Note: Descriptions are shown in the official language in which they were submitted.
W 095/11028 2 1 7 4 5 52 PCT/GB94/02292
FLAVIN DERIVATIVES AS ANTI-VIRAL AGENTS
The present invention relates to anti-viral agents and
their use in the treatment of human and animal patients to
alleviate or cure the ill-effects caused by viral
infection, especially HIV. A detailed study of compounds
according to the invention has been carried out to evaluate
their efficacy against infection from several strains of
HIV-l. The compounds have similar activity against HIV in
both acutely and chronically infected cells. This is a
dual property only ocassionally associated with other
compounds which are in current use in the therapy of HIV
infection although de nova (acute) infections of cells may
be treated by compounds which act early in the replication
cycle of HIV to block integration of vDNA into the host
chromosome. It is this integration which signifies entry
of the infection into the chronic state. Compounds which
act post-integration of HIV are therefore inhibitors of
chronically infected cells. Zidovudine (AZT) for example
is only active against de nova infection of HIV and has no
significant activity against chronically infected cells.
Inhibitors of gene expression of HIV (which is a positive
strand RNA virus) would therefore be active in HIV
chronically infected cells.
HIV is a positive strand RNA virus which affects humans.
The virus attaches to cell membranes by virion adsorption
to CD4 surface receptor. The virion then passes through
WO95/11028 2 1 7 ~ PCT/GB94/02292
the cell membrane penetratively and enters the cell
cytoplasm. Uncoating of the virion then takes place in the
cytoplasm whereby the viral envelope and the protein coat
of the genome release the viral RNA into the cytoplasm.
Reverse transcription therein produces a double-stranded
DNA transcript from host cell genetic material. This
invades the host cell nucleus and integrates with the host
cell chromosomal DNA. Transcription follows to produce a
vRNA replicate which is translated in the cytoplasm to
produce new viral proteins. The latter then assembles with
vRNA at the inner cell surface to produce new virus
particles which are released from the host cell.
HIV is normally associated with an initial asymptomatic
phase. This initial asymptomatic phase may last a number
of years before the early signs of HIV disease occur.
A number of ideas causing cell death are proposed.
Apoptosis is one of these. It is a morphologically
distinctive form of programmed cell death involved in many
physiological and pathological processes including cellular
processes which seek to maintain appropriate intracellular
oxidant-antioxidant balance. Cell death in T-cells is
closely associated with this balancing process. Infection
with HIV is thought gradually to disturb the balance in
favour of cell death. Another critical factor in
determining whether cells will grow and divide in a normal
fashion is intracellular ATP concentration. Low
WO95/11028 21 7 4 ~ ~ 2 PCT/GB94/02292
intracellular levels of ATP are associated with ischemic
death. T-lymphocytes are especially vulnerable to
depletion of intracellular ATP levels. HIV infection may
disturb cellular oxidative phosphorylation which is the
cellular process responsible for ATP levels in the cell.
Cell death from whatever cause will eventually lead to cell
depletion to a level that induces AIDS.
Much of the current work in the field of antiviral research
is concerned with targeting specific viral encoded enzymes.
Compounds discovered from this research, in principle,
should have low toxicity on cellular processes. The long
term use of compounds in clinical trials in HIV infection
treatment has not given the degree of benefit initially
expected, and new approaches are needed.
Riboflavine is a known compound, which is also variously
known as:
ElOl;
Lactoflavin;
Riboflavin;
Riboflavinum;
Vitamin B2;
Vitamin G;
7,8-Dimethyl-lO-(l'-D-ribityl) isoalloxazine; and
3,lO-Dihydro-7,8-dimethyl-lO-(D-ribo-2,3,4,5-tetra-
hydroxypentyl) benzopteridine-2,4-dione.
