se
lf-ad
min
istration
•M
ay d
irectly a
ttack a p
roxim
ate cau
se of h
um
an
m
orb
idity/mo
rtality u
nique
to the a
vian influe
nza
vira
l ge
netic m
achin
ery
•P
ote
ntia
l to im
pro
ve the
secon
dary im
mu
ne
dysre
gulation
from in
flue
nza in
fectio
n w
hich m
ay
contrib
ute to th
e severity o
f the
dise
ase
proce
ss
•P
osse
sses nea
r term
availab
ility by d
elivering
50
to 10
0 millio
n do
ses m
anu
factu
red
in fa
cilities a
lrea
dy ce
rtified b
y the F
DA