AP620A

AP620A - Uses of a flavin in the treatment of ill-effects caused by viral infections.

Info

Publication number: AP620A
Application number: APAP/P/1994/000695A
Authority: AP
Inventors: Ayuko Washington Odur Dr
Original assignee: Radopath Ltd
Priority date: 1993-10-19
Filing date: 1994-10-19
Publication date: 1997-10-14
Also published as: JO1866B1; CZ113796A3; PE5596A1; CO4520283A1; NO961547L; HUT76322A; AP9400695A0; EE9600057A; AU7943794A; OA10579A; NO961547D0; MA23356A1; BG100599A; EP0739208A1; GB9321558D0; MD960168A; HRP940688A2; IL111338A0; GB9421099D0; JPH09505804A

Abstract

Various flavin derivatives are disclosed for administration to mammalian subjects as an anti-viral agent. Riboflavin and riboflavin derivatives are given as particular examples which may be preferred.

Description

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 KIV. A detailed study of compounds according to the invention has been carried cut in terms of their efficacy againsr infection frcm several strains of Kiv-i. The compounds have broadly uniform activity against both acutely and chronically infectad cells. This is a dual property not associated with other compounds which are in current use although some agents against acute KIV infection have de minimis activity against chronic infection. A.2T, for example, acts as a reverse transcription. blocker in the ce.nstic activity cf the virus intracellularlv. The druc addresses acute infections of

KIV, and can prevent the onset of chronic infection, but has no effect cn calls which are airaadv chronically

AP/P/ »4/00695

KIV is an RiVA. virus which affects the mammalian species. In humans, tha virus attaches tc cell membranes by virion adsorption tc CD4 surface antigsr.. The virion then passes through the cell membrane penetratively anc enters the cell cytoolasm. t'nccatinc cf the virion than takes place in. the cytoplasm whereby the viral envelope and the protein coat cf the genome release the vira] R>i.-. into the cytoplasm. Reverse transomiction therein crcduces a double-stranded

AP. 00620

DNA transcript iron best cell genetic material. This invades the host cell nucleus and integrates with the host cell chromosomal DNA. Transcription follows to produce a vP.NA replicate which precesses from the host cell nucleus and translates in the cytoplasm to produce viral protein. The latter then assembles to the vRNA. replicate whereafter the assembly matures to produce a complete viral replicate which penetrates the cell membrane to exit the host cell.

HIV is normally associated with an initial period of asymptomatic infection. The virus enters the bloodstream but dees net penetrate the membranes cf cells within the body and thus also does net replicate. This initial asymptomatic phase may last a number of years. Acute infection then normally follows, ths virus penetrating the cell membranes and' interfering with cellular metabolism. Chronic infection may then follow in a matter cf hours.

tr.

cn <r c?

a

AF/P/⁹ *

Viral infection changes metabolism, and unless erad in cell death. Apoptosis is form of programmed cell physiological and pathologies processes which sea?, to main oxidant-antioxidant balance, closely associated with this with HIV is thought gradual favour of cell death.

many aspects cf cellular icated will ultimately result, a morphologically distinctive death involved in many processes including cellular

tain appropriate intracellular
Call death in	T-cells is
balancing process.	Infection
ly to disturb the	balance in
Another critical	factor in

in a normal

-² AP. 0 0 6 2 0 determining whether cell . will grow and divide fashion is intracellular ATP concentration. Low intracellular levels of ATP are associated with ischemic death. T-lymphocytes are ' especially vulnerable to depletion of intracellular AT? levels. HIV infection imposes a viral negative stimulus on cellular oxidative phosphorylation which is the cellular process ' responsible for AT? levels in the call. Csill ceatn and failure to divide, from whatever cause, will eventually lead to cell depletion to a level that induces AIDS.

Much of 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 only a limited effect on normal cellular precesses. The long term use of known compounds for use in HIV infection treatment has not given the degree of benefit initially expected, and new approaches are needed.

