Potent selective inhibitors of protein kinase C.

A series of potent, selective inhibitors of protein kinase C has been derived from the structural lead provided by the microbial broth products, staurosporine and K252a. Our inhibitors block PCK in intact cells (platelets and T cells), and prevent the proliferation of mononuclear cells in response to interleukin 2 (IL2).

A novel conformationally restricted protein kinase C inhibitor, Ro 31-8425, inhibits human neutrophil superoxide generation by soluble, particulate and post-receptor stimuli.

A novel, bis-indolylmaleimide, Ro 31-8425, bearing a conformationally restricted side chain, inhibits protein kinase C isolated from rat brain and human neutrophils with a high degree of selectivity over cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase. It also inhibits phorbol ester-induced intracellular events known to be mediated by protein kinase C (p47 phosphorylation in intact platelets, CD3 and CD4 down-regulation in T-cells). Ro 31-8425 inhibited superoxide generation in human neutrophils activated by both receptor stimuli (formyl-methionyl-leucylphenylalanine, opsonized zymosan, IgG and heat aggregated IgG) and post-receptor stimuli (1,2-dioctanoylglycerol and fluoride). The compound also blocked antigen driven, but not IL-2 induced, T-cell proliferation. These results support a central role for protein kinase C in the activation of the respiratory burst and antigen-driven T-cell proliferation.

Inhibitors of protein kinase C. 1. 2,3-Bisarylmaleimides.

The design and synthesis of a series of novel inhibitors of protein kinase C (PKC) is described. These 2,3-bisarylmaleimides were derived from the structural lead provided by the indolocarbazoles, staurosporine and K252a. Optimum activity required the imide NH, both carbonyl groups, and the olefinic bond of the maleimide ring. 2,3-Bisindolylmaleimides were the most active, and the potency of these was improved by a chloro substituent at the 5-position of one indole ring (compound 28, IC50 0.11 microM). In a series of (phenylindolyl)maleimides, nitro compound 74 was most active (IC50 0.67 microM). Naphthalene 19 and benzothiophene 21 showed greater than 100-fold selectivity for inhibition of PKC over the closely related cAMP-dependent protein kinase (PKA).

Inhibitors of protein kinase C. 3. Potent and highly selective bisindolylmaleimides by conformational restriction.

The protein kinase inhibitor staurosporine has been used to design a series of selective bisindolylmaleimide inhibitors of protein kinase C (PKC). Guided by molecular graphics, conformational restriction of the cationic side chain has led to ATP competitive inhibitors of improved potency and selectivity. Two compounds have been further evaluated and were shown to inhibit PKC of human origin and prevent T-cell activation in a human allogeneic mixed lymphocyte reaction. One of these compounds was orally absorbed in mice and antagonized a phorbol ester induced paw edema in a dose-dependent manner. This compound also selectively inhibited the secondary T-cell mediated response in a developing adjuvant arthritis model in rats and provides evidence for the potential use of PKC inhibitors as therapeutic immunomodulators.

Synthesis and antiviral activity of metabolites of rimantadine.

The hydroxy metabolites of rimantadine (3-5) were synthesized and compared to amantadine (1) and rimantadine (2) for their ability to inhibit the replication of influenza viruses in vitro. All three metabolites were inhibitory to wild-type influenza A viruses (H3N2 and H1N1). In particular, 2-hydroxyrimantadine (3) showed similar activity to amantadine, but the 3- and 4-hydroxy metabolites (4 and 5, respectively), both of which are found in rimantadine-treated patients, showed only modest inhibitory activity. A rimantadine-resistant isolate of influenza A virus exhibited cross-resistance to amantadine and to each of the metabolites 3-5. None of the compounds were effective against influenza B virus.

Inhibitors of protein kinase C. 2. Substituted bisindolylmaleimides with improved potency and selectivity.

