Study of antiangiogenic agents with possible therapeutic applications in neoplastic disorders and macular degeneration.

Using a previously developed method (Ambrus, et al., 1991), we found that pentoxifylline and thalidomide potentiate each others antiangiogenic effect induced by human malignant melanoma cells in the cornea of Macaca arctoides monkeys.

Removal of non-transferrin-bound iron from blood with iron overload using a device with immobilized desferrioxamine.

An extracorporeal hollow-fiber device with immobilized desferrioxamine (DFO) was developed for the removal of nontransferrin-bound iron (NTBI) from blood, without the toxicity of parenteral chelation. When blood circulates through the fibers having pores with 30 kD cut-off, non-transferrin-bound-iron (NTBI) crosses the fiber pores and is chelated by the immobilized desferrioxamine. Removal of circulating iron stimulates iron release from larger proteins and tissue stores, establishing continuous iron flow to the immobilized chelator. During in vitro circulation through a device, iron removed from blood of hemodialysis or sickle cell patients was proportional to, but always in less than 50% of the initial iron level. We attribute the inability to remove more serum iron to irreversible iron binding by transferrin. To investigate where removable and fixed iron was bound, iron binding proteins were analyzed in sera from six patients with genetic anemias and iron overload. Sera separated by sieving chromatography contained 1-14% of the iron in the < 30 kD protein pool, 26-48% was in the combined non-transferrin pools. Sera from hemochromatosis patients without iron overload did not contain NTBI. Circulation of hemochromatosis blood through the device removed one third of the iron, this came from all molecular weight fractions. Iron removal by the device from the < 30 kD pool appears to establish a disequilibrium, that stimulates continuous iron release from ligands with low iron affinity, renewing the pool in the < 30 kD range, which includes potentially toxic NTBI. Therapy with the chelator device having immobilized desferrioxamine should be beneficial for treatment of patients with iron overload.

Total joint arthroplasty and the immune response.

Total joint replacement arthroplasty has proved highly successful in the management of osteoarthritis and rheumatoid arthritis. The cause of aseptic loosening of prosthetic joint replacement components is unclear. Early experience with total joint arthroplasty was plagued by a number of problems that no longer exist as major impediments to long-term success. Improvement in the operating room environment and the use of prophylactic antibiotics have substantially reduced the high incidence of infection to less than 1%. Implant materials have long been considered biologically inert, but recent studies indicate that inflammatory reactions directed against the implanted materials may contribute to aseptic loosening. Currently, particulate debris from cement or polyethylene causing loosening of the prosthesis is the major problem in total joint arthroplasty. Significant data suggest a progression from a simple inflammatory reaction to complex immune responses against the biomaterials. The cellular responses to particles of polymethylmethacrylate, ultra-high-molecular-weight polyethylene, and alloys of cobalt-chromium and titanium have been assayed in vitro in patients with osteoarthritis, rheumatoid arthritis, and avascular necrosis who had total joint replacement. Elevated immunologic cell proliferation responses to both acrylic and cobalt chromium were observed in patients with aseptically loosened prostheses. These findings suggest that the development of a cellular response to particulate debris may be significant in the pathogenesis of aseptic loosening.

Potentiation of thrombolytic therapy by enzyme combinations and with aspirin or pentoxifylline.

Radioactively labeled human fibrin clots were placed into veins of Macaca arctoides monkeys. Thrombolysis was recorded by the disappearance of radioactivity and by angiography. Streptokinase (SK) and urokinase (UK) induced thrombolysis was potentiated by low dose aspirin (ASA) and pentoxifylline (PE). Studies on the mechanisms of action revealed that PE inhibits platelet aggregation, releases tissue plasminogen activator (t-PA) from the endothelium, increases red cell deformability and inhibits white cell adhesion. Thrombolysis by pro-urokinase (pro-UK) was potentiated by low dose SK probably because of streptokinase-plasmin activation of pro-UK to UK. Platelet aggregation inhibitory effects, disaggregation of platelet aggregate inducing effects, and the t-PA releasing activity of PE was demonstrated in patients with obstructive cardiovascular disease. Pharmacodynamic studies suggested that PE metabolites one and five are most effective from this point of view. These metabolites are currently studied in combination with thrombolytic enzymes.

Influence of cold storage altered red cell surface on the function of platelets.