Ribcflavine is a known compound, which is also variously known as:

E101; ' ···

Lactoflavin;

Riboflavin;

P.ibof lavinum; vitamin c₂;

Vitamin G;

7,3-Dimethy1-10-(1'-D-ribityl) isca11 oxazine ; and

3,lO-Dihydro-7,S-dimethyl-10-(D-ribc-2,3,4, hydroxypcntyl) benzopteridmc-2 , 4 -clone .

AP/P/ 94/00695

5-tetra4AP.00620

Riboflavine is commercially available as such or as its sodiun phosphate or t^trabutyrate. salt, typically in the former instance as the dihydrate salt. It is also •available in various mixtures with other vitamins, all essentially being tor 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 l.S mg cf riboflavine is recommended that is to say the basic recommended intake of riboflavine is 550 μς per <200 kj (1000 kcal) cf diet - Report cf a Joint FAO/WHO Expert Group, Tech. Rep. Ser. Vid 111th Org. No. 352, 1S57.

O’ c* c>

o’ c*

The estimated acceptable daily intake cf riboflavine is up 20 to 500 pg per kg body weigh! - see Thirteenth Report of

FAO/VHO Expert Committee cn feed Additives, Tech. Rep. Ser. WHO . No . 4 *i 5 , 19 71.

Riboflavine, which is a water-soluble vitamin, is essential 25 fcr the utilisation of energy from food. The active, chosphorylated forms, flavine mono-nucleotide and flavine adenine dinucleotide, ar involved a; cc-erW/n.s:

in

AH . 0 0 6 2 0 oxidative/reductive metabolic reactions.

Various other flavins id derivatives thereof are also know, mainly as flavouring agents.

It has new 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 compcunc indicates involvement in tne process of oxidative phosphorylation within cells. It is possible that tne compounds of the invention preferentially tercet the same target as HIV and so resist cr prevent the manifestations cf infection including ths procreative capacity cf the virus.

Acccromgly, the present invention in one aspect previces 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.

is ϊ*· Ο ΪΓ O ^- 2 — _t ίϊ·3θί 21* £2 C 2 ΰ ¹ p if ^ ^* '·** J, ** 3 * —, thereof are net know- as pharmaceuticals, even in a general sense as with riboflavine (known as an enzyme ce-facter vitamin), the invention in a second and broader ascsct provides such certain flavins o* cerivat'teof fr use as anti-viral ace.nts.

If*

AP/P/ 9 4 / 0 0 69

If· tne use a co or-mg to tm invent, ion riboflavine cr ether flavin may be used an such cr as a derivative and the flavin derivative may fce anv derivative wh > ch is safe for human or animal use. Prefer a b’v h c w p v e — 'n the case c r riboflavine ths 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 contamination with spurious ingredients should be avoided.

AP.0 0 6 2 0

In more general terms, the flavin or derivative for use in accordance with the invention may bo defined as a compound of the formula (1), namely: ξ

ch₂

OH OH OH I I I - c - c—c I I I Η Η H

OH OH OH I i I

X is -CH_?-C-C-C-CH₇OH III ^L , ..

Η Η Η (rιbofίavme)

-COH (schizoflavin SF₂J

OH OH OH

- CH₂ — c-c —C —COOH

Η Η H (schizof lav in SFJ

OH OH OH Ho 0 ! I i f H —ch₂ — c—c—c—c - 0—P

ONa

OH

Η Η H

AP.00620

(Ib) m* (flavin-adenine dinucleotide)

or CHj {lur.if levin) .

		&
In addition, in the above formula (I)	the group χ may be	£
alkyl, or Ii 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:

(A) lur.ichroGie cf the forr.ula:

(III

AP.00620 (3) Roseoflavin of the formula:

where in s 6 9 i) 0 / * 6 /d/dV

R is ribitvl, alkyl, or E;

X is OH, Br, Cl, -SE, OAlk cr

Some Examoles of the above are:

R = alkyl ribitvl £

cr rib-P

AP.0 0 6 2 0 (8-hydroxy-FMN)

R-Rib-P-AMP (3-hydrcxy-FAD) cA

AP/P/ 94/00695

wherein ?. is as above.

AP.00620 (D) 8α-Ν(3)-histidylflavin;

H₃ H-CH-COO (V) wherein P. denotes the. ribityl side chain for the riboflavin derivative .