A hypothetical mode of inhibition of protein kinase C (PKC) by the natural product staurosporine has been used as a basis for the design of substituted bisindolylmaleimides with improved potency over the parent compound. Structure-activity relationships were consistent with the interaction of a cationic group in the inhibitor with a carboxylate group in the enzyme, and the most potent compound had a Ki of 3 nM. The inhibitors were competitive with ATP but inhibited cAMP-dependent protein kinase (PKA) only at much higher concentrations despite the extensive sequence homology between the ATP-binding regions of PKA and PKC. Three compounds were evaluated further and found to inhibit a human allogeneic mixed lymphocyte reaction pointing to the potential utility of PKC inhibitors in immunosuppressive therapy. One of these compounds was orally absorbed in the rat and represents an attractive lead in the development of PKC inhibitors as drugs.

Biological interaction of selenium with other trace elements in chicks.

Studies were conducted to determine whether or not elements whose valence shell of electrons was similar to that of selenium would reverse the toxicity of selenium to chicks. The elements studied were arsenic, tellurium, tin, and lead. Each of these elements, when added to the diet of chicks, reversed the toxicity of 25 ppm selenium as measured by weight gain. In spite of the protection afforded by these elements, there was no decrease in liver concentration of selenium except with levels of arsenic higher than that needed for reversal of toxicity.

Ocular manifestations of leprosy in a noninstitutionalized community in the United States.

OBJECTIVE: Our goal was to delineate the epidemiologic and clinical patterns of ocular leprosy in an outpatient setting in the United States. DESIGN: Examinations were performed in 61 consecutive outpatients seen in a Midwestern leprosy clinic. PATIENTS: Forty-three male and 18 female patients were examined. The patients' origins included Southeast Asia (24 patients [39%]), Latin America (23 patients [38%]), India (nine patients [15%]), Europe or North America (two patients [3%]), Africa (two patients [3%]), and the Middle East (one patient [2%]). RESULTS: Thirty-nine percent of patients were classified as having polar lepromatous leprosy; 18%, borderline lepromatous leprosy; 3%, borderline borderline leprosy; 36%, borderline tuberculoid leprosy; 2%, polar tuberculoid leprosy; and 2%, indeterminate leprosy. Ninety-six percent of patients had a best-corrected visual acuity of 20/40 or better. Ocular findings included madarosis (28 patients [46%]), subconjunctival fibrosis (18 patients [30%]), punctate epithelial keratopathy (17 patients [28%]), posterior subcapsular cataract (10 patients [16%]), corneal hypesthesia (10 patients [16%]), lagophthalmos (seven patients [11%]), corneal pannus (six patients [10%]), entropion (five patients [8%]), prominent or beaded corneal nerves (four patients [7%]), iridocyclitis (four patients [7%]), focal avascular keratitis (three patients [5%]), scleritis (three patients [5%]), interstitial keratitis (two patients [3%]), iris pearls (two patients [3%]), and ocular clofazimine crystals (two patients [3%]). Madarosis, corneal hypesthesia, and posterior subcapsular cataracts were significantly associated with disease duration (P < .05). CONCLUSION: We report herein a relatively low frequency of visual impairment attributable to leprosy in our series compared with that seen among institutionalized leprous patients. However, since 48% of subjects had one or more sight-threatening complications as a result of their disease, a program of regular ophthalmic follow-up is strongly advocated for all patients with leprosy.

Interactions of vitamin C with lead and mercury.

Ascorbic acid has been found to interact with several elements in such a manner as to render them less available for animals. This property of the vitamin has a negative effect on the animals fed a copper-deficient diet, but a positive effect on those fed toxic levels of copper, selenium, vanadium, and cobalt. The effect of ascorbic acid in alleviating cadmium toxicity has been attributed to the effect of the vitamin on iron metabolism, since ferrous iron will also alleviate cadmium toxicity in the Japanese quail. The results of studies reported here indicate that iron will alleviate lead toxicity but ascorbic acid is ineffective. Ascorbic acid will alleviate mercury toxicity, but iron exacerbates this condition. For these two elements, the effects of iron and ascorbic acid are independent of each other.

