CA2102221A1 - Abrogation of viral resistance to nucleoside analogues by double-stranded rnas - Google Patents

Abstract

The rate of viral resistance developed during the course of treatment with antiviral nucleoside analogues is reduced by administering dsRNAs early in the treatment of the infection or in later stages when viral genetic mutation has occured to restore susceptibility of the virus to otherwise ineffective antiviral agents.
Delaying and/or reducing the appearance of nucleoside analogue resistant retroviruses, particularly HIV, is achieved with mismatched dsRNAs notably in the peripheral blood mononuclear cells, especially the CD4 lymphocytes.

Description

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 -. ~:',':,~

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 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 ; ~

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. -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. ~
,`'` ' ,::

.. . .
'''' ''''';'.''' ',."'~ `"'.'''.

''', ' .~'' :'~.:

WO 92/18~04 PCI`/US92/01972 21~2~ ~

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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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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Claims (13)

WHAT IS CLAIMED IS:

1. A combinational regimen which ameliorates the appearance of nucleoside analogue resistant virus and/or ameliorates the morbidity, especially on immunological cells, of previously existent nucleoside resistant virus.

2. A method of delaying, reducing or both delaying then reducing the appearance of nucleoside analogue resistant virus in a patient having an HIV
infection, comprising administering to s aid patient an effective amount of a mismatched dsRNA prior to therapy with a nucleoside analogue antiretroviral agent.

3. A method of ameliorating the morbidity of the peripheral blood mononuclear cells of a patient infected with a nucleoside analogue resistant HIV, comprising administering to said patient an effective amount of a mismatched dsRNA.

4. The method of claim 2, wherein the patient is administered a nucleoside analogue antiretroviral agent subsequent to therapy with said mismatched dsRNA.

5. The method of claim 3, wherein said cells are CD4 lymphocytes.

6. The method of claim 2 or 3, in which the mismatched dsRNA is a polyadenylic acid complexed with polyuridylic acid.

7. The method of claim 6, in which the mismatched dsRNA is a complex of polyinosinate and polycytidylate containing from 1 in 5 to 1 in 30 uracil or guanidine bases.

8. The method of claim 7, in which the mismatched dsRNA is rIn?r(C11-14'U)n or the mismatched dsRNA contains regions of bond breakage and exhibits the favorable therapeutic ratio property of rIn?r(C11-14'U)n.

9. The method of claim 6, in which the amount of mismatched dsRNA administered results in a level of from 2 to 1,000 micrograms of the mismatched dsRNA
per milliliter of the patient's systemic blood circulation.

10. The method of claim 2 or 3, in which the dsRNA is a short oliglnucleotide of defined structure of the formula:

5'lock-(I)n-lock 3' 3'lock-(C)m-lock 5' where m and n are each more than 5 and less than 100, I is inosine monophosphate, C is cytidine monophosphate, or 5'lock-[(I)xA]j-lock 3' 3'lock-[(C)yU]k-lock 3' where x and y are each more than 5 and less than 25, j and k each at least 1 and less than 10, I and C are as identified above, A
is a nucleotide which is not I, and U is a nucleotide which base pairs with A, or 5'(I)n-hinge-(C)m3' where n, m, I and C are as defined above, provided that the locks in one strand are complementary to locks in the opposite strand.

11. The method according to claim 10, in which the oligonucleotide is stabilized by internal registers of complementary heteropolymer and the lock or hinge or both contain regions of complementary heteropolymer.

12. The method of claim 2, 3 or 4, wherein said nucleoside analogue antiretroviral agent is zidovudine, dideoxyinosine, dideoxycytidine or combinations thereof.

13. The method of claim 2 or 3, wherein said mismatched dsRNA is rIn?(11-14'U)n.