Note: Descriptions are shown in the official language in which they were submitted.
WO 9~J18004 P/C~/VSg2/01972
..... 1 '
~ROGATI~N OF VIlR~L RE:Sl:ST~NC:E TO NUCLEOSIDE
ANAL~GUES BY D~UBLE- STRANDED RNAs
Nucleoside analogues are colTunonly employed
antiviral agents, particularly against retroviruses. ~ .
Viruses undergo genetic c:han~es, or mutations,
l~ading to r~lative re~i~taIlce to th~ antiviral ~.
agent~. When resi~ance o~curs, tAe ~riruses multiplv
more quic:kly and the underlying disease accelerates. -~
By deployin~ dsRNAs relatively early in the
infection, the rate of emergen~e of viral resistance
is reduced. 13ven later in the infectic)n when genetic .
mutation has already occllrred, d~NA~ restore
susceptibility of the viruæes to ot~e~wise
inefective antiviraI agents~ T:his can be seen
clinically by the unexpected resul~ that long term
use of the two modalitie~, in combination, causes
~reater recovery of host immune function, and less
dete table virus, than is seen with either class of
anti~iral agent applied by i~elf.
Prolonged therapy of viral diseases,
particularly retro~iral disorders, i~ associated with ~`
emergence o viral resistance ~references 1 and 2~. ..
Ideally, therapeutically employed the nucleoside ~ 5i~
analogues are incorporated into viral genetic :
informatiorl which thereby becomes faulty or
incomplete, leading to a reduction i~ eficiency of ;
the viral growth cycle. Defective, or incomplete,
viral pro~eny are formed of reduced infectivity
"'`-"""~'
.., ,,, ~
.. ~
"'"~`~'
WO 92~180V4 PCr/l)~i92/~1972
~ ~ 2
potential. ~Iowever, by modifying its genetic makeup,
the virus may emerge relatively resistant, thereby
generating infectious prog~ny even in the presence of - :
nucleoside analogues. Typical genetic charlges occur
in the polymera~e gene ( i . e ., the viral eomponent
which directs incorporation of the antiviral
nucleoside in the first place) allowing the virlls to
escape from th~ forms of antiviral blockade. The
best studied ca e to date is the n~eraction between ~:
retroviru~ses, e. g., HIV (human immunode~ici~ncy ~.
virus3 and 3 ' -azido-3 1 -d~oxythmidine, also called AZT
or zidovudine. The mutation(s~ leading to resistance
to AZT often confe~s simultaneous resistance to
various other drugs, such as (bult not limited to),
dideoxyinosine (DDX ) and dideox~ycytidine (DDC~
Di~closed are pros:~edure~ fo:r delaying, reducing
or both delaying and reducing the appearance of
nucleoside analogue resistant ~i:rus in a patient
having an HIV infection (HIV po~itive) by
administering an effectiYe amount over a suitable
time of a mismatched dsRNA prior to therapy with a -.. ~.;.``
nucleoside analogue. These pr~cedures ~erve to
sensitize the patient to the later, in terms of the `~
cour~e of the HIV infection, administration of a ;~
nucleoside analogue antiretroviral agent if and when `
it is r~quired.
Also described are therapeutic procedu~es or
ameliorating the morbidity of the peripheral blood ~-`
mononuclear blood cells ~PBMC), notably the T4 ox CD
4 lymphocytes, of a patient i~fected with a
:,;'." "~' ,`
wo s2rlsoo4 Pcr/l~ss2/ols72
2 1 ~ ~ ~ 2 1
retrovirus that ha6 become resistant to nucleoside ~ :
analogues by administering an effective amoun~ of a
mismatched dsRNA to the ~a~ient.
