Anti-tumor activity of motesanib in a medullary thyroid cancer model.

BACKGROUND: Medullary thyroid cancer (MTC) is frequently associated with mutations in the tyrosine kinase Ret and with increased expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Motesanib is an investigational, orally administered small molecule antagonist of VEGFR1, 2, and 3; platelet-derived growth factor receptor (PDGFR); Kit; and possibly Ret. AIM: The aim of this study was to investigate the effects of motesanib on wildtype and mutant Ret activity in vitro and on tumor xenograft growth in a mouse model of MTC. METHODS/RESULTS: In cellular phosphorylation assays, motesanib inhibited the activity of wild-type Ret (IC(50)=66 nM), while it had limited activity against mutant Ret C634W (IC(50)=1100 nM) or Ret M918T (IC(50)>2500 nM). In vivo, motesanib significantly inhibited the growth of TT tumor cell xenografts (expressing Ret C634W) and significantly reduced tumor blood vessel area and tumor cell proliferation, compared with control. Treatment with motesanib resulted in substantial inhibition of Ret tyrosine phosphorylation in TT xenografts and, at comparable doses, in equivalent inhibition of VEGFR2 phosphorylation in both TT xenografts and in mouse lung tissue. CONCLUSIONS: The results of this study demonstrate that motesanib inhibited thyroid tumor xenograft growth predominantly through inhibition of angiogenesis and possibly via a direct inhibition of VEGFR2 and Ret expressed on tumor cells. These data suggest that targeting angiogenesis pathways and specifically the VEGF pathway may represent a novel therapeutic approach in the treatment of MTC.

Effect of short-term treatment with a monoclonal antibody to P-selectin on balloon catheter-induced: intimal hyperplasia, re-endothelialization, and attenuation of endothelial-dependent relaxation.

The effects of an anti-P-selectin monoclonal antibody (MAb, PB1.3; Cytel Corporation) on neoendothelialization; neoendothelial function, as evidenced by acetylcholine-induced relaxation (nitric oxide formation); and intimal hyperplasia following embolectomy catheter-induced injury to the rabbit thoracic aorta were investigated. Catheter injury was induced in two groups of New Zealand White rabbits. One group received no treatment, while the second group received short-term treatment with the MAb (i.p., immediately before and 12 h after induction of catheter injury). A third group underwent a sham operation and served as uninjured controls. Following sacrifice at 2 weeks after injury, aortic rings were assessed for degree of intimal hyperplasia, neoendothelial morphology (scanning electron microscopy), and acetylcholine-induced relaxation. Aortic tissue from catheter-injured animals that received treatment exhibited improved neoendothelial morphology, as compared with tissue from untreated but catheterized animals; however, no statistically significant attenuation of the hyperplastic response or improvement in the attenuated neoendothelial-dependent acetylcholine-induced relaxant response that is characteristic of neoendothelium that forms after catheter denudation was observed. These data suggest that short-term attenuation of P-selectin-mediated polymorphonuclear leukocyte (PMN)/endothelium, PMN/platelet interactions, and/or thrombin formation beneficially affects neoendothelialization of the vascular wall following balloon catheter-induced injury.

A quantitative evaluation of Fourier components in transparent and opaque calf cornea.

Fourier transform methods were applied to STEM (scanning transmission electron microscopy) images to detect and quantify the subtle differences between the structure of normal transparent calf cornea and opaque calf cornea. In order for a tissue to be transparent, it can scatter or absorb only a small amount of light. Light scattering is minimized when the principal Fourier components of the spatial fluctuations in the index of refraction have wavelengths which are small relative to the wavelength of light (Benedek, 1971). Corneal opacity was produced as a result of high intraocular pressure (100-150 mmHg) when liquid was injected into calf eyes (0-2 weeks old). Pressurization created large structural defects and slight disruptions in the organization of the collagen fibers. Although the fiber organization appeared similar in the micrographs of both opaque and transparent corneas, Fourier analysis of STEM images collected at 50K magnification identified statistically significant differences. Far fewer Fourier components with wavelengths in the light scattering range (200-1100 nm) were observed in the transparent corneas than in the pressurized corneas as predicted by Benedek's theory. It was of interest that corneas treated with 100% glycerol prior to pressurization remained transparent at high intraocular pressures, possibly because glycerol stabilized the structure of the corneas and maintained a uniform index of refraction across the corneal stroma. The results demonstrate the effectiveness of Fourier analysis in detection and quantification of slight changes in structure at the electron microscopic level.