(E) ScN(1)-histidylflavins:

derivative .

(Vl)

AP/P/9 4 / 0 0 6 9 5 (?) Sc-Cysteinylf 1 avin thioethers:

(C-) 6-S-cysteinylflavin thioethers:

AP.00620 (H) Luraiflavins:

(Vila) c

(I) 5-Deazaflavins:

These ray be illustrated by the folloving formula:

c o

- 2 0

R₂ 1 wherein the substituent groups ere as defined belov:

AP/PZ 9 4/ 0 0 69 5

CE.

n-C-E_z

CHJ n-C^H,

CHCH, ^C2^H5

P?

H

E

Ii

CH.

J n-Cj!i_? ¹¹ AP . 0 0 6 2 0

CK₃ K	n-C_tH, Cn₃	H 7,2-(CH₃)
rl	D-ribity1	7,8-(CHj)
H	C ^ve_r._s	7 Ctl, J
ch₃	c ^vu_r·.	7-c:-;₃
ch₅	D-ribityl	7,S-(Cii₃)

and derivatives thereof such as:

(J) 5-Carba-5-deaza riboflavin, FI-ίΝ, (IX)

AP/P/ 9 4 / 0 0 6 9 5 ch₂ococh₃ and l-carba-l-dea. za analogs and F.-.D.

of

These may be illustrated by riboflavin analogs (X), 5carba-5-deazariboflavin analogs (XI) and l-carba-1deazaribof lavir. analogs (XII), that is:

0 6 2 0

I ί

(Xll (Xll) (K) Flavin 1, N⁴-Et.hyanoadsnina dir.ucleotide

H h₃i: ’

P/P/ 9 4/ 00695

HqC ^{1 11} H

I II I I /^cv^c

'4AP .00620

AP/P/ 9 4/ 0 0 6 9 5 ¹⁵ AP . 046 2 Ο (I.) Sc'nizoflavins and derivatives.

	CH2OH \|	CHO j	COOH 1
	1 HCOH I	HCOH I	1 HCOH
5	1 HCOH J	1 - HCOH I	1 HCOH
	ί HCOH I	1 HCOH \|	1 HCOH t
	1 ch₂ Ϊ	1 CHj I	1 CK₂
	7,8-dinethyI-	1 7,8-dimethyl-	7,8-dimethy1-
	isoalloxazine	isoalloxazine I	isolloxazine I
10	j Riboflavin	1 sf₂ .	1 SF,

The above are chemical structures of schizoflavins and show their formation from riboflavin. SF2 and SF, can be identified as 7,8-dimethvI-lo-(2,3,4-trihydroxv-415 formylbutyl) isoalloxazine and 7,8-dimethyl-10-(2_z3,4~ trihydroxy-4-carboxybutyl) isolloxazine, respectively.

o

Other flavins nay be illustrated by:

c .20 3-oarbcxymethylriboflavin

3-carboxymcthy1 FMN

7-a-inc-10-(l'-D-ribityl)isoalloxazine 2-anino-7,10-dimcthy1isoallcxazine So(S-Mercaptoprcpionic acid) riboflavin

So(S-Mercsptopropionic acid) FMN

S a (N-.-.minchexy 1) FMN 9-Azobenzcyl FMN i i - ((..•-carboxy a Iky 1) -7 , 8-dimet.hylisoa 1 loxazine

AP/P/ 9 4 / 0 0 6 9 5

I&

AP.00620

In the use according to the invention the flavin such as riboflavine, or derivative thereof, is preferably employed at a high dose level significantly in excess of the cosas currently used cr recommended. Thus, typically the riboflavine or ether flavin is used in the present invention at a dosage regime Oj. at leas^ abcu<. 1 to abouk.

100 to more (eg i₀ 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 acministrable form, most preferably capsule form.

