Note: Descriptions are shown in the official language in which they were submitted.
- l 1340S13
The present invention relates to 3 -azido-3 -deoxythymidine
and its use in the treatment or prophylaxis of human retroviral infections.
This Application is a divisional of Canadian Patent Application Serial No.
556,981 filed January 20, 1988.
Retroviruses form a sub-group of RNA viruses which, in order to replicate, must
first 'reverse transcribe' the RNA of their genome into DNA ('transcription'
conventionally describes the synthesis of RNA from DNA). Once in the form of
DNA, the viral genome is incorporated into the host cell genome, allowing it to
take full advantage of the host cell's transcription/translation machinery lor the
purposes of replication. Once incorporated, the viral DNA is virtually
indistinguishable from the host's DNA and, in this state, the virus may persist
for as long as the cell lives. As it is vircually invulnerable to attack in this form,
any treatment must be directed at another state of the life cycle and will, of
necessity, have to be continued until all virus-carrying cells have died.
HTLV-I and HTLV-II are both retroviruses and are known to be causative agents
of leukaemia in man. HTi_V-I infections are especially widespread and are
responsible for many deaths world wide each year.
A species of retrovirus has now been reproducibly isolated from patients with
AIDS. This virus is now identified as Human Immunodeficiency Virus (referred to
herein as HIV) and has been shown preferentially to infect and destroy T-cells
bearing the 01~4 surFace marker. The virus is now generally accepted as the
aetiologic agent of AIDS. The patient progressively loses this set of T-cells,
upsetting the overall balance of the immune system, reducing his ability to
combat other infections, and predisposing him to opportunistic infections which
frequently prove fatal. Thus, the usual cause of death in AIDS victims is by
opportunistic infection, such as pneumonia or virally induced cancers, and not as
a direct result of HIV infectian.
Recently, HIV has also been recovered from other tissue types, including B-
cells expressing the T4 marker, macrophages and non-blood associated tissue in
the central nervous system (CNS). This latter infection has been discovered in
13~0~19
patients expressing classical AIDS symptoms and is associated with progressive
demyelination, leading to wasting and such symptoms as encephalopathy,
progressive dysarthria, ataxia and disorientation.
There are at least four clinical manifestations of HIV infection. In the initial'carrier' state, the only indication of infection is the presence of anti- HIV
antibodies in the blood-stream. It is believed that such 'carriers' are capable of
passing on the infection, e.g. by blood transfusion, sexual intercourse or used
syringe needles. The carrier state may of~en never progress to the second stage
characterised by persistent generalised Iymphadenopathy (PGI_). It is currently
estimated that about 20% of PGL patients progress to a more sdvanced condition
known as 'AIDS related complex' (ARC). Physical symptoms associated with
ARC may include general malaise, increased temperature and chronic
in~ections. This condition usually progresses to the final, fatal AIDS condition,
when the patient completely loses the ability to fight infection.
The existence of these human retroviruses and others has only recently been
recognised and, as the diseases with which they are linked are of a life-
threatening nature, there exists an uryent need to develop ways to combat these
viruses.
Various drugs have now been proposed as 'cures' for AIDS. These includeantimoniotungstate, suramin, ribavirin and isoprinosine, which are either
somewhat toxic or have shown no mart<ed anti-retroviral activity. As the HIV
genome is incorporated into the host cell DNA after infection and is virtually
invulnerable to attack in this ~tate, it will persist 8S long as the host cell
survives, causing new infection in the meantime. Thus, any treatment of AIDS
would have to be for an extended period, possibly life, requiring substances with
an acceptable toxicity.
Reports have described the testing of compounds against various retroviruses for example
Friend Leukaemia Virus (FLV) a murine retrovirus. For instance Krieg et al. (Exp. Cell Res.
