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Lactate dehydrogenase (LDH) inhibition is considered a worthwhile attempt in the development of innovative anticancer strategies. Unfortunately, in spite of the involvement of several research institutions and pharma-companies, the discovery of LDH inhibitors with drug-like properties seems a hardly resolvable challenge. While awaiting new advancements, in the present review we will examine other pathologic conditions characterized by increased glycolysis and LDH activity, which could potentially benefit from LDH inhibition. The rationale for targeting LDH activity in these contexts is the same justifying the LDH-based approach in anticancer therapy: because of the enzyme position at the end of glycolytic pathway, LDH inhibitors are not expected to hinder glucose metabolism of normal cells. Moreover, we will summarize the latest contributions in the discovery of enzyme inhibitors and try to glance over the reasons underlying the complexity of this research.

Most cancer cells use aerobic glycolysis to fuel their growth and many efforts are made to selectively block this metabolic pathway in cancer cells by inhibiting lactate dehydrogenase A (LDHA). However, LDHA is a moonlighting protein which exerts functions also in the nucleus as a factor associated to transcriptional complexes. Here we found that two small molecules which inhibit the enzymatic activity of LDHA hinder the transcription of histone 2B gene independently from the block of aerobic glycolysis. Moreover, we observed that silencing this gene reduces cell replication, hence suggesting that the inhibition of LDHA can also affect the proliferation of normal non-glycolysing dividing cells.

Lactate dehydrogenase A (LDH-A) binds single stranded DNA (ssDNA) and stimulates cell transcription. Binding is prevented by NADH, suggesting that the coenzyme site is involved in the interaction LDH-A/ssDNA. We recently identified an inhibitor of LDH-A enzymatic activity (Galloflavin, GF) which occupies the NADH site. In the experiments reported here we studied whether GF can also hinder the binding of LDH-A to ssDNA and investigated its effects on RNA synthesis in cultured cells. Using a filter binding assay we observed that 4 μM GF inhibited the binding of human LDH-A to a single stranded [(3)H]DNA sample by 50%. After only 0.5-1h, 50-100 μM GF inhibited RNA synthesis in SW620 cells maintained in a medium in which galactose substituted glucose. In these culture conditions, SW620 cells did not produce lactic acid and effects caused by the inhibition of the enzymatic activity of LDH-A could be excluded. Novel LDH-A inhibitors which hinder aerobic glycolysis of cancer cells are at present actively searched. Our results suggest that: (i) inhibitors which bind the NADH site can exert their antiproliferative activity not only by blocking aerobic glycolysis but also by causing an inhibition of RNA synthesis independent from the effect on glycolysis; (ii) GF can be a useful tool to study the biological role of LDH-A binding to ssDNA.

In the attempt of developing innovative anticancer treatments, growing interest has recently focused on the peculiar metabolic properties of cancer cells. In this context, LDH, which converts pyruvate to lactate at the end of glycolysis, is emerging as one of the most interesting molecular targets for the development of new inhibitors. In fact, because LDH activity is not needed for pyruvate metabolism through the TCA cycle, inhibitors of this enzyme should spare glucose metabolism of normal non-proliferating cells, which usually completely degrade the glucose molecule to CO2. This review is aimed at summarizing the available data on LDH biology in normal and neoplastic cells, which support the anticancer therapeutic approach based on LDH inhibition. These data encouraged pharmaceutical industries and academic institutions in the search of small-molecule inhibitors and promising candidates have recently been identified. The availability of inhibitors with drug-like properties will allow the evaluation in the near future of the real potential of LDH inhibition in anticancer treatment, also making the identification of the most responsive neoplastic conditions possible.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. There is a substantial need for new chemotherapeutic drugs effective against this tumor. Doxorubicin (DOXO), used for chemoembolization of HCCs, is poorly efficacious when administered systemically at conventional doses; dose escalation is hindered by unacceptable toxicity. Here, we review preclinical experiments showing that the efficacy of DOXO against HCCs and its safety increased following conjugation to lactosaminated human albumin (L-HSA). L-HSA-DOXO was initially prepared to improve the anticancer activity of the drug on well-differentiated HCCs, which actively internalize L-HSA by means of the asialoglycoprotein receptor. Unexpectedly, it was found that the conjugate enhanced DOXO concentrations in all forms of HCCs, independently of their differentiation grade.

