Efficacy of doxorubicin coupled to lactosaminated albumin on rat hepatocellular carcinomas evaluated by ultrasound imaging.

BACKGROUND/AIMS: Doxorubicin was conjugated with lactosaminated human albumin, a hepatotropic drug carrier, in order to increase its efficacy in the treatment of hepatocellular carcinoma. In rats bearing hepatocellular carcinomas induced by diethylnitrosamine, lactosaminated human albumin coupled doxorubicin enhanced the drug concentrations in the tumours and lowered those in extrahepatic tissues. The aim of the present study was to investigate the effects of lactosaminated human albumin coupled doxorubicin on the growth of established rat hepatocellular carcinomas induced by diethylnitrosamine. METHODS: Lactosaminated human albumin coupled doxorubicin and the free drug were i.v. administered to rats twice a week for 4 weeks at the single dose of 1 microg/g. Growth of individual tumours was followed through time by ultrasonography. RESULTS: In the control animals injected with saline the mean area of the tracked tumours significantly increased during the whole period of treatment. In the group of rats treated with lactosaminated human albumin coupled doxorubicin the mean area of the followed hepatocellular carcinomas remained practically unchanged. The free drug inhibited tumour growth only in the first period of drug administration. Lactosaminated human albumin coupled doxorubicin also hindered the development of new neoplastic nodules, which was unaffected by the free drug. CONCLUSIONS: The results support lactosaminated human albumin coupled doxorubicin as a promising agent for a systemic chemotherapy of hepatocellular carcinomas to treat noncurable patients.

Doxorubicin coupled to lactosaminated albumin: Effects on rats with liver fibrosis and cirrhosis.

BACKGROUND: The conjugate of doxorubicin with lactosaminated human albumin has the potential of increasing the doxorubicin efficacy in the treatment of hepatocellular carcinomas expressing the asialoglycoprotein receptor. However, coupled doxorubicin also accumulates in the liver, which might damage hepatocytes. AIMS: To verify whether coupled doxorubicin impairs liver function in rats with liver fibrosis and cirrhosis. METHODS: Coupled doxorubicin was administered using the same schedule which exerted an antineoplastic effect on rat hepatocellular carcinomas (4-weekly injections of doxorubicin at 1 microg/g). Liver fibrosis/cirrhosis was produced by carbon tetrachloride (CCl4) poisoning. Liver samples were studied histologically. Serum parameters of liver function and viability were determined. RESULTS: In normal rats, administration of coupled doxorubicin neither caused microscopic changes of hepatocytes nor modified serum liver parameters. In rats with fibrosis/cirrhosis, although a selective doxorubicin accumulation within the liver followed coupled doxorubicin administration, the drug did not have a detrimental effect on the histology of the liver and, among serum liver tests, only alanine aminotransferase and aspartate aminotransferase levels were moderately modified. CONCLUSIONS: Coupled doxorubicin can be administered to rats with liver fibrosis/cirrhosis without inducing a severe liver damage. If further studies will confirm the efficacy and safety of this compound, coupled doxorubicin therapy may open a new perspective in the treatment of hepatocellular carcinoma.

Targeting of antiviral drugs by coupling with protein carriers.

Side effects of antiviral drugs might be circumvented by their selective delivery into infected cells. This targeting can be obtained by conjugation of the drugs to macromolecules which are taken up specifically by the infected cells. The experiments reviewed, on this approach to antiviral chemotherapy, are mainly directed at improving the chemotherapeutic index of adenine arabinoside (ara-A) in the treatment of chronic hepatitis B by its coupling to galactosyl terminating glycoproteins.

Galactosylated poly(L-lysine) as a hepatotropic carrier of 9-beta-D-arabinofuranosyladenine 5'-monophosphate.

D-Galactopyranosyl residues were coupled to poly(L-lysine) and the antiviral agents arabinofuranosyladenine 5'-monophosphate (ara-AMP) and acyclovir were conjugated with this glycosylated polymer. In mice the ara-AMP conjugate accomplished a selective drug delivery to liver cells.

Targeting of antiviral drugs bound to protein carriers.

The value of antiviral drugs would be improved by their selective delivery into infected cells. This goal can be achieved by conjugating the drug to a protein which is taken up specifically by the cells where the virus grows. Conjugates will be pharmacologically effective in these cells if the bond linking the drug to the carrier does not suppress the activity of the drug or if the bond is broken down with consequent intracellular release of the drug. Experiments on this approach to antiviral chemotherapy have been directed mainly at selectively delivering antiviral agents into hepatocytes in order to increase their chemotherapeutic index in the treatment of chronic hepatitis B.

Drug targeting in antiviral chemotherapy. A chemically stable conjugate of 9-beta-D-arabinofuranosyl-adenine 5'-monophosphate with lactosaminated albumin accomplishes a selective delivery of the drug to liver cells.

