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loirrrta!oJVirnl H?pfltitis. 1997.4, 363-370

REVIEW

Liver targeting of antiviral nucleoside analogues through the

L. Fiume,' G. Di Stefano,' C. Busi,' A. Mattioli,' F. Bonino,2 M. Torrani-Cerenzia,2 G. Verme,2 M. Rapicetta, M. Bertini* and G. B. Gervasi4 'DiprtimetIto di Pntologiu Sperirnetitnle, Utriversitli di B O ~ O ~ I I ~ . Bologt1n. my. 'Ospedde Mo~~r~ette, Torino. Itafg. 31stitiito ~iiperiore di Snnitci. Rumn. l tn~y nnd bornto tori Bff~dncci, Pisn, ltnfg

3

SUMMARY. In order to reduce the extrahepatic side- effects of antiviral nucleoside analogues in the treatment of chronic viral hepatitis, these drugs are conjugated with galactosyl-terminating macromolecules. The conjugates selectively enter hepatocytes after inter- action of the carrier galactose residues with the asialoglycoprotein receptor present in large amounts and high affinity only on these cells. Within hepatocytes the conjugates are delivered to lysosomes where enzymes split the bond between the carrier and the drug, allow- ing the latter to become concentrated in the liver. The validity of this chemotherapeutic strategy has been endorsed by a clinical study. Adenine arabinoside monop~osphate (ara-A~F), conjugated with lactosaminated human serum albumin (GHSA) and administered to hepatitis B virus (HBV)-infected patients for 28 days, exerted an antiviral activity to the same extent as the free drug without producing any clinical side-efTects.

including the severe neurotoxicity caused by the free drug. Preclinical studies are now underway with conjugates obtained using lactosaminated poly-tlysine (Lac- poly(Lys)) as the hepatotropic carrier. These new conjugates have some advantages over those prepared with L - X S A they can be administered by the in~amuscular route: they are obtained entirely by chemical synthesis, thus eliminating the problems involved in the use of haemoderivatives: they have a heavy drug load, which permits administration of smaller quantities of conjugate that are more easily digested in lysosomes; and they enable higher quantities of drug to be introduced into hepatocytes. The results of the experiments with two Lac-poly(Lys) conjugates, one with ara-AMP and one with ribavirin, are reported in this review.

Kqwords: antiviral nucleoside analogues. asialoglycoprotein receptor, liver targeting of drugs. viral hcpatitis.

I ~ ~ R ~ D U C T I O N

The side-effects of antiviral nucleoside analogues can be reduced by selectively transporting them to the infected cells. This goal can be obtained by adopting the tysosomotropic approach to chemotherapy in which the drug is coupled to a macromolecule that is selectively taken up by the target cells and delivered to lysosomes [ 1-31, Lysosomal enzymes can attack the

Abbreviations: AF. desialylated fetuin: HBV, hepatitis B virus: HSA. human serum albumin: IFN-a. interferon-% LSA. lactosaminated serum albumin: MHV, murine hepatitis virus; RIBV. ribavirin: WHV, woodchuck hepatitis virus.

Correspondence: Prof. Luigi Fiume. Dipartimento di Patologia Sperimentale. Via S . Giacomo 14.40126 Bologna. Italy.

conjugate and release the drug. If the drug is resistant to lysosomal enzymes and acidic pH and crosses the lysosomal membrane it becomes concentrated in the cells into which it was transported. This strategy can be particularly appropriate for reducing the e ~ r ~ h e p a t i c side-effects of nucieoside analogues in the treatment of human viral hepatitis. In viral hepatitis the bulk of virus replicates in hepatocytes, and these cells specifi- cally internalize and deliver to Iysosomes galactosyl- terminating macromolecules which can be used as selective hepatotropic vectors of drugs. These molecules enter parenchymal liver cells after inter- action with a high-a~nity receptor for asialoglycoproteins 141 that is present in large numbers only on these cells and which recognizes the clustered and exposed galactose residues on the asialoglycoprotein

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[reviewed in 51. Conjugates can freely come into contact with the surface of hepatocytes as liver sinusoids do not form a barrier for proteins.

ASIALOFETUIN CONJUGATES

The first hepatotropic conjugates of drugs were prepared by using desialylated fetuin (AF) as a carrier t6.71. Fetuin is a glycoprotein from fetal calf serum which, after desi~lylation, exposes gafactosyl residues. It had been previously used to deliver proteins [8] and liposomes f9] to liver cells, The nucleoside analogues trifluorothymidine, adenine arabinoside (ara-A) and its monophosphate (ara-AMP), coupled to AF and administered to mice with Ectromelia virus hepatitis, were selectively concentrated inside hepatocytes in a pharmacologically active form [6,7]. However, AF conjugates induce antibodies [l 01, which can inactivate the conjugates and produce allergic lesions.