The bisindolylmaleimide protein kinase C inhibitor, Ro 32-2241, reverses multidrug resistance in KB tumour cells.

Ro 32-2241 is a bisindolylmaleimide that selectively inhibits protein kinase C (PKC) as compared with other protein kinases. Experiments were carried out to examine its potential as a multidrug resistance-reversing agent. Ro 32-2241 inhibited efflux, and increased accumulation, of [3H]-daunomycin in multidrug-resistant (MDR) KB-8-5 and KB-8-5-11 cells and had no effect on drug-sensitive KB-3-1 cells. Ro 32-2241 completely reversed the doxorubicin resistance of KB-8-5 and KB-8-5-11 cells, showing no effect on the sensitivity of drug-sensitive KB-3-1 cells. The potency of Ro 32-2241 was comparable with that of cyclosporin A and better than that of verapamil, known modulators of multidrug resistance. Ro 32-2241 also completely reversed the taxol resistance of KB-8-5 cells and partially reversed the resistance of KB-8-5-11 cells. Vinblastine resistance was also partially reversed. Mechanistic experiments were carried out to determine whether Ro 32-2241 interacted with P-glycoprotein (Pgp) directly. Increased efflux of [14C]-Ro 32-2241 was seen with the more resistant KB-8-5-11 cells (although the percentage effluxed was very low as compared with [3H]-daunomycin), suggesting that Ro 32-2241 can act as a substrate for Pgp. Direct interaction of Ro 32-2241 with Pgp was confirmed by demonstration that it inhibited binding of [3H]-azidopine to Pgp in KB-8-5-11 membranes. In conclusion, Ro 32-2241, acting directly on Pgp (rather than, or in addition to, an effect on PKC), is effective in reducing or reversing resistance to doxorubicin, taxol and vinblastine in human tumour cells with a clinically relevant degree of MDR. However, results of in vivo experiments conducted to investigate the effects of Ro 32-2241 on resistance to doxorubicin suggest that it may not be possible to achieve sufficiently high levels of Ro 32-2241 in vivo to modulate MDR.

Vanadium stimulates immunological responses of chicks.

In a continuation of studies on the interaction of dietary phosphorus (P) and vanadium (V) levels, studies have directed toward an examination of this interaction on the immune system of chicks. Antibody titers to sheep red blood cells (SRBC) were increased at 7 days post-inoculation (PI) by as little as 10 mg V/kg diet in the P-deficient group, while 50 mg V/kg was required in the P-supplemented group. At 14 days PI, only the 50 mg V/kg was significantly higher in both P-deficient and P-supplemented groups. At 21 days PI, vanadium had no significant effect. P-deficiency resulted in a decrease in the percentage of phagocytic macrophages obtained from the abdominal cavity and a decrease in the number of intracytoplasmic SRBC per phagocytic macrophage. These two criteria were increased by vanadium in both the P-deficient and P-supplemented animals. In P-supplemented animals, the CD4/CD8 ratios of lymphocytes obtained from the blood and spleen were increased by the inclusion of 50 mg V/kg diet. The IL-1-like activity of macrophage supernatants was not significantly affected by dietary V, but IL-6 activity was increased. Densitometric analysis of lysates of macrophages isolated from control and V-fed chicks for anti-protein-tyrosinephosphate (PTP) bands indicate that dietary V increased PTP. While the evidence is not clear that there is a P x V interaction in the immune system studies, it is clear that dietary V at the levels used results in a positive immune response of chicks, possibly mediated through increased PTP.

Tongue lesions in children.

A review of tongue lesions in children showed that there is a great variety requiring operative intervention, often in infancy. While the presenting symptoms may be related to dysphagia and dyspnea, the aim of operative intervention should not only be to salvage life by restoration of breathing and swallowing, but also to leave a tongue capable of adequate speech, taste, sensation, and normal orofacial development. Intimate knowledge of lingual anatomy and function is necessary to allow selection of the ideal procedure and appropriate timing of the therapy. While careful observation and nonoperative approach may be indicated in non-neoplastic macroglossia, early intervention is often necessary in diffuse neoplastic lesions such as lymphangioma, fibromatosis, or fibrolipomatous dysplasia. While malignant tumors are rare in childhood, they do occur and have to ruled out.