WO95/11028 PCT/GB94/02292
2t 74552
Riboflavine is commercially available as such or as its
sodium phosphate or tetrabutyrate salt, typically in the
former instance as the dihydrate salt. It is also
available in various mixtures with other vitamins, all
essentially being for the treatment of, inter alia, vitamin
B deficiency. In such mixtures the dose of riboflavin
varies between 0.5 and 10 mg, with a maximum recommended
daily dose being 30 mg.
No adverse effects have been reported with the use of
riboflavine. However, significant doses of riboflavine
result in a bright yellow discoloration of the urine which
may interfere with certain laboratory tests.
The riboflavine requirement of humans is often related to
the energy intake, but it appears to be more closely
related to resting metabolic requirements. A daily dietary
intake of about 1.3 to 1.8 mg of riboflavine is recommended
that is to say the basic recommended intake of riboflavine
is 550 ~g per 4200 kj (1000 kcal) of diet - Report of a
Joint FAO/WHO Expert Group, Tech. Rep. Ser. Wld 111th Org.
No. 362, 1967.
The estimated acceptable daily intake of riboflavine is up
to 500 ~g per kg body weight - see Thirteenth Report of
FAO/WHO Expert Committee on Food Additives, Tech. Rep. Ser.
WHO. No. 445, 1971.
wo 95"1028 2 1 7 4 5 5 2 PCT/GB94/02292
Riboflavine, which is a water-soluble vitamin, is essential
for the utilisation of energy from food. The active,
phosphorylated forms, flavine mono-nucleotide and flavine
adenine dinucleotide, are involved as co-enzymes in
oxidative/reductive metabolic reactions.
Various other flavins and derivatives thereof are also
known, mainly as flavouring agents.
It has now been found surprisingly that the administration
of riboflavine, as well as other flavins and derivatives
thereof, at doses far higher than previously used or
recommended can be highly effective in the management and
treatment of viral infections, in particular HIV. The
structure of the compound indicates involvement in the
process of oxidative phosphorylation within cells. It is
possible that the compounds of the invention preferentially
target the same target as HIV and so resist or prevent the
manifestations of infection including the procreative
capacity of the virus.
Accordingly, the present invention in one aspect provides
the use of a flavin, especially riboflavine, or a
derivative thereof for the manufacture of a medicament for
the management and treatment of viral infection.
Moreover, insofar as certain flavins and derivatives
WO95/11028 2 1 7 4 5 5 2 PCT/GB94/02292
thereof are not known as pharmaceuticals, even in a general
sense as with riboflavine (known as an enzyme co-factor
vitamin), the invention in a second and broader aspect
provides such certain flavins or a derivative thereof for
use as anti-viral agents.
In the use according to the invention riboflavine or other
flavin may be used as such or as a derivative and the
flavin derivative may be any derivative which is safe for
human or animal use. Preferably, however, in the case of
riboflavine the derivative is a riboflavine salt and more
preferably the riboflavine salt is riboflavine sodium
phosphate or riboflavine tetrabutyrate. Most preferably,
the flavin or derivative should be of high purity and
lS contamination with spurious ingredients should be avoided.
In more general terms, the flavin or derivative for use in
accordance with the invention may be defined as a compound
of the formula (I), namely:
X
H~C~N'~ N H ~ I )
0
wherein:
WO95/11028 2 1 7 4 5 5 2 PCT/GB94/02292
OH OH OH 7
X iS--CH2 - C - C-C- CH20H
H H ~ Iriboflavine)
OH OH OH
1 1 1
- CH2--C- C--C--COOH
H H H ~schlzoflavln SFli
OH IH CH
- CH2 - C - C--C CO H
H H H (schizoflavin SF2~
OH OH OH H2 ONa
--CH2--C--C--C--C--O--P ~
~ O H
H H H
(riboflavine-5'-phosphate sodium salt dihydrate)
OH OH
-ribose-O-P-O-P-O-C117
\~H
HO OH
(flavin-adenine dinucleotide)
WO95/11028 PCT/CD~ 2292
21 74552
or CH3 (lumiflavin).
