Carolina Polvani

Liver targeting of autologous erythrocytes loaded with doxorubicin

ERYTHROCYTES HAVE been proposed in animal models as carriers of cytotoxic drugs or as bioreactors converting prodrugs to active drugs [ 1,2]. Doxorubicin has been encapsulated in human and animal erythrocytes [3-71. Treatment of the doxorubicin-loaded erythrocytes with glutaraldehyde, a bifunctional reagent, prevented the fast efflux of doxorubicin from the cells and produced their specific targeting to the liver of mice and dogs [4-71. The present research aimed to extend these experimental studies to the clinic. A 51-year-old male had a colorectal carcinoma, massively metastatic to the liver (60% tumour involvement). The primary tumour was removed surgically and, at the time our study started, the disease was confined to the liver and slowly progressing. The patietit had an ECOG performance status of 2. He had previously failed three lines of chemotherapy (5fluorouracil, fotemustine, mitomycin), either systemic or locoregional, through a Port-a-cath implanted in the hepatic artery at the time of surgery. Informed consent was obtained from the patient. Freshly drawn erythrocytes were loaded with doxorubicin by simple diffusion [6]; the yield of encapsulation of the drug was 2 mg/ml of packed cells. Glutaraldehyde treatment (0.15% and 0.30% final concentration) was performed after encapsulation of doxorubicin [6]. This treatment substantially reduced the rate of doxorubicin efflux in vitro (more than 70% of the drug retained after 210 min at 37°C). For in viva distribution studies erythrocytes were labeled with 99mTc [8] prior to encapsulation. All manipulations were performed under sterile pyrogen-free conditions. Figure 1 shows the time-radioactivity curves over the regions of liver and heart, after administration through hepatic artery of 99mTc labeled native (a) and unloaded GA treated (b) erythrocytes. Unlike the native cells, showing a rapid transit from the liver to systemic circulation (a), the glutaraldehyde treated erythrocytes were almost completely retained by the liver (b), up to 24 hours after injection (not shown). No accumulation of

Enhanced antitumor activity of adriamycin by encapsulation in mouse erythrocytes targeted to liver and lungs.

Adriamycin was encapsulated within human and murine (B6D2F1 female mice) erythrocytes using a procedure based on hypotonic hemolysis followed by isotonic resealing and reannealing. Following drug encapsulation the murine erythrocytes were treated with glutaraldehyde to obtain: a) control of Adriamycin efflux from loaded erythrocytes, b) appropriate hepatic and pulmonary targeting of the in vivo re-infused cells. The antitumor effect of equivalent amounts of bolus (i.v.) administered Adriamycin, 1) free, 2) encapsulated within erythrocytes, 3) encapsulated within glutaraldehyde-treated erythrocytes, was compared using an in vivo model of metastasis based on selective hepatic and pulmonary dissemination of intrasplenically injected L1210 cells in B6D2F1 mice. The therapeutic index (TI) of Adriamycin encapsulated within glutaraldehyde-treated erythrocytes increased by more than two-fold over that of the free drug.

Use of Glutaraldehyde Treated Autologous Human Erythrocytes for Hepatic Targeting of Doxorubicin

Systemic chemotherapy has only a marginal role in the treatment of unresectable hepatocellular carcinomas or secondary liver metastases from gastrointestinal malignancies, producing a 20% response rate at best. In the attempt to improve the efficacy of the chemotherapeutic agents against these diseases, several drugs have been directly administered via the hepatic artery instead of intravenous injection.1–3 The rationale behind this strategy lies in the finding that hepatic malignancies receive their blood supply primarily from the hepatic artery, while normal hepatocytes mainly derive it from the portal vein.4 Thus, hepatic artery administration allows increased tumor exposure to the drug and lower peripheral blood levels are obtained for those agents that are metabolized by the liver, when compared to the systemic administration.2 Accordingly, tumor cell killing will be enhanced and drug toxicity reduced.

Multiple small molecular weight guanine nucleotide-binding proteins in human erythrocyte membranes.

Native membranes from human erythrocytes contain the following G proteins which are ADP-ribosylated by a number of bacterial toxins: Gi alpha and Go alpha (pertussis toxin), Gs alpha (cholera toxin), and three proteins of 27, 26 and 22 kDa (exoenzyme C3 from Clostridium botulinum). Three additional C3 substrates (18.5, 16.5 and 14.5 kDa) appeared in conditions of unrestrained proteolysis during hemolysis. SDS-PAGE separation of erythrocyte membrane proteins followed by electroblotting and incubation of nitrocellulose sheets with radiolabeled GTP revealed consistently four GTP-binding proteins with Mr values of 27, 26, 22 and 21 kDa. Although a 22 kDa protein was immunochemically identified as ras p21, the C3 substrate of 22 kDa is a different protein probably identifiable with a rho gene product. Accordingly, at least five distinct small molecular weight guanine nucleotide-binding proteins, whose functions are so far undetermined, are present in native human erythrocyte membranes.

Glycoxylic acid prevents NAD+ and NADH depletion in K562 cells cultured at limiting dilution

K562 erythroleukemic cells cultured at low population density in the absence of serum die within 12-24 hours, unless 0.1 mM glyoxylic acid is added to the culture medium. Earlier events, preceding cell death and occurring within 2 hours culture, are: a) a marked drop of both the NAD+/NADH ratio and the NAD+ concentration, which is prevented by 10mM benzamide, b) an increased biosynthesis of NAD+, leading to extensive depletion of cellular ATP. In the presence of 0.1 mM glyoxylic acid the NAD+/NADH ratio as well as their absolute concentrations remain unchanged, while NAD+ biosynthesis is absent. A NAD+/NADH glycohydrolase activity is present in the cell extract, inhibited by 10 mM benzamide and with a higher affinity for NADH than for NAD+. Preservation of a high NAD+/NADH ratio by glyoxylic acid apparently prevents enzyme activity and the related loss of pyridine nucleotides.