Novel, potent and selective inhibitors of protein kinase C show oral anti-inflammatory activity.

Clarification of the precise role of protein kinase C (PKC) in cellular functional responses has been hampered by a lack of potent, selective inhibitors. The structural lead provided by staurosporine, a potent but non-selective protein kinase (PK) inhibitor, was used to derive a series of bis(indolyl)maleimides of which the most potent, Ro 31-8425 (I50: PKC = 8 nM) showed 350-fold selectivity for PKC over cAMP-dependent protein kinase. Ro 31-8425 antagonised cellular processes triggered by phorbol esters (potent, specific PKC activators) and inhibited the allogeneic mixed lymphocyte reaction, suggesting a role for PKC in T-cell activation. Methylation of the primary amine in Ro 31-8425 produced an analogue. Ro 31-8830 which, when administered orally, produced a dose-dependent inhibition of a phorbol ester-induced paw oedema in mice (minimum effective dose = 15 mg/kg). Ro 31-8830 also selectively inhibited the secondary inflammation in a developing adjuvant arthritis model in the rat. The results presented here suggest that these selective inhibitors of PKC may have therapeutic value in the treatment of T-cell-mediated autoimmune diseases.

Interaction of vanadium and phosphorus in chicks.

Studies were carried out to determine the effect of dietary vanadium on chicks fed phosphorus deficient and control diets. Vanadium at 50 mg/kg of diet decreased growth of both control and deficient chicks. The high mortality among the phosphorus deficient chicks was significantly alleviated by the presence of vanadium. The increased relative ventricular weights found among the deficient chicks was also alleviated by the presence of dietary vanadium. Vanadium fed at 10 or 20 mg/kg diet did not reduce growth rate but significantly reduced mortality among chicks fed the deficient diet and decreased the relative ventricle weights. Time course studies revealed that chicks are hatched with high relative ventricular weights (.83% of body wt) and remain at that level among chicks fed the phosphorus deficient diet. The addition of vanadium or phosphate to the diet resulted in a progressive decrease in relative ventricular weights. The inclusion of vanadium in the diet resulted in increased serum phosphorus levels among the deficient chicks that may be related to the decrease in mortality and relative ventricle weights.

Interaction of vanadate and chloride in chicks.

Six experiments have been conducted examining the interaction of dietary sodium chloride levels and vanadate toxicity in chicks. Increasing the dietary supplement of NaCl from 0.5 to 2.0% resulted in amelioration of vanadate toxicity, as measured by growth rate. The amelioration was found to reside in the chloride ion. Hepatic, renal, and femur vanadium concentrations were usually reduced at the higher levels of NaCl supplementation, but there was little correlation between these reductions and the reversal of vanadate toxicity.

The effect of dietary mercury on vanadate toxicity in the chick.

Three experiments were conducted investigating the interaction of dietary vanadate and mercury on the growth of chicks. The growth-retarding effect of 30 mg vanadium/kg diet was completely overcome by the inclusion of 500 mg mercury/kg diet. Restricting the feed intake of the mercury-supplemented animals to approximately those receiving vanadate alone still resulted in an amelioration of the growth retarding effect of vanadate. Analyses of femurs and kidneys revealed that mercury added to a vanadium-containing diet increased the vanadate concentration of the femur and had no effect on the vanadium concentration in the kidney. As little as 25 mg mercury/kg diet significantly reduced the growth retarding effect of vanadium. The inclusion of 100 mg mercury/kg in the diet resulted in a significant increase in renal glutathione concentration.

Effect of dietary copper on vanadate toxicity in chicks.