In addition, in the above formula (I) the group X may be
alkyl, or H or an aromatic or other cyclic hydrocarbon
group.
Thus, and furthermore, the use of the invention may be
realised with flavins or derivatives such as:
tA) lumichrome of the formula:
H~C~,N N ~
~C ~ IIII
(B) Roseoflavin of the formula:
y
IHO - C-H~
~N~ o
H~C ~ lIIII
(C) B-Hydroxyflavine, alloxazines and other derivatives
WO95/11028 2 1 74 5 52 P~~ 2292
theref:R
X~~ O
H3C ~ (IV I
wherein
R is ribityl, alkyl, or H;
X is OH, Br, Cl, -SH, OAlk or SAlk.
Some Examples of the above are:
R = alkyl
ribityl
~3c ~
0 (8-hydroxy-FMN)
C2 H5 R=Rib-P-AMP
, ~ ~ ~ (8-hydroxy-FAD)
WO95/11028 2 1 7 4 5 5 2 PCT/GB9~ 2292
C2 H5
CH30~N~N~0
H3C N~NH
H
H0 ~X~
H3C o
H~C~N~NH
wherein R is as above.
(D) 8~-N(3)-histidylflavins
l~N--H2C ~No~O
CH2 H3C 6 NH
H3 N-CH-COO lVI
wherein R denotes the ribityl side chain for the riboflavin
W095tllO28 ~ 1 74 ~ 52 PCTIGB94/02292
derivative.
(E) 8~N(l)-histidylflavins:
5H3N--CH-COO
CH2 R
o N N-H2C tNH
wherein R denotes the ribityl side chain for the riboflavin
derivative.
(F) 8~-Cysteinylflavin thioethers:
(G) 6-S-cysteinylflavin thioethers:
(H) Lumiflavins:
Rl CH~
22 ~N~O lV II I
~4
- wherein Rl=R4=H, R2=R3=CH3 for lumiflavin itself.
(I) 5-Deazaflavins:
WO95/11028 PCT/GB94/02292
2t 7~2
These may be illustrated by the following formula:
Rl--N~--R3
o~N N (Vllll
12
wherein the substituent groups are as defined below:
Rl R2 R3
H CH3 H
H C2H5 H
H n-C3H7 H
H n-C4Hg H
CH3 CH3 H
CH3 C2Hs H
CH3 n C3H7 H
CH3 n~C4Hs H
H CH3 7,8-(CH3)2
H D-ribityl 7,8-(CH3)2
H C2H5 7 CH3
CH3 C2H5 7-CH3
CH3 D-ribityl 7,8-(CH3) 3
and derivatives thereof such as:
W095/1l028 2 ~ 7 4 5 5 2 PCT/GB94/02292
I~N~CH3
o ~N~NI ~\CH3 1 IX I
CHOCOCH3
CHOCOCH3
CH20COCH3
(J) 5-Carba-5-deaza and 1-carba-1-deaza analogs of
riboflavin, FMN, and FAD.
These may be illustrated by riboflavin analogs (X), 5-
15 carba-5-deazariboflavin analogs (XI) and 1-carba-1-
deazariboflavin analogs (XII), that is:
N N ~0
~ N ~ ~ H lx~ ~
~N N~O
J~NH IXI)
WO95/11028 2 1 7 4 5 5 2 PCT/GB94/02292
14
~1~
(K) Flavin 1, N6-Ethyenoadenine dinucleotide
H O
H3C~C~C~,N~C,C~
~C~c`N'c`N'c~
H :-H HC NH
OH HC~C~C~ ~CH
C--O- '-C-'-O-CH2 o
H L H H ~<~ (Xlll~
HO OH
(L) Schizoflavins and derivatives.
