OA10579A

OA10579A - Flavin derivatives as anti-viral agents

Info

Publication number: OA10579A
Application number: OA60815A
Authority: OA
Inventors: Washington Osur Ayuko
Original assignee: Radopath Ltd
Priority date: 1993-10-19
Filing date: 1996-04-18
Publication date: 2002-06-19
Also published as: HRP940688A2; AP9400695A0; ZA948191B; NO961547D0; MD960168A; CO4520283A1; AU7943794A; EE9600057A; BG100599A; CZ113796A3; AP620A; IL111338A0; HU9601006D0; NO961547L; CA2174552A1; HUT76322A; GB2283913A; PE5596A1; CN1140992A; CA2123825A1

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 présentthair use in alleviate ex infection, es

FLAylN bfeCV^TIYES ΛδΑΝΤί-Υ·*? AL AGENTS invention 4o .anti-viral a^gitts <Ma"^ the treatment of human and animal patients 4ocure the ill-effects caused' by viral^ecially HIV, A detailed study of compounds dccording to :he invention has been carried out to evaluatetheir etficacy against infection from sovcral strains ofompounde hâve cimilar activity against HIV inand chronically iufecued cells. Tftis is aonly ocassionally associated uith other HTV-i, The cboth acutclydual propei'tÿ compounds which are in current use in the therapy of Hiv

inrection altl·be treated bycycle of HIV chromosome, ough de nova (acute) infections of cells maycompounds which act early in the réplicationto bloch intégration of vDNA into the host

• I ::t is this intégration which signifies entry of the infection into the chrnnic. State, Compounds whichact post-intetrration of HIV are therefore inhibitors ofchronically irfected cells. Zidovudine (AZT) for exampieagainst de nova infection ot HIV and has notivity agair.st chronically infected cells.

gene expression of Hiv (which is a positiveirus) woulc. therefore be active in HIV is only activesignificant acInhibitors of strand RNA v chronically inicçted celle rive strand RHA.-virus which affects humons./ · :hes to cpII membranes by virion adsorption

Hiv is a posiThe virus atta to CD4 surface rêCeDtor. The virion t-h»n i.h’rhiinh 2 η » η !? ~ V 1 17 *;Λ / '·* ν -* ν 10 cytoplasm vheiof thc genome the cell membrane penetratively and enters the ccllcytoplasm. îlncoating of the virion then takcs place in the:eby the viral envelope and the protein coatreleaae the viral RNA irito the cytoplasm.Reverse transciipiion therein produces a double-strandedDNA transcrip1: irom host cell genet-ic material. Thisinvades the hast cell nucléus and integratcc vith the hostcell chromosomal DNA. Transcription follovs to produce avRNA replieatc which is translated in the cytoplasm toproduce new viral ptuteins. Thé latter then assembles vithvRNA at the inner cell surface to produce neu virusparticles whicl· are releesed from the host cell. 15 20 y aasociated with an initial asymptomaticnitial asyinptumaLic phase may last a numberthe early signs of Hiv disease occur. cell death are proposed. It is a morphologically cell death involved in many HIV is normalphase. This iof years before A number of

Apoptosjs is pne of thèse,distinctive £ physiological &^d pathological processes including cellularseek tn main-tain appropriate intracellular processes vhicn oxidant-antioxidant balance

Cell dcath in T-cells is 23 cloçely associavith HIV is th :ed vith this balancing pxueess. iniectionjought gradually tu disturb the balance infavour of cell death. Another critical factor in t ’ detemiiininy whether cells will grow and divide in a normalfashion is intracellular atp concentration. Low * t 010579 déplétion u£

Cell death ft)ô:déplétion to intracellulaxl levels of ATP are. apcociated with ischémiedcath. T-lymphocytes are especially vulnérable tointiacellular ATP levels. HIV infection may disturb cellbiar oxidative phosphorylation which io thecellular process responsible for ATP levelc in the cell. uhatôVèr cause will eventually leed to celllovel that induces AIDS. 10