Additionally or alternatively the invention includes a pharmaceutical cr veterinary composition for use in the management and treatment cf viral infections and in unit dosage,.form, which composition comprises a unit dose of at least about 35mg, for example, 50mg or mere (eg 50 to 300mc, such as 50 to 200mg or 50 to lOOmg) iboflavino or derivative therecr

94/00695 cf a flavin such as r _2S described cr defined herein, o- v=tnrir.a ril'·' acceptable diluent, excipisnt or carrier locetr.er wit.n a

0. a pharmaceutically

A composition aooording to the invention is preferably one wherein trie unit cose is from about 35 mg to about 1000 mg. More crszerably, the unit dose is iron, about 250 tc 500 me.

ir, addition, a co-position according to the invention is ./ oral or

F-^ej-^ebu-.y in/ injectable form. Within that context a preferred composition, is cne as a solution in sterile .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 ribof lavir.e , or a derivative thereof for anti-viral use.

/7

Ar. m ζ o

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.

Tn addition, the invention flavin such as riboflavine, anti-vj.ral agent, together anti-viral activity as simultaneous, separate or therapy .

Again, a product according one which includes cna cr features of the invention defined herein.

includes a product containing a or a derivative thereof, as an with another compound(s) having a combined preparation for sequential use in anti-vjral to the above definition may be more of the ether specific

S 6 9 0 0 / * 6 /d/dV

The invention further includes a method for the treatment of viral injection, vhich method comprises parenterallv administering an effective amount of a flavin sucn riboflavine, or a derivation thereof.

Pre f erably in amount admin is a method according to the invention, the ,1 to 100 (eg , . _ hn^v ered is'at least ιΰ)mg/kg c: pa<-ien- bcc_ weight.

Furthermore, the method is particular;/ useful when the virus is human immunodeficiency virus, KIV.

AP . Ο ο 6 2 ο

Once again, a method according to the invention may include one or more of the other specific features of the invention defined herein.

Mont preferably, the invention is carried out with one cr mere of ribofiavine, ribofiavine sodium phosphate, flavinadenine cinucleotide, lumiflavin, lumichrome, or especially riboflavin tetrabutyrate, whose formula is set forth below:CH, - 0 butyrate

H - C - O butvrate I

H - C - 0 butyrate

Jn Vitro Assav

AP/P/ 9 4 / 0 0 6 9 5

The following in vitro assays vers used to investigate the anti-viral activity against HIV of compounds in accordance with the invention:19

AP.00620

Acute Infection Assavs

1·2 Standard Acute Assay

Kich titre virus stocks of the human immunodeficiency virus KIV-l (HTLV-lllB) were grown in H9 cells with

?.??!! 1S40 supplemented 10% fetal calf serum as growth medium. Cell debris was removed by low speed centrifugation and the supernatant stored at -70’C until required. In a typical assay, C3166 Tlymphcblastoid cells were incubated with 10TCID_<0 KIV1 at 37 °C for 90 minutes anci then washed three times , t with phosphate buffer saline (PBS). Aliquots of 2 x 10^s cells were resuspended in 1.5ml of growth medium in 6ml culture tubes, and test compound at log dilutions frcm 0.2 to 200μ^:< was added immediately.

The test compound was dissolved ih 70% ethanol and the final concentration cf alcohol in the culture was 1%. Cultures were incubated at 37^aC for 72 hours in 5% CC₂. 200pI of supernatant was taken frcm each culture and assayed by optical density measurement at 450nm for HIV p24 core antigen (Xinchincton et al 1539, Roberts et al 1990) usinc a commercial ELISA which recognises all the core proteins equally (Coultar Electronics Ltd, Luton, LX). To determine the IC_5Q values standard curves were drawn from unti'eated cultures containing 1% alcohol. A2T and ddc were used as internal controls. Assays were carried cut i.n cupl icate.

AP/P/ 94/00695

AP.00620 i .2 Deolet&d Mecium Assay

In the standard assay, cel] cultures were harvested, split ana fed with fresh medium approximately 25 hours before assay. Addition of fresh media stimulates the cells to enter a new cell cycle. To investigate the effect of ceils reaching confluence in conditions of depleted media, cell cultures were fed and solit at 72, 4c ar.d 24 hours before be:ing subjected to standard acute assay.

I .3 Light P.sciztior, Lxcosure Assay

A. freshly	dissolved sample of tesi	t compound	was	split
into two	aliquots. They were	placed	eiths	r in
daylight	or the dark for two	hours bed	ore	being
subjected	to standard acute assay.