LACTOSAMINATED ALBUMIN CONJUGATES

The problem of immunogenicity was overcome by using lactosarninated serum albumin (LSA) as the hepatotropic drug carrier [ 11,121. Lactose is a disac- charide co~prising glucose and galactose. In the preparation of LSA the aldehyde group of the glucose is linked by reductive amination [ 133 to a lysine amino group of albumin. The galactose moiety of lactose remains unmodified and can therefore interact with the asialoglycoprotein receptor [14.15]. An albumin molecule, to which 20-30 lactose molecules have been bound. selectively enters hepatocytes. When prepared with homologous album~n [lo] and injected intravenously f l6f L-SA conjugates are not immunogenic.

L-HSA-nm-AMP: esperirnerzts in nrziriinls

The majority. of animal experiments were performed using a L-H(human)SA conjugate of ara-AMP. This drug exerts a marked antiviral activity in chronic hepatitis €3 [I 71 but the severe neurotoxic side-effects hindered its clinical use [ 171 and led to its withdrawal in both the UK and the USA.

Ara-AMP was conjugated with LHSA by means of a water-soluble carbodiimide [ 1 1,121. In the conjugate, the ara-AMP molecules are linked by phosphoamide bonds with lysine and histidine residues of HSA [18]. The conjugate is composed of the monomer as well as

polymers of LHSA that were formed by the carbodiimide as a side reaction 1121.

In contrast to the free drug, ara-AMP conjugated with LHSA is not deaminated in the bloodstream [19]. The conjugate is selectively taken up by the liver [12.20]. The bonds between ara-AMP and L-HSA, which are stable in blood [12,21], are rapidly broken down in liver [18]. In mice with Ectromelia virus hepatitis, ara-AMP conjugated with L-HSA inhibited viral DNA synthesis in the liver without producing significant inhibition of ceIIular DNA synthesis in bone marrow and in intestine; this was in contrast to free ara-AMP that affected DNA synthesis in all three organs [ 11.12 3. In woodchucks with hepatitis caused by woodchuck hepatitis virus (WHV), ara-AMP coupled to LHSA at a daily dose of 1.5mgkg-' produced a decrease in viraemia equal to or greater than that brought about by the free drug at doses of 10 or 20 mg kg-' 1221.

LHSA-nm-AMP: clinical st irdies

In preliminary clinical studies, 1 3 patients with chronic hepatitis B virus (HBV) infection were treated with L-HSA-ara-AMP for a maximum of 7 days 123,241. The conjugate was administered by a 30-min in~ravenous infusion at a daily dose of 3 5 m g kg-' body weight. corresponding to 1.5 m g ara-AMP kg-' . By comparison. free ard-AMP is usually given for 28 consecutive days at a daily dose of 10 mg kg-' for the h s t 5 days and 5 mg kg-' for the remaining 13 days. I,-HSA- ara-AMP consistently decreased the serum levels of HBV DNA in all treated patients. indicating that coupled ara-AMP can inhibit HBV reptication at a daily dose 3-6 times lower than that of the free drug. Subsequently, L-HSA-ara-AMP was administered to eight patients for 28 consecutive days at the dosage used in the previous studies [2 51. During the treatment. the conjugate produced the same degree of inhibition of viral replication as the free drug. but it cawed neither the severe neurotoxic manifestations that usually appear 3 weeks after admi~istr~tion of the free ara- AMP (261 nor other clinical side-effects. The conjugate produced a significant increase in serum alkaline phosphatase activity and in platelet number, and a small but significant decrease in erythrocyte number. These laboratory parameters returned to normal levels within 2 months after treatment completion. The increased alkaiine phosphatase level was not caused by

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Liver targeting ofantiviral drugs 365

cholestasis. as the levels of gamma-glutamyl trans- ferase and j'-nucleotidase were not changed by the treatment. The increased alkaline phosphatase level was probably caused by the carrier moiety of the conjugate as, in a toxicological study using Cynomolgus monkey, serum alkaline phosphatase was increased in the animals injected with L-HSA-ara-AMP or L-HSA but not in monkeys administered the free drug [27] . The increased platelet counts and the decreased erythrocyte number could be explained, at least in part, by the sizeable blood samples (approximately 10 ml) withdrawn daily during treatment. The conjugate did not-affect the number of lymphocytes and this may be an important advantage of L-HSA-ara-AMP over free ara-AMP. which produces a marked lympho- cytopenia [26,28], as successful antiviral treatment requires the co-operation of the immune system. The conjugate induced the production of small amounts of antibodies (approximately 1.7 pg IgG ml-' serum) in one patient only.