FH2OH CHO fOOH
HCOH HCOH HCOH
HlOH HCOH HCOH
HCOH HFOH HFOH
2 5CH2 CH2 CH2
7,8-dimethyl- 7,8-dimethyl-7,8-dimethyl-
isoalloxazine isoalloxazineisolloxazine
Riboflavin SF2 SFl
WO95/11028 2 1 7 4 ~ ~2 PCT/GB94102292
The above are chemical structures of schizoflavins and show
their formation from riboflavin. SF2 and SFl can be
identified as 7,8-dimethyl-l0-(2,3,4-trihydroxy-4-
formylbutyl) isoalloxazine and 7,8-dimethyl-l0-(2,3,4-
trihydroxy-4-carboxybutyl) isolloxazine, respectively.
Other flavins may be illustrated by:
3-carboxymethylriboflavin
3-carboxymethyl FMN
7-amino-l0-(l'-D-ribityl)isoalloxazine
8-amino-7,l0-dimethylisoalloxazine
8~(S-Mercaptopropionic acid) riboflavin
8~(S-Mercaptopropionic acid) FMN
8~(N-Aminohexyl)FMN
9-Azobenzoyl FMN
l0-(~-carboxyalkyl)-7,8-dimethylisoalloxazine
In the use according to the invention the flavin such as
riboflavin, or derivative thereof, is preferably employed
at a high dose level significantly in excess of the doses
currently used or recommended. Thus, typically the
riboflavin or other flavin in the clinical trial is used in
the present invention at a dosage regime of at least about
l to about l00 or more (eg l0 or above) mg/kg of body
weight per day. In addition, use according to the
invention preferably is one wherein the medicament is in
orally administrable form, especially as a capsule (eg two-
part).
WO95/11028 PCT/CB91/~2252
2t ~55~
16
Additionally or alternatively the invention includes a
pharmaceutical or veterinary composition for use in the
management and treatment of viral infections and in unit
dosage form, which composition comprises a unit dose of at
least about 35 mg such as 50mg or more (eg 50 to 300 mg,
such as 50 to 200 or 50 to lOOmg) of a flavin such as
riboflavine or derivative thereof as described or defined
herein, together with a pharmaceutically or veterinarily
acceptable diluent, excipient or carrier.
A composition according to the invention is preferably one
wherein the unit dose is from about 35 mg to about 1000 mg.
More preferably, the unit dose is from about 250 to 500 mg.
In addition, a composition according to the invention is
preferably in oral or injectable form. Within that context
a preferred composition is one as a solution in sterile
water.
The invention also includes a process for the manufacture
of a medicament for use in the management and treatment of
viral infections, which process comprises formulating a
flavin such as ribo1avine, or a derivative such as the
tetrabutyrate salt thereof for anti-viral use.
As will be appreciated, a process according to the above
definition may be carried out using one or more of the
additional features mentioned herein.
WO95/11028 2 1 74552 PCT/GB94/02292
In addition, the invention includes a product containing a
flavin such as riboflavine, or a derivative thereof, as an
anti-viral agent, together with another compound(s) having
anti-viral activity as a combined preparation for
simultaneous, separate or sequential use in anti-viral
therapy.
Again, a product according to the above definition may be
one which includes one or more of the other specific
features of the invention defined herein.
The invention further includes a method for the treatment
of viral infection, which method comprises orally or
parenterally administering an effective amount of a flavin
such riboflavine, or a derivation thereof.
Preferably in a method according to the invention, the
amount administered is l to l00 (eg at least l0) mg/kg of
patient body weight.
Furthermore, the method is particulary useful when the
virus is human immunodeficiency virus, HIV.
Once again, a method according to the invention may include
one or more of the other specific features of the invention
defined herein.
WO95/11028 2 1 74 5 5 2 PCT/GB94/02292
18
Most preferably, the invention is carried out with one or
more of riboflavine, riboflavine sodium phosphate, flavin-
adenine dinucleotide, lumiflavin, lumichrome, or especially
riboflavin tetrabutyrate, whose formula is set forth
below:-
CH2 - O butyrate
H - C - O butyrate
H - C - O butyrate
H - C - O butyrate
H - C - H
H3C
In Vitro Assay
The following in vitro assays were used to investigate the
anti-viral activity against HIV of compounds in accordance
with the invention:-
Acute Infection Assays
1.1 standard Acute Assay
High titre virus stocks of the human immunodeficiency
virus HIV-l (HTLV-lllB) were grown in H9 cells with
WO95/11028 2 1 7 4 5 5 2 PCT/GB94/02292
.