The dsRNA may be a complex of a polyinosinate
and a polycytidylate containin~ a proportion of
uracil bases or guanidine bases, e.~., from 1 in 5 to
1 in 30 ~uch bases (poly I ~ poly(C4 2~x>U or G3). ~ :
The dsRN~ may be of ~he genexal formula
n 11-14 )n rIn r~C12,U3n Other suitable
examples of dsRNA are discussed b~low. :
By "mismatched dsRNA" ar m~ant those in which .~
hydrogen bonding ~base ~tacking) betwee~ the :. -
csunterpart strands is relati~el~y intact, i.e., is ` ::;
interrup~ed on average less than one base pair in ;.~
. - . . .
every 29 consecutive base pair rlesidues. The term `~
"mismatch~d dsRNA" should be und~erstood accordingly~
The mismatched ~RNAs preerred for use in the ~.
present invention are based on copoly~u~leotides
selected from poly (Cn,U) ~nd poly (Cn,G) in which n
is an integer having a value of from 4 to 29 and are
mismatched analogs of complexes of polyriboinosinic
and polyribocytidilic acids, formed by modifying
rIn-rCn to incorporate unpaired bases ~uracil or :~
guanidine) along the polyribocytidylate (rC~)
... ...
~trand. Alternatively, the ds~NA may be deri~ed from
poly(I)rpoly(C) ds~NA by modifying the ribosyl
~"-: ;'
W09~ 004 P~T/US92/0197
~ 4
backbone of polyriboinosinic acid (rIn), e.g., b~
including 2 t -0-methyl ribosyl residues. The
mismatched complexes may be complexed with an
~N,A-stabilizin~ polym.er such as lysine and
cellulose. These mismatched analogs of rIn~rCn, ~-
preferred ones of which are of the general formula
rIn-(C~ 4,U)n or rInl~r(C29,G)n, are described by
Carter and T~'o in U.S. P.at~nts 4,130,641 and
4,024,2~2 the disclosures of which are hereby : .
incorporated by re~erence. The dsRNAs described
therein genex~ally are suitable for use accord}ng to ~::
the present invention. The preferred mismatched
dsRNA is rIn~ (C~ ,4~U)n
Researchs Inc. of Rockville, MD, USA, available a~ a
lyophilized powder.
Other examples o mismatched dsRNA for use in
the invention include~
poly ( I ~ poly (C4,U)
poly ( I ) - poly ~ C7, U )
poly ( I ) ~ poly ( C13 j U )
poly (I) poly ~C~22,U)
poly (I) poly (C20,G! `
poly 5 I ) poly (C~g~G3 and ;~
poly ( I ) poly Cp23 G~p
Another class of ds ~ As æuited to the practice - ~:
of this învention are short dsRNAs of defined :~
",,. ,,." .,~ ~,",,~ ,1" " ~ ~ ' " '~' , , . ~ , . ' ." ! "
W~92/180~ PCT/US~2/~1~72
2~2221
.
structure, for example oligonucleotides of the ~ ::
formula: ~
:..
5'lock~ n-lock 3'
3'lock-(C)m-lock 5'
where m an~ n arei each more than S and less than lOO, ~ ~
I is inosine monophosphate, C is cytidine ~ :
monophosphat~, and wher~ the lock~ i~ one strand are
compl~mentary to locks in the opposite ~trand, or~an
oligonucleotide of the structure:
...... ..
5Cloc~-[(I~xA]j-lock 3' ~-.
3'lock-[(C)yUlk-lock 3' ,~rS~
where x a~d y are each ~lore than 5 and less tha~:25,
j and k each at least 1 and le~s than lO, I and C are ` ;`
~s ldentifie~ a~ove, A is a nucleotide which is not
I, and U is a n~cleotide which base pairs with A. .
Alternatively, the short oli~onucleotide may '-!.
have the struc~ure~
''.: ~, ~.
".......
n m .
where n, m, I and C are as defined above.
. ,. ~:
These oligonucleotides may have substitutions in
one strand not complementary to nucle~tides i~ t~e
opposite strand. Preferably these o~igonucleotides -;~ .
~:
::.
,
,': ' .'
WO92~1~0~ PCT/US92/0197~
~ ~ 6 ~-
are stabilized by internal r~gist~rs of complementary
heteropolymer and desirably the lock or hinge or both
contain regi~ns of complem~ntary heteropolymer.