Up-regulation of glycolysis, a well recognized hallmark of cancer cells, was also found to be predictive of poor chemotherapy response. This observation suggested the attempt of sensitizing cancer cells to conventional chemotherapeutic agents by inhibiting glucose metabolism. Lactate dehydrogenase (LDH) inhibition can be a way to hinder glycolysis of cancer cells without affecting the metabolism of normal tissues, which usually does not require this enzymatic activity. In this paper, we showed that two LDH inhibitors (oxamate and galloflavin) can increase the efficacy of cisplatin in cultured Burkitt's lymphoma (BL) cells and that this potentiating effect is not exerted in proliferating normal lymphocytes. This result was explained by the finding that in BL cells LDH inhibition induced reactive oxygen species (ROS) generation, which was not evidenced in proliferating normal lymphocytes. In BL cells treated with the association of cisplatin and LDH inhibitors, these ROS can be a further cause of DNA damage, to be added to that produced by cisplatin, leading to the failure of the response repair. At present LDH inhibitors suitable for clinical use are actively searched; our results can allow a better understanding of the potentiality of LDH as a possible target to develop innovative anticancer treatments.

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 μg/g). Liver fibrosis/cirrhosis was produced by carbon tetrachloride (CCl 4 ) 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.

The expression of the asialoglycoprotein receptor on the cells of the large majority of the well differentiated hepatocellular carcinomas can be exploited to improve the chemotherapy of these tumours by coupling anticancer agents to macromolecules taken up by the receptor. In line with this approach, in previous experiments we coupled doxorubicin (DOXO) to lactosaminated human albumin (L-HSA) using the (6-maleimidocaproyl)hydrazone derivative of the drug as an acid sensitive linker. Encouraging results were obtained in laboratory animals using L-HSA-DOXO. This conjugate, however, has the disadvantage of a difficult synthesis, which requires protein thiolation with iminothiolane and can hinder its preparation on a large scale. Here we describe a very simple method of coupling. The HS-groups required for the reaction with the maleimide moiety of DOXO-EMCH are made available in L-HSA by a cleavage of the protein disulphides achieved with tris(2-carboxyethyl) phosphine (TCEP). Contrary to thiolic reducing agents, the use of TCEP eliminates the need of an inert atmosphere and allows a one-step coupling reaction, without purification of the reduced protein before the addition of DOXO-EMCH. As the previous L-HSA-DOXO conjugate, the new conjugate accomplishes a very efficient liver targeting of the drug. This novel method of synthesis should facilitate the preparation of L-HSA-DOXO in the amounts required for clinical studies.

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.

Introduction: This review deals with the use of serum albumin (SA) as a carrier for the selective delivery of drugs to liver cells. Areas covered: The synthesis and properties of the SA onjugates prepared to enhance the performance of the drugs used in the treatment of viral hepatitis, hepatocellular carcinoma (HCC), liver micrometastases and hepatic fibrosis are reported. Expert opinion: Studies in humans and laboratory animals demonstrated the capacity of SA conjugates to accomplish a liver targeting of the drugs, but at the same time underscored their limits and drawbacks, which can explain why to date these complexes did not reach a practical application. The major drawback is the need of administration by intravenous route, which prevents long-term daily treatments as required by some liver pathologies, such as chronic virus hepatitis and fibrosis. At present, only a conjugate carrying doxorubicin and addressed to the treatment of HCC showed in laboratory animals a solid potentiality to improve the value of the coupled drug. In the future, conjugation to SA could remain a successful strategy to permit the administration of drugs with rapid resolutive effects inside liver cells without causing severe extrahepatic adverse reactions.