13-cis retinoyl-beta-glucuronide in lacrimal gland fluid of rabbits treated with 13-cis retinoic acid.

When rabbits and humans are treated with orally administered 13-cis retinoic acid, the retinoic acid and a more polar retinoid metabolite appear in tears and lacrimal gland fluid. This study tested the hypothesis that this metabolite was a retinoyl-beta-glucuronide. Lacrimal gland fluid from rabbits treated with a pharmacologic dose of 13-cis retinoic acid was analyzed. Based on chromatographic retention time, absorbance maximum, and degradation to 13-cis retinoic acid by the enzyme beta-glucuronidase, it was shown that this retinoid metabolite is 13-cis retinoyl-beta-glucuronide. The 13-cis retinoyl-beta-glucuronide was also extracted from the lacrimal gland itself. It is concluded that 13-cis retinoyl-beta-glucuronide is a major metabolite of 13-cis retinoic acid in the rabbit and that this retinoid is secreted by the lacrimal gland.

Electrolyte composition of lacrimal gland fluid and tears of normal and vitamin A-deficient rabbits.

Rabbit tears and lacrimal gland fluid were collected simultaneously during pilocarpine stimulation with the goal of comparing the ionic composition of these fluids at various flow rates. Ions measured were sodium, potassium, calcium, magnesium, zinc, chloride, and bicarbonate. Human tears were also analyzed for purposes of comparison. Generally, tears and lacrimal gland fluid do not differ in ionic composition except for zinc and bicarbonate, which are in higher concentration in tears than in lacrimal gland fluid. The ionic composition of tears and lacrimal gland fluid of vitamin A-deficient rabbits was also analyzed. The maximal flow rate of lacrimal gland fluid was decreased in vitamin A-deficient rabbits as were calcium levels in tears and lacrimal gland fluid, as compared with controls. Concentrations of other ions generally did not differ from normal levels, indicating that vitamin A deficiency has only moderate effects on lacrimal gland function in the rabbit.

The relationship between secretion of retinol and protein by the lacrimal gland.

Secretion of retinol and protein by the rabbit lacrimal gland appear to be closely related, suggesting that they are secreted by the same mechanism. Pilocarpine and vasoactive intestinal peptide (VIP) both stimulate protein and retinol secretion rate in a dose-dependent manner, but the concentrations of retinol and protein in the lacrimal gland fluid are independent of fluid flow at flow rates in excess of 1 microliter/min. Under all conditions of stimulation with pilocarpine and VIP that were studied, the retinol:protein ratio in lacrimal gland fluid of normal rabbits remained constant at 3.3 ng retinol/mg protein. This correlation between retinol and protein secretion by the lacrimal gland suggests that retinol is protein-bound in lacrimal gland fluid. To identify this protein, vitamin A-deficient rabbits were treated orally with 3H-retinyl acetate. Lacrimal gland fluid was collected and analyzed for 3H-retinol and protein. The 3H-retinol in lacrimal gland fluid was identified by reverse-phase HPLC and analysis of protein by gel filtration chromatography showed that this 3H-retinol was associated with protein which eluted from the columns in the 20 kD range.

Vitamin A is stored as fatty acyl esters of retinol in the lacrimal gland.