The~e oligonucleotid~ desirably have single-stranded
tails. These oligonucleotides are de~cribed in
PCT/US89/0~172.
Patients ar~ treated with intravenous infusions
of 200 to 700 mg of rI~r(C~ 4,U~ as r~quired, e;g., -
once a week to as often as daily in accordance with
their clinical i~provem~nt. The amount of dsRNA ;i~
administered and the frequency of administration will ~:.
provide a level of from 0.01 to 1,000 micrograms of
dsRNA per milliliter of ~he patient's æystemic blood -~
circulation immediately following administration ;~
measured at a point distal from the point of infusion.
Illustrative nucleo~ide analo~ue antiviral ~ ;~
agents include Zidovudine (azidothymidine, RETROVAR~
or AZT as commonly used herein) which is
3'-azido-3'-deoxythymidine, a nucleoside analogue
antiviral for the systemic treatment of ac~uired ~-
i~munodeficiency syndrome (AIDS) and AIDS-related .;
compIex (ARC) caused by human immunodeficiency ~irus
(HIV~ ~TLV-I, HTLY-II, HTLV-III, LAV, ARV and the
Iike designators for various strains). The usual
adult dose i 5 200 mg every ~our hours, around the
clock. For a 70 kg patient, the corresponding dose :~
is 2,9 mg per kg of body weight every four hours~
Doses up to 60 mg per kg of body weight daily h~ve
~ . .;'
; ;'"
WO ~2/1801M PCr/US92/Olg72
2 ~. Q 2 ~
been used. Usually less than the normal and :
cu~tomary ~nounts of the nucleoside analog retroviral
is used when coadminstered with a mismatched dsRNA/
as illustrated fur~her in the disc:ussion that follows. ~:
BRIEF DESCRIPTION OF T~ I~RAWIN~S `-:
.
`"' ~
Fig. 1 i~ a table showing the time in months to
cleath or "full blown" AIDS for 298 patien~s ins terms
of proportion of the patients free of critical
I~IV-related events comparing with AZT tr~atment - -
"early" t s~uare boxes) before symptoms occurred or
" late" ~ circles ) after symptoms occurred .
Fig. 2 compares the early u~;e of a dsRNA or its
concurrent use with AZT in controlling retroviral .
growth as compared to rapid ret~oviral growth in the
case of AZT alone~ :
Fig. 3 compares the relative effects of a d~RNA
and AZT as monotherapies with placebo and dsRNA and
AZT as a combinational therapy ~ in long term ;~
maint~nance of CD4 cells i:n ~IIV diseas~3 in median
change in CD4T lyznphocytes over the indicated number -~
of weeks. .
Fig. 4 relates the number of days of
combinational (AZT and dsRNA~ therapy to the percent ~:
change in mean T4 level ~howin~7 that the`dsRNA
Arnpligen~ extends the period of T4 cell ~tabilization
in HIV di~ease over that expected with AZ~ alone.
Median does for e ch meimber o.f the combination are
indicated . .:
`'"~. ~`'
W092~18M04 PCT~US92/01972
Eig. 5 is a graph relating the number of day~ on
the eombined Rmpligen~ and AZT regime~ to the mean
percent change (increase) in T4 cells showing that ;~
A~pligen~ increases and/or stabilize~ T4 cell levels
in HIV disease beyond the time period in which AZT is
effective.
Fig. 6 is a graph.relatin~ the proportion of HIV -~
patient~ free of critical events over a period of 12
mo~th~ for place~o, AZT alone, ~mpli~en~ mo~otherapy ~ :
and the combinational therapy of ~mpligen0 and AZT. ;~
In Fi~ures 4 a~d 6 the number of patientæ is
indicatad near the relewant data point in
parenthe~is, in Figures 3 and 5 it is indicated by N=.