In conclusion, this study demonstrated that in chronic HBV the chemotherapeutic index of ara-AMP was increased by coupling to GHSA, and supported conjugation with galactosyl-termina ting carriers [h,l1,29,30] as a way for improving the value ofthose drugs that are active against human hepatitis viruses but cause extrahepatic side-effects. A multicentre Phase I11 clinical trial is underway in France to assess the long-term efficacy of the conjugate. L-HSA-ara- AMP is being administered to patients who did not respond to interferon-a (IFN-a) and have low levels of serum HBVDNA. In fact, ara-AMP was found to be able to produce a complete and sustained inhibition of HBV replication in patients with low viraemia [3 11.

LACTOSAMINATED POLY-L-LYSINE C~NJUGATES

A drawback of conjugates prepared with L-SA is that they have to be administered intravenously on account of the volume to be injected and because of their immunogenicity when administered by other routes [ 161. Conjugates injectable by the intramuscular (i.m.) route are now being studied. These can be obtained by using lactosaminated poly-L-lysine (Lac-poly(Lys)) as the hepatotropic carrier 1291. Nucleoside analogues are first phosphorylated in the primary hydroxyl group of the sugar moiety and are then linked to Lac-poly(Lys~ [ 32,331 by the same phosphoamide bond that is formed

between ara-AMP and the lysine residues in L-HSA- ara-AMP, Many molecules of drug (>loo) can be bound to a single carrier molecule because of the high number of amino groups in Lac-poIy(Lys). The high drug load and solubility of conjugates prepared with L- poly(Lys) enables their pharmacological doses to be injected in a small volume, compatible with the i.m. route.

Substitution of a high percentage (>70) ofthe amino groups of poly(Lys) completely eliminates the toxic effects of this polymer [33,34], which are associated with its polycationic character and are caused by its binding to the electronegative charges on the cell membranes [35]. Coupling of lactose by reductive amination preserves the cationic character of Iysine amino groups [ 131, but the bound lactose, together with the coupled phosphorylated nucleoside analogues (which are negatively charged), hinder the binding of the conjugate to the electronegative groups of the cell membrane [34].

In addition to being injectable i.m. Lac-poIy(Lys) conjugates have other advantages over those prepared with L-HSA

1 they are obtained entirely by chemical synthesis, thus eliminating the problems involved in the use of haernoderivatives;

2 because of the heavy drug load they permit administration of smaller quantities of conjugate, more easily digested in lysosomes; and

3 they enable higher quantities of drug to be introduced into hepatocytes.

Preclinical studies (discussed below) have been conducted using two Lac-poly(Lys) conjugates, one with ara-AMP and one with ribavirin,

~ ~ ~ - ~ ~ J ~ (Lgs) - ~ r a - ~ P

Injected by the i.m. route into mice, this conjugate was shown to enter the liver seIectively E33.341. Its antiviral activity was studied in woodchuck carriers of WHV f34.361. Free ara-AMP injected i.m. at a daily dose of 2.5 mg kg-' had no antiviral effect whereas Smgkg-I produced a decrease of viraemia. Lac- poly(Lys)-ara-AMP given i.rn. at a daily dose of 4.2 mg kg-' (equivalent to 1.5 mg kg-I ara-AM~) or 7 mg kg-' (equivalent to 2.5 mg kg-' ara-AMP) strongfy decreased the viral DNA levels. In the animals injected with the higher dose, viraemia fell to

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undetectable levels during treatment. Assuming that the same doses will also be active in HBV-infected patients, the amount of Lac-poIy(Lys)-ara-AMP (soluble at 200 mg kg-') required by a 70 kg patient will be contained in a 1.5-2.5 rnl volume, compatible with the i.m. route. The possibility of administering the conjugate by thc ism. route was supported both by an eye irritation study in rabbits [33] and a 4-week intramuscular toxicity study in rats [36]. In both studies the concentrated conjugate solution did not produce any irritation at the site of administration.

