Gut, 1985, 26, 745-751

Clinical trialRandomised controlled trial of adenine arabinoside5'-monophosphate (ARA-AMP) in chronic hepatitis Bvirus infectionI V D WELLER, ANNA S F LOK, A MINDEL, P KARAYIANNIS,SARAH GALPIN, J MONJARDINO, SHEILA SHERLOCK, AND H C THOMAS

From the Departments of Medicine and Physiology, Royal Free Hospital and School of Medicine andAcademic Department of Genito Urinary Medicine, Middlesex Hospital Medical School

SUMMARY A randomised controlled trial was conducted in 29 HBV carriers who had been HBsand HBe antigen positive for more than six months. Fifteen patients were treated withARA-AMP 10 mg/kg/day given as intramuscular injections 12 hours apart for five days followedby 5 mg/kg/day for 23 days. The 14 controls received no treatment. Serum HBV-DNApolymerase, and HBV-DNA decreased in all patients during therapy. Six treated patients lostserum HBV-DNA polymerase, HBV-DNA and HBeAg, HBsAg concentrations decreased, andfive developed anti-HBe. One of these six patients lost HBsAg and developed anti-HBs. No suchchanges were observed in the control group over a similar 18 month period of observation. A fourweek course of ARA-AMP inhibits HBV replication and in a significant minority of patients thisis long lasting and is associated with a reduced level of inflammatory activity in the liver.

Adenine arabinoside (ARA-A) is a synthetic purinenucleoside with anti-viral activity against a largenumber of DNA viruses.1 It has been used, largelyin uncontrolled studies, in patients with chronic liverdisease caused by hepatitis B virus (HBV) infection.In the majority of those treated, a transient decreasein HBV-DNA polymerase (HBV-DNAp) has beenobserved with no change in HBsAg concentration,HBeAg status, liver function tests or histology. In aminority of patients, however, inhibition of HBV-DNAp is prolonged beyond the treatment periodwith a decrease in HBsAg concentration, loss ofHBeAg and the development of anti-HBe.2- Simi-larly, in uncontrolled studies with ARA-A and/orhuman leucocyte interferon, 37% of treated patientshave shown this prolonged inhibition of HBVreplication with HBeAg to anti-HBe seroconver-sion, accompanied by an improvement in liverfunction tests and histology.6 7 Spontaneous HBeAgto anti-HBe seroconversion does occur, however,with a variable frequency in different populationsand may occur following withdrawal of immuno-Address for correspondence: Prof H C Thomas, Academic Department ofMedicine, Royal Free Hospital, Pond Street, London NW3 2QG.Received for publication 24 August 1984.

suppressant therapy.-'2 Controlled trials are there-fore required in chronic HBV infection to show withcertainty the long term effects of any drug. ARA-Aalone, in a randomised controlled study, inducedHBe antigen to anti-HBe seroconversion with lossof HBV-DNAp, a decrease in HBsAg concentra-tion and aspartate transaminase in three out ofseven treated patients and no such changes occurredin the control group.' 3Adenine arabinoside 5'-monophosphate (ARA-

AMP) is the synthetic monophosphate ester ofARA-A, is at least 400 times more water solubleand therefore suitable for intramuscular administra-tion. A three to five week course of ARA-AMP,given twice daily intramuscularly, to three consecu-tive HBeAg positive patients was associated withloss of HBV-DNAp, serum HBV-DNA, HBeAg toanti-HBe seroconversion and a decrease in HBsAgconcentration. 14The aim of this study was to determine, in a

randomised controlled trial, whether ARA-AMPhad a permanent effect on the level of viralreplication in patients with HBsAg/HBeAg positivechronic liver disease and if so to assess the effects ofthis on liver function and histology.