Much of the is concernedCompounds diehould hâve terri use of tieatment ha^expected, and 15 5 current Work in the field of antiviral resesrehUith targ^ting spécifie viral oncoded enzymes.;:covered from this re3earch, in principïe,toxicity on cellular processes. The long|owpuunds in clinical-trials in Hiv infectionnot given the degree nf benefit initiallynew apprnaches are needed. c a known compound, whiuh is also variously 10

Riboflavine i known as: E101;

Lactoflavlin;

Riboflavin;

Riboflavifrum; vitamin vitamin 7,8-Dimet^iyl 10-(l ' -D-iibityl) isoailoxazine; and / · 3,10-Dihÿdi-û“7,8-dimethyl-10-( D-r ibo-2 , T , 4,5-tetra-hydroxypentyl) benzopteridine-2, 4-dione. 010579

Riboflavine i:; commercially available as such or as its sodium phosphate or tetrabutyrate sait, typically in the ce as the dihydrate sait. It is also various mixtures with other vitamins, ail former mstan available in essentially being for the treatment of, inter alia. vitamin B deficiency.varies betweer

In such mixtures the dose of riboflavin0.5 and 10 mg, with a maximum recommended daily dose being 30 mg. 10

No adverse riboflavine. resuit in a b:may interfère hâve been reported with the use ofHowever, significant doses of riboflavinerlight yellow discoloration of the urine whichwith certain laboratory tests, effects 15 20

The riboflavin» requirement: of humans is often related tothe energy intake, but it appears to be more closelyrelated to resting metabolic requirements. A daily dietaryintake of about: 1.3 to 1.8 mg of riboflavine is recommendedthe basic recommended intake of riboflavine4200 kj (1000 kcal) of diet - Report of a

Joint FAO/WHO $xpert Group, Tech. Rep. Ser. Wld lllth Org.NO. 362, 1967. that is to sayis 550 pg per 25 cceptable daily intake of riboflavine is up kg body weight - see Thirteenth Report of* ·

Committee on Food Additives, Tech. Rep. Ser.

The estimated <,to 500 /ig perFAO/WHO Expert * Λ «« 10 15 20 à**.·: n»;*5 ,*f

Riboflavine, for the utiphosphorylatejdadenine din which îs a vater-soluble vitamin, is essentiaisation of energy frotn food. The activaforns, flavine mono-nucleotide and flavin ncleotide, are involved as co-en2ymes i: ,ctive metabolic reactions. oxidative/redu

Various otheknown, mainly flavins and dérivatives thereof are als;as flavouring agents.

It has now béen found surprisingly that the administratiorof riboflavine, as well as other flavins and dérivativesthereof, at doses far higher than previously used orrecommended c^n be highly effective in the management anc treatment of structure of viral infections, in particular HIV. ‘ ' The• ’* 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 proereative capacity of ths virus.

Accordingly, the présent invention in one aspect providesthe use of a flavin, especially riboflavine, or adérivative theteof for the manufacture of a médicament for 25 6 10 15 20 010579 ’ ? thcrcof are n aense as vitvitamin), meprovicies such pt knowp as phainuaceuticals, even in .a generali xibuflavine (knovn as an enzyme co-factorinvention in a second and broader aspectcertain flavins or a dérivative thereof for use as anti-viral agents.

In the use ae;ording tu Lhe invention riboflavine or otherflavin î»ày bt used as such or as a dérivative and theflavin deriva^ive may be any dérivative uhich io safe for1 use. Preferably, however, in the case ofriboflavine trie dérivative is a riboflavine sait and morepreferably ths riboflavine sait is riboîlavine sodiumriboflavine Letrabutyrate. Most preferably,dérivative should be nf higb purity and phosphate orthe flavin oi contamination vi'th spurious ingrédients should bc avoided.

In more généraaccordance vitof th© formula (I), iiainely: L terras, the flavin or dérivative for use inî the invention may be defined as a compound

U) 25 vherein: 010579. ©r CHj (lumiflavin) .

In addition, inalkyl, or H pr group.