6 9 0 0 / V 6 /d/dV

7.4 Preincubetion Assav »

Target ceils were prsir.curaf.ee with test compound at loc dilutions cf 200 ~o 0.2u>i for 18/24 hours before infection with HZ7-1. Each sample concentration was then treated individually as in the standard acute assav.

2. i

AP. 00620

Assays for Chronically Infected Cells

2.J. Standard Chronic Assay

H9 cells chronically infected with the EIV-lrf (H9rf) were washed three times with medium to remove extracellular virus and incubated with test compound (2C0 to 0.2μΜ) for three days. p24 antigen was then determined by optical density measurement at 450nm as in the acute infection standard assay. To determine

the IC_S0	values	standard	curves were era	w'n frem
untreated	cultures	containing	Γί alcohol. RO	31-SS59
(Roche Pr	ote ir.ase	inhibitor)	was used as en	internal
control.	Assays were carried	out in duplicate.

1t

2.2 Depleted Medium Assav

In the standard assay, cell cultures were harvested, split and fed with fresh medium approximately 25 hours before assay. Addition of fresh media stimulates the cells to enter a new cell cycle. To investigate the » effect cf cells reaching confluence in conditions of depleted media, cell cultures were fed anc split at 72, 43 and 24 hours before being subjected tc standard

AP/P/ 94/00695 acute assav.

12.

AP.00620

Ljg.ht P.adistzon Exposure Assay

A freshly into two daylight subjected dissolved sample of test compound was solit aliquots. They were placed either in or the dark for two hours before being to standard chronic assay.

2. < Preincubation Assay

Target cells were preincubated with test compound at log dilutions cf 200 to 0.2μΜ for 1S/24 hours before infection with HIV-1, . Each sample concentration was then treated individually as in the standard chronic assav.

Toxicity Assav

AP/P/ 9 4 / η o

Tc test for compound toxicity, aliguo	OS Of 2 X 10^s
uninfected cells wsre cultured with test	compound at the
same log dilutions for 72 hours. The	cells were then
washed with medium and resuspended in	200ul cf grower·
5 Λ medium conasinine C ’ protein hydrolysate.	The cells were

4 harvested after IS hours and the C ’ incorporation measured. >

Untreated cells were used as controls.

The assavs ware aocliec to the compounds identified in

Table 1 below:13 ΑΡ.00620

In vitro Studies

Table l © 10

Code Compound

Fl Riboflavine 5'phosphate

F2 Riboflavine

F3 Flavine adenine dinucleotide

F4 Lumifiavin

F5 Lumichrome

FS Riboflavin tetrar.icot inate

F7 Riboflavin tetrabutvrat«

Initial assays were carried out in relation to the various compounds mentioned in Table 2 to achieve preliminary results. The IC_c_ results in Table 2 are subject to confirmation; they, are not consistent witn re-run assavs conducted tc date.

Assay results are hewn in the graphs fermin the follcwinc

AP/P/ 9 4/ 0 0 6 9 5 drawings and in Tables 2 to 10 which folio'.-;:1/4ΑΡ , 0 0 6 2 0

Figure

F igure e

rigure

Figure 4 l: Antigen as optical density (CD) for Compounds F2,

F4 (first antigen assay) and F5 at 450 nm versus concentration (μΜ) · The dotted line at OD 0.371 represents IC_5Q (active).

Antigen optical density (OD) for Compounds Fi and F3 at 450 nm versus concentration (μΜ.) . The dotted line at OD 0.371 represents IC_5Q (active).

Toxicity as C¹’ uptake (dpm) versus concentration (μΜ) for Compounds F2, F3 , F4 (first toxicity assay) and F5. The dotted line at 6035 dom represents CC₅₀ (non-toxic).

Toxicity as c¹' uptake (den) versus concentration (μΜ) for Compound FI. The dotted line at 6035 com represents CC₅₀ (non-toxic).

AP/P/ 9 4 / 0 0 6 9 5

Antigen	optica1	density (CD) for	Compound F4
(second	antigen	assay) at 450	nm versus
concentr	atior. (μΜ	) . The dotted line	at CD 0.371

represents IC_5C (active).