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.

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.

The expression of the asialoglycoprotein receptor (ASGP-R) on human hepatocellular carcinoma (HCC) cells might be exploited to reduce the extrahepatic toxicity of DNA synthesis inhibitors by their conjugation with galactosyl-terminating peptides. In the present study we first assessed the frequency of ASGP-R expression in 60 HCCs. Secondly, we investigated whether the receptor was maintained on the plasma membranes of DNA synthesizing cancer cells. Needle biopsies of HCC were evaluated. Diagnosis and grading of HCC were performed on routine haematoxylin and eosin-stained sections according to Edmondson and Steiner (1953). Thirty-five tumours were grade I and II and were classified as well differentiated, while 25 tumours were grade III and IV and were classified as poorly differentiated. Sections from formalin-fixed, paraffin-embedded samples were incubated, after antigen retrieval, with an anti-ASGP-R monoclonal antibody revealed by secondary biotinylated antibody and streptavidin–biotin–peroxidase–diaminobenzidine reaction. A clear immunolabelling of plasma membranes of HCC cells was observed in 28 out of 35 (80%) well differentiated (grade I and II) and in five out of 25 (20%) poorly differentiated (grade III and IV) HCCs. The presence of the ASGP-R on the surface of DNA synthesizing cancer cells was also investigated after in vitro bromodeoxyuridine (BrdU) labelling of HCC samples by immunohistochemical visualization of both the ASGP-R and incorporated BrdU on the same section. The results obtained clearly demonstrated that DNA synthesizing cancer cells expressed the ASGP-R on their surface. The presence of ASGP-R on cell plasma membrane in the majority of differentiated HCCs and its maintenance on proliferating cells encourages studies in order to restrict the action of the inhibitors of DNA synthesis of HCC cells by their conjugation with galactosyl-terminating carriers internalized through this receptor. © 1999 Cancer Research Campaign

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.

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 μg 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).

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 45000 and 65000 Da guarantees high solubility and low renal elimination of the conjugates. Conjugate preparations with these properties, intramuscularly administered to woodchuch 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.

A conjugate of adenine arabinoside monophosphate (ara-AMP) with the liver-targeting molecule lactosaminated human serum albumin (L-HSA) was administered by intravenous infusion for 28 days to eight patients with chronic type B hepatitis. The daily dose varied among the patients, ranging from 34 mg/kg to 53 mg/kg (equal to 1.5 and 2.3 mg/kg ara-AMP, respectively). Pharmacokinetic analysis indicated that, at every dose tested, the conjugate was disposed of without accumulation. Viral DNA serum levels fell markedly during treatment; values rose again when treatment was ceased. The L-HSA-ara-AMP conjugate did not cause either the neurotoxic side effects of free ara-AMP or other adverse clinical reactions. It produced a significant increase both in serum alkaline phosphatase activity and platelet number, and a small but significant decrease in erythrocyte number. These laboratory parameters returned to normal levels within 2 months after treatment. The conjugate induced the production of small amounts of antibodies (approximately 20 pmol of conjugate bound by 1 mL of serum) in one patient only. In conclusion, the present results indicate that the L-HSA-ara-AMP conjugate can exert the antiviral activity of ara-AMP in chronic type B hepatitis patients without producing the neurotoxic side effects which hamper a 4-week period of treatment with the free drug.

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.

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.

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.