Thus, and furjtrealised with the above formula (I) the group x may tean aromatic or other cyclic hydrocarbon nermore, tne use ol the invention way be'lavins nr dérivatives such as: 10 (A) lumichrome of the formula: lb 20 h3c h3c

(B) Roseoflavir of the formula:

H (HO-Ç-H)t

(II) (II) (III) (C) B-Hydroxyf ).avine, alloxazines and other dérivatives 10 15 20 25

οι esn

10 15 010579

(K) Flavin 1, H3c-

(XIII Ί -Ethyenoadenine dinucleotide

H 0

11 H il « i i ζΤίχΓ"«"^«'[<!Ό

H C-H

C-OHI

H3C

C-OH l

C-OH

K[^KH 0 0 |( Il ·<

[XI11J

C-O-P-C-P-O-CHj .Q

I 1 1jj OH QH

\ό OH (L) Schizoflavilns and dérivatives, 20 ch2oh 25

HCOH

I

HCOH

I

HCOH

I CHj 7,9-dimethyl-isoalloxazine

CHO

I

HCOH

I HCOH ’

I

HCOH I· CH, 7,8-diraethyT- isoalloxazine

(jOOH

HCOH

I

HCOH

HjZOH ch2 7,8-dimethyl-isolloxazine 10 15 20 25 010579 .-.:¾¾¾.. · .· .“· J · , 15

The above aro Chemical structures of schi2oflavins and shotheir -format identifiedformylbutyl)trihydroxy-4-

Other flavins ion from riboflavin, as 7, 8-dimethyl-10-(2,3,4-trihydroxy-4 SF2 and SFj can t isoalloxa2ine and 7,8-dimethyl-10-(2,3,4carboxybutyl) isolloxa2ine, respectively. may be illustrated by:

3-carboxymethylriboflavin3-carboxymethyl FMN 7- amino-io-(l'-D-ribityl) isoalloxazine 8- amino-7,lO-dimethylisoalloxasine

8a(S-Meroaptopropionic acid) riboflavin8a(S-Merzaptopropionic acid) FMN8a(N-Amilohexyl)FMN

9- A2obensoyl FMN 10- (ω-carboxyalkyl)-7,S-dimethylisoalloxazine-

In the use azcording to the invention thê flavin such ariboflavin, cr dérivative thereof, is preferably employerat a high dose level significantly in excess of the dosescurrently us Bd or recommended. Thus, typically th* riboflavin orthe présent i 1 to about other flavin in the clinical trial is used i:nvention at a dbsage régime of at least about100 or more (eg 10 or above) mg/kg of bod; weight per day. In addition, use according . to the invention pre

Eerably is one ’ÿherein the médicament is ir orally administrable form, especially as a capsule (eg tuo- 010579

Additionally pharmaceutica 16 or alternatively the invention includes al or veterinary composition for use in the management ard1 treatment of viral infections and in unit 10 15 20 25 dosage form,least about 3 such as 50 t riboflavine o herein, toget which composition comprises a unit dose of at5 mg such as 50mg or more (eg 50’ to 300 mg,o 200 or 50 to lOOmg) of a flavin such asr dérivative thereof as described or definedher with a pharmaceutically or veterinarily acceptable diluent, excipient or carrier. A compositionuherein the uMore preferably according to the invention is preferably oneijit dose is from about 35 mg to about 1000 mg., the unit dose is from about 250 to 500 mg.

In addition,preferably ina preferred c water. a composition according to the invention isoral or injectable form. Within that contextomposition is one as a solution in stérile

The invention of a medicamenviral infectidflavin suchtetrabutyrate as also includes a process for the manufacturet for use in the management and treatment ofns, which process comprises formulâting ariboflavine,, or a dérivative such as the àalt thereof for anti-viral use.

As wili be apdéfinition mayadditions 1 f«=ar· preciated, a process according to the above/ * be carried out using one or more of the 17 0Î0579 . · i.............. -1...-’ 10

In addition,· the invention includes.a product containingflavin such sis riboflavine, or a dérivative thereof, as a.anti-viral agent, together with another compound(s) havincanri-viral activity as a combined préparation fo:sinultaneous, separate or seguential use in anti-vira:therapy.