Figure 6:

Toxicity as C¹’ upcake (μΜ) for Compound F4 The dotted line at 603 (dcra) versus concentration (second toxicity assay).

don represents CC θ (non toxic).

Figure 7: Antigen optical density (OD) for Compounds FS and

F7 at 450 nm versus concentration (μΜ) . The dotted line at OD .0.371 represents IC_5Q (active).

U •Z

ΑΡ.00620

Figure 2: Toxicity as C¹ uptake (dpm) versus concentration (μΜ) for Compounds F6 and F7. The dotted line at 6035 dpm represents CC_SQ (non-tcxic).

Figure 9: Antigen control (ddC)

As shown by the Tables, the test compounds were evaluated for activity against cells both acutely and chronically ir cr.

infected with HIV. Antiviral (IC₅₀) and toxicity (CC

-so^J exoerim.ent was carried cut to investigate the efface:

^AP/p/94/oo_fi data is, shewn below. In another series of experiments, compounds were tested in cell cultures in which fresh media was added at 72, 4S and 24 hours prior to infection. This cf the compounds cn cells in actively dividing and quiescent states. This data indicates that cells may be more sensitive to the test compounds when quiescent. Tne efrecc cf light cn stability, preinoubaticn cf target cells, and the activity against an African KIV-l isolace were also investigated. Exposure to light, for two hours had no effect on the activity of the compound. Preinoubaticn with the target cells enhanced its activity and it showed sicnificant activity acainst the Africa KIV-1 isolate.

AP. Ο Ο 6 2 Ο

Table 2 (Ficures 1 to 4} - Acute Infection Standard Assay (1.1)

Conoound Assav No	No/	— 50 (Fxcu	ires 1 and '2)	-=50 (Fioures 3 and 4)	SI
Fl/1		1 to	20	>200	-
Fl/2		<0.4		>400	>1000
Fl/3		0.1	(Figure 2)	>200 (Figure 4)	>3000
F2		u	(Figure 1)	>200 (Figure 3)	>60
F 3 *		0.8	(Figure 2)	>200 (Figure 3)	>200
F4		1	(Figure 1)	150 (Figure 3)	150
F5		3	(Figure 1)	>200 (Figure 3)	>60
Table 3	(Figures 7	and 8) - Acute	Infection Standard	Assay
			(1-1)	f
Conoound	No /
Assav No		^ICSO	CC s *
F7/1		27 (	Figure 7) 130	(Figure 8) 5
F7/2		57	>200	>4
F7/3		10	70	7
F7/4		2 5	140	6

AP/P/9 4/ 0 0 6 9 5

Table 4 - Chronic Infection Standard Assay (2.1)

Conoound No/	1^50	^50	ΞΙ
Assav No
F7/1	0.2	7	35
F7 /2	>20	>20
F7/2	10	>200	>20
F7/4	4	7 5	IS
F7/5	26	>200	>7

AP.00620

Table _5 - Acute Infection Depleted Medium Assay (1.2)

Compound 72 hours 48 hours 24 hours

No -

—50	CC —so	i£so ££50	£S₅₀
F7 10	160	21 100	lio 160
Table .6’- Chronic Infection Depleted Medium	Assay (2.2)
Compound 72	hours	48 hours	24 hours
No
^50	CC —50	^50 ^50	^50 ££SO
F7 40	75	90 250	60 101

Table 7 - Acute Infection Light Radiation Exposure Assay (1-3)

Compound No Davlioht

Darkness

F7 ^SQ —50 >200

CC —50 >200

S 6 9 0 0 / > 6 /d/dV

Table S - Acute Infection Preincubation Assay (1-4)

Preincubation of target cells vith. test compound for 24 hours before infection

Compound No IC₅₀ —so —

F7

120

Table 9 (Figures 5 to 8) - Acute Infection Standard Assay α-i) :

□β ΑΡ.00620

Comoound	^No IC₅₀	CC —50	SI
F4	13 (Figure 5)	150 (Figure 6)	12
F6	30 - 60 (Figure 7)	>200 (Figure S)	min 3 -6

Table 10

Acute Infection Standard Assay (1.1)

Assay applied to C8166 Cells (T-lymphoblastoid cells trans formed and immortalized by F.TLV) with an African Kiv Isolate (HIV-1 C5L4)

Comoound No IC.-₀

CC so

SI

F7

150

The variation in the end points observed with Compound F7 nay be cue to the properties cf the target lynphoblestoid calls. Even in synchronized cultures there may be subtle changes in the metabolic state cr sub-populations cf 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 cuiescent states mav have different sensitivities to tha test compound.