One of the most prominent alterations in cancer cells is their strict dependence on the glycolytic pathway for ATP generation. This observation led to the evaluation of glycolysis inhibitors as potential anticancer agents. The inhibition of lactate dehydrogenase (LDH) is a promising way to inhibit tumor cell glucose metabolism without affecting the energetic balance of normal tissues. However, the success of this approach depends chiefly on the availability of inhibitors that display good selectivity. We identified a compound (galloflavin, CAS 568-80-9) which, in contrast to other inhibitors of human LDH, hinders both the A and B isoforms of the enzyme. To determine the mechanism of action, we collected LDH-A and -B inhibition data in competition reactions with pyruvate or NADH and evaluated the results using software for enzyme kinetics analysis. We found that galloflavin inhibits both human LDH isoforms by preferentially binding the free enzyme, without competing with the substrate or cofactor. The calculated Ki values for pyruvate were 5.46 μM (LDH-A) and 15.06 μM (LDH-B). In cultured tumor cells, galloflavin blocked aerobic glycolysis at micromolar concentrations, did not interfere with cell respiration, and induced cell death by triggering apoptosis. To our knowledge, the inhibition of LDH is, to date, the only biochemical effect described for galloflavin. Because galloflavin is not commercially available, we also describe herein a procedure for its synthesis and report its first full chemical characterization.

Protein kinase inhibitors are a relatively new class of promising anticancer drugs, most of which exert their action by binding to the ATP site on the targeted kinases. We hypothesized that a decrease in ATP levels in neoplastic cells could reduce the competition for the same enzymatic site, thus increasing the efficacy of kinase inhibitors. Using oxamic acid, an inhibitor of lactic dehydrogenase (LDH) which hinders aerobic glycolysis, we decreased ATP levels in PLC/PRF/5 cells (a line from a hepatocellular carcinoma). We found that in these cells oxamic acid potentiated the antiproliferative activity of sorafenib, imatinib and sunitinib, three kinase inhibitors. When aerobic glycolysis was shut down by culturing the cells in the absence of glucose, oxamic acid did not reduce the ATP levels, suggesting that in normal tissues, which do not rely on aerobic glycolysis for their ATP synthesis, the block of LDH should not impair cellular metabolism. In conclusion, the inhibition of LDH could enhance anticancer activity of sorafenib, imatinib and sunitinib without increasing their side effects on normal cells, which in conditions of normal functional activity and sufficient oxygen supply do not need the activity of this enzyme.

Background/Aims:By reducing the number of ATP molecules produced via aerobic glycolysis, the inhibition of lactic dehydrogenase (LDH) should hinder the growth of neoplastic cells without damaging the normal cells which do not rely on this metabolic pathway for their energetic needs. Here, we studied the effect of oxamic and tartronic acids, 2 inhibitors of LDH, on aerobic glycolysis and cell replication of HepG2 and PLC/PRF/5 cells, 2 lines from human hepatocellular carcinomas. Methods:Aerobic glycolysis was measured by calculating the amounts of lactic acid formed. The effect on replication was assessed by culturing the cells in both standard conditions and glucose-deprived medium, which was used to shut down aerobic glycolysis. Results: The oxamic and tartronic acids inhibited aerobic glycolysis, impaired the growth of both cell lines and also induced an increased expression of p53-upregulated modulator of apoptosis, a signal of cell death. A strong impairment of cell replication by oxamic acid was only found when the cells were cultured in the presence of glucose, indicating that it was for the most part owing to inhibition of aerobic glycolysis. Conclusions:Inhibition of aerobic glycolysis achieved by blocking LDH could be useful in the treatment of human hepatocellular carcinomas. Without interfering with glucose metabolism in normal cells, it could hinder cell growth by itself and could also enhance the chemotherapeutic index of associated anticancer agents by decreasing the levels of ATP selectively in neoplastic cells.

A selective delivery of drugs to liver can be obtained by conjugation with galactosyl terminating macromolecules. The conjugates selectively enter hepatocytes after interaction of the carrier galactose residues with the asialoglycoprotein receptor (ASGP-R) present only on these cells. Within hepatocytes the conjugates are transported to lysosomes where the drug is set free from the carrier, becoming concentrated in liver cells. The present article reviews the liver targeting of drugs obtained with lactosaminated albumin (L-SA), a neoglycoprotein exposing galactosyl residues. We report: (1) experiments which demonstrate the antiviral efficacy of the L-H(human)SA-ara-AMP conjugate in laboratory animals and in humans with viral hepatitis; (2) the property of a L-HSA conjugate with fluorodeoxyuridine to produce concentrations of the drug higher in hepatic sinusoids than in systemic circulation, with the potential of accomplishing a loco-regional, noninvasive treatment of liver micrometastases; (3) the increased anticancer activity of doxorubicin (DOXO) when coupled to L-HSA on all the forms of chemically induced rat hepatocellular carcinomas including those which do not express the ASGP-R.