19
RPMI 1640 supplemented 10% fetal calf serum as growth
medium. Cell debris was removed by low speed
centrifugation and the supernatant stored at -70C
until required. In a typical assay, C8166 T-
lymphoblastoid cells were incubated with lOTCID50 HIV-
1 at 37C for 90 minutes and then washed three times
with phosphate buffer saline (PBS). Aliquots of 2 x
105 cells were resuspended in l.Sml of growth medium
in 6ml culture tubes, and test compound at log
dilutions from 0.2 to 200~M was added immediately.
The test compound was dissolved in 70% ethanol and the
final concentration of alcohol in the culture was <1%.
Cultures were incubated at 37C for 72 hours in 5% C02.
200~1 of supernatant was taken from each culture and
assayed by optical density measurement at 450nm for
HIV p24 core antigen (Kinchington et al 1989, Roberts
et al 1990) using a commercial ELISA which recognises
all the core proteins equally (Coulter Electronics
Ltd, Luton, UK). To determine the IC50 values standard
curves were drawn from untreated cultures containing
<1~ alcohol. AZT and ddC were used as internal
controls. Assays were carried out in duplicate.
l.Z Depleted Medium Assay
~ In the standard assay system, cell cultures were
harvested, split and fed with fresh medium
approximately 18 to 24 hours before the start of
WO95/l1028 PCT/GB~ 2292
217~5~2
assay. Addition of fresh medium stimulates the cells
to enter a log phase of growth. To investigate the
effect of cells reaching confluence in conditions of
depleted media, cell cultures were fed and split at
72, 48 and 24 hours before being used in a standard
acute assay.
1.3 Liqht Exposure Assay
A freshly dissolved sample of test compound was split
into two aliquots. They were placed either in
daylight or the dark for two hours before being
subjected to standard acute assay.
1. 4 Preincubation Assay
Target cells were preincubated with test compound at
log dilutions of 200 to 0.2~M for 18/24 hours before
infection with HIV-1. Each sample concentration was
then treated individually as in the standard acute
assay.
2 Assays for Chronically Infected Cells
2.1 Standard Chronic Assay
H9 cells chronically infected with HIV-lrf (H9rf) were
washed three times with medium to remove extracellular
WO9S/11028 2 1 74 5 52 PCT/GB94/02292
virus and incubated with test compounds (200 to 0.2~M)
for three days. p24 antigen was then determined by
optical density measurement at 450nm as described for
the acute infection standard assay. To determine the
IC50 values standard curves were drawn from untreated
cultures containing 1% alcohol. R0 31-8959 (Roche
Proteinase inhibitor) was used as an internal control.
Assays were carried out in duplicate.
2.2 Depleted ~lediuJn Assay
In the standard assay, cell cultures were harvested,
split and fed with fresh medium approximately 18 to 24
hours before assay. Addition of fresh medium
stimulates the cells to enter a log phase of growth.
To investigate the effect of cells reaching confluence
in conditions of depleted media, cell cultures were
fed and split at 72, 48 and 24 hours before being used
in a standard acute assay.
2 . 3 Liqht Exposure Assay
A freshly dissolved sample of test compound was split
into two aliquots. They were placed either in
daylight or the dark for two hours before being
- subjected to standard chronic assay.
W095/11028 2 1 7 ~ 5 5 2 PCT/GB94/02292
3 Toxicity Assay
To test for compound toxicity, aliquots of 2 x 105
uninfected cells were cultured with test compounds at the
same log dilutions for 72 hours (1.1 and 2.1). The cells
were then washed with medium and resuspended in 200~1 of
growth medium containing clg protein hydrolysate. The cells
were harvested after 5 or 20 hours and the c1g incorporation
measured. Untreated cells were used as controls.
