Alessandro Mattioli

INHIBITION OF HEPATITIS B VIRUS REPLICATION BY VIDARABINE MONOPHOSPHATE CONJUGATED WITH LACTOSAMINATED SERUM ALBUMIN

Vidarabine (ara A) produces severe dose-dependent side-effects. To examine whether its monophosphate ester (ara-AMP) can be effective in the treatment of chronic hepatitis B when given in reduced dosage as a conjugate with lactosaminated human serum albumin (L-HSA), which selectively enters hepatocytes, five patients with chronic type B hepatitis (HBsAg/HBV-DNA positive for at least 2 years) were treated with the conjugate. The daily dose of conjugate given (35 mg/kg) contains 1.5 mg ara-AMP, whereas the usual daily dose of free ara-AMP is 5-10 mg/kg. In three patients HBV-DNA fell to undetectable levels and remained negative in two; in one of them anti-HBe developed. In the other two patients HBV-DNA decreased but was detectable during treatment--one received three cycles of therapy, and became HBV-DNA negative and anti-HBe positive 45 days after the end of treatment; the other remained HBeAg/HBV-DNA positive. No adverse effects were observed, and biochemical variables (including aminotransferases) remained unchanged or decreased with viraemia. No antibodies (IgM and IgG classes) that bound the conjugate were detected. Thus L-HSA-ara-AMP inhibits HBV replication as well as free ara-AMP but at a third to a sixth of the dose.

Ribavirin conjugated with lactosaminated poly-L-lysine: selective delivery to the liver and increased antiviral activity in mice with viral hepatitis.

Ribavirin (RIBV) is a useful drug in the treatment of chronic type C hepatitis but displays a toxicity for red blood cells (RBC), which limits its dosage and necessitates withdrawal in some patients. Selective concentration of RIBV in liver should improve therapeutic results. Liver targeting can be achieved by coupling the drug to galactosyl-terminating peptides, which specifically enter hepatocytes. In the present work, we conjugated RIBV to lactosaminated poly-L-lysine (L-Poly(Lys)), a hepatotropic carrier enabling intramuscular (IM) administration of conjugates. The L-Poly(Lys)-RIBV conjugate had a heavy drug load (312-327 microg of RIBV in 1 mg of conjugate) and was very soluble in 0.9% NaCl (200 mg/mL). The conjugate was devoid of acute toxicity in mouse. When incubated with human or mouse blood, it did not release the drug. After IM administration to mice, the conjugate was selectively taken up by the liver, where the drug was released in a pharmacologically active form. This was demonstrated using mice infected with a strain of murine hepatitis virus (MHV) sensitive to RIBV. Coupled RIBV, IM injected, inhibited MHV replication in liver at a daily dose two to three times lower than that of the free drug. In mice IM injected with a conjugate tritiated in the RIBV moiety, the ratios between the levels of radioactivity in liver and RBC were two times higher than in animals injected with free tritiated RIBV. In conclusion, the present results support the possibility that the chemotherapeutic index of RIBV in chronic type C hepatitis can be increased by conjugation with L-Poly(Lys).

Liver targeting of antiviral nucleoside analogues through the asialoglycoprotein receptor.

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 interaction 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, allowing the latter to become concentrated in the liver. The validity of this chemotherapeutic strategy has been endorsed by a clinical study. Adenine arabinoside monophosphate (ara‐AMP), conjugated with lactosaminated human serum albumin (L‐HSA) 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‐effects, including the severe neurotoxicity caused by the free drug. Preclinical studies are now underway with conjugates obtained using lactosaminated poly‐L‐lysine (Lacpoly(Lys)) as the hepatotropic carrier. These new conjugates have some advantages over those prepared with L‐HSA: they can be administered by the intramuscular 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.

Selective delivery to the liver of antiviral nucleoside analogs coupled to a high molecular mass lactosaminated poly-l-lysine and administered to mice by intramuscular route

In order to obtain hepatotropic conjugates of antiviral drugs suitable for intramuscular administration, three nucleoside analogs (adenine arabinoside monophosphate, ribavirin and azidothymidine) were coupled to a high molecular mass lactosaminated poly-L-lysine. The conjugates had a high molar ratio drug/conjugate and after intramuscular administration to mice, were selectively taken up by the liver and eliminated by the kidney only in minute quantities. The high molar ratio and low renal elimination are important properties not possessed by conjugates previously prepared by using a small molecular mass lactosaminated poly-L-lysine. The conjugate with adenine arabinoside monophosphate (ara-AMP) was found to be devoid of acute toxicity for mice and in spite of its high molecular dimension (Mn = ca. 72,500) did not induce antibodies in this animal after repeated intramuscular injections. This conjugate could have two advantages over a similar complex of ara-AMP with lactosaminated human albumin currently under clinical trials for the treatment of chronic type B hepatitis which must be injected intravenously: it might provide better patient compliance since it is injectable intramuscularly and could introduce larger amounts of ara-AMP into hepatocytes due to its higher drug/carrier molar ratio.