In Figure 2, ~IV co-culture was performed a~
described in reerence 3 using peripheral blood :~
mononuclear (PBMC) cells from ca~;~s l and 3. After
4, 7 and 14 days of co-culture, PBMC were harvested
and dissolved,in a guanidine ~yanate solution u~-d to :~
dissolv~ cells and release viral materials. ~IV RN~
was m~asured by molecular hybridization as described
in more detail below. -:
: Fi~ure 1 shows the development of critical
event~ (development of full blown AIDS or death) in a
well-publicized Veterans' ~roup o~ HIV infected :~
subjects treated either "early" (e.g., before
symptoms occurred such as weight loss, night sweats,
etc.) or "late" (i.e., aftar such symptoms occurred
including HIV~associated infections caused by fungi ::
or bacteria~ with AZT.
... . . . . .. . . .
WO 92/lB004 PCI`~V$92/01972
21~22~
9 , . .
.
It is apparent from Fig. 1 that the "early" . ~ ~
taking of AZT does not prolong life. Samples of ~:
blood taken from individuals who developed critical
events show an enrichment in concentration of AZT
re~istant virus (hereafter referred to as AZTR~
whereas patien~s who remained fr~e of critical events
during the approximately 40 month observation
interval gen~rally had more AZT sensi~i~e IAZT )
viru~
Below, I charact~rize more fully the relative ~
AZT sen~itivi.ties of typical EIV isolates taken from .::
individuals whose disease progre~ses despite AZT
therapy and compare thes~ ~esults with viral is~lates .
~: ~
including hepatitis ~irus ~rom patient~ whose disease ::~
is under relatively ~etter control. It is apparent
that emergence of AZTR vlru~;contributes a .
~oreshorten~d life span. :Typica.~ human retroviruses
i cl;lding HTLV-~, HTLV 2 and HTLV-3 and viruses which
multiply by similar mechanisms include certain
hepatitis viruses.
I describe herein a~ i~v~ntion which ameliorates
this presently unsolvable problem. The invention can - :
be practiced in multiple ways including: ~a) reducing
the emergenc~ of AZT by utilizing speclfi~c clas~es
of dsRNAs before exposure of the virus to AZT or
other analogues an~ (b) overcoming the lethal :: ~-~
properties o AZTR HIV (or other virus rssistant
analogues~, by adding back dsRNA to the regi~en. The~.;
ds~NA/nucleoside analogue regimen shows unexpected
therapeutic synergy against AZT HIV without a `.`
~- ~......
~`"'',;.'..,."
~`.'. '''''
WO 92~18004 PCI/U~;92~01972 .
corresponding synergistic toxicity and thus is a
truly unique and unexpectedly us~ful combination o
drugs which becomes life saving when us~d correctly. ~ ~
Insight as to how combined ~npli~en(ÉD-AZT ~: :
treatment reduce~ H~V-viral burden was obtained by my
studie~ in which cell free virus obtained from
patient~; receiving either ~mpligen0 or AZT ~ ~;
moslotherapy or Ampli~en0-~ZT com~inational treatment ~ ~
were us~d to in~ec:t freæh human peri~eral blood ~:
cells which w~re pr2viously expo~ed to Ampligen~,
AZT~ or Ampli~en~9 and AZT. Vinl5 rom the patient
who re~eiYed only AZT monotherapy for more than one
year was insensitive to A2T (O~5,uM AZT - 4% ~ ::
inhibition; ED5 ~ < 5~M) but was as sensitive to
Ampligen~9 alone as wild type ~IIV-vir~as ~ 5~1g,~ml . ~ .
~npligen~ -- 80% inhibi1:~on; E:D5 0 ~ C3. 5~g/ml . ) . In
additiorl, the Asnpligen~-AZT combination produced
greater i~ibition of HIV (93%) than Ampligen~
alone. Virus from ~he patients who received
~mplig~n~9 aloIle or the Ampligen~9-AZT combination
showed relative resistance to AZT sensitivity to ~-
Ampligen~9 and even greater sensitivity to the .