Again, a product according to the above définition may beone which irjcludes one or more of the other spécifiefeatures of trie invention defined herein. 15

The invention of viral infparenterallysuch riboflavi

Preferably in further includes a method for the treatment ection, which method comprises orally or dministering an effective amount of a flavin• *’ ne, or a dérivation thereof. a method according to ' the invention, the amount administered is 1 to 100 (eg at least 10) mg/kg of 20 patient body v

Furthermore, virus is human 3Îght. ^he method is particulary useful when theimmunodeficiency virus, HIV. 25

Once again, a one or more of defined herein, nethod according to the invention may. includethe other spécifie features of the invention 0 î 05 79 18 y, the invention is carried out with one oraviné, riboflavine sodium phosphate, flavin- eotide, lumiflavin, lumichrome, or especiallyàtrabutyrate, whose formula is set forth

Most preferabmore of ribofï adenine dinuc] riboflavin t below:-

10 15 20 25 ·; - ’ί/·'.· 1 ί'\ h-!?-? . 0a,.

astoid celle were incubated with 10TCIDSQ HIV 19 RPMI 16^0 supplemented .10¾ fêtai calf sérum as growt medium. Cell débris vas removed by Ιου spee centrifugation and the supernatant stored at -70°

until tj-equired. In a typical assay, C8166 T lymphobl 1 at 37 ’C for 90 minutes and then washed three timeswith phosphate buffer saline (PBS). Aliquots of 2 >:10s cells were resuspënded in 1.5ml of growth mediu:in 6ml culture tubes, and test compound at loçdilutions from 0.2 to 200μΜ vas added immediately.The test compound vas dissolved in 70% éthanol and thefinal concentration of alcohol in the culture vas <1%Cultures 200μ1 of) supernatant was taken from each culture ancassayed by optical derisity measurement at 4 50nm forHIV p24 core antigen (Kinchington et al 1989, Robertset al 1990) using a commercial ELISA vhich recognises were incubated at 37 ’C for 72 hours in 5% co2. ail the core proteins equally (Coulter Electronics

Ltd, Lutgn, UK). To détermine the ICg0 values standardcurves wjere dravn from untreated cultures containinc<1% alcohol. A2T and ddC were used as internaiControls J Assays were carried out in duplicate. 1,2 Deo lleted Medium Assay

In the standard assay,-system, cell cultures wereharvested, split and fed with . fresh medium 010379' • 20 assay. édition of fresh medium stimulâtes the' cellsto enter a log phase of growth. To investigate theeffect of cells reaching confluence in conditions ofdepleted media, cell cultures were fed and split at72, 4 8 a!nd 24 hours before being used in a standard acute assay 1.3 Lighfc Exposure Assay A freshlyinto twodaylightsubjected dissolved sample of test compound vas splitaliquots. They were placed either inor the dark for two hours before being to standard acute assay. 1.4 Preincubation Assay

Target ce

Lis were preincubated with test compound at log dilutions of 200 to 0.2μΜ for 18/24 hours beforeinfection with HIV-l. Each sample concentration wasthen treased individually as in the standard acuteassay. 2.1 Standard Chronic Assay H9 cells c ironically infected with HIV-lrf (H9rf) were 2 Assays for

Chronicallv Infected Cells nd virus a for thr£opticalthe a eut icso vai| 10 15 20 21 010579 incubated vith test compounds (200 'to · 0.2μΜe days. p24 antigen vas then determined kdensity measurement at 450nm as described fce infection standard assay, To détermine th ues standard curves were drawn from untreatecultures) containing 1% alcohol, RO 31-8959 (Roch,Proteinajse inhibitor) vas used as an internai control.Assays wjere carried out in duplicate. 2 .2 Pedleted Medium Assav

In the standard assay, cell cultures were harvestedsplit and fed vith fresh medium approximately 18 to 2<hours before assay. Addition of fresh mediu:stimulâtes the cells to, enter a log phase of grovth.To investigate the effect of cells reaching confluencin conditions of depleted media, cell cultures vertfed and split at 72, 4 8 and 24 hours before being userin a standard acute assay. 2.3 Lia. ht Exposure Assay A freshlÿ dissolved sample of test compound vas splitinto tvo aliquots. They were placed either irdaylight or the dark for tvo hours before beincsubjectec to standard chrônic assay. 25 22 010579