Lac-poly(Lys)-ara-AMP is practically devoid of acute toxicity. A bolus intravenous injection of 1.5 g conjugate per kg body weight in mice and rats produced no toxic effects [ 33,361. Furthermore, Lac-poly(Lys)-ara- AMP did not produce toxic effects in a subehronic study in rat [36] . Lac-poly(Lys)-ara-AMP injected i.m. into rats for 4 weeks, at a daily dose 20 times higher than that active in woodchucks, produced neither signs of clinical toxicity nor changes at the histological level. HaematoIogical and laboratory parameters were not modified. L-HSA-ara-AMP produced vacuoles in hepatic cells when given at a dose five times higher than that capable of inhibiting virus replication in woodchucks and in HBV-infected patients. The vacuoles were caused by accumulation of incomplete degradation products of conjugate in lysosomes [3 71. The absence of vacuolation in liver cells of rats treated with Lac-poly(Lys)-ara-AMP was probably the result of more rapid digestion of the carrier, which was administered in smaller amounts. By comparison, 1 mg ma- AMP is transported by 1.2-1.9 mg carrier in Lac- poly(Lys)-ara-AMP and by 22 mg in L-HSA-ara-AMP, respectively.

~ , ~ C - ~ O ~ ~ ~ ~ ~ i S } - ~ i ~ v Ribavirin (RIBV). is a broad-spectrum antiviral nucleoside analogue which, when administered to patients with chronic hepatitis C in association with EN-a , has been found to cause a sustained biochemical and viro- logical response in a number of patients that is signifr- cantly higher than in patients treated with IFN-a alone [ 38421. A drawback of R B V is its accumulation in red blood cells (KBC) C43.441, which produces haemolysis 138-441. This side-effect not only causes the withdrawal of the drug in some patients, but also increases the iron concentration inside hepatocytes to levels 645.461 that might decrease the efficacy ofIFN-a [47].

Therefore, reduction of haemolysis, which might be obtained by a liver targeting of the drug, could result in an even higher percentage of long-term responders to F N s r associated with w 3 V . The RIBV was coupled to Lac-poly(Lys); the conjugate had a high drug load (3 15-320 pg RIBV mg-I conjugate) f48] and was very soluble in NaClO.9% (450 rng ml-') (G. Di Stefano et nl.. submitted). It was devoid ofacute toxicity in the mouse. After i.m. administration, to mice, of a ['*C]Lac- poly(Lys)-RIBV conjugate, labelled in the lactose moiety, the levels of radioactivity in liver were more than 10 times higher than in the other organs [48]. In liver, RIBV was released in a pharmacologically active form. This was demonstrated using mice infected with a strain of murine hepatitis virus (MHV) sensitive to this drug. Coupled RIBV, i.m. injected, produced a 50% inhibition of M H V replication in liver at a daily dose two times lower than that of the free drug. In mice injected im. with a conjugate tritiated in the RIBV moiety, the ratios between the levels of radioactivity in liver and REX were two or five times higher than in animals that received the free radioactive drug given i.m. or orally, respectively (G. Di Stefano et nf., submitted).

These data support the possibility that conjugated RIBV might display the same antiviral activity against hepatitis C virus (HCV) in liver as the free drug, but with reduced haemolysis.

LACTOSAMINATED LIPOPROTEIN COMPLEXES

After lactosamination of the protein moiety, the lipoproteins interact with the asialoglycoprotein receptor and selectively enter hepatocytes [49,50]. Tritiated iododeoxyuridine (IDU), rendered lipophilic by derivati- zation with oleoyt residues through an acid-~abile link- age, was incorporated into lactosarninated high- density lipoproteins. The fevels of radioactivity in liver were several times higher in rats injected intravenously (i.v,) with this complex than in animals treated with an equal dose of free J3H]IDU. However, no evidence was obtained to suggest that this complex was pharmacologically active in liver [ 5 1)-

ARABINOGALACTAN CONJUGATES

Arabinogalactan is an arabinose/galactose polysac- charide displaying high affinity for the asialoglycoprotein receptor [ 301. An arabinogalactan-ara-AMP conjugate (with a molar ratio drug:carrier=8) i.v.

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injected into WHV-infected woodchucks at a daily dose of 50 mg kg-' (corresponding to 3 mg kg-I ara-AMP) produced a more marked decrease of viraemia than the free drug at 15 rng kg-lday-l [ 5 2 ] . In acute and sub- chronic (30 days) toxicity studies, this conjugate administered to mice and rats at doses several t i e s higher than that pharmacologically active in woodchucks did not produce either toxic symptoms or modifi- cations in clinical biochemistry and haematology [52].