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.

The finding that imatinib enhances the drug transport from bloodstream to neoplastic cells suggested a possible role of this drug as an adjuvant to the chemotherapeutics given in the treatment of solid malignancies. The present experiments aimed to verify whether imatinib can selectively increase the penetration of a doxorubicin–lactosaminated human albumin conjugate (L-HSA-DOXO) in chemically induced rat hepatocellular carcinomas (HCCs). We observed that imatinib increased the uptake of L-HSA-DOXO by HCCs but at the same time caused a similar enhanced penetration of the conjugate in liver and bone marrow. To our knowledge, this is the first demonstration that the enhancing effect of imatinib on interstitial drug transport is not restricted to the tumors, but can be also displayed in normal tissues. This observation casts some doubts about the possibility that the value of anticancer agents with toxic side effects on liver and bone marrow can be improved by imatinib.

In order to improve the efficacy of doxorubicin (DOXO) in the treatment of hepatocellular carcinomas, the drug was conjugated with lactosaminated human albumin (L-HSA), a hepatotropic drug carrier. Conjugation was performed using the (6-maleimidocaproyl)hydrazone derivative of the drug (DOXO-EMCH). The maleimide group of DOXO-EMCH reacts with the aminoacidic residues of the carrier forming stable bonds, whereas the hydrazone bond is rapidly hydrolysed in the acidic endosomal and lysosomal compartments of the cells allowing the intracellular release of DOXO. To identify the amino acids of L-HSA involved in the bond with DOXO-EMCH, in the present study we synthesized this compound with the 2,3 carbon atoms of the maleimide moiety enriched in the (13)C isotope and used this labelled DOXO-EMCH to prepare two types of L-HSA conjugate. Type I was prepared in analogy to those studied in the anticancer experiments using tris(2-carboxyethyl)phosphine (TCEP) to reduce l-cysteine disulfides and make the sulfhydryl groups available for the reaction with DOXO-EMCH; type II was synthesized omitting TCEP. By (13)C NMR spectroscopy we could demonstrate that in type I conjugate cysteine was the only amino acid residue that reacted with DOXO-EMCH, whereas in type II conjugate lysine was the only amino acid in the reaction with DOXO-EMCH. When hydrolysed in an acidic medium to cleave the hydrazone bond, type I conjugate released only DOXO, whereas type II conjugate also released a derivative of the drug.

Coupling to lactosaminated human albumin (L-HSA) makes doxorubicin (DOXO) an effective drug against chemically induced rat hepatocellular carcinomas (HCCs). In the conjugate there is a large heterogeneity in the number of DOXO molecules bound to one L-HSA molecule. After lyophilization, the molecules with the higher DOXO load form large complexes (C-DOXO (L) ), whereas those with low drug load (C-DOXO (S) ) have the size of the carrier L-HSA. In the present experiments, we demonstrated that in C-DOXO (L) the molecules are not linked by covalent bonds, but are strongly aggregated probably because of mutual drug–drug interaction between the DOXO residues. In healthy rats and in animals with HCCs which received the same dose (1 μg/g) of DOXO injected in C-DOXO (L) or in C-DOXO (S) forms, penetration of the drug in tumors and in tissues was more rapid after administration of the former complex. Three hours after injection of both conjugate forms the intracellular release of DOXO from the carrier was completed. The AUCs from 0.5 to 4 h of the levels of the released DOXO in HCCs, surrounding liver and bone marrow of animals injected with C-DOXO (L) were similar to those calculated in animals given C-DOXO (S) . This suggests that after administration of the dose of DOXO used in the present experiments the conjugate molecules with lower or higher drug load can exert comparable pharmacological and toxic effects.