Taxicity Assay Το test for uninfected cesame log dilu were then vasgrowth medium were harvested measured. Unt compound toxicity, aliquots of 2 x io5ls were eultured with test compounds at the:ions.for 72 hours (l.l and 2.1). The cellsied with medium and resuspended in 200μ1 of containing C14 protein hydrolysate. The cells « after 5 or 20 hours and the C14 incorporationreated cells were used as Controls. 10

The assays were applied to the compounds identified inTable 1 belowxt

Table 1 15 20 25

Code

Fl F2 F3 F4 F5 F6 F7

Compound

Riboflavine 5'phcsphate

Riboflavine

Flavine adenine dinucleotide

Lumif Lavin

Lumichrome ’

Ribofiavin tetranicotinateRibof;,avin tetrabutyrate

Initial assays were carried out in relation to the various 110579 23 50 results in Table 2 are subject t

The IC they are not consistent with re-run assaydate. Assay results are shown in the graphCollowing drawings and in Tables 2 to 10 whic results. confirmation conducted to forming the follov:- 10

Figure 1: An F4 cou re t igen as optical density (OD) for Compounds F2(first antigen assay) and F5 at 450 nm versucentration (μΜ). The dotted line at OD 0.37 resents IC5O (active) .

Figure 2: Antigen optical density (OD) for Compounds Fi an F3 at 450 nm versus concentration (μΜ) .

Th dotjted line at OD 0.371 représente IC50 (active). 15

Figure 3: Toxicity as C14 uptake (dpm) versus concentratic(μΜ for Compounds F2, F3, F4 (first toxicit asspy) and F5- The dotted line at 6035 dprepjresents CC5£J (non-toxic) . 20

Figure 4 : Toxji(μΜdpm city as C14 uptake (dpm) versus concentraticfor Compound Fl. The dotted line at 603 represents CC5Û (non-toxic). 25

Figure 5: Ant :.gen optical density (OD) for Compound ’ I(second antigen 'assay) at 450 nm versxconcentration (μΜ) . The dotted line at OD 0.3: 24 •5

Figure 6: Toxi . (MK)

The toxi 10 15 20 25 010579 city as C1’ uptake (dpro) versus concentrationfor Compound F4 (second toxicity assay).dotted line at 6035 dpm represents CCSQ (non- =) -

Figure 7: AntigF7 en optical density (OD) for Compounds F6 andt 450 nm versus concentration (μΜ). Thedotted line at OD 0.371 represents IC5Q (active).

Figure 8: Toxic: (μΜ) 6035 ity as C14 uptake (dpm) versus concentrationfor Compounds F6 and F7. The dotted line atdpm represents CC50 (non-toxic).

Figure 9: Antigen control (ddC)

As shown by th 5 Tables, the test compounds were evalua.ted for activity against cells both acutely and chronically infectéd vith data is shown IV. Antiviral (IC50) anâ toxicity (CC5Q)belov. in another sériés of experiments, compounds were tested in cell cultures in which fresh media was added at 72experiment vas , 48 and 24 hours prier to infection. This1 carried out to investigate the effects of the compounds oi cells in actively dividing and quiescent

States. This sensitive to theof light on sta data indicates that cells may be more • « test compounds when quiescent. The effectbility, preincubation of target cells, and 010579 10 15 20 25 30 investigated.effect on the •the targetsignificant a ‘ . 25

Exposure to light for two hours had neactivity of the compound. Preincubation with délie enhanced its activity and it showedctîvity against the Africa HlV-i 'isolate.