With the aim of improving the chemotherapeutic index of 9-beta-D-arabinofuranosyl-adenine 5' monophosphate (ara-AMP) in the treatment of chronic hepatitis B, this drug was conjugated with lactosaminated serum albumin (L-SA), a neoglycoprotein which only enters into hepatocytes. We used a L-SA-ara-AMP conjugate which, in contrast to those previously employed, has the advantage of remaining soluble after lyophilization. We found in mice that: (I) this new conjugate was quite stable in the bloodstream where only a small part of ara-AMP was released; (II) after administration of the conjugate labelled in the drug moiety both acid insoluble and soluble radioactivities were several times higher in liver than in other organs; (III) in mice with Ectromelia virus hepatitis, the conjugate inhibited virus DNA synthesis in liver without affecting cellular DNA synthesis in intestine and bone marrow; (IV) the conjugate did not display any recognizable sign of acute toxicity even at doses several fold higher than those pharmacologically active; and (V) when prepared with homologous albumin it was not immunogenic.

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.

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.

Enhanced liver blood concentrations of adenine arabinoside accomplished by lactosaminated poly-L-lysine coupling: implications for regional chemotherapy of hepatic micrometastases.

Conjugates of antiviral and antiblastic nucleoside analogs (NAs) with galactosyl-terminating peptides selectively enter hepatocytes after binding of the carrier galactose residues to the asialoglycoprotein receptor. Since NAs, when set free from the carrier within hepatocytes, partly exit from these cells into the bloodstream, we considered the possibility that administration of galactosyl-terminating conjugates of NAs could result in plasma concentrations of these drugs that would be higher in liver sinusoids than in capillaries of other organs. In the present study we demonstrated the validity of this hypothesis. We injected rats with a conjugate of adenine arabinoside (ara-A) with lactosaminated poly-L-lysine and found that the plasma concentrations of ara-A were >2-fold higher in blood of liver than in systemic circulation. Liver blood was collected from the inferior vena cava after closing below and above the outflows of the hepatic veins. The present result suggests that conjugation with galactosyl-terminating peptides might be a way to selectively increase the concentrations of NAs not only in hepatocytes, which have the asialoglycoprotein receptor, but also in cells infiltrating the liver, such as neoplastic cells of micrometastases nourished by hepatic sinusoids.

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).

Coupling of 5-fluoro 2'-deoxyuridine to lactosaminated poly-l-lysine: an approach to a regional, non-invasive chemotherapy of liver micrometastases.

Nucleoside analogs conjugated with galactosyl-terminating peptides selectively enter liver cells and after intracellular release from the carrier partly exit into bloodstream, resulting in higher concentrations in liver blood than in systemic circulation. The aim of the present experiments was to ascertain whether, in mice injected with non-toxic doses of a 5-fluoro 2'-deoxyuridine (FUdR) conjugate with lactosaminated poly-L-lysine (L-poly(LYS)), the drug was released by hepatic cells in high enough amounts to be pharmacologically active on neoplastic cells infiltrating the liver. We observed that L-poly(LYS)-FUdR inhibited the growth of hepatic metastases induced by intrasplenic administration of murine colon carcinoma C-26 cells. L-poly(LYS)-FUdR was not toxic for C-26 cells in vitro, was selectively taken up by mouse liver, and was stable in mouse blood, indicating that the effect on the metastases was due to FUdR (and/or its active metabolites) released in liver blood after the conjugate was taken up by the hepatic cells. These results suggest that L-poly(LYS)-FUdR might be useful in adjuvant chemotherapy of tumors giving liver metastases. The drug released from hepatic cells into liver blood following conjugate administration via the peripheral venous route might accomplish a locoregional, non-invasive treatment of micrometastases nourished by liver sinusoids.

Inhibition of [3H]thymidine incorporation into DNA of rat regenerating liver by 2',2'-difluorodeoxycytidine coupled to lactosaminated poly-L-lysine.

The expression of asialoglycoprotein receptor (ASGP-R) on human hepatocarcinoma cells might be exploited to reduce the extrahepatic toxicity of DNA synthesis inhibitors by their conjugation with galactosyl- terminating peptides. We conjugated 2',2'-difluorodeoxycytidine (dFdC), an inhibitor of DNA synthesis active on solid tumors, with lactosaminated poly-L-lysine (L-poly(LYS)). In experiments in vitro, L-poly(LYS)-dFdC inhibited proliferation of Hep G2 cells, a human hepatocarcinoma cell line which maintains the ASGP-R. Inhibition was rescued by asialofetuin. To study the pharmacological action of the conjugate in vivo, we used rats 18-24 hr after 2/3 hepatectomy and observed that regenerating hepatocytes expressed ASGP-R on their surface and internalized L-poly(LYS)-dFdC. Conjugate uptake by bone marrow, spleen, and intestine was negligible. We also found that L-poly(LYS)-dFdC inhibited [3H]thymidine incorporation into DNA of regenerating liver. These results indicated that hepatectomized rats were a suitable animal model to study the pharmacological action, on DNA-synthesizing hepatocytes, of conjugates binding to ASGP-R and carrying inhibitors of DNA synthesis. L-poly(LYS)-dFdC also inhibited [3H]thymidine incorporation in bone marrow, spleen, and intestine. Evidence was obtained that inhibition of DNA synthesis in extrahepatic tissues was a consequence of drug release from hepatocytes into blood-stream after the bond with the carrier has been broken down within liver cells. Possible ways of reducing the exit of dFdC from liver cells, thereby obtaining an inhibition of DNA synthesis restricted to dividing hepatocytes, were discussed.