Several attempts have been made to enhance doxorubicin (DOXO) concentrations in tumour cells by drug conjugation with human albumin (HSA). HSA-DOXO has the drawback of causing DOXO accumulation in spleen and bone marrow, with a consequent leucopoenia not produced when lactose molecules are coupled to the carrier protein. In the present experiments we demonstrated that the effect of HSA lactosamination is not a consequence of a more rapid disappearance from the bloodstream of the lactosaminated conjugate (L-HSA-DOXO), which is rapidly internalized by the liver through the asialoglycoprotein receptor, but is due to a hindered uptake by spleen and bone marrow cells caused by the coupled lactose molecules. Experiments in vitro showed that HSA-DOXO produced an inhibition of murine macrophage proliferation not caused by L-HSA-DOXO. This result can be explained by higher amounts of the former conjugate entering in these cells and suggests macrophages as the cell type responsible for the spleen and bone marrow internalization of HSA-DOXO hindered by lactose coupling. Importantly, lactosamination of HSA did not reduce the marked uptake of HSA-DOXO by chemically induced rat hepatocellular carcinoma. L-HSA-DOXO, by avoiding DOXO accumulation in bone marrow is an attractive candidate for clinical trials against tumors which were found to actively internalize this conjugate in laboratory animals, such as hepatocellular carcinoma.

Background/Aims: The hepatocyte receptor for asialoglycoproteins (ASGP-R) internalizes macromolecules exposing galactosyl residues (MEGRs) which can be used as liver-addressed drug carriers. This receptor was also found on the cells of the large majority of well differentiated hepatocarcinomas (HCCs). The aim of the present experiments was to ascertain whether ASGP-R of HCCs is functionally active and these tumors can internalize higher quantities of MEGRs than extra-hepatic tissues. Methods: We injected radioactive lactosaminated human albumin (L-HSA) in rats with HCCs produced by nitroso-diethylamine and measured the radioactivity of tumors, surrounding liver, heart, intestine and kidney. L-HSA is a MEGR successfully used in humans as a hepatotropic drug carrier. Results: The levels of radioactivity of HCCs were two to three times lower than those of surrounding liver, but several times higher than those of extra-hepatic tissues. L-HSA accumulation in the tumors mainly occurred via the ASGP-R, as indicated by the 20 times lower penetration of non-lactosaminated HSA. L-HSA uptake by the well-differentiated tumors were four times higher compared with that by the poorly differentiated forms. Conclusions: The present results suggest that in the chemotherapy of HCCs expressing the ASGP-R the extra-hepatic toxicity of anticancer agents can be reduced by conjugation to L-HSA.

BACKGROUND/AIMS: The hepatocyte receptor for asialoglycoproteins internalizes galactosyl terminating macromolecules which can be used as hepatotropic drug carriers. Since this receptor is also expressed on the cells of well differentiated human hepatocellular carcinomas (HCCs), we studied whether conjugation of doxorubicin (DOXO) with lactosaminated human albumin (L-HSA) increases the drug efficacy on HCCs induced in rats by diethylnitrosamine (DENA). METHODS: DENA was given in the drinking water for 8 weeks. One week after the last day of DENA administration, animals were randomly assigned to three groups. Each group was administered with either saline, free or coupled DOXO (1 microg/g). Rats received 4 weekly intravenous injections. One week after the last administration, rats were killed and HCC development was evaluated by counting the tumor nodules on the surface of hepatic lobes. RESULTS: In rats treated with L-HSA coupled DOXO the number of neoplastic nodules was significantly lower (P < 0.05) than that counted in animals injected with saline or with free DOXO. Coupled DOXO did not decrease body rat weight, which was markedly reduced by the free drug. CONCLUSIONS: Conjugation with L-HSA increased the antineoplastic efficacy and decreased the systemic toxicity of DOXO administered to rats with HCCs produced by DENA.