Table 2 (Figures 1 to 4) - Acute Infection Standard Assay

Compound No Z

Assav No

Fl/1

Fl/2

Fl/3 F2 F 3 F4 F5 (Figures 1. and 2) 1 to 20 <0.4 0.1 (Figure 2) 3 (Figure 1)0.8 (Figure 2) 1 (Figure 1) 3 (Figure l};· cc

SI (Figures 3 and 4) >200 >400 >800 (Figure 4)>200 (Figure 3)>200 (Figure 3) 150 (Figure 3)>200 (Figure 3) >1000 >8000 >60 >200 150 >60

Table 3 (Figuras 7 and 8} - Acute Infection Standard Assay(1.1)

Compound No./

Assav No —50 CC -50- 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 ' « r · 010579 26

Table 4 - Chrojnic Infection Standard Assay (2.1)

Cotnpound No/

Assav No 1^50 ^50 si 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 - Acuta Infection Depleted Medium Assay (1.2)

No —50 —SO F7 10 160 Table S -* Chroi ic Infecti ComDOund 72 ? ours No ^50 cc —50 F7 40 75 Table 7 - Acute Infection • Coïrrpound No Oavlioht ICg CC —50 F7 60 >200

IC $0 48 hours 24 hours 1^50 21 ^50 ^50 cc so 100 110 160 ic Infection Depleted Medium Assay (2.2) 48 hours 50 cc 50 250 24 hours ^50 ^50 60 101

Infection Light Radiation Exposure Assay

Darkness

IC 50 >60

CC —50 >200 010579 27

Table 8 - A eu te Infection Preincubation Assay (1.4) of target cells vith test compound for 24infection

Preincubationbours before

Compound No F7 I£so 5 —50 120

SI 24

Table 9 (Figures 5 to 8) - Acute Infection Standard Assay(1.1)

Compound N_o £ F4 F6

ccE

SI 50 —50 lj} (Figure 5) 150 (Figure 6) 12 3b - 60 (Figure 7) >200 (Figure 8) min 3 -6

Table 10 - Acute Infection Standard Assay (1.1)

Assay applieptransformed aIsolate (ffJV- to C8166 Cells (T-lymphoblastoid cellsnd immortalized' by XTLV) vith an African HivCBI4)

Compound No F7

IC 50 cc —50 150

SI 37.5

Table 11 (Figures 10 to 12) - Acute Infection StandardAssay (1.1)

Compound No F7 dde (control)

The variation ^50 32 0.2 ^50 200

SI 6.3 in the end points observed vith Compound F:may be due to the properties of the taroet lvmnhohiaet-^·;. 010579 cells. Evenchanges in theThis is reflegin the pairedassay (Table 3indicate chatmay hâve diffe 23 in synchronized cultures there may be subtlemetabolic State of sub-populations of cells.ted in the shift in the end points observedantiviral and toxicity values from assay to ). The results tabulated in Tables 5 and 6cell culture in active or quiescent States :rent sensitivities to the test compound.

Patient Treatment 10

Thirty-five pfollow up medid ajtients were placed on therapy. Thirty hadal visits. i) General Condition of the Patients 15

Twenty patientsreported an in out of thirty who came for follow-up visits 20 provement in their general condition. Themajority of thèse reported improvement insofar as malaise,appetite and w^ight gain vas concerned. Two patients alsoement in skin rash with régression of skinone reported no new skin lésions developed 25 reportéd impro\lésions while while on therapy. One patient also reported improvement inimpotence (vhich had been présent for three months prior toonset of therapy), while two other patients reportedcessation of loig standing coryza. 010579 29 10 15 20 25 ii) Sick visits

Few patients 1. One patlarthritis wh: attended clinic for unscheduled sick visits: ent had récurrent abscesses as well as septich persisted even on therapy. 2. Two patients had récurrent lower respiratory tracinfections vith one developing frank broncho-pneumoni^during second week of therapy. Repeated smears for AAFE-hâve continued to be négative. 3. Two patients had pyrexia vith no localizing signs an·,repeated blood smear for malarial parasites were negativ-and no signii’icant growth on blood culture. One of théspatients respanded well to amoxycillin and is now afebrile. 4. One patient had gastroenteritis during the third vee^of therapy. 5. Oral anc vulvo-vaginal candidiasis were reported b·. two patients récurrent as tablets vas completed with the vulvo-vaginal candidiasis beinesoon as a coufse of Nystatin pessaries anc 6. Two patients also reported récurrent attacks of herpes . . . *simplex genitalis. 010579 iii) Toxicitv 30

Most of the c first two wee ks ases of toxicity reported occurred during theof therapy and hâve been transient.

