Selection of therapeutic agents for intraocular proliferative disease 3. Effects of fluoropyrimidines on cell-mediated contraction of human fibroblasts.

We evaluated the efficacy of 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUR) as anticontractile agents in an in vitro model that measures the ability of human and rabbit fibroblasts to contract a collagen gel. Human fibroblasts were more contractile than rabbit fibroblasts, required higher concentrations of 5-FUR for inhibition of contraction, and were able to tolerate higher doses of the drug. 5-FU failed to inhibit contraction of human fibroblasts at concentrations up to 1.5 mM (200 micrograms ml-1), but significantly inhibited rabbit fibroblast contraction, although to a lesser degree than 5-FUR under similar conditions. 5-Fluorouridine (100 microM, 26 micrograms ml-1) inhibited contraction of human fibroblasts by more than 80%, whereas only 10 microM (2.6 micrograms ml-1) 5-FUR was required for 90% inhibition of rabbit fibroblast contraction. 5-FUR was cytotoxic to rabbit fibroblasts at concentrations of 200 microM (52 micrograms ml-1) or greater, but 800 microM was non-toxic to human fibroblasts after 96 hr exposure. At concentrations of 1.5 mM, 5-FU had no significant effects on the viability of either human or rabbit fibroblasts. These results suggest that 5-FUR may prove useful for the treatment of human ocular proliferative disorders in which contraction of fibrocellular elements is thought to play a part. They also suggest that fluoropyrimidine dosages found to be effective in a rabbit model of proliferative vitreoretinopathy may be less effective in humans and that 5-FUR levels shown to be toxic to rabbit-derived cells may be tolerated by humans cells.

The effect of beta irradiation on monkey Tenon's capsule fibroblasts in tissue culture.

Fibroblast proliferation with subsequent bleb scarring is a major cause of filtering surgery failure. To investigate possible antiproliferative effects of beta radiation, owl monkey Tenon's capsule fibroblasts in tissue culture were irradiated to doses of 30, 100, 300, 1,000, and 3,000 rads with a linear accelerator and to doses of 95,285, and 950 rads with a Strontium-90 applicator. The irradiated cell proliferation expressed as the percentage of the non-irradiated control growth on the third and seventh days, respectively, after beta irradiation was: 97% and 96% after 30 rads; 72% and 90% after 100 rads; 48% and 44% after 300 rads; 39% and 14% after 1,000 rads; and 39% and 14% after 3,000 rads. Similar effects on cell proliferation were observed with the Strontium-90 applicator. The inhibitory effect of beta irradiation on fibroblast proliferation in tissue culture suggests that beta irradiation after filtering surgery may reduce postoperative bleb scarring.

Selection of therapeutic agents for intraocular proliferative disease. II. Differing antiproliferative activity of the fluoropyrimidines.

We confirm the potent antiproliferative effects of the fluoropyrimidines on cellular proliferation in vitro in three different nonmalignant cell types. All fluoropyrimidines tested, except for fluorocytosine, decrease proliferation of human dermal fibroblasts, bovine aortic vascular endothelial cells, and human retinal pigment epithelial cells in vitro. Fluorouridine, an intracellular metabolite of fluorouracil, is nearly 100-fold more potent than fluorouracil and its deoxymetabolite. Human dermal fibroblasts are more sensitive to the inhibitory effects of deoxymetabolites than the cells of either human retinal pigment epithelium or bovine aortic vascular endothelium. Fluorouridine and other fluoropyrimidines may prove to be valuable second-generation drugs in the treatment of intraocular proliferative disorders.

Trifluorothymidine and 5-fluorouracil: antiproliferative activity in tissue culture.

The antiproliferative activity of trifluorothymidine (F3TDR) and 5-fluorouracil (5-FU), two cell-cycle-phase-specific antimetabolites, was compared in a tissue culture model of human scleral fibroblasts and rabbit corneal epithelial cells. The mean concentrations required to inhibit human scleral fibroblast proliferation to 50% of the control rate (ID50) after 5 days of exposure were 8.50 micrograms/mL for F3TDR and 0.43 microgram/mL for 5-FU. The corresponding ID50 levels for rabbit corneal epithelial cells were 0.24 microgram/mL and 0.42 microgram/mL. The ID50 of F3TDR was significantly higher than that of 5-FU in human scleral fibroblasts (p less than 0.001) but not in rabbit corneal epithelial cells. F3TDR appears to have no clinical advantage over 5-FU in minimizing corneal epithelial toxicity when given to prevent fibroblast proliferation after glaucoma filtering surgery.

Light ON depresses and light OFF enhances the release of dopamine from the cat's retina.

The release of [3H]dopamine from the cat's retina was determined by measuring the level of radioactivity in a series of vials in which the retina was incubated. At light ON; there was a sustained decrease and at light OFF there was an enhanced release of dopamine. The results also demonstrate that dopamine is released continuously in the dark.

In vitro inhibition of ocular cell proliferation with ara-C: blockage of the antiproliferative effect with 2'-deoxycytidine.

Cytosine arabinoside (ara-C), a cell cycle-phase specific antimetabolite, was tested in a tissue culture model of human scleral fibroblasts (HSCF), and human retinal pigmented epithelium cells (HRPE), to investigate possible applications in the modulation of wound healing after glaucoma filtering surgery and in the postoperative management of proliferative vitreoretinopathy. Cell proliferation after a 72 hr incubation in the presence of ara-C was inhibited to 50% of the control growth rate (ID50) with calculated doses of 60 ng/ml in HSCF and 4 and 5 ng/ml in HRPE cultures. 2'-deoxycytidine, a competitive inhibitor of ara-C action, completely eliminated the cell growth retardation of both cell types produced by ara-C after 72 hr incubation.