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Methods

PATIENTSBetween August 1980 and November 1981, allpatients who had been HBsAg and HBeAg positivefor more than six months attending the Departmentof Medicine at the Royal Free Hospital and theDepartment of Genito Urinary Medicine at theMiddlesex Hospital, were considered for entry. Thefollowing were excluded: those under 18, women ofchildbearing potential, patients with clotting ab-normalities preventing liver biopsy, renal failure ordecompensated cirrhosis, those receiving immuno-suppressant or anti-viral therapy less than sixmonths before entry, those with an alcohol con-sumption great than 80 g per day and those patientsfrom overseas who would not be available for longterm follow up. Fifteen patients were excluded onthese grounds. Twenty nine patients were random-ised using sealed envelopes to receive no treat-ment (n=14) or ARA-AMP (n=15) given intra-muscularly twice daily for 28 days; 10 mg/kg/day forfive days and then 5 mg/kg/day for 23 days. Themajority of patients were British and white otherthan two Asians in the control group and in thetreatment group; one Asian, two Spanish, oneItalian, one North American and one South Afri-can, both white. Treated patients were seen weeklyduring therapy and for four weeks afterwards andthereafter monthly for 18 months. Control patientswere followed up monthly for a similar period.Blood was taken at each visit for full blood counts,prothrombin time, liver function tests (aspartatetransaminase, billirubin, alkaline phosphatase andalbumin), urea, creatinine and electrolytes and asample of serum stored at -20°C for viral markers.Patient characteristics are shown in the Table. Onlythree patients had ever received antiviral therapy.Two patients in the control group had receivedARA-AMP for 10-15 days, 15, and 17 monthsrespectively before entry into the trial. One patientin the treatment group had received ARA-A for 10days two years before entry.HBsAg was detected and quantified using a

monoclonal antibody based radioimmunoassay(RIA) which has been shown to be as sensitive asthe Ausria-IL (Abbott).'6 Serial duplicate, 10-folddilutions of HBsAg positive specimens were used. Astandard curve was plotted using a pooled serumstandard of mixed subtupe which was standardisedagainst the British standard and contained 85 ug/mlof HBsAg. All serial samples on each patient wererun within the same assay. HBeAg and anti-HBewere detected by RIA (Abbott Laboratories).HBV-DNAp was measured by using 200 gul of

serum layered over 600 Al of 30% (w/v) sucrose

Table Patient characteristics on entry - (all male)

Treatment Control

Patients (no) 15 14Age 30 (22-46) 32 (20-46)Homosexual 7 7Intravenous drug abuse 2 0Others 6 7Total 15 14HBsAg (,g/ml) 240 (30-3800) 260 (50-1000)HBeAg + +HBV-DNA polymerase 3614 (1108-10 158) 3281 (204-6166)(dpm/200 j.l)HBV-DNA 1668 (32-2556) 1236 (28-2133)(cpm/200 g1)Aspartate trans- 67 (36-179) 59 (34-95)aminase (IU/l)HistologyCPH 2 3CPH/CAH I ICAH 12 9Cirrhosis 0 1

CPH=Chronic persistent hepatitis; CAH=Chronic activehepatitis.Values expressed as median with range.

containing TNEM-BSA.'7 Methyl thymidine 5'-tri-phosphate was used as the tritiated base. After theDNAp reaction, samples were TCA-precipitatedand washed individually in petri dishes. The upperlimit of the normal background count was 568dpm/200 ,ul of serum (mean of 47 negative controls+ 2SD). HBV-DNAp positive samples from 22HBeAg positive patients were analysed and thenstored at -20°C for a mean of 10 months (range4-16 months) and retested. There was no significantdifference between the values before and afterstorage at -20°C for this period of time (Wilcoxon'smatched pairs signed ranks test). Serial samplesfrom the trial patients were run within the sameassay. Within assay variance was studied by analys-ing 41 duplicates of different samples (five to sevenduplicates in each of eight assays) and the overallcoefficient of variation for a single sample found tobe +16%.Serum HBV-DNA was measured by molecular

hybridisation using 32p- labelled cloned HBV-DNAusing a method previously described.'8 This wasmodified to improve the non-uniformity of spots.Phenol extraction was carried out in microfuge-centrifuge reaction vials (LIP Ltd) and the aqueouslayer separated by centrifugation in an eppendorfcentrifuge (Anderman). The HBV-DNA in 50 ,ul ofthe aqueous phase was denatured by adding it to 100,ul of 0 15 M NaOH/1 5 M NaCl. The mixture wasvortexed and left for five minutes at room tempera-ture and then neutralised by adding 100 ,ul of 0-15 NHCl/2 1 M NaCl and vortexing. These 250 Ftl

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samples were then pipetted into the wells of theBRL 'hybridot' manifold containing a nitrocellulosefilter.19 Hybridisation was carried out as previouslydescribed. The HBV-DNA positive spots werequantified after 18 hours autoradiography by pun-ching out the corresponding spots on the nitro-cellulose filter with a 6 mm diameter cork-borer.These discs were then counted in 4 ml scintillationfluid in a Philips liquid scintillation counter. Theamount of HBV-DNA in the samples was thenexpressed as counts per minute (cpm) per 200 gl ofserum minus background. The highest negativecontrol on the filter was usually 2-3 cpm.The possibility of intercurrent infection during the

study period with either hepatitis A virus (HAV) ordelta agent was studied. All patients' sera wereexamined for anti-HAV by radioimmunoassay(HAVAB, Abbott) on entry and at the end of thestudy period. All positive sera were examined forIgM anti-HAV (HAVAB-M, Abbott). Similarlypaired sera spanning the study period were ex-amined for antibodies to delta agent detected by asolid-phase bocking RIA by Dr M Rizzetto, Turin.20