Two patients axperienced pruritus which averaged four daysduring first week of therapy and cleared spontaneouslywithout any supportive médication. 10

Four patients reported moderate diarrhoea during the first two weeks of therapy- This has averaged four days. This has been a d being a side

:.fficult symptom to attrîbute as between itaffect or a natural manifestation of the HIV infection. However, the consistency of its appearance in 15 the first week of therapy, and its transient nature makes it reasonable to suppose it is a side effect. reported drowsiness and another reportedr urine. MSU vas normal. 20

One patientdarkening of he

Two patients reported abdominal discomfort. iv) Labaratory

Results 25

Three patientsthe second to t

Of liver dise had transient rises in liver enzymes duringlird week of therapy, with no clinical signs4se. However, the enzyme levels always 010579 31

The-above clinic trial reports are the preliminary resuitof a clinicul trial vhich bas currently been in progrèsweeks using Compound F7 administered orally i(the capsules are as described in Example for severalcapsule forn below) dosage was:-

Dose 10

Dose

Dose 15

Dose

Dose level 1 level 2 level 3 level 4 : level 5: level 6: lmg/kg body weight per day orally in tw divided dosages 2mg/kg body weight per day orally in tw divided dosages long/kg body weight per day orally in tw divided dosages 15mg/kg body weight per day orally in tWf divided dosages 20mg/kg • body weight per day orally in tw, divided dosages 30mg/kg body weight per day orally in two tl 20

Dose level 7: 25

Dose

The level 8; level 9: folloving three divided dosages 40mg/kg body weight per day orally in two tethree divided dosages 50mg/kg body weight per day orally in two tethree divided 'dosages lOOmg/kg body weight per day orally in tweto three divided dosages spécifie Exemples illustrate compositions 010579 A formulation 32

Example 1 ;an be prepared from the following: riboflavi le-S-phosphatestérile wàter to provide aadministration infection. 10 mg 2 ml unit dosage of 10 mg of riboflavine foronce per day in the treatment of viral

Examole 2 A formulation can be prepared from the following: riboflavin »-5-phosphate stérile water to provide aadministration infection. 30 mg 2 ml anit dosage of 30 mg of riboflavine foronce per day in the treatment of viral

Example 3

Similar , formulations to those of Examples i and 2- can beprepared at doses of; 010579 33 10 15 20 vS·? 25 mg

50 mg P ml, and fer ml pfer respectivelystérile watetf in either a unit aroount of 2 ml or 5 ml cand based on an active ingrédient vhich is:

Riboflavine 5'phosphate

Ribof lav]

Flavine

Lumif lavlin

Lumichrome or a mixture thereof. me jadenine dinucleotide

The following

Sizesi 25 Type;

Composition:

Example 4 • * [capsules were formulated:- 25mg 50mg îoomg 200mg 400mg ’ 2-part hard gelatin • « Z ’ 3orapound· F7 in admixture with 010579 34 166.4/156.7/118.6/108.7/50wg to give capsuleweights of 191-4/206.7/218.6/ 308.7/450mg.

Claims

010579 35 CLAIMS

1. Use of a flsvîn compound or s mixture comprising two or more thereoffor the manufacture of a médicament for the trestment by prophyîsxis ortherapy of disease caused by HIV infection, characterized în that the flavincompound is ribofIavine-5’-phosphate, flavine adenîne dinucleotide,lumrriavin, lumichrome, rîboflavîn fetrsnicotinate or riboflavin tetrabutyrate.

2. Use of a flavin, flsvîn dérivative or a mixture comprising two or morethereof for the manufacture of a médicament for the ireatment by prophyiaxîsor therapy of disease caused by virai infection.

3. Use as clsîmed in Clsim 2 wherein the riboflavin dérivative is ariboflavin sait.

4. Use as claimed in any one of Claims 1 îo 3 wherein the riboflavin saitis riboflavin sodium phosphate or riboflavin tetrabutyrate.