Arnpligen~-AZT co~nbialation. When results with many :~
diffe~:ent viral isolates were compared from patients
on different s~hedules of therapy, it becarne apparent
that virus from patients who received Ampligen~
before exposura to AZT were more sensitive to AZT
( and ~ther nucleosidé analogues as well ) than the
viruses derived ~rom patients who received AZT first. ~ .
-. . :
WO 92~18004 2 1 ~ 2 ~ 2 .L PCI/IJS92/01972
11
-. ~:',':,~
T~33L13 1 DRUG RESISTANC3: QF HIY ISOI~TES
Anpligen~ AZT
HIV ED5 o ED5 o Combination AZT
I solate ( llq/ml ) ( ~la~mol~ar )In exa Phenot~,rpe `
E1112-~ 2 . 03 O. 5 sensitive ~~ ~
69ïO-6 3 ~ 0.2 resistant ~ ?
Cas~ #l 1 0 . 6 0 . 4 partl ally `~
r~si stant
Case #2 l 0 .2 0. 2 partially
resistant
C~ase #3 . 5 ~5 0 . 2 resistant
: ~ase~ #4 1 >5 0.8 resistant
ase *5 . 5 ~5 0 . 2 resi stant
Case # 6 2 >5 0 . 3 resis~ant
a ~ AZT-Arnpligen~ interaction was det:e~mined by :
isobole analysis~ (reference 4). The Combination
Index (CI ) is defined as CI + (AC/As? + (Bc/s5j,
wh~re Ac and Bc are the concentr~tions of the dru~s
used in the comk)1nation treatment, and As and Bs are
the concentrations of the drugs which, when ~ u:sed
alone, give the same e~fect as the combined
treatment. Wh~n CI ~ 1, the drug~ are synerg~ stic, - ~ .
when C:I = l, the dru~s are additive, and CI > l, the ~ -
drug~3 are antagonistic. ED5 0 is the in~ vitro dose
which inhi~its virus multiplication by 50%. --
., , ~
~ ~ .
:: :
WS:~ 92J1~004 Pclr/usg2/~l~72
?~
In Fig. 2, I show a typical elTbodiment of the
invention whereby the early use of ~npli~en~, or its
use concurrently with AZT, in f act results in
substantial benefit in terms of lowering the bodily
concentration of harmful virus such as r~ltrovirus.
The rapidly increasincJ productir: n curve of viral RNA
~ ribonucleic acid~ shown with the open cîrcles is
indicative of uncontrolled viral growth, whereas ~he
two flat lines illu~;trate good control o viru~
production by the combinational approach. The ope~n
circles in Eig. 2 illustraltive of rapidly developing
P . .
P~ZT` virus, are thus syanbolic of the typical patient ~ :
in Fig. 1 who, when ~iven AZT alone, proceeds to
advance into the more te~ninal ~tayes of di sease .
Certain dæRNAs, notably mi ~;matched d:~;RNAs,
showed synergistic i~ibition of retro~iruses, when
combined with P~:~T ( or other rlucle~o~ide analogues )
regardl~s~ of the drug resi~tance! phenotype (Ta}~
1 ) . H112 -2 and 6910-6 are well charact~rized HIV ~ ~ :
prototy~e clones displaying t~ical serlsitivity and
re~istancef respectively. Whereas, cae~ #1 through
#6 are vixuses isolatedL from speci~ic patients ~. -
exposed tc) various re~imens ~e.g., pati~nts ~ 1, 2, ;: :~
. ~ .
and 3 are the same as ths: se in Fig. 2, and pa~ients
4, 5 and 6 were treated initially with AZT alone as ~. -
suggested by Fiy. 13. .