Fluorouracil (5-FU) and cytarabine (ara-C) inhibition of corneal epithelial cell and conjunctival fibroblast proliferation.

We evaluated the antiproliferative properties of fluorouracil (5-FU) and cytarabine (ara-C) in a tissue culture model of rabbit corneal epithelial cells and conjunctival fibroblasts. Cells were treated with fluorouracil or cytarabine for five days. Corneal epithelial cell proliferation was inhibited to 50% of control proliferation by 0.6 mg/L of fluorouracil or 30 micrograms/L of cytarabine. Proliferation of conjunctival fibroblasts was inhibited to 50% of control by 0.5 mg/L of fluorouracil or 50 micrograms/L of cytarabine. The antiproliferative effect of 10 mg/L of fluorouracil was not blocked by thymidine. The inhibition of proliferation produced by 100 or 1,000 micrograms/L of cytarabine was blocked by 2'-deoxycytidine at concentrations of 10(-5) M and 10(-4) M, respectively. The antiproliferative effects of fluorouracil and cytarabine appear similar for both cell types. Blockage of cytarabine-related ocular toxic effects may be possible with 2'-deoxycytidine.

Selection of therapeutic agents for intraocular proliferative disease. Cell culture evaluation.

A variety of antimetabolites, steroids, and nonsteroidal anti-inflammatory agents were tested for their ability to inhibit rabbit dermal and conjunctival fibroblast proliferation in cell culture. Doxorubicin hydrochloride and fluorouracil produced notable inhibition in concentrations of less than 1 mg/L. Meclofenamate sodium and indomethacin produced notable inhibition at concentrations of 11 and 40 mg/L, respectively. Dexamethasone sodium phosphate and triamcinolone acetonide produced inhibition at 200 and 150 mg/L, respectively, but paradoxically increased proliferation almost two-fold at concentrations ranging from 1 to 30 mg/L under identical culture conditions. Methotrexate sodium demonstrated only limited effectiveness. This assay system may be a useful approach to drug selection in the treatment of a variety of ocular proliferative disorders. Fluorouracil may prove to be of significant value in the treatment of intraocular proliferative disorders.

Demonstration and partial characterization of insulin receptors in human platelets.

Recently, evidence has been reported to suggest that human platelets like several other circulating blood cells may bind insulin. To examine whether human platelets contain specific insulin receptors, washed human platelets suspended in Hepes buffer were incubated at 24 degrees C with 125I-insulin in the presence and absence of unlabeled insulin and specific insulin binding was determined. Insulin binding by platelets increased progressively with time of incubation to reach a maximum at 3 h and was proportional to the number of platelets in the incubation mixture. Maximum insulin binding was observed at pH 8. Insulin degradation by platelets as assessed by TCA precipitability and reincubation studies was minimal. Scatchard analysis of the binding data and dissociation studies revealed evidence of negative cooperativity of the platelet insulin receptor. A high affinity dissociation constant of approximately equal to 3 X 10(9) M-1 was determined and the concentration of platelet insulin receptors was estimated as 25 binding sites/micron2 platelet surface area. Binding of 125I-insulin by platelets was inhibited by unlabeled porcine insulin and to a lesser extent by catfish insulin and porcine proinsulin but not by glucagon, prolactin, growth hormone, and thrombin. The findings indicate that human platelets contain specific insulin receptors. The significance of the platelet insulin receptor, particularly with respect to altered platelet function in diabetes mellitus, remains to be determined.

Characterization of a protein in mid-term human amniotic fluid which reacts in the somatomedin-C radioreceptor assay.

Amniotic fluid contains materials other than insulin which react in a somatomedin C radioreceptor assay using human placental membranes. The material in mid-gestational amniotic fluid which reacted with the somatomedin C radioreceptor assay eluted slightly after albumin from a Sephadex G-150 column equilibrated with 0.1M NH4HCO3. Neither boiling nor treatment of this fraction with 1% formic acid yielded small molecular weight somatomedin-like peptides. Separation of the somatomedin reactive material (Sm RM) from albumin was achieved by gel filtration through Ultrogel AcA44 in 3.1M NH4HCO3. The active product had an apparent molecular weight of 33,000 to 35,000 Daltons; its isoelectric point determined by focusing was between 4.1 and 5.1. The purified amniotic fluid protein displaced somatomedin C in the somatomedin C radioreceptor assay but did not compete with insulin in the insulin radioreceptor assay. Sm RM produced only a slight stimulation of thymidine uptake in human fibroblasts but was inactive in stimulating sulfate uptake in hypox rat costal cartilage. In human fibroblast cultures Sm RM inhibited the stimulation of thymidine uptake induced by human serum and by purified rat somatomedin. When Sm RM was added to the 125I somatomedin C, some of the radioactivity eluted from gel filtration at pH 8.6 was converted to a molecular weight complex of about 43,000. We conclude that the material which we have isolated from mid-gestational amniotic fluid is a protein which may bind somatomedin and make it unavailable to the somatomedin receptor of human placenta and human fibroblasts.