The assays were applied to the compounds identified in
Table 1 below:-
Tabl e
Code ComPound
F1 Riboflavine
5'phosphate
F2 Riboflavine
F3 Flavine adenine
dinucleotide
F4 Lumiflavin
FS Lumichrome
F6 Riboflavin tetranicotinate
F7 Riboflavin tetrabutyrate
Initial assays were carried out in relation to the various
compounds mentioned in Table 2 to achieve preliminary
WO95/11028 2 1 7 4 5 5 2 PCT/GB94102292
results. The IC50 results in Table 2 are subject to
confirmation; they are not consistent with re-run assays
conducted to date. Assay results are shown in the graphs
forming the following drawings and in Tables 2 to l0 which
follow:-
Figure l: Antigen as optical density (OD) for Compounds F2,F4 (first antigen assay) and F5 at 450 nm versus
concentration (~M). The dotted line at OD 0.371
represents ICso (active).
Figure 2: Antigen optical density (OD) for Compounds Fl and
F3 at 450 nm versus concentration (~M). The
dotted line at OD 0.371 represents IC50 (active).
Figure 3: Toxicity as C14 uptake (dpm) versus concentration
(~M) for Compounds F2, F3, F4 (first toxicity
assay) and F5. The dotted line at 6035 dpm
represents CC50 (non-toxic).
Figure 4: Toxicity as C14 uptake (dpm) versus concentration
(~M) for Compound Fl. The dotted line at 6035
dpm represents CC50 (non-toxic).
5 Figure 5: Antigen optical density (OD) for Compound F4
(second antigen assay) at 450 nm versus
concentration (~M). The dotted line at OD 0.371
represents ICso (active).
WO95/11028 2 1 7 4 5 5 2 PCT/~91~!~2292
24
Figure 6: Toxicity as C14 uptake (dpm) versus concentration
(~M) for Compound F4 (second toxicity assay).
The dotted line at 6035 dpm represents CC50 (non-
toxic).
Figure 7: Antigen optical density (OD) for Compounds F6 and
F7 at 450 nm versus concentration (~M). The
dotted line at OD 0.371 represents ICso (active).
Figure 8: Toxicity as C14 uptake (dpm) versus concentration
(~M) for Compounds F6 and F7. The dotted line at
6035 dpm represents CC50 (non-toxic).
lS Figure 9: Antigen control (ddC)
As shown by the Tables, the test compounds were evaluated
for activity against cells both acutely and chronically
infected with HIV. Antiviral (IC50) and toxicity (CC50)
data is shown below. In another series of experiments,
compounds were tested in cell cultures in which fresh media
was added at 72, 48 and 24 hours prior to infection. This
experiment was carried out to investigate the effects of
the compounds on cells in actively dividing and quiescent
states. This data indicates that cells may be more
sensitive to the test compounds when quiescent. The effect
of light on stability, preincubation of target cells, and
the activity against an African HIV-l isolate were also
WO95/11028 2 ~ 7 4 5 5 2 PCT/GP~ 2292
investigated. Exposure to light for two hours had no
effect on the activity of the compound. Preincubation with
the target cells enhanced its activity and lt showed
significant activity against the Africa HIV-1 isolate.