Targeting of antiviral drugs to the liver using glycoprotein carriers

Abstract In order to improve the efficacy and to reduce the side-effects of antiviral drugs in the treatment of chronic hepatitis B, a lysosomotropic approach through covalent linkage of the drugs to appropriate glycoproteins was adopted. Antiviral nucleoside analogs were coupled to asialofetuin and to lactosaminated albumin (L-SA) (two galactosyl-terminating glycoproteins). The conjugates were selectively taken up by liver cells where they released the drugs in a pharmacologically active form. L-SA conjugates showed the advantage of being non-immunogenic when prepared with homologous albumin and injected intravenously. The majority of the experiments reported in this article were performed employing a conjugate of L-SA with ara-AMP, a drug active against hepatitis B virus (HBV). The results of animal studies warranted a 7-day administration of L-HSA-ara-AMP to patients with chronic hepatitis B. Conjugated ara-AMP was shown to inhibit HBV growth at a dose 3–6 times lower than that of the free drug.

Interaction of ADP-ribosylated aminoacyl-transferase II with GTP and with ribosomes

Abstract Rat liver aminoacyl-transferase II has been converted in the presence of diphtheria toxin and [ adenine - 3 H] NAD in the inactive form of the enzyme which bears covalently linked to the molecule the ADP-ribose moiety of NAD. The resulting [ 3 H] ADP-ribosyl-transferase II has been purified by electrofocusing procedures and its interaction with GTP and ribosomes has been investigated. While the inactive form of the enzyme binds GTP with a K d (1.09 · 10 −7 M) very close to that of the active enzyme (1.1 · 10 −7 M), it appears to be unable to interact with ribosomes.

A conjugate of lactosaminated poly-l-lysine with adenine arabinoside monophosphate, administered to mice by intramuscular route, accomplishes a selective delivery of the drug to the liver

A conjugate of the antiviral agent adenine arabinoside monophosphate (ara-AMP) with a low molecular mass lactosaminated poly-L-lysine, administered to mice by i.m. route, selectively delivers the drug to the liver. In mice the conjugate is devoid of acute toxicity even at high dose (1.3 mg/g) and injected i.m. for 20 days does not induce antibodies. Moreover it is highly soluble in water; this means that a pharmacologically active dose may be administered in a small volume compatible with the i.m. route. Compared to the similar ara-AMP complex with lactosaminated albumin which must be injected intravenously, the present conjugate might assure a better compliance of patients with hepatitis B virus infection for a long lasting, liver targeted antiviral treatment.

Hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly-l-lysine: Synthesis of the carrier and pharmacological properties of the conjugate

BACKGROUND/AIMS The hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly-L-lysine (L-Poly(Lys)) must have a high solubility in order to be injected in a small volume compatible with the intramuscular route. In this paper the molecular weights of Poly(Lys) which allowed the synthesis of conjugates with the properties of high solubility and limited loss by the kidney were determined and a procedure for obtaining Poly(Lys) preparations with the required range of polymerization has been described. METHODS Conjugates were prepared using Poly(Lys) of different molecular weights obtained by the procedure described here or purchased from a commercial source. Their solubility and renal loss in mice was determined. RESULTS Poly(Lys) with molecular weights ranging from 45,000 and 65,000 Da guarantees high solubility and low renal elimination of the conjugates. Conjugate preparations with these properties, intramuscularly administered to woodchuck hepatitis virus-infected woodchucks for 37 days at a daily dose of 5.8 mg/kg exerted a strong antiviral activity. These preparations were devoid of acute toxicity in rat and caused no toxic effects when injected intramuscularly daily for 28 days at a dose ten times higher than that active in woodchucks. CONCLUSIONS The results support the possibility of a clinical use of L-Poly(Lys) to obtain liver targeting of adenine arabinoside monophosphate for the treatment of chronic hepatitis B virus infection.

The pathogenesis of vacuoles produced in rat and mouse liver cells by a conjugate of adenine arabinoside monophosphate with lactosaminated albumin

A conjugate of adenine arabinoside monophosphate with lactosaminated albumin produced vacuoles in hepatic cells of rats and mice when given at doses 5-10 times higher than that (35 mg/kg) capable of inhibiting hepatitis B virus replication in patients with chronic hepatitis B. The vacuoles were due to the swelling of secondary lysosomes probably caused by incapacity of the lysosomal enzymes to rapidly digest large amounts of conjugate into products able to cross the lysosomal membrane. Although vacuoles progressively disappeared when conjugate administration was discontinued, the present observation suggests caution in giving the conjugate to man at daily doses higher than 35 mg/kg.

Studies on diphtheria toxin the effect of GTP on the toxin-dependent adenosine diphosphate ribosylation of rat liver aminoacyl transferase II

Abstract 1. (1) Diphtheria toxin catalyzes the transfer of ADP-ribose from NAD to aminoacy transferase II (T2). The resulting ADP-ribosyl-T2 complex is devoid of enzymatic activity: both the translocase and the ribosome-dependent GTPase activities are inhibited. GTP protects T2 from ADP-ribosylation. 2. (2) By equilibrium dialysis the dissociation constants and the molarity of the T2 protein groups interacting with ADP-ribose and with GTP have been established. The same number of binding sites is present for the two ligands. The K d of the GTP-T2 complex is 4.2·10 −7 M and that of the ADP-ribosyl-T2 complex is 4.9·10 −7 M . 3. (3) While GTP lowers the affinity of T2 for ADP-ribose, the presence of diphtheria toxin and NAD does not affect the binding of GTP to T2; thus the binding sites for GTP and for ADP-ribose are not the same. 4. (4) GTP binds to diphtheria toxin with a K d = 5.9·10 −6 M and behaves as a competitive inhibitor of the toxin-NAD interaction. Considering that GTP binds both to diphtheria toxin and to transferase, the mechanism by which the guanosine nucleotide protects T2 from ADP-ribosylation is discussed.