116 (1978) 21-29) found that 3'-azido-3'-deoxythymidine affected the release of C-type particles
and increased the formation of A-type particles of a FLV complex in in vitro experiments and
Ostertag et al. (Proc. Nat. Acad. Sci. (1974) 71 4980-85) stated that on the basis of antiviral
activity related to the above FLV complex and a lack of cellular toxicity 3'-azido-3'-
deoxythymidine "might favourably replace bromodeoxyuridine for medical treatment of ~liceases
caused by DNA viruses". However De Clerq et al. (Biochem. Pharm. (1980) 29 1849-1851)
~- ~'' ~hed six years later that 3'-azido-3'-deoxythymidine had no
,11 /7~t.h F~hr~ l~rv 1 9R~
--3--
appreciable activity against any viruses used in their tests, including vacclnia, 1 9
HSVI and varicella zoster virus (VZV). Glinski et al. (J. Org. Chem. (1973), 3~,4299-4305) discloses certain derivatives of 3'-azido-3'-deoxythymidine ~ and
their ability to block mammalian exoribonuclease activity.
We have now discovered that 3'-azido-3'-deoxythymidine has a surprisingly
potent activity against human retroviruses, with a particularly high activity
against HIV as demonstrated by the experimental data referred to below. Such
activity renders the compound useful in the therapy of human retroviral
infections.
.
Thus, in a first aspect of the present invention, there is provided a compound of
formula (I): ~
'~ ~
. ~ N (I)
Ho
~7
(i.e. 3'-azido-3'-deoxythymidine)
for use in the treatment or prophylaxis of human retrovirus infections.
The compound of formula (I) is
hereafter referred to as the compound according to the invention.
Activity of 3'-azido-3'-deoxythymidine against human retroviruses has been
established in various in vitro assay systems. For example, infection of the H9
human iymphoblastoid cell-line by HIV is effectively prevented by
concentrations of 3'-azido-3'-deoxythymidine as low as 0.013 mcg/ml up to 20
hours after infection. HIV infection of U937 human lymphoblastoid cells, PHA-
stimulated white blood cells and cultured peripheral blood lymphocytes is also
prevented at similarly low concentrations. In addition, 10-day challenge
experiments using up to 5000 HIV virions per cell and cloned T4, tetanus-
specific, T-helper lymphocytes, showed no decrease in cells treated with 3'-
azido-3'-deoxythymidine, while untreated cells had decreased ~-fold. Cytopathic
effects were also completely blocked in the same cell-line transformed by
HTLV-I and super-infected with HIV .
Other studies using purified HIV reverse transcriptase have shown that the
activity of this enzyme is blocked by the triphosphate of 3'-azido-3'-
deoxythymidine by a competitive inhibition mechanism.
HDL/LMJ/26th February 1986
, . . .... .
1 3 4 0 ~ 1 g
Phase I clinical trials have also shown that 3'-azido-3'-deoxythymidine is capable
of crossing the blood/brain barrier in clinically effective quantities. This
property is both unusual and valuable for the treatment and prophylaxis of CNS
infections caused by human retroviruses.
The ability of 3'-azido-3'-deoxythymidine tn modify the cnurse of retrovirus-
lnduced malignancy has been demonstrated in a mouse model, whereby
administration of 3'-azido-3'-deoxythymidine prevented splenomegaly caused by
intravenously administered Rauscher Murine Leukaemia Virus, the murine
equivalent of HTLV-I. In further experiments, 3'-azido-3'-deoxythymidine has
been shown to inhibit the in vitro replication of HTI V-I at concentrations as low
as û.8 mcg/ml.
Thus, in a further, preferred aspect of the present invention, there is providedthe use of a compound according to the invention in the manufacture of a
medicament for the treatment or prophylaxis of human retrovirus infections.
The present invention further provides a method for the treatment or prophylaxisof AIDS in a human subject which comprises administering to the said human
subject an effective amount of a compound according to the invention.
The present invention also includes a method for the treatment or prophylaxis ofinfections caused by retroviruses in a human subject, which comprises
administering to the said human subject an effective antiviral amount of a
compound according to the invention.
The present invention further provides a compound according to the invention foruse in the treatment or prophylaxis of AIDS or a virus infection as identified
above.
Fxamples of human retrovirus infections which may be treated or prevented in
accordance with the present invention include T-cell lymphGtropic retroviruses
(HTLV), especially HTLV~I, HTLV-II and AIDV (HTLV-III). Clinical conditions
that may be treated or prevented in accordance with the invention include AIDS,
AIDS-related complex and HTLV-I positive leukaemia and lymphoma. Sui~able
patients for treatment also include those having antibodies to HIV, AIDV C~IS
infections, PGL and ARC.