Liver specimens were obtained by percutaneousliver biopsy within six months of entry into the trialand after 12 months of post-treatment follow up inthe treated group and at the same time in the controlgroup. Assessment of portal and periportal inflam-matory activity and lobular inflammation and spottynecrosis was made.

Statistical evaluation was carried out using Wil-coxon's rank sum test or a two-tailed Fischer's exacttest.

Results

On entry to the trial, there were no statisticallysignificant differences between patients in the treat-ment and control group in terms of age, proportionof homosexuals, levels of markers of HBV replica-tion (HBsAg, HBV-DNAp and HBV-DNA), aspar-tate transaminase and the histological spectrum ofliver disease on the initial biopsy (Table). Twopatients in the treatment group were identical twins,abused intravenous drugs and shared needles. Ofthe 13 non-homosexual, non-intravenous drug abus-ers, five were health care workers and three lived orhad lived in parts of the world with a highprevalence of HBV infection. In the remaining fivepatients risk factors for hepatitis B were notidentified. The most frequent histological findingwas chronic active hepatitis.

HBV REPLICATIONTwo patterns of response to ARA-AMP were seen(Fig. 1). During the treatment period all patients

showed a decrease in HBV-DNAp and HBV-DNA.In 11 patients these returned immediatley to pre-treatment levels and HBeAg remained positive.Four patients showed a prolonged inhibition ofHBV-DNAp and HBV-DNA after treatmentand this was associated with the development ofanti-HBe six to eight months after therapy. Afurther two patients lost HBV-DNA, and DNApolymerase and HBe between 12 and 18 monthsafter treatment and one of these seroconverted toanti-HBe. There were no consistent changes inHBV-DNAp or HBV-DNA in the control groupand all patients remained HBeAg positive up to 18months post treatment.

LIVER FUNCTION TESTFour of the patients in the treatment group whodeveloped anti-HBe showed three to nine-foldincreases in aspartate transaminase five to sevenmonths after treatment. In one homosexual, thisoccurred three weeks after a trip to Amsterdam. Hedeveloped nausea, loss of appetite and jaundice.Serum IgM anti-HAV was positive (Fig. 2). Thisacute hepatitis A was associated with a rise inalkaline phosphatase and bilirubin as well as trans-aminase, loss of HBV-DNAp, HBV-DNA, HBeAgand seroconversion to anti-HBe. Furthermore,HBsAg disappeared from the serum followed by thedevelopment of anti-HBs.Both the identical twin intravenous drug abusers

developed an asymptomatic rise in transaminasefive months after therapy (Fig. 3). Inhibition ofHBV-DNAp and HBV-DNA had persisted for atleast three to four months post-treatment in bothtwins before this rise in aspartate transaminase.Serum antidelta, which was negative in both onentry into the trial, became positive at the time ofelevation in aspartate transaminase. Delta antigenwas detected by direct immunoflourescence in thenuclei of hepatocytes in both liver biopsies 12months after therapy.

After loss of HBeAg in the six treated patients,the aspartate transaminase showed improvement. Inthe patients without delta infection, the trans-aminase returned to within the normal range. In thetwo delta infected intravenous drug abusers, aspar-tate transaminase decreased but remained above thenormal range.Of those who remained HBeAg positive, one

patient in the treatment group and one controlshowed asymptomatic three- to four-fold rises inaspartate transaminase during the follow up period.In neither case was this associated with a decrease inthe level of HBV replication, HAV or deltainfection. There were no consistent changes in liverfunction tests in the other patients in either the

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2

1-

Treatment (n-15)uARA-AMP

600 a

200- 0 1

-1 0 1 2 3 5 7 9 11 8

Contmrs (n-14)

7I 0 U1 I3 5 7 I 1113

I I I I IIIII Ii~~-Mi-*-*i**-1 0 1 2 3 57 91113

Time (months)Fig. 1 Serum HBV-DNA polymerase, DNA and HBsAg concentration responders.-- - - -non-responders.HBV-DNAp is expressed as dpmxlO3W1200 ,ul ofserum, HBV-DNA as cpmxJO31200 ml serum and HBsAg as uglml.

treatment or control groups. Fourteen patientsshowed evidence of past HAV infection on entryand those negative for anti-HAV remained sothroughout the study period. No other patientsshowed serological evidence of delta infection.