5. Use as claimed in Clsim 2 wherein the flsvîn or flavin dérivative is acompound of one of the foiiowing general formuiae:- 5.1 A compound of the general formula:- Ri

Rù (Vlla) 0 010579 36 Wherein R is hydrogen or aîkyï; Ri and R; are, each independently,hydrogen, alkyl, hydroxy, halo, slkoxy, alkylthîo, thio or an optionally 5 substîtuted aromatic or non-aromatic nitrogen heterocyci, and X is:- i) hydrogen, ribîtyl, alkyl, hydrogen or an aromstîc or non-aromatic carbocycle (iï) a group of the general formule:-10 -CH2-(CHOH),.-Y in whîch n is an integer of 3 or 4 and Y is -CH2OHtCOOH or -CÛH or s group of the formula:- •15 OK GHI l - rib g se - 0“ P-G—P-0 H II0 G

(Ib) 2 0 wherein R is hydrogen or alkyl; and wherein Wi and W2 are, each independently,alkyl, hydroxy, halo, slkoxy, alkylthîo, thio or an optionally substîtuted sromstic ornon-arorriâtic nitrogen heterocyole

52. A compound of the general formula: 25 010579 37

whereîn X is (i) hydrogen, ribttyl, alkyl, hydrogen or an sromatic or non-arornatiecsrbocycfe (i!) a group of the general formula:·' -CKr(CHOH)n-Y in whîch n is sn integer of 3 or 4 and Y is -CH2OHrûOOH or -COH or a group of the formula:-

v/herein R ts hydrogen oralkyl; and wherein W-t and W3 ara, each independenîly,aIkyl. hydroxy, halo, alkoxy. alkylthio, thio or an optionally substituted sromatic ornon-arornatie nitrogen heterocyde. 5.3. A compound of the general formula:· 010579 33 *1

(VIII) whersin Ri îs hydrogen or sn £îkyl grcup, R3 îs sn alkyl group or s ribityl croup,and Rs represents hydrogen or mono- or di-substîtution of the outer carbocyclicring with εη sîkyl group.

6. Use ss dsimed in CIsim 2, wherein the flavin or flavin dérivative isiumrchrome; rosefîsvin; a hydroxyftsvin; εη alloxazine or dérivative thereof; an8a.-N(3)-histidyîflsvin; en Sa-N(l)-hîstidyl flavin; an 8&<ysteîny[ thioether; an 6a-S-cysfeinyl thicether, a lumiflavin; s S-deezaflsvin; a 5carbs-5-deazs or 1-csrba-'-deaza analog of ribcflsvin, FMN or FAD; flaviM.^-sthenoadeninedinuclectide; S-methylflsvin; 9-phenylflavin; 9-benzylflavin; 9-cyclohexyIflavin;e.S-dimethyiflsvîn; 6,7,9-tnmethylflavîn; S-oxyethylflavin; S-dioxypropylflavin;6,8,9-trïmethylfisvin; Iscroflavin; flavin-9-carfcoxylic acid; 6,7-dimethylflsvin-S-carboxylic acid: or a schizaflavin. ?. A phsrrnsceuticsi composition for the treaîment by prophylaxis or therapyof dlsease causea by viral infection, the composition beina characterized in thsi it ·comprises s flavin or flsvin dérivative. 010579 S. A composition as claimed in Ciaîm 13 v/herein the flavin or flavindsrrvstive is as defined in any one of daims 3 to 6. 5 9. A composition es claimed in Claim 7 or daim 3 which composition comprises a unit dose of at least about 35 mg of a flavin or flavin dérivativetogether \vith a pharmaceutically or veterinarily acceptable diluent, excipient or carrier. 10 10. A rnethod for ths trestment by prophylaxis or therapy of disease caused by viral infection which methori comprises administering îherapeutically to apatient suffering from such disease an effective amount of a flavin or a flavindenvative or administering praphyiacticaHy to a patient at risk of viral infection aneffective amount therecf, the effective amouni being 100 mg/kg body weight/day. 15 20