Arabinogalactan has an advantage over the other hepatotropic carriers because it does not require chemical changes in order to bind to the asialoglycoprotein receptor. On the other hand it must be chemically modified in order to acquire amino groups to which ara-AMP or other phosphorylated nucleoside analogues can be bound.

DRA~BACKS IN LIVER TARGET IN^ OF NUCLEOSIDE ANALOGUES

Penetrntion ofconjugates in cells other than hcpatocytes

In liver, the GHSA-ara-AMP conjugate not only enters hepatocytes, but also the sinusoidal cells [20,53]. This observation agrees with the finding that liver sinusoidal cells take up chemically modified albumin [54,55]. Delivery of ara-AMP to sinusoidal cells (and probably to other cells of the reticuloendothelial system) is not expected to decrease the chemotherapeutic index of the conjugate as these cells are not damaged by ara-AMP (or by the other nucleoside analogues studied).

Rediiced n ~ f ~ b ~ r ~ ~ r e c e ~ ~ o r ~ o ~ e c u l e s on ~ B V - i n f ~ c ~ e d he~Rto&~tes

Liver uptake of asialoglycoproteins is slower in patients with chronic HBV than in healthy subjects or laboratory animals, probably because of a diminished number of receptor molecules [56-581. In HBV- infected patients, liver uptake of L-HSA-ara-AMP was approaching saturation at a daily dose of only 42 mg kg-"25], which is close to the therapeutic dose of 35mgkg-'. This suggests that nucleoside analogues, which are pharmacologically active at doses greater than ara-AMP, can only be targeted to the liver by using conjugates with a high molecular ratio of drugxarrier, such as those prepared with Lac- POlY(LYS).

Liver-targeted antiviral treatment requires that repeated administration of hepatotropic conjugates does not cause downregulation of the asialoglycoprotein receptor, thereby reducing, still further, the liver capacity to internalize them. In this context, an important finding was that liver uptake of L-HSA-ara-AMP by HBV-infected patients was not substantially altered after the 28-day treatment [ 2 5 ] .

Release of nucleoside analogues from liver cells into the bloodst ream

After their release from the carrier within hepatocytes, nucleoside analogues partly exit from these cells into the bloodstream and redistribute in the organs [6,20,48]. This loss decreases the eficacy of the liver targeting. In fact, 3 h after administration to rats of a L- HSA-ara-AMP conjugate labelied in the protein molecule with [3H]sucrose (a sugar that remains within lysosomes [59]), radioactivity in the liver was 93 , 23 and five times higher than in intestine, spleen and kidney respectively, whereas 3 h after administration of the same dose of a LHSA-ara-AMP conjugate labelled in the drug moiety, liver radioactivity was only six, seven and three times higher than in the intestine, spleen and kidney, respectively [20]. (For comparison. in rats injected with free tritiated ara-AMP. radioactivity was equally distributed in liver, spleen and intestine with higher values in kidney [20].)

HBV a id HCV rc~lica~~ur7 in eells otlier than ~ i ~ ~ ~ ~ ~ ~ , ~ ~ ~ s

A limitation of the efficacy of the hepatic-targeting approach might be the capacity of HBV and HCV to replicate in cells other than hepatocytes, which do not take up the conjugates. However, non-conjugated nucleoside analogues whose antiviral activity has been studied on hepadnavirus replication in extrahepatic sites have been shown to be ineffective [60.61]. PossibIy in these sites they are not metabolized to phospho~lated derivatives, which are the antiviral active forms [61]. Extrahepatic viral replication should not therefore reduce the advantage of using he~atotropic conjugates over unconjugated nucleoside analogues in HBV infection. Moreover, it should be stressed that:

1 in chronic B and C hepatitis the viral load in hepatocytes is far higher than in other cells; and

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368 L. F ~ ~ ~ e et al.

2 the task of the antiviral treatment is basically to achieve a drastic inhibition of viral replication in the organism in order to limit the infection of new cells, thus facilitating elimination of infected cells by the immune system,

CONCLUSIONS

The clinical study with the LHSA-ara-AMP conjugate has demonstrated that conjugation with galactosyl- terminating camers can maintain the antiviral activity of nucleoside analogues in liver and abolish or reduce their extrahepatic toxic effects. Hepatic targeting will aIIow, first, a higher and therefore more efficient concentration of these drugs in liver and. second, enable administration of new powerful anti-hepatitis nucleoside analogues, whose use, as free drugs, might be prevented by extrahepatic side-effects.

The author's work reported in this review was supported by grants from MURST, CNR, ARC. the Istituto Superiore di Sanita (Progetto Epatite Virale) and the University of Bologna (funds for selected research topics).

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