Table 2 (Figures 1 to 4) - Acute Infection Standard Assay
(1 . 1)
ComPound No/ IC$o CC50 SI
Assay No (F1qures l and 2) (Flqures 3 and 4)
F1/1 1 to 20 >200
F1/2 <0.4 >400 ~1000
F1/3 0.1 (Figure 2)>800 (Figure 4) >8000
F2 3 (Figure 1)>200 (Figure 3)>60
F3 0.8 (Figure 2)>200 (Figure 3) >200
F4 1 (Figure 1)150 (Figure 3)150
F5 3 (Figure 1)>200 (Figure 3)>60
20 Table 3 (Figures 7 and 8) - Acute Infection Standard Assay
(1 ~ 1)
ComPound Nol
AssaY No IC50 CC50 SI
F7/1 27 (Figure 7)130 (Figure 8) 5
F7/2 57 >200 >4
F7/3 10 70 7
F7/4 25 140 6
WO9S/11028 2 1 7 4 5 5 2 PCT/GB94/02292
Table 4 - Chronic Infection Standard Assay (2.1)
Compound No/
S AssaY NoICso CCso
F7/1 0.2 7 35
F7/2 >20 >20
F7/3 10 ~200 >20
F7/4 4 75 19
F7/5 26 >200 >7
Table 5 - Acute Infection Depleted Medium Assay (1.2)
Compound 72 hours 48 hours 24 hours
No
IC50 CCso IC50 CCso IC50 CCso
F7 10 160 21 100 110 160
Table 6 - Chronic Infection Depleted Medium Assay (2.2)
Compound 72 hours 48 hours 24 hours
No
ICso CCso IC50 CC50 ICso CC50
F7 40 75 90 250 60 101
Table 7 - Acute Infection Light Radiation Exposure Assay
(1.3)
Compound No Dayliqht Darkness
IC50 CC50 IC50 CC50
F7 60 >200 60 >200
Wo95/11028 2 1 7 4 5 5 2 PCT/GB94/02292
Table 8 - Acute Infection Preincubation Assay (1.4)
Preincubation of target cells with test compound for 24
hours before infection
Compound No ICso CCso SI
F7 5 120 24
Table 9 (Figures 5 to 8J - Acute Infection Standard Assay
(1 .1)
ComPound No IC50 CC50 SI
F4 13 (Figure 5) 150 (Figure 6) 12
F6 30 - 60 (Figure 7) >200 (Figure 8) min 3 -6
Table 10 - Acute Infection Standard Assay (1.1)
Assay applied to C8166 Cells fT-lymphoblastoid cells
transformed and immortalized by HTLV) with an African HIV
Isolate (HIV-l CBL4)
ComPound No IC50 CC50 SI
F7 4 150 37.5
Table 11 (Figures 10 to 12J - Acute Infection Standard
Assay (1.1)
Compound No IC50 CC50
F7 32 200 6.3
ddC (control) 0.2
The variation in the end points observed with Compound F7
may be due to the properties of the target lymphoblastoid
WO95/11028 2 1 74~5~ PCT/GB94/02292
28
cells. Even in synchronized cultures there may be subtle
changes in the metabolic state of sub-populations of cells.
This is reflected in the shift in the end points observed
in the paired antiviral and toxicity values from assay to
assay (Table 3). The results tabulated in Tables 5 and 6
indicate that cell culture in active or quiescent states
may have different sensitivities to the test compound.
Patient Treatment
Thirty-five patients were placed on therapy. Thirty had
follow up medical visits.
i) General Condition of the Patients
Twenty patients out of thirty who came for follow-up visits
reported an improvement in their general condition. The
majority of these reported improvement insofar as malaise,
appetite and weight gain was concerned. Two patients also
reported improvement in skin rash with regression of skin
lesions while one reported no new skin lesions developed
while on therapy. One patient also reported improvement in
impotence (which had been present for three months prior to
onset of therapy), while two other patients reported
cessation of long standing coryza.
WO95111028 2 1 7 4 5 5 2 PCT/GB94/02292
29
ii) sick Visits
Few patients attended clinic for unscheduled sick visits:-
1. One patient had recurrent abscesses as well as septicarthritis which persisted even on therapy.
2. Two patients had recurrent lower respiratory tract
infections with one developing frank broncho-pneumonia
during second week of therapy. Repeated smears for AAFBS
have continued to be negative.