--5--
13~0~1~
~xperiments have shown that 3'-azido-3'-deoxythymidine is converted, in vo, by
the action of cellular enzymes into the 5'-monophosphate. The monophosphate is
then further phosphorylated by other enzymes to form the triphosphate via the
diphosphate, and other studies have demonstrated that it is the triphosphate
form of 3'-azido-3'-deoxythymidine which is believed to be the effective chain
terminator in the reverse transcription of HIV, as evidenced by its effect on
avian myeloblastosis virus and MoLoney murine leukaemia viru3. This form also
inhibits HIV reverse transcriptase in vitro whilst having a negligible ef,~ect on
human DNA polymerase activity.
--6--
13~0513
3'-Azido-3'-deoxythymidine
(hereafter referred to as the active ingredient), may be administered to humans
for prophylaxis or treatment of retroviral infections by any suitable route
including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and
parenteral (including subcutaneous, intramuscular, intravenous and intradermal).It will be appreciated that the preferred route wiLl vary with the condition andage of the recipient, the natùre of the infection and the chosen active
ingredient.
In general a suitable dose will be in the range of 3.0 to 120 mg per kilogram body
weight of the patient per day, preferably in the range of 6 to 90 mg per kilogram
body weight per day and most preferably in the range 15 to 60 mg per kilogram
body weight per day~ The desired dose is preferably presented as two, three,
four, five, six or or more sub-doses administered at appropriate intervals
throughout the day. These sub-doses may be administered in unit_dosaye forms,
for example, containing 5 to 1500 mg, preferably 10 to 1000 mg, and most
preferably 20 to 700 m~ of active ingredient per unit dosage form.
Fxperiments with 3'-azido-3'-deoxythymidine suggest that a dose should be
administered to achieve peak plasma concentrations of the active compound of
from about 1 to about 75 IIM, preferably about 2 to 50 IIM, most preferably
about 3 to about 30 lIM. This may be achieved, for example, by the intravenous
injection of a 0.1 to 5% solution of the active ingredient, optionally in saline, or
orally administered as a bolus containing about 1 to about 100 mg/kg o' the
active ingredient. Desirable blood levels may be maintained by a continuous
infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent
infusions containing about 0.4 to about 15 mg/kg of the active ingredient.
While it is possible for the active ingredient to be administered alone it is
preferable to present it as a pharmaceutical formulation. The formulations of
.~
1340~19
the present invention comprise at least one active ingredient, as above defined,togethsr with one or more acceptable carriers thereof and optionally other
therapeutic agents. Fach carrier must be "acceptable" in the sense of belng
compatible with the other in~redients of the formulation and not injurious to the
patient. Formulations include those suitable for oral, rectal, nasal, topical
(including buccal and sublingual~, vaginal or parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) administration. The ~ormulations
may conveniently be présented in unit dosage form and may be prepared by any
methods well known in the art of pharmacy. Such methods include the step of
bringing into association the active ingredient with the carrier w hich constitutes
one or more accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association the active ingredient with
liquid carriers or finely divided solid carriers or both, and then if necessary
shaping the product.
Formulations of the present invention suitable for oral administration may be
presented as discrete units such as capsules, cachets or tablets each containing a
predetermined amount of the active ingredient; as a powder or granules; a~ a
solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water
liquid emulsion or a water-ln-oil liquid emulsion. The active ingredient may also
be presented as a bolus, electuary or paste. Oral formulations may further include
other agents conventional in the art, such as sweeteners, flavouring agent~ and
thickeners.
A tablet may be made by compression or moulding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared by compressing in a
suitable machine the active ingredient in a free-flowing form such as a powder
or granules, optionally mixed with a binder (e.g. povidone, gelatin,
hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,
disintegrant te.g- sodium starch glycol!ate, cross-linked povidone, cross-linkedsodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded
tablets may be made by moulding in a suitable machine a mixture of the
powdered compound moistened with an inert liquid diluent. The tablets may
optionally be coated or scored and may be formulated so as to provide slow or
controlled release of the active ingredient therein using, for example,
hydroxypropylmethylcellulose in varying proportions to provide the desired
release profile.