Background/Aims: The conjugate of doxorubicin (DOXO) with lactosaminated human albumin (L-HSA) has the potential of improving DOXO efficacy in the treatment of hepatocellular carcinomas (HCCs) expressing the asialoglycoprotein receptor (ASGP-R). In view of an adjuvant chemotherapy with L-HSA–DOXO after the surgical removal of the tumour, in the present experiments we verified whether DOXO accumulation produced by the conjugate can impair the liver regeneration following hepatic resection in non-cirrhotic liver. Methods: Using saline-injected hepatectomised rats as controls, we studied the effects of the conjugate on the ultrastructure of regenerating hepatocytes and evaluated [3H]thymidine incorporation, mitotic index and rate of DNA recovery in the liver remnant. Results: L-HSA–DOXO caused a selective drug accumulation in liver remnant, with low DOXO levels in extra-hepatic tissues. It did not change the ultrastructure of hepatocytes and did not increase serum alanine aminotransferase. It decreased [3H]thymidine incorporation and mitotic index, causing a moderate delay in hepatic DNA recovery. Conclusions: The experiments indicate a substantial resistance of rat regenerating hepatocytes to high intracellular concentrations of DOXO. They support the possibility of using L-HSA–DOXO in an adjuvant chemotherapy after the surgical removal of HCCs which maintain the ASGP-R.

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.

Nucleoside analogs (NAs) conjugated with galactosyl terminating peptides selectively enter hepatocytes via the asialoglycoprotein receptor and, after intracellular release from the carrier, partly exit from these cells into the bloodstream, resulting in higher concentrations in liver blood than in systemic circulation. Therefore, conjugates of anticancer NAs can be exploited to accomplish a loco-regional noninvasive treatment of liver micrometastases. In the present experiments we studied whether the enhancement of drug levels in liver blood achieved when NAs are given in the coupled form depends on the rate of drug elimination from the bloodstream. Three NAs, adenine arabinoside (ara-A), 5-fluoro-2'-deoxyuridine (FUdR), and 2',2'-difluorodeoxycytidine, were coupled with lactosaminated human albumin, a galactosyl terminating carrier. In rats that received an intravenous bolus injection of these conjugates, we compared the drug concentrations in liver blood to those in the systemic circulation. We found that enhanced levels of NAs in liver blood were only achieved by administering the conjugates of the drugs (ara-A and FUdR), which are rapidly cleared from the bloodstream. Increased drug levels also were obtained when ara-A and FUdR conjugates were slowly infused (a way of administration often used for anticancer drugs). The experiments also showed that galactosyl terminating conjugates of NAs might have the potential to produce a therapeutic effect only when the coupled drugs are active at low blood concentrations, since the amounts of drugs introduced into hepatocytes and released by these cells in the bloodstream cannot be increased when the receptor for the hepatic uptake of galactosyl terminating peptides is saturated.

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.

We prepared a hepatotropic conjugate, suitable for intramuscular (IM) injection, of lactosaminated poly-L-lysine with adenine arabinoside monophosphate (ara-AMP), a drug active against hepatitis B virus (HBV). We studied its organ distribution in mice and its antiviral activity in woodchucks that are carriers of woodchuck hepatitis virus (WHV). In mice, after IM administration of a conjugate tritiated in the drug moiety (5.2 micrograms/g equal to 2 micrograms/g of ara-AMP) radioactivity in liver was three times greater than in kidney, spleen, and intestine. On the contrary, after IM injection of unconjugated, tritiated, ara-AMP (5 micrograms/g) the amounts of radioactivity in liver, spleen, and kidney were similar. Unconjugated ara-AMP and the conjugate were administered IM to woodchucks for 13 days. Unconjugated ara-AMP decreased viremia at the daily dose of 5 mg/kg but was ineffective at 2.5 mg/kg. The conjugate at the daily doses of 4.2 and 7 mg/kg (equal to 1.5 and 2.5 mg/kg of ara-AMP, respectively) markedly lowered the viremia, which decreased to undetectable levels in the animals treated with the higher dose. Assuming that in HBV-infected patients the same doses will be active, then the amount of conjugate (soluble at 200 mg/mL) required by a 70-kg patient will be contained in a volume of 1.5 to 2.5 mL, compatible with the IM route. Compared with a similar ara-AMP complex with lactosaminated human albumin, currently being studied in clinical trials for the treatment of chronic type B hepatitis, which must be infused intravenously, the present conjugate might provide more patient compliance because of IM administration.