The addition of copper to a corn-soybean diet at levels of 200 mg/kg and above lessened the growth-retarding effect of vanadate for chicks. This interaction between vanadate and copper was evident in both ad libitum-fed chicks and chicks in which feed consumption was restricted to approximately equal amounts. The ameliorating effect of copper was not accompanied by changes in the femur levels of vanadium nor by changes in the hepatic or renal glutathione concentrations. Zinc added at 515 mg/kg of diet had no effect on the toxicity of vanadium. Sodium sulfate added at a level to supply the same amount of sulfate, as supplied by 500 mg/kg copper sulfate, was without effect on the vanadate-induced growth depression. The underlying mechanism of the interaction of copper and vanadium is not known, but it does not lie in changes in feed consumption or organ burdens of vanadium, as represented by the femur vanadium concentrations.

Studies on the role of iron in zinc toxicity in chicks.

The interaction of dietary iron and zinc was studied in chicks. Zinc was found to be more toxic in iron-deficient animals than iron-supplemented animals as measured by hemoglobin concentrations and growth. Analyses of the kidney and liver for iron and zinc were carried out. As the level of iron was increased from 0-1000 ppm supplementation, the concentration of liver zinc increased. The organ levels of iron were decreased as the dietary zinc levels were increased from 0-5000 ppm. Radioisotope studies using 65Zn revealed that the iron content of the diet did not affect absorption of zinc. Administration of the isotope, either in an intestinal segment or intravenously, resulted in more zinc being taken up by the liver in the iron supplemented animals. This was especially noted when the ratio of the isotope in liver to that in the blood was compared. Gel chromatography of kidney and liver homogenates revealed that iron deficiency resulted in less zinc being eluted in a volume characteristic of metallothionein compared to homogenates of organs from iron supplemented animals. The results indicate that iron-supplemented animals have a greater capacity for sequestering zinc on metallothionein than do iron-deficient animals. Conversely, iron-deficient chicks were more susceptible to the effects of zinc toxicity than are iron-adequate chicks.

Studies on the role of iron in the reversal of cadmium toxicity in chicks.

Studies were conducted to determine the effect of dietary iron (Fe) levels ranging from a deficiency to an excess on the toxicity of cadmium (Cd) in chicks. In Fe-deficient animals, cadmium was found to be more toxic than in Fe supplemented animals as measured by growth. The liver Cd burdens were increased significantly in the presence of dietary Fe supplementation, and there was a significant Cd-Fe interaction in the Cd concentration of the kidney, indicating that iron deficiency increased the concentration of Cd in the kidneys of those chicks receiving this element. Cd tended to reduce the Fe concentration in both the liver and kidney. The absorption of Cd as measured by the amount of 109Cd that disappeared from an isolated duodenal segment in one h was not affected by the Fe content of the diet, but the amount of isotope appearing in the liver compared to the amount present in the blood was increased in the Fe supplemented chicks. Separation of the Cd binding ligands by column chromatography revealed that more of the Cd in the liver, but not the kidney, was associated with ligands which eluted in a column volume that contained metallothionein in those chicks receiving Fe than in the livers from Fe deficient animals. The inverse relationship between the amount of Cd bound to the metallothionein containing fraction and toxicity may be related causally.

Studies on the role of iron in the reversal of vanadium toxicity in chicks.

The interaction of dietary iron levels on vanadium toxicity was studied in chicks. Dietary iron levels ranged from a deficiency, ca. 10 ppm, to an adequacy, 100 ppm supplemental iron. to an excess, 1000 ppm supplemental iron. Vanadium was fed at 10, 20, and 40 ppm. Vanadium toxicity as measured by chick growth was more severe in the iron-deficient animals than in those receiving supplemental iron. The increase in degree of toxicity in the iron-deficient animals was accompanied by an increase in the liver vanadium, both total and concentration. The addition, of vanadium to the diet did not influence the iron concentration of the liver or kidney. Radioisotope, studies with(48)V revealed that the absorption of vanadium was not influenced by the iron concentration of the diet, but that the iron-deficient animals retained more vanadium in the blood and liver and less in the bone than did the iron supplemented animals. It is proposed that the degree of iron saturation of transferrin and ferritin to which vanadium can bind is a possible explanation for the results obtained.