Floxuridine coupling with lactosaminated human albumin to increase drug efficacy on liver micrometastases.

BACKGROUND: Conjugates of nucleoside analogues with galactosyl terminating peptides selectively enter hepatocytes through the asialoglycoprotein receptor. After intracellular release from the carrier, the drugs partly exit from hepatic cells into hepatic blood. AIMS: To establish whether administration of a conjugate of floxuridine with lactosaminated human albumin selectively enhances drug concentrations in hepatic blood. Floxuridine is a fluoropyrimidine active on human colorectal cancer, a tumour which metastasises first to the liver. METHODS: In rats injected with free or conjugated floxuridine, plasma levels of the drug were determined in hepatic veins and in inferior vena cava, in order to measure drug concentrations in hepatic blood and in the systemic circulation, respectively. RESULTS: Ratios between floxuridine levels in hepatic veins and those in systemic circulation were found to be seven times higher in rats injected with the conjugate (p=0.000). CONCLUSIONS: The present results suggest that coupling to lactosaminated albumin might improve the effect of floxuridine in adjuvant chemotherapy of colorectal cancer by exposing the cells of liver micrometastases (nourished by hepatic sinusoids) to enhanced drug concentrations.

Doxorubicin coupled to lactosaminated human albumin remains confined within mouse liver cells after the intracellular release from the carrier.

BACKGROUND: The hepatocyte receptor for asialoglycoproteins, which binds and internalises galactosyl terminating peptides, was found to be expressed also on the cells of the majority of hepatocarcinomas. AIMS: To verify whether doxorubicin coupling to lactosaminated albumin, a galactosyl terminating neoglycoprotein, produces selective drug accumulation in hepatocytes with reduced concentrations in extra-hepatic tissues, thus facilitating the use of the drug in hepatocarcinoma treatment. METHODS: Doxorubicin concentrations were measured in organs of mice injected with the free or coupled drug. RESULTS: In mice injected with the coupled drug, the ratios between doxorubicin concentrations in liver and those in heart, intestine, spleen and kidney were 8-14 times higher than in animals that received the same dose of the free drug. CONCLUSIONS: Due to the very efficient liver targeting of doxorubicin, the lactosaminated human albumin-doxorubicin conjugate appears to have the potential of improving the chemotherapy of hepatocellular carcinomas through the asialoglycoprotein receptor.

Prony-Householder method applied to 31P NMR signals: II. Study of conjugates of ara-AMP with lactosaminated albumin.

Conjugates of 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) with lactosaminated albumin (L-SA), obtained by using two different coupling procedures, produce antibodies in rats and mice which display only a small cross-reactivity. 31P NMR signals from the two conjugates have been examined to clarify whether different lysine and histidine residues are involved in the two reaction pathways. The occurrence of different chemical shifts and linewidths between the two conjugates, as evidenced by processing the signals with the Prony-Householder method, indicates the formation of two different complexes.

Galloflavin, a new lactate dehydrogenase inhibitor, induces the death of human breast cancer cells with different glycolytic attitude by affecting distinct signaling pathways.

Galloflavin (GF), a recently identified lactate dehydrogenase inhibitor, hinders the proliferation of cancer cells by blocking glycolysis and ATP production. The aim of the present experiments was to study the effect of this compound on breast cancer cell lines reproducing different pathological subtypes of this tumor: MCF-7 (the well differentiated form), MDA-MB-231 (the aggressive triple negative tumor) and MCF-Tam (a sub-line of MCF-7 with acquired tamoxifen resistance). We observed marked differences in the energetic metabolism of these cell lines. Compared to MCF-7 cells, both MDA-MB-231 and MCF-Tam cells exhibited higher LDH levels and glucose uptake and showed lower capacity of oxygen consumption. In spite of these differences, GF exerted similar growth inhibitory effects. This result was explained by the finding of a constitutively activated stress response in MDA-MB-231 and MCF-Tam cells, which reproduce the poor prognosis tumor forms. As a further proof, different signaling pathways were found to be involved in the antiproliferative action of GF. In MCF-7 cells we observed a down regulation of the ERα-mediated signaling needed for cell survival. On the contrary, in MCF-Tam and MDA-MB-231 cells growth inhibition appeared to be contributed by an oxidative stress condition. The prevalent mechanism of cell death was found to be apoptosis induction. Because of the clinical relevance of breast cancer forms having the triple negative and/or chemoresistant phenotype, our results showing comparable effects of GF even on aggressively growing cells encourage further studies to verify the potential of this compound in improving the chemotherapy of breast cancer.