HDL/I_MJ/26th February 1986
-a-
1340~19
Formulations suitable for topical administration in the mouth include lozenges
comprising the active ingredient in a flavoured basis, usually sucrose and acacia
or tragacanth; pastilles comprising the active ingredient in an inert basis such as
gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
Formulations for rectal administration may be presented as a suppository with a
suitable base comprising for example cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as pessaries,
tampons, creams, gels, pastes, foams or spray formulations containing in addition
to the active ingredient such carriers as are known in the art to be apprnpriate.
Formulations suitable for parenteral administration include aqueous and non-
aqueous isotonic sterile injection solutions which may contain anti-oxidants,
buffers, bacteriostats and solutes which render the formulation isotonic with the
blood of the intended recipient; and aqueous and non-aqueous sterile sus;oensions
which may include suspending agents and thickening agents. The formulations
may be presented in unit-dose or multi-dose sealed containers, for example,
ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Fxtemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and tablets of the
kind previously described.
Preferred unit dasage formulations are those containing a daily dose or unit,
daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an
active ingredient.
The administered ingredients may also be used in therapy in conjunction with
other medicaments such as 9-L[2-hydroxy-1-(hydroxy-
methyl)ethoxy]methyl]guanine, 9-(2-hydroxyethoxymethyl)guanine (acyclovir), 2-
amino-9-(2-hydroxyethoxymethyl)purine, interferon, e.g., a-interferon,
interleukin II, and phosphonoformate (Foscarnet) or in conjunction with other
immune modulating therapy including bone marrow or Iymphocyte transplants or
medications such as levamisol or thymosin which serve to increase Iymphocyte
r~umbers and/or function as is appropriate.
It should be understood that in addition to the ingredients particularly mentioned
.
HDL/I_MJ/26th February 1986
- 9 -
13~0~19
above the formulations of this invention may include other agents conventional
in the art of formulation.
The compound of formula (I)
may be prepared in conventional manner, for example as described in the
following references, or by methods analogous thereto:J.R. Horwitz et al., J.
Org. Chem. 29, (July 1964) 2076-78; M. Imazawa et ai., J. Org. Chem, 43 (15)
(1978) 3044-3048; K.A. Watanabe e~ al., J. Org. Chem., 45, 3274 (19B0); and
R.P. Glinski et al., J. Chem. Soc. Ghem. Commun., 915 (197û~, as well as the
processes described in Reference Example 8 hereinafter.
The following Fxamples are intended for illustration only and are not intended to
limit the scope of the invention in any way. The term 'active ingredient' as used
in the Examples means a compound of formula (I) or a pharmaceutically
acceptable derivative thereof.
Reference Example 1: Tablet Formulations
The followiny formulations A to C were prepared by wet granulation of the
ingredients with a solution of povidone, followed by addition of magnesium
stearate and compression.
mq/tablet~ mq/tablet
Formulation A
(a) Active ingredient 250 250
(b) Lactose B.P. 210 26
(c) Povidone B.P. 15 9
(d) Sodium Starch Glycollate 20 12
(e) Magnesium Stearate 5 3
500 300
HDL/I_MJ/26th February 19~6
-10-
Formulation B 13 ~ O ~;19
mq/tabletmQ/tablet
(a) Active ingredient 250 250
(b) I_actose 150 -'
(c) Avicel*PH 101 60 26
(d) Povidone B.P. 15 9
(e) Sodium Starch Glycollate 20 12
(f) Magnesium Stearate 5 3
500 300
Formulation C.
mq/tablet
Active ingredient 1nO
Lactose 200
5tarch 50
Povidone 5
Magnesium stearate 4
359
The following formulations, D and E, were prepared by direct compression of the
admixed ingredients. The lactose used in formulation F was of the direct
compression type (Dairy Crest - "Zeparox").
Formulation D
m~/tablet
Active Ingredient 250
Pregelatinised Starch NF15 150
400
Formulation F
mq/tablet
Active Ingredient 250
Lactose 150
Avicel * 100
500
Formulation F (Controlled Release Formulation~
The formulation was prepared by wet granulation of the ingredients (below) with
a solution of povidone followed by the addition of magnesium stearate and
compression.
I IDLhMJ/26th February 1986 *trade mark
.