AP/P/ 94/00695

AP . Ο Ο 6 2 Ο

Patient Treatment

Thirty-five patients were ..placed on therapy. Thirty had follow up medical visits.

ί) General Condition of the Patients ζ*

12. Η

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 (vhich had been present for three months prior to onset of therapy), while two other patients reported cessation of long standing coryza.

ii) Sick visits

Few pa ts attended clini for unscheduled sick visits·.AP/P/ 9 4/ 0 0 69 5

1. One patient had recurrent abscesses as well as septic arthritis which persisted even on therapy.

2. Twc patients had recurrent, lower respiratory tract infections with cne developing frank trcr.cho-pneumcnia

Repeated smears for AAF5S during second week cf therapy.

so ΑΡ. 0 0 6 2 0 have continued to be negative.

3. Two patients had pyrexia with no localizing signs and repeated B/S for MPS -ve and no significant growth on blood culture. One of these patients responded well to araoxvl and is now afebrile.

4. One patient had gastroenteritis during the third week of therapy.

5. Oral and vulvo-vagina 1 candidiasis were reported by two patients, with the vulvo-vacinal candidiasis being recurrent as soon as a course of Nystatin pessaries and tablets was completed.

tt

6. Two patients also reported recurrent attacks of herpes simplex genitalis.

iii) Toxicltv

Most of the cases cf toxicity reported occurred during the first two weeks cf therapy and have been transient.

Two patients experienced pruritus which averager four cays durinc first week of therapy and clearer spontaneously without any supportive medication.

ΛΡΙΡΙ94/00695

Four patients reported moderate diarrhoea curing the firs

AP. Ο Ο 6 2 Ο 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. HSU was normal.

Two patients reported abdominal discomfort.

iv) Laboratory Results

Three patients had transient rises in liver enzymes during the second tc third ’week of therapy, with no clinical signs of liver disease. However, the enzyme levels always returned to normal.

AP/P/ 9 4 / 0 0 69 5

In addition, the results were clotted as relevant graphs and reference is made tc the accompanying drawings which :

in

F’gur*³ 1 i« a graph of optical density OD at 450n«. ^vscde named F2, F4 and concentration in for the compounds c F5 against antigen (Ag). The dotted line a t CD 0.37 recressnts I<

'SC'

Ficure 2 is a similar gra;

i-ure 1 for the compounds ash to ngu

AP.00620 code named fl and F3;

Figure 3 is a graph of C^!‘ uptake in dpm vs. concentration in μΜ for the compounds code named F2 to F5 against toxicity. Again, the dotted line is IC_5(J and the compounds are clearly all non-toxic; and

Figure 4 is a similar graph to Figure 3 for the compound code named FI. Again, clear non-toxicity is demonstrated.

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 * /	leve 1	1:	lmg/kg body weight per day orally in two divided dosages
Dose	levs 1	2 :	2mg/kg body weight per day orally in two divided dosages
Dose	level	O . o ·	lOmg/kg body weight per cay orally in two divided dosages
Dose	level	4 :	15mg/kg body weight per day orally in two divided dosages
Dose	level	5 :	20mg/kc body weight per day orally in two divided dosages
Dose	level	6:	30mg/kg bcdv 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 cosages
Dose	level	£ :	50mg/kg body weight per day orally in two to three divided dosages
Do s e	level	9 :	IOOmg/kc bcdv weight per day orally in two to three divided dosages

6 9 0 0 / V 6 /d/d¥

The following specific	Examples	illustrate compositions
formulated in accordance	with the	invention.