In previous studies, we have shown that in the hemoocclusive crisis of sickle cell disease, red blood cells (RBC) develop reversible membrane changes which may represent a thrombogenic surface for platelets. As a result there is platelet activation and platelet derived procoagulant activity. Platelet activation is also enhanced when platelets come into contact with stored RBC in comparison with freshly obtained red blood cells from normal individuals. Pentoxifylline (P, Trental), a trisubstituted xanthine derivative, acts on the red cell membrane and decreases its effect on platelet activation. In addition, it may have direct platelet aggregation inhibitory effects. By decreasing red cell "stiffness", it may also increase the circulating life span of stored red blood cells. It is suggested that pentoxifylline may be explored as an additive in the preservation of RBC.

Pentoxifylline and meclofenamic acid treatment reduces clinical manifestations in a murine model of AIDS.

C57/BL/6 mice infected with LP-BM5 MuLV virus developed an AIDS-like disease (MAIDS) with splenomegaly, leukopenia, thrombocytopenia, anemia, decreased numbers of helper/inducer and suppressor/cytotoxic T-cells and decreased production of interferon alpha. We have shown previously that HIV-associated Kaposi's sarcoma tissue contains high levels of prostaglandin E2 (PgE2), and this inhibits interferon synthesis through a cAMP-dependent second-messenger process. In this study we treated groups of MAIDS-infected mice with combinations of pentoxifylline, an agent which increases cAMP and inhibits phosphodiesterases, and sodium meclofenamic acid, a PgE2 inhibitor. Treated mice showed: 1) significantly higher total leukocyte and platelet counts, 2) higher total L3T4+ (helper/inducer) and Lyt-2+ (suppressor-cytotoxic) T-cell population. Pathologic examination also showed significantly less hepatosplenomegaly and lymphadenopathy in animals treated with pentoxifylline and meclofenamic acid. Partly, PgE2-induced suppression of interferon alpha production may mediate expression of retrovirus infection in this murine model of AIDS.

Effect of red blood cells from patients with sickle cell disease on platelet factor 3 release.

Red cells from two SS genotype and two SA genotype patients were sampled. When the samples were deoxygenated and mixed with platelet-rich plasma, they caused the release of platelet factor 3 as recorded in a coagulometer. This phenomenon was not present in control blood samples from normal individuals. Membrane changes in abnormal red cells during hypoxia may be responsible in part for platelet activation and its role in vasoocclusive crisis. Vasoocclusive crisis could be prevented by increasing the red cell membrane fluidity and inhibiting the platelet aggregation with pentoxifylline.

Studies on tumor induced angiogenesis.

Methods were developed to test angiogenic response to human tumor implants and various biologic agents in the cornea of rabbits and non-human primates (Macaca arctoides). Crude PDGF preparations were found to have significant angiogenic effect. Purified, recombinant PDGF preparations were also effective inhibitors (e.g. pentoxifylline (Px) (which also were found to release PgI2 and t-PA) inhibited human tumor implant induced angiogenesis and reduced spontaneous metastases in 3 transplantable murine tumors (Furth-Columbia Wilms' tumor in Furth-Wistar rats, C-1300 neuroblastoma in A/J mice and HM-Kim mammary carcinoma in Wistar rats) but not in the NIH adenocarcinoma in Balb/c mice. Sodium diethyldithiocarbamate (DDTC), a metal complexing agent with special affinity to copper and anti-thyroid as well as, immune stimulating activity was shown to be anti-angiogenic and to potentiate the effect of Px. The anti-fibrinolytic agents epsilon amino caproic acid (EACA) and tranaxamic acid (t-AMCHA) were anti-angiogenic. DDTC and Px were synergistic from this point of view.

Studies on tumor induced angiogenesis.

Methods were developed to test the angiogenic response to human tumor implants and various biologic agents in the cornea of rabbits and non-human primates (Macaca arctoides). Human malignant melanoma tissue and crude platelet derived growth factor (PDGF) preparations had significant angiogenic effects. Purified, recombinant PDGF preparations were also effective initiators. Hemorheologic agents which also inhibit platelet aggregation [e.g. pentoxifylline (Px) (Trental) (also found to release PgI2 and tissue plasminogen activator (t-PA)] inhibited human tumor implant-induced angiogenesis. Sodium diethyldithiocarbamate (DDTC), a metal complexing agent with special affinity to copper and anti-thyroid as well as immune stimulating activity, was shown to be anti-angiogenic and to increase the effect of Px. The anti-fibrinolytic agents epsilon amino caproic acid (EACA) and tranexamic acid (t-AMCHA) were anti-angiogenic.