Inflammatory activity and degree of hepatocytenecrosis was reduced in two patients who showedlong term inhibition of HBV replication withoutdelta superinfection. In the twins who had super-infection with the delta agent, both spotty necrosisand periportal and portal inflammatory activity wereincreased. The remaining two patients sero-converted after the follow up biopsy. There were nosignificant changes in the level of inflammatory

activity in the untreated and treated non-respondergroups.

Fourteen of the 15 treated patients complained ofincreasing fatigue during the treatment period. Infour, this was associated with loss of appetite and intwo, nausea. Six patients complained of either lowerlimb pain or muscle cramps. In three patients thesesymptoms persisted throughout therapy and in onepatient they were intermittent. In one case thesesymptoms appeared during the initial five dayperiod on 10 mg/kg/day of ARA-AMP and in onethey developed during the week following therapy.In no patient were these symptoms associated withneurological deficit on clinical examination. None of

748

HBV-DNApolymerase

HBV- DNA

0'1000-

800-I

HBsAg

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[0ARA-AMP]

.I.- ~-I

anti- 6-vel anti- 6aveV100L- I

m' 50 - :t2S* l

iBeAg.vel I anti -HBe +ve

I

I9

i I

-i 6 1 2 3 5 7 9 11 13Time in months

I 2 3 5 7. 9 11 13Time (months)

Fig. 2 ARA-AMP and acute HAV infection. Aspartatetransaminase is expressed as IUIl (normal range 5-40);HBsAg as uglml, HBV-DNAp as dpm x 031200 ulofserum.

the above side effects necessitated analgesics or thecessation of therapy.One patient developed acute right loin pain

radiating to the groin five days after therapy beganand after a day of sunbathing. This was associatedwith haematuria and crystalluria microscopicallyand an urgent intravenous urogram (IVU) revealedan obstructed right pelvicalyceal system. Serumcalcium and 24 hour urine calcium, phosphate, uricacid and oxalate were all normal. The 24 hour urinexanthine excretion was 235 mg (normal 10-20 mg).A stone was not recovered with sieving of the urine.ARA-AMP was continued at the 5 mg/kg/day doseand a [99Tc]-Sn-DTPA scan after 48 hours and a

repeat IVU one week later showed normal kidneyswith no evidence of obstruction.

Full blood count, urea, creatinine and electrolytesremained normal throughout the treatment period.

Discussion

ARA-AMP given twice daily by intramuscularinjection for four weeks inhibited HBV replicationduring therapy as shown by the decrease in HBV-DNAp and HBV-DNA in treated patients when

Fig. 3 ARA-AMP and delta infection in two patients.Aspartate transaminase is expressed as IUll, HBsAg asuglml, HBV-DNAp as dpm x1031200 ,ul and HBV-DNA ascpm x 103200 ,ul ofserum.

compared with controls. Similar changes have beenshown in uncontrolled studies. 14 21 In four of the 15treated patients this inhibition was prolonged andaccompanied by HBeAg to anti-HBe sero-conversion six to eight months after therapy. Afurther two patients lost HBe antigen between 12and 18 months after therapy. No such changesdeveloped in the control group. This difference inseroconversion from HBeAg to anti-HBe betweentreated patients and controls was statistically signifi-cant using a two tailed Fischer's exact test, p<O,3.In four of these six patients there were three- tonine-fold rises in aspartate transaminase precedingthe seroconversion. In two patients intercurrentinfection with the delta agent and in one HAVoccurred during the follow up period. In the twointravenous drug abusers the delta hepatitis occur-red four to five months after HBV-DNAp andHBV-DNA had become negative after ARA-AMPtherapy. A mean interval of three months has beenpreviously observed between therapy and loss ofHBV-DNAp and HBV-DNA and six months beforeloss of HBeAg, after ARA-A and ARA- AMP. 14 22In HBV carrier chimpanzees it has been shown thatinoculation with delta agent is followed by ahepatitis, intrahepatic expression of delta antigenafter three weeks and the development of antideltaafter eight to nine weeks. This superinfection is

a; _sF F

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750 Weller, Lok, Mindel, Karayiannis, Galpin, Monjardino, Sherlock, and Thomas

associated with transient inhibition of HBV replica-tion as shown by a decrease in serum HBsAg andHBeAg and HBcAg display in the liver demons-trated by immunofluoresence.23 Similar decreasesin HBsAg have been observed in human HBVcarriers superinfected with delta agent but pro-longed inhibition with HBeAg to anti-HBe sero-conversion has not been described.24 If the deltavirus incubation period is similar in the chimpanzeeand human HBV carrier it would seem that in thetwo intravenous drug abusers inhibition of HBVreplication was well established after ARA-AMPbefore delta infection occurred.