ΑΡ. Ο Ο 6 2 Ο

Example 1

A formulation can be prepared from the following:

ribof lavir.e-5-phosphate sterile vater mg ml co provide a unit dosage of 10 mg of riboflavine for administration once per day in the treatment of viral infection .

• υχ

Example. 2

A formulation can be prepared from the following:

riboflavine-5-phosphate sterile vater

0 mg ml to provide a unit dosage of 30 mg of riboflavine for administration once cer day in the treatment of viral infection .

Example 3

Similar formulations to those of Examples 1 and 2 can be prepared at doses of:

AP/P/ 94/00695

10	10	mg	per	ml,
	2 5	mg	per	ml, and
	50	mg	per	ml

34AP. Ο Ο 6 2 Ο respectively, in either sterile water and based a unit amount of 2 ml or 5 ml of on an active ingredient which is:

P.iboflavine 5'phosphate Riboflavine

Flavine adenine dinucleotide

Lumiflavin

Lumichrcme or a mixture thereof.

Example 4

The following capsules were formulated:Sizes: '> 2 5mg □ Cmg lOOr.o 2 00mg 4 OCmg

Type: 2-parr hard culatin

Composition: Compound F7 in admixture with microcrystalline cellulose ?h. Eur 1SS.4/15S.7/113.6/103.7/5Cmc to give capsule weights cf 191.4/206.7/21S.6/ 3 03. >/4 oOng .

The compounds have similar activity against Kiv in both acutely and chronically infected cells. This is a dual property only occasionally associated with other compounds which are in current use although denova (acute) infections of cells may be blocked by compounds which act early in the replication cycle of KI^V snd

AP/P/ 9 4/ 0 0 69 5 consequently block integration of vDN.a into the host chromosome (this is the·chronic stats). Compounds which act post-integration cf KIV are therefore inhibitors cf chronically infected cells. Zidovudine (AZT) for example is only active against denova infection of KIV and has no significant activity against chronically infected culls. Inhibitors of gene expression cf KIV (which is a positive strand RNA virus) would therefore be active in κίν chronically inferred cells.

A number of ideas causing Apoptosis is one of these.

cell death are proposed. 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 associe.ted 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 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.

As will be appreciated, the invention is net limited to the specific details exemplified above and numerous variations and modifications may be made within the spirit and scope of the claims which follow.

Claims

1. Use of a flavin for the manufacture of a medicament for the treatment by 5 prophylaxis or therapy of disease caused by HIV, the flavin being one of those listed below or a mixture of two or more thereof: lumichrome; roseoflavin; a 8-Hydroxyflavin, alloxazine a derivative thereof; a 8aN(3)-histidylflavin; a 8a-N(l)-hystidyl flavin; a 8a-cysteinyl thioether, a 6a-S10 cysteinyl thioether; a lumiflavin; a 5-deazaflavin; a C-carba-5-deaza or 1-carba-1deaza analog of riboflavin, FMN or FAD; flavin-1, N^e-ethenoadenine dinucleotide; a schizaflavins; 9-methylflavin; 9-phenylflavin; 9-benzylflavin; 9-cyclohexylflavin;

6.9- dimethylflavin; 6,7,9-trimethylflavin; 9-oxyethylflavin; 9-dioxypropylflavin;

6.8.9- trimethylflavin; lacroflavin; flavin-9-carboxylic add; 6,7-dimethylflavin-915 carboxylic acid; riboflavin tetrabutyrate.

2. Use according to Claim 1 at a dosage regime of at least about 10 mg/kg of body weight per day.

20

3. Use according to Claim 1 or Claim 2 wherein the medicament is one in injectable form.

4. Use as claimed in any one of Claims 1 to 3 wherein the flavin is formulated to the form of a composition which comprises a unit dose of at least

AP/P/ 94/00695

AP.00620 about 35 mg of flavin together with a pharmaceutically or veterinarily acceptable diluent, excipient or earner.

5. Use as claimed in Claim 4, wherein the unit dose is from about 35 mg to 5 about 1000 mg.

6. Use as claimed in Claim 5 wherein the unit dose is from about 250 to 500 mg.

10 7. Use as claimed in any preceding claim wherein the flavin is formulated to the form of a composition in the form of a solution in sterile water.