3. Two patients had pyrexia with no localizing signs and
repeated blood smear for malarial parasites were negative
and no significant growth on blood culture. One of these
patients responded well to amoxycillin and is now afebrile.
4. One patient had gastroenteritis during the third week
of therapy.
S. Oral and vulvo-vaginal candidiasis were reported by
two patients, with the vulvo-vaginal candidiasis being
recurrent as soon as a course of Nystatin pessaries and
tablets was completed.
6. Two patients also reported recurrent attacks of herpes
simplex genitalis.
WO95/11028 2 1 7 4 5 5 2 PCT/GB94/02292
iii) Toxicity
Most of the cases of toxicity reported occurred during the
first two weeks of therapy and have been transient.
Two patients experienced pruritus which averaged four days
during first week of therapy and cleared spontaneously
without any supportive medication.
Four patients reported moderate diarrhoea during the first
two weeks of therapy. This has averaged four days. This
has been a difficult symptom to attribute as between it
being a side effect or a natural manifestation of the HIV
infection. However, the consistency of its appearance in
the first week of therapy, and its transient nature makes
it reasonable to suppose it is a side effect.
One patient reported drowsiness and another reported
darkening of her urine. MSU was normal.
Two patients reported abdominal discomfort.
iv) Laboratory Results
Three patients had transient rises in liver enzymes during
the second to third week of therapy, with no clinical signs
of liver disease. However, the enzyme levels always
returned to normal.
W095/11028 2 1 7 4 5 5 2 PCT/GB94/02292
The above clinic trial reports are the preliminary results
of a clinical trial which has currently been in progress
for several weeks using Compound F7 administered orally in
capsule form (the capsules are as described in Example 4
below) dosage was:-
Dose level 1: lmg/kg body weight per day orally in twodivided dosages
0 Dose level 2: 2mg/kg body weight per day orally in two
divided dosages
Dose level 3: lOmg/kg body weight per day orally in two
divided dosages
Dose level 4: 15mg/kg body weight per day orally in two
divided dosages
Dose level 5: 20mg/kg body weight per day orally in two
divided dosages
Dose level 6: 30mg/kg body weight per day orally in two to
three divided dosages
Dose level 7: 40mg/kg body weight per day orally in two to
three divided dosages
Dose level 8: SOmg/kg body weight per day orally in two to
three divided dosages
Dose level 9: lOOmg/kg body weight per day orally in two
- to three divided dosages
The following specific Examples illustrate compositions
formulated in accordance with the invention:-
WO95/11028 2 1 7 4 ~ S 2 PCT/GB94/02292
Example 1
A formulation can be prepared from the following:
riboflavine-5-phosphate 10 mg
sterile water 2 ml
to provide a unit dosage of 10 mg of riboflavine for
administration once per day in the treatment of viral
infection.
Example 2
A formulation can be prepared from the following:
riboflavine-5-phosphate 30 mg
sterile water 2 ml
to provide a unit dosage of 30 mg of riboflavine for
administration once per day in the treatment of viral
infection.
Example 3
Similar formulations to those of Examples 1 and 2 can be
prepared at doses of:
10 mg per ml,
-W095/11028 2 1 74 5 52 PCT/GB94/02292
-
25 mg per ml, and
50 mg per ml
respectively, in either a unit amount of 2 ml or 5 ml of
sterile water and based on an active ingredient which is:
Riboflavine 5'phosphate
Riboflavine
Flavine adenine dinucleotide
10Lumiflavin
Lumichrome
or a mixture thereof.
Exam~le 4
The following capsules were formulated:-
Sizes: 25mg
50mg
lOOmg
20Omg
40Omg
Type: 2-part hard gelatin
Composition: Compound F7 in admixture with
microcrystalline cellulose Ph. Eur
WO95/11028 2 1 7 4 5 5 2 PCT/GB94/02292
34
166.4/156.7/118.6/108.7/50mg to give capsule
weights of 191.4/206.7/218.6/ 308.7/450mg.