-11-
mq/tablet
(a) Active Ingredient 50013 4 0 5 13
(b) Hydroxypropylmethylcellulose 11Z
(Methocel~4M Premium)
(c) Lactose B.P. 53
(d) Povidone B.P.C. 28
(e) Magnesium Stearate
700
Drug release took place over a period of about 6-8 hours and was complete after
12 hours.
Reference ExamPle 2: C~rsu'Q Formulations
Formulation A
A capsule formulation was prepared ~y admixing the ingredients of Formulation
D in Example 1 above and filling into a two-part hard gelatin capsule.
Formulation B (infra) was prepared in a similar manner.
Formulation B
mq/capsule
(a) Active ingredient 250
(b) Lactose B.P. 143
(c) Sodium Starch Glycollate 25
(d) Magnesium Stearate 2
420
~ormulation C
mq/capsule
(a) Active ingredient 250
(b) Macrogol*4000 BP 350
600
Capsules were prepared by melting the Macrogol*4000 BP, dispersing the active
ingredient in the melt and filling the melt into a two-part hard gelatin capsule.
*trade mark
HDL/I_MJ/26th February 1986
- ~ -
-12-
1340513
Formulatlon D
m~/capsule
Active ingredient 250
Lecithin 100
Arachis Oil 100
450
Capsules were prepared by dispersing the active ingredient in the lecithin and
arachis oil and filling the dispersion into soft, elastic gelatin capsules.
Formulation F (Controlled Release Capsule~
The following controlled release capsule formulation was prepared by extruding
ingredients a, b and c using an extruder, followed by spheronisation O r the
extrudate and drying. The dried pellets were then coated with release-
controlling membrane (d) and filled into a two-piece, hard gelatin capsule.
mq/capsule
(a) Active lngredient 250
(b) Microcrystalline Cellulose 125
(c) Lactose BP 125
(d) Ethyl Cellulose 13
513
Reference ExamPle 3: Iniectable Forrnulation
Formulation A.
Active ingredient 0.2009
Hydrochloric acid solution, 0.1M q.s. to pH 4.0 to 7.0
Sodium hydroxide solution, 0.1M q.s. to pH 4.0 to 7.0
Sterile water q.s. to 10ml
The active ingredient was dissolved in most of the water (35 ~-40-C) and the pH
adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as
appropriate. The batch was then made up to volume with the water and filtered
through a sterile micropore filter into a sterile 10ml amber glass vial (type 1) and
sealed with sterile closures and overseals.
HDL/I_MJ/26th February l9a6
-13-
Formulation B. 1 ~ 4 0 ~19
Active ingredient 0.125 9
Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to 25 ml
Reference ExamPle 4: Intramuscular iniection
Active Ingredient 0.20 9
Benzyl Alcohol 0.10 9
Glycofurol 75 1.45 9
Water for Injection q.s. to 3.00 ml
The active ingredient was dissolved in the glycofurol. The benzyl alcohol was then
added and dissolved, and water added to 3 ml. The mixture was then filtered through a
sterile micropore filter and sealed in sterile 3 ml amber glass vials (type 1).
Reference ExamPle 5: In~redients
Active ingredient 0.2500 9
Sorbitol Solution 1.5000 9
Glycerol 2.0000 9
Sodium Benzoate; 0.0050 9
Flavour, Peach 17.42.3169 0.0125 ml
Purified Water q.s. to 5.0000 ml
The active ingredient was dissolved in a mixture of the glycerol and most of thepurified water. An aqueous solution of the sodium benzoate was then added to thesolution, followed by addition of the sorbitol solution and finally the flavour. The
volume was made up with purified water and mixed well.
Reference ExamPle 6: SuPPositorY
mq/suppository
Active lngredient (63~m)* 250
Hard Fat, BP (Witepsol**H15 - Dynamit Nobel) 1770
2020
**trade mark
.
-14- 1340513
*The active ingredient was used as a powder wherein at least 90% of the particles
were of 631~m diameter or less.
One-fifth of the Witepsol Hl5 was melted in a steam-jacketed pan at 45-C maximum.