When I utilized the combination of drugs
together ~linically, the results were again
unexpected and not at all predictable from ~he ~;
observed clinical results of the two dru~s given
~''
;
,:
WQ9~ 00~ 2 1 0 2 ~ ~ 1 PCT/US92/01972
13
singly ~monotherapy)~ For example, AZT (and other
analogues) given alone results in a transitory
increase in certain immune cells (reference 5~,
termed CD4 cells, which are among ~he i~mune cells
most favored for attack by certain retroviruses and
certain herpes viruses (especially HHV 6). After the
transitory increase (~een at around 12-16 weeks), the ;~
immune cell nu~ber deteriorates ~s HIV proliferates : ::
~, . . .
a~d AZT~' HIV e~erges (Fig. 3) The number in - :~
p~renthes~$ r~fers to nu~ber of ~ubject~ ~tudied. By
comparison, Ampligen~ alon~ causes a horizontal
"line" of CD4 cell number over time (Fig. 30, e.g.,
the imm~ne cel1 ~umber nei~her increases nor
decr~a~es (in other words, cell number îs stabilized) ~-
at Am~ligen~ doses of approxima~ely 400 to 500 mg `~
given twice weekly by IV infu~ion.
Patien~s receiving AZT mono~herapy took 200 mg.
orally every 4 hours, daily (120~ mg./day)~ Patients
in the ~mpligen~ monoth~rapy group received a minimum
o two IV infusions weekly ~mean dose range of ~:~
463-555 mg. weekly~. [Patients receiving ~he .
combinational treatment show~ in Fig. 4 were also
infused with typical Ampligen6~ doses of 400 mg. twice
weekly with an average concomitant AZT dai ly dose
ranging between 300 and 540 mg
The trends in th~ median change in T4 .; . :~
lymphocyte for ~he placebo and AZT monotherapy ~:
treatment (after 16 week53 groups are consistent wi~h
evidence that the cour5e of the di ~ease i s inexorably
downhill. Without effective therapeutic
;~
:
WO 92/1X004 PCF~VS92/01972
~L~ 14 -
intervention, the median change in placebo patient
lymphocyte counts declines. After an initial
increase, even patients recei~ing AZT monother~py ~ :
experi~nced a deterioration in their median T4 cell
counts and AZTR ~IIV app~ars. This decline from Wee~ -
12 onward appear~ to parallel that seen with the
placebo patientæ .
Compari~on of the slopes by the statistical ;
method of linear r~gre~ )n lin~s constructed frorn
the change in median T4 ce~ls from week 12 onward for
.. the AZT monotherapy and placebo grotlps showed they -:
~ere significantly different ~p ~ 0 . 01 and p < 0. 01,
respectively3 than a slope of O which represents "no
change" in rnedian T4 cell l~vels. "No change"
typically relprese~ts a stabiliza~ion o~ immunols:~gic
disease, a "negati~re slope" represents disease ;~:
deterioration, but th~ mo~t desira~le outcom~ is a
positive slope suggestive of di~ea~e ~immune~
recovery, and this could not be achieved by the
monotheraPy regimen~ studied.
Th~ A~npligen~ alone abrogates the expected CD4 ;
decline after twelve weeks of AZT Monot~erapy of .
Placebo. The slope of ~he serial median CD4 level ;~
reyression line during A~npligen~ treatment i s not
skatistically different frvm a hori~ontal line ` ~ -,
reflecting no CD4 cell loss over time . :;;;
Slope Comparison With
HorizoIltal (no change) p Value ;~ .
Ampligen~9 not si~nificant p > O.2
WO 92/lgO04 P~/lJS92~01972
21~222 1 ; ~
1 5
AZT significant p < 0 . 01 ; ~`
Placebo significant p < 0. 01
Ampligen~ monoth~rapy ( Fig . 3 ) abrogate~ the
severe decline in median T4 l~nphocyt~s seen in both ::
placebo patients and in AZT treated patients (after a ;
transitory twelve week rise~ but th~ avarage T4 level :
did not increase. Moreover, in the Ampligen~9 group,
the small ~ statistically insi~nificant) decline in..
median T4 lymphocyt~s observ~d at one ysar ~Fiq. 3 ) ~ .
c:an be readily r~v~rsed thereb~ bringing ~out an
~.