In the patient who had acute HAV infection,inhibition of HBV-DNAp and HBV-DNA wasprolonged beyond the treatment period but thenreturned towards pretreatment levels. An acutehepatitis A seven months after therapy was followedby loss of HBV-DNAp and HBV-DNA and HBeAgto anti-HBe seroconversion. It would seem from thetemporal relationship of the hepatitis and the loss ofHBV markers that in this patient acute HAVinfection contributed to the inhibition of HBVreplication.25 Similar rises of transaminase associ-ated with loss of HBeAg and development ofanti-HBe have been observed in other studies at asimilar time after therapy with ARA-A and ARA-AMP and without concurrent hepatitis virus infec-tion.14 21 22 In addition, similar changes have beendescribed after therapy with human leucocyte andlymphoblastoid interferon,4 6 7 after immuno-suppressant withdrawal in HBeAg positive patientsand during immunostimulant therapy with BCG.5 Ithas been suggested that this rise in transaminases ismediated by an enhancement of the immune re-sponse to the virus or virally determined antigensperhaps facilitated by a preceeding lowering of viralload.'4 Because the acute HAV infection in ourpatient occurred at a time when loss of HBeAgmight be expected after antiviral therapy the extentof its role in the inhibition of HBV replication isdifficult to evaluate.

Seroconversion from HBeAg to anti-HBe afterantiviral therapy and that which occurs spon-taneously is often associated with a decrease intransaminases and hepatic inflammatory activ-ity.7 8 10 14 Of the five treated patients who de-veloped anti-HBe, transaminases returned to nor-mal in three and in two of these hepatic inflamma-tory activity was considerably reduced. In the twointravenous drug abusers who had superinfectionwith the delta agent, transaminases remained raisedand inflammatory activity persisted. This observa-tion is consistent with evidence that in chronic HBVcarriers with concomitant delta infection there is amore rapid progression of chronic liver disease.26 27

Thus the continuing inflammation in the two deltainfected patients was probably attributable to thissuperinfection and not to continuing HBV infection.Liver histology showed no significant change in theuntreated and treated non-responder groups.Three of the four 'early' responders were hetero-

sexual and the fourth patient, who suffered theacute HAV infection, was homosexual. The two'late' responders were homosexual. Although this isnot significant with such small numbers this data andthe observation that the responders in our previousstudies14 were all heterosexual raises the possibilitythat there may be different responses to treatment inhomosexual and heterosexual HBV carriers.28 Thismay relate to the coexistence of other infectiveagents and immunodeficiency in the homosexualgroup.

Mild side effects were seen in the majority of theARA-AMP treated patients. Fatigue was present inall patients and in a minority, cramps and paraesthe-sia occurred late in the course of the treatment, andin some cases, after treatment.ARA-AMP has been shown to be effective in

producing inhibition of HBV replication and, in asignificant proportion of patients, this effect is longlasting, and associated with a reduced level ofinflammatory necrosis of liver cells. In two patientsshowing long term inhibition of HBV replication,intercurrent delta infection occurred and at thisstage, the transaminase and hepatic histologyshowed an increased level of inflammatory activity.Further controlled studies are in progress to deter-mine whether a longer course of ARA-AMP isassociated with a higher rate of seroconversion fromHBeAg to anti-HBe.

IVDW was an MRC training fellow, and is nowlecturer in the Academic Department of Genito-urinary Medicine, Middlesex Hospital MedicalSchool, ASFL was supported by the University ofHong Kong and HCT was a Senior WellcomeFellow of Clinical Science. ARA-AMP used in thisstudy was supplied by Warner Lambert - Parke-Davis. The authors would like to thank Professor MAdler for his help in the clinical trial, Professor P JScheuer and Dr I Lindsay for reviewing the liverbiopsies, Dr M J F Fowler for his help in theHBV-DNA assays and Mr D Hindley for statisticaladvice.

References

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