The woodchuck was selected to study the efficacy of liver-targeted antiviral drugs on hepadnavirus replication. Nineteen woodchucks chronically infected with woodchuck hepatitis virus were treated with adenine arabinoside monophosphate or acyclovir monophosphate, either free or conjugated with the liver-targeting molecule lactosaminated human serum albumin. Circulating woodchuck hepatitis virus DNA levels remained unchanged in untreated animals and in those receiving the carrier lactosaminated human serum albumin alone; in contrast, they were consistently lower after 5 days of treatment with the antiviral drugs. Free and conjugated adenine arabinoside monophosphate were active at doses of 10 and 0.75 mg/kg, respectively, and free and coupled ACVMP were active at doses of 20 and 2.6 mg/kg, respectively. These results indicate that the dosages of adenine arabinoside monophosphate and acyclovir monophosphate required to inhibit hepadnavirus growth can be sharply reduced by coupling the drugs to lactosaminated human serum albumin. (HEPATOLOGY 1991;14:16–24.)

With the aim of improving the chemotherapeutic index of 9-β- 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.

This review gives a comprehensive account of the molecular toxicology of the bicyclic peptides obtained from the poisonous mushrooms of the genus Amanita. The discussion of the biochemical events will be preceded by a consideration of the chemistry of the toxic peptides. The structural features essential for biological activities of both the amatoxins and the phallotoxins will be discussed, also including the most important analytical data. Similar consideration will be given to antamanide, a cyclic peptide, which counteracts phalloidin. In addition, the phallolysins, three cytolytic proteins from Amanita phalloides will be discussed. The report on the biological activity of the amatoxins will deal with the sensitivity of the different RNA-polymerases towards the toxins and with their action on various cell types. Consideration will also be given to systems in which alpha-amanitin was used and can be used as a molecular tool; in the past, many investigators used the inhibitor in molecular biology, genetics, and even in physiological research. As for the phallotoxins, discussion of the affinity of these toxins for actin is provied. Further discussion attempts to understand the course of intoxication by filling in the gap between the first molecular event, formation of microfilaments, and the various lesions in hepatocytes during the intoxication.

Poisoning by cytotoxic mushrooms (Amanita phalloides and related species) is associated with severe morbidity and a high mortality rate. Due to the difficulty of performing controlled studies and to the poor knowledge of the pharmacodynamics of toxins in human poisoning, there is considerable debate about appropriate treatment, particularly the feasibility and the efficacy of detoxification. Because circulating amatoxins can be detected in the serum of poisoned patients as long as 30 h after ingestion, a detoxification treatment should ideally increase the rate of toxin elimination in order to minimize the toxic exposure of highly susceptible cells, such as hepatocytes. We found forced diuresis to be the most effective procedure for toxin removal. Other techniques, such as plasmapheresis and peritoneal dialysis, proved much less useful for this purpose. The administration of cathartics, adsorbent agents, and gastroduodenal lavage, are indicated for preventing further absorption of toxins from the gut. An important part of therapy is early and vigorous volume replacement, to correct the severe hypovolemia which results from massive fluid loss during the cholera-like phase of intoxication. Use of this therapeutic approach in 53 patients with amatoxin poisoning resulted in a high survival rate and a low incidence of severe liver injury.