The active ingredient was sifted through a 200)Jm sieve and added to the molten base
with mixing, using a silverson fitted with a cutting head, until a smooth dispersion was
achieved. Maintaining the mixture at 45 ~C, the remaining Witepsol Hl5 was added to
the suspension and stired to ensure a homogenous mix. The entire suspension was
passed through a 250~m stainless steel screen and, with continuous stirring, wasallowed to cool to 40~C. At a temperature of 38~C to 40-C 2.029 of the mixture was
filled into suitable plastic moulds. The suppositories were allowed to cool to room
temperature.
Rerer,:~ce ExamPle 7: Pessanes
mq/pessary
Active ingredient 63~m 250
Anhydrate Dextrose 380
Potato Starch 363
Magnesium Stearate 7
1000
The above ingredients were mixed directly and pessaries prepared by direct
compression of the resulting mixture.
Z'HDL/I_MJ/26th February l9a6
-15-
Reference ExamPle 8: 3'-Azido-3'-deox~thymidine
1~40513
a) 2l3'-Anhydrothymidine
.
Thymidine (85.4 9: 0.353 mol) was dissolved in 500 ml dry DMF and added to N-
(2-chloro-1,1,2-trifluoroethyl)diethylamine (100.3 9; 0.529 mol) (prepared
according to the method of D.~. Ayer, J. Med. Chem. 6, 608 (1963)). This
solution was heated at 70~C for 30 minutes then poured into 950 ml ethanol
(FtOH) with vigorous stirring. The product precipitated from this solution and
was filtered. The FtOH supernatant was refrigerated then filtered to yield the
title compound. mp. = 228 -230~C.
b) 3'-Azido-3'-deoxythymidine
2,3'-0-Anhydrothymidine (25 9: 0.1115 mol) and NaN3 (29 9, 0.446 mol) was
suspended in a mixture of 250 ml DMF and 38 ml water. The reaction
mixture was refluxed for 5 hours at which time it was poured into 1 liter nf
water. The aqueous solution was extracted with FtOAc (3 x 700 ml). The
FtOAc extracts were dried over Na2SO4, filtered and the FtOAc was
removed in vacuo to yield a viscous oil. This oil was stirred with 200 ml
water providing the title compound as a solid which was collected by
filtration. mp = 116-118 ~ C
HDL/LMJ/26th February 1986
.
-
-16-
Example : Antiviral Activity
1340513
(a) (i) Retrovirus - Induced Maliqnancy
3'-Azido-3'-deoxythymidine was administered to female BALB/c mice infected
with 1.5X104 Pfu of the RVB3 strain of Rauscher Murine Leukaemia Virus.
Treatment was started 4 hours af ter infection at dosages of 80 mg/kg
intraperitoneally every 8 hours or 0.5 or 1.0 mg/ml orally in drinking water.
Such treatment was found to prevent infection of spleen cells and subsequent
development of splenomegaly and also suppressed viraemia.
(ii~ HTLV-I
TM-11 cells (T-cell clone susceptible to HTl_V-I infection) were co-cultivated
with irradiated, HTLV-I producer MJ-tumour cells as follows:
a) TM-11 cells only;
b) TM-11 cells and MJ-tumour cells
c) TM-11 cells, MJ-tumour cells and 3'-azido-3'-deoxythymidine (31-M);
d) TM-11 cells, MJ-tumour cells and 3'-azido-3'-deoxythymidine (91JM);
e) TM-11 cells, MJ-tumour cells and 3'-azido-3'-deoxythymidine (271JM).
On day 1~, total DNA was extracted from each culture and digested with Bam H1 togenerate a fragment of the HTLV-I genome, independent of any host flanking sequence
and having a standard molecular weight of 3.3 kD. The digest was then probed with
radio-labelled lambdha MT-2, a standard probe recognising the Bam H1 fragment ofHTLV-I.
No hybridisation was observed for a), indicating a lack of virus in the uninfected
control. A strong signHl was seen for b), the untreated, infected control. A weak
signal was observed with c), indicating incomplete eradication of the virus, and no
hybridisation was noted in d) or e) indicating complete extermination of the virus.
Fach culture was also probed with a probe for T-cell receptor ~ chain, with a strong
signal being generated for all cultures, showing the continued presence of TM-11 for
the duration of the experiment.
HDl_/LMJ/26th February 19û6
~.. _.