Many tissues which require vitamin A store the vitamin as long-chain fatty acyl esters of retinol. As part of a study designed to characterize vitamin A metabolism in the lacrimal gland, which transports retinol from blood to lacrimal gland fluid, extracts from lacrimal glands of rabbits and rats were analyzed by non-aqueous high performance liquid chromatography. Retinyl linoleate, retinyl palmitate, and retinyl stearate were identified in these extracts by their co-elution with standards, their retention time relative to retinyl palmitate, and their susceptibility to hydrolysis by saponification. Retinyl palmitate was present in rabbit lacrimal gland at 51.0 +/- 10.1 ng/g tissue. After treatment of vitamin A-deficient rabbits with orally administered [11,12-3H] retinyl acetate, the radiolabeled esters retinyl linoleate, palmitate, and stearate were extracted from the lacrimal glands. These data show that the lacrimal gland stores vitamin A as fatty acyl esters of retinol.

Tear secretion and lacrimal gland function of rabbits treated with isotretinoin.

Adverse ocular reactions including dry eye symptoms and blepharoconjunctivitis are common side effects of treatment with isotretinoin (13-cis-retinoic acid). However, there is little agreement in the literature on the effect of this drug on the tears. Because we have previously shown that the lacrimal gland secretes isotretinoin, we conducted a study of the effect of isotretinoin on lacrimal gland function. Rabbits were treated with isotretinoin for 5 months. Throughout the study tear secretion was monitored by the Schirmer test. At the end of the study lacrimal gland function was assessed by measurement of fluid and protein secretion rates and secretion of retinol in response to a pilocarpine stimulus. Lacrimal gland function was not affected by isotretinoin as compared with a group of age-matched control rabbits; however, Schirmer test scores were significantly increased in the treated animals as compared with control values. We conclude that isotretinoin is not toxic to the lacrimal gland of rabbits. This suggests that ocular irritation in patients treated with isotretinoin is not caused by decreased tear secretion during therapy.

Inhibition of phase separation by reagents that prevent X-irradiation cataract in vivo.

Galactose, and the phosphorothioates, WR-77913 and WR-2721, which inhibit cataract produced by X-irradiation, were evaluated for their effects on the phase separation temperature, Tc, of calf lens homogenate. The reagents were added to nuclear homogenate and the change in Tc per mol, dTc/dC, was measured. Galactose decreased Tc, -65 degrees C mol-1, WR-77913 decreased Tc, -28 degrees C mol-1 and WR-2721 decreased Tc, -76 degrees C mol-1. These are the first phase separation inhibitors that can be used in vivo to study the inhibition of lens opacification.

Inhibition of lens opacification in x-irradiated rats treated with WR-77913.

Radiation induced cataracts are models for studying mechanisms of lens opacification. WR-77913, S-3-(amino-2-hydroxypropyl) phosphorothioate (NCS-318809), has been identified as a radioprotective agent. Injection of WR-77913 (1160 mg/kg, i.p.) 15 to 30 min before exposure to 15.3 gray of x-irradiation inhibited rat lenses from developing radiation cataracts. Irradiated rats which did not receive the drug developed dense cataracts. Lenses from control rats which received no radiation remained transparent. Individual lenses were weighed, homogenized, and assayed for protein content using the Lowry method. The molecular weight distribution of soluble protein was determined by HPLC. Mean lens weights were: controls 48.2 mg; irradiated, drug-treated 45.9 mg; and irradiated, nontreated 45.5 mg. Protein accounted for over 40% of the lens weight in control and drug-treated rats and less than 20% for the nontreated cataractous lenses. Water was less than 60% of the lens weight in control and drug-treated rats and over 80% in cataractous lenses. Insoluble protein ranged from 12 to 17% of the total lens weight for each group. The ratio of insoluble to soluble lens protein was 0.40 for control, 0.65 for drug-treated, and 11.28 for cataractous rat lenses. HPLC confirmed a dramatic loss of soluble protein and a complete absence of protein below 25K daltons in cataractous lenses. Proteins below 25K daltons accounted for over 25% of the soluble protein in control and drug-treated rat lenses. WR-77913 stabilizes protein composition and appears to be an effective inhibitor of radiation cataractogenesis.