even more durable T4 stabiIization l~y increa~ing the
dose to above 200 mg . twice weekly t data no~ showr~
The slope of a regression line ~onLstructed from the
change in median T4 cell~ observed ~rom week 12
onward in the Ampligen~D monotheraLpy group ~;was not
s:ignificantly di:fferent ~rom 0 ther~by indicati~re of ~ -
di sease stabilization . The P~mpli.gen~ monotherapy, : -
AZT monotherapy, or placebo tr~ated groups shown in ~ -
Fig. 3 had approxlmately e~ival~3nt median and mean
absolute T4: ~(also called CD4) lymphocyte :Levels. I ~ .
then compared them with a ~new group that received
combined Ampli~en~ and AZT treatment. ~: The~ new group ~-
had in ~act lower ( approximately 33% ) medi an and ~
absolute levels o~ thi s prognostic indicator of the ;:
progression of HIV infection, thu~ indicating they
were at greater risk of death vr other "critical
events".
, ;' .,.' ;;~ :,
'' :~' '
WO 92/18004 PCT/l~Sg2/Og972
?~
.
The relative effects oiE Ampligen~\ and AZT in
long term maintenance of- CD4 cell~ per mm3 in HIV :
di~ease is shown in the following table. -
TABLE 2
Baselirle Median Absolute ~:
Number of CD4 Cel l Change
Txeatment Patients M~3dian Mean at :1 Year
Asnpligen0 + AZT 11 2Q1 24B +15
Ampligen~ alone 54 311 350 -15 :~
~ZT alone 260350* 355 -28*
Placebo 253350* 356 -~g*
'
:~
*E8timate based on summary data in Re . 5 ~ '
,; .~ :.
As noted in Fig. 3, on completion of 1 year of `~
treatment, median ab~olut~3 T4 lymphocyte counts
declined 59 celIs ( approximately 4~ 9 cells per month) ;~
in the patients who: received placebo; declined 28
cells (~pproximately 2.3 cell~ per mont:h~ in patients
who received AZT monotherapy; and declir~ed slightly,
15 <::ell~; ~ approximately 1. 25 cells per month~, in .
patients who received Arinpli~en~ monotherapy.
However, the group that received the combinational `~
Ampligen~D and AZT therapy, which had the lowest
pretreatment median and mean absolut~ T4 lyTnphocyte ~; ~
levels (therefore being at the greatest risk) ` ~:
WOg2/180~ P~T/US92/0197t
2~ ~2~2 1 ~:
actually experienced an increase in median absolute
T4 cell level of 15 cells even after one year o
combination regimen. These results are summarized in
Table 2 and provided in more detail in Fig. 4.
Figure 4 demonstrates the serial changes in
median T4 lymphocyte levels during the 1 year of ~
c3mbination treatment which should be compared with ~:
Amplige~ m~notherapy, AZT monotherapy, and placebo :~
~ffect~ o~ Fig. 3.. The ba~eline for comparison is
calculated from th~ avera~2 of the patients' s~rial
abæolute T4 lymphocyte counts m~asured during the
three month period immediately before starting the ~:
cnmbined therapy.
The increase observed in the first ninety day
.period is con~istent wi~h that previously reported `~
for the initiation of AZT monotherapy as o~se~ved in
Figure 3 above. However, the subsequent in~reases,
seen from l80 to 540 days of the combined therapy,
are statistically signifi~ant and are not observed in
patient~ re~eiving AZT monotherapy. ~A
non-parametric analysis confirmed the statistical1y
signifi~ant (p<0.05) increase in T4 counts at 1
year. This analysis was performed because o~ the
possibility that the T4 l~vels were not normally
distributed due to the relatively small sample ~ :
~ize.) Thus, the duration o~ the T4 lymphocy~e
incr~ase is longer than the 3-6 month transient
increase e~pected from AZT monotherapy and must be
attributed to ~he pr~ence of dsRNA. In add1tion,
this effect was seen with relatively low doses of
WO g2/18~04 PClr/VS~2/01972
~s~ 18 ~;
both dsRNA and AZT. This observation is consistent
with previou~ findings that at a dose as low as 200
mg. IV twîce weekly for 1 to 4 months, Ampligen~
- monotherapy sta~ilized the T4 cell decline expec:ted
in untreated EIIV infected patients (T4 ~ 60-3C)0 :
cells/nun3 ), and that this effect was sustained when ~ ~:
patien~s continued this treatment for 5-8 months
~Fig. 3).