-17-
(b) HIV 13 l~S~3
(i) Reverse Transcriptase Activity
3'-Azido-5'-triphosphate-3'-deoxythymidine was tested in vitro agalnst HIV
transcriptase (HIV RT)
HIV RT was purified from pelleted and extracted HIV by elution through
DFAE and phosphocellulose columns. The enzyme activity was linear through 60
minutes and stable for at least 2 months when stored in 60% glycerol and 1 mg
bovine serum albumin per ml. Using rA-odT (12 18) as the template-primer,
HIV RT had a pH optimum of 7.0 to 7.3, a MnCl2 optimum of 0.3 mM and a
MgCl2 optimum of 5 mM. The activity in the presence of 5 mM MgC12 was 10-
fold greater than the activity in the presence of 0.3 mM MnCl2. Maximal
enzyme activity was also found in 80 to 140 mM KCl and 60 to 100 mM NaCl.
Incorporation of [3H] dTTP was linear with respect to enzyme concentration.
When tested, 3'-azido-5'-triphosphate-3'-deoxythymidine was found to be a
competitive inhibitor of HIV RT, giving a Ki of 0.04 I~M when using rA-odT (12
18) as the template-primer. The enzyme had a Km for dTTP of 2.811~M,
suggesting that 3'-azido-5'-triphosphate-3'-deoxythymidine binds tighter to the
enzyme than does dTTP. Further experiments with the RT's of avian
myeloblastosis virus, Moloney murine leukemia virus and HIV, showed 3'-azido-
5'-triphosphate-3'-deoxythymidine to be a terminator of DNA chain elongation.
(ii~ In Vitro Anti-HlV Activity
3'-Azido-3'-deoxythymidine was tested and found to possess activity in a number
of in vitro assay systems. Drug effects were measured by assaying reverse
transcriptase (RT) activity in the supernates from infected, uninfected, and drug
treated cells. 3'-Azido-3'-deoxythymidine effectively blocked the infection by
HIV of the H9 and U937 human Iymphoblastoid cell lines at concentrations from
2.7 to 0.0013 mcg/ml. Similarly, infection of normal PHA stimulated white
blood cells and cultured peripheral blood Iymphocytes was inhibited at drug
concentrations as low as 0.013 mcg/ml. Drug addition and subtraction
experiments in H9 cells revealed that 3'-azido-3'-deoxythymidine was most
effective when present at the time of virus infection of susceptible cells, but
still retained most of its sntiviral activity even when added as late as 20 hours
after initial HIV infection. Inhibition of viral replication was also evident when
the drug was present in the media only during the 20 hour period of virus
HDL/LMJ/26th February 1986
., ,
-18- 1340~13
absorption. Effects were seen at 0.13 and 0.013 mcg/ml. 3'-Azido-3'-
deoxythymidine exhibited no direct anti-RT activity against purified HIV
virions. Similarly, the drug had little or no effect on the production and release
of virions from the chronically infected H9 HIV cell line.
(iii) Preventinq Infection bY HIV
The ability of 3'-azido-3'-deoxythymidine to block infection of cells by HIV wasdetermined as follows.
Cloned T4 positive tetanus specific T helper Iymphocytes were infected with a
pool of HIV isolates [at challenge doses of up to 5000 virions/cell] and cell
survival after infection was monitored. After 10 days in culture no viral
cytopathic effects were seen in infected T cells treated with 8.8 and 1.3 mcg/ml3'-azido-3'-deoxythymidine, while untreated, infected cells were 5-fold
decreased. Cell survival was also evaluated in an HTI V-I transformed, HIV
superinfected cell line derived from the cells above. 3'-azido-3'-deoxythymidineat concentrations of 2.7, 0.27 and 0.13 mcg/ml totally blocked cytopathic effects
at 7 days. Protective effects were seen in infections induced by both cell free
virions and cell associated virus. 3'-Azido-3'-deoxythymidine at 0.27 mcg/ml
concentraion also effectively prevented cytopathic effect induction by a less
related Haitian isolate of HIV
Example 18: Toxicity Assay
3'-Azido-3'-deoxythymidine was administered to both mice and rats.- The LD50
value was in excess of 750 mg/kg in both species.
HDL/LMJ/26th February 1986