Radioprotection against cataract formation by WR-77913 in gamma-irradiated rats.

Protection by WR-77913 against radiation-induced cataract formation in rats was observed following intraperitoneal (i.p.) administration of drug (1160 mg/kg) 15-30 min before exposure to 15.3 Gy of Cs-137 whole head irradiation. Control groups included irradiated, non-protected animals, and sham-irradiated aging controls. Protection was documented photographically and by analysis of eye lens constituents. All non-protected irradiated animals developed dense cataracts throughout the lens between 90-120 days post-irradiation, while WR-77913 protected animals developed minimal lens opacification through 200 days post-irradiation. No opacification in aging controls was seen. Lens protein analysis by Lowry assay and size exclusion HPLC showed radioprotected and aging control animals were similar in protein content, distribution of total and soluble protein, and degree of lens hydration. This contrasted significantly with cataractous lenses of non-protected animals. In cataractous lenses, the soluble protein concentration in the 25-43 K dalton range was approximately 10% of that found in radioprotected or aging control lenses. Hydration was substantially higher in cataractous lens. These results indicate that WR-77913 protects against lens opacification, protein insolubilization, and hydration in lenses of irradiated animals. Biodistribution studies with [S-35]-WR-77913 showed ocular uptake of drug within 15 minutes after i.p. injection, which remained relatively constant through 60 min. The relative order of drug concentration for individual eye components was: globe greater than total eye approximately equal to humor greater than lens. Although the mechanism of radioprotection observed remains to be elucidated, WR-77913 clearly prevents radiation-induced cataracts in rats. The potentially significant clinical use for this radioprotective compound is being investigated further.

The DST in psychiatric outpatients with generalized anxiety disorder, panic disorder, or primary affective disorder.

The authors administered the dexamethasone suppression test (DST) to outpatients, who were free from psychoactive drugs for at least 10 days before the test, with primary affective disorder (N = 60), generalized anxiety disorder (N = 26), panic disorder (N = 22), and agoraphobia with panic attacks (N = 13). With a cortisol value of 5 micrograms/dl considered nonsuppression, there were no significant differences in dexamethasone nonsuppression rates among the diagnostic groups. Scores on the Hamilton Rating Scale for Depression and a melancholia subscale were significantly higher in the depressed group than in the anxiety disorder group. The findings raise questions concerning the specificity of the DST for primary affective disorder in relationship to anxiety disorders.

Gas-liquid chromatographic determination of cholesterol and other sterols in foods.

A gas-liquid chromatographic (GLC) method, using the butyryl esters of sterols, has been developed for the measurement of cholesterol, stigmasterol, sitosterol, and campesterol in foods. An immobile phase of 1% SE-30 coated on 100-120 mesh Gas-Chrom Q packed in a 6 inch X 4 mm id glass column operated at 255 degrees C was the most satisfactory of 7 column packings evaluated. Extraction with chloroform-methanol gave 98.7% recovery with a coefficient of variation of 1.8% for cholesterol added to a variety of foods. When cholesteryl palmitate was added to vegetable oil and the butyryl derivative was prepared, followed by GLC analysis, the recovery was 99.3% with a coefficient of variation of 0.9%. Amounts as low as 1 mg/100 g food can be detected with a precision of 2.5%. The results of the analysis of a variety of foods for cholesterol, campesterol, sitosterol, and stigmasterol are given.

Linkage studies in Lenz microphthalmia.

Glucose-6-phosphate dehydrogenase (G6PD) phenotype studies were done on a black family with X-linked heredofamilial bilateral microphthalmia (HBM). Three crossovers and three non-crossovers were detected in three informative matings of four generations yielding a recombination value of 0.5. These findings do not provide evidence for linkage between the G6PD and HBM loci, suggesting either that the G6PD and HBM loci are far apart on the X chromosome or that HBM in this family is inherited as an autosomal dominant male sex-limited trait.