A regression line ~ Fiçlure 5 ~ was constructec~
f rom the mean percenta~e change in T4 Lymphocyte
counts oVer 135 to 630 days of eombined dsRNA and AZT
therapy. The avera~e of each patient' s serial T4 ~ :~
lymphocyte counts during 91-180 days of the combined
treatment regimen served as the baseline or
determinin~ the percentag~ chang~. This baseline
was selected a~ a point a~ter which AZT~ s anticipate~
effect on T4 levels typically has dissipated.
Using statisti~al methods, the positive slope
of the regression line and the slopes of the 95%
confidence limits indicate that the combined
Ampligen~i~ and A:ZT therapy successfully abrogated the
long-term T4 cell decline ~xpected in untre~ted : .
patients (4-6 cells/mon~h) or those on long term (~ 6 ~ :~
month) AZT monotherapy. Thus the c:ombination
treatment in act sustained T4 levels indef.initely :
and or muc:h longer time period~ than previously
observed with AZT monotherapy and this illustrates a
basic utility of my invention or~ nucleoside resistant ;~
Yiruses, e~;pecially retroviruses. ~
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WO 92/18~04 PCI`/US92/01972
21~2~ ~
19
Finally, Figure 6 corroborate~ the utility of
my invention by presenting the proportion of patients
progressing to an AIDS-deinirlg opportunistic
infectionflymphoma ( "cr~ tical event" ~ while receivirlg
placebo, AZT monotherapy, Ampligen0 monotherapy, or ~:
Ampligen~9 and AZT combi~ational therapy.
As indicated in Figure 6, approximately 10% o ;~
the patients recei~ing placebo experierlced a critical
event during the 12-month ob~ervation period. The
number of pati~nts at ~he beginning, month 6 and :.
month 12 of the study are indic:ated parenthetically.
Of the patients receiving AZT, approximately 4% `; ~:
experienced a critical even during that period. Of
particular not~, was the avoidance of th~ expected
tumor formation seen with AZT given alone ( re~erence
6 ~ by th~ combined treatment . Further no patient in
this study with an absolute T4 1 ym~hocyte count
~reat~r than 115 cells/mm~ on entry experien~ed a
critical event while receivirlg Ampligen~ with
concomitant AZT, even though they were at gre~t risk
to do so due to markedly deteriorated i~une systems
be~ore beginning the co~nbinational regimen having a ,.
median CD4 level of only 201 ~t baseline ~Table 2 ) .
In concert with practicing this invention,
lymphokines such as intexleukins and interferons may `
be judicious~y added after ths foundation of control
o~ viral replication i s in place .
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WO 92/18004 PC~/V!i9~/01972 `:
REE~CES CITED
1. Larder, E~. A. et aL pp. 436-4g1 Antimicrobia1
Agents and Chemotherapy. Vol. 34, March 19gO.
2. Larder, B. A. et al. pp. 1731-1734, Science, ``~`:
Vol. ~43, 1g89.
3. Jackson, J.B. et al. pp. 1416-1418. Journal
Clin. Microbio1Ogy, Vol. 216, 1990; Thompson,
J. D. et al. pp. 371-378, Analytical Biochem.
1 . 182 ~ 1989 .
4 . Beren~aum, M. C . pp . ~69-333 . Advances in Cancer
Research Vol. 35, ~981~
5. Fischl, M. et al. p. 727-733, ~nnals of
Internal Medic:ine Vo1. 112, 1990.
6. P1uda, 3. M~ et al pp.: 276-282
~nals of Internal Medicine, Vol. :113 ~nu~er
4, 1990 ~.; ;
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