Indoline derivatives as 5-HT(2C) receptor agonists.

A series of 1-(1-indolinyl)-2-propylamines was synthesised and evaluated as 5-HT(2C) receptor agonists for the treatment of obesity. The general methods of synthesis of the precursor indoles are described. The functional efficacy and radioligand binding data for all of the compounds at 5-HT(2) receptor subtypes are reported. A number of compounds were found to reduce food intake in rats after oral administration.

Discovery of nonxanthine adenosine A2A receptor antagonists for the treatment of Parkinson's disease.

During a program to investigate the biochemical basis of side effects associated with the antimalarial drug mefloquine, the authors made the unexpected discovery that the (-)-(R,S)-enantiomer of the drug is a potent adenosine A2A receptor antagonist. Although the compound was ineffective in in vivo animal models of central adenosine receptor function, it provided a unique nonxanthine adenosine A2A receptor antagonist lead structure and encouraged the initiation of a medicinal chemistry program to develop novel adenosine A2A antagonists for the management of Parkinson's disease (PD). The authors have synthesized and screened more than 2,000 chemically diverse and novel adenosine A(2A antagonists. Early examples from two distinct chemical series are the thieno[3,2-dy]pyrimidine VER-6623 and the purine compounds VER-6947 and VER-7835, which have high affinity at adenosine A2A receptors (K(i) values 1.4, 1.1, and 1.7 nmol/L, respectively) and act as competitive antagonists. In particular, VER-6947 and VER-7835 demonstrate potent in vivo activity reversing the locomotor deficit caused by the D2 receptor antagonist haloperidol, with minimum effective doses comparable with that of KW6002 (0.3 to 1 mg/kg). In conclusion, the authors have discovered potent, selective, and in vivo active nonxanthine adenosine A2A antagonists that have considerable promise as a new therapy for PD.

An analysis of UK waste minimization clubs: key requirements for future cost effective developments.

The UK waste strategy is based upon use of the best practicable environmental option (BPEO), by those making waste management decisions. BPEO is supported by the use of the waste hierarchy, with its range of preferable options for dealing with waste, and the proximity principle, where waste is treated/disposed of as close to its point of origin as possible. The national waste strategy emphasizes the key role of waste minimization and encourages industry, commerce and the public to move towards sustainable waste management practice for economic and environmental reasons. Waste minimization clubs have been used, since the early 1990s, to demonstrate to industry/commerce that reducing waste production can lead to significant financial savings. There have been around 75 such clubs in the UK and they receive support from a wide range of agencies, including the Environmental Technology Best Practice Program. The early Demonstration Clubs had significant savings to cost ratios, e.g. Aire and Calder at 8.4, but had very high costs, e.g. Aire and Calder at 400,000 pounds. It is acknowledged that the number of clubs will have to be approximately doubled in the next few years so as to have an adequate coverage of the UK. There are at present, marked regional variations in club development and cognizance needs to be taken, by facilitators, of the need for extensive coverage of the UK. Future clubs will probably have to operate in a financially constrained climate and they need to be designed to deliver significant savings and waste reduction at low cost. To aid future club design, final reports of all projects should report in a standard manner so that cost benefit analysis can be used to inform facilitators about the most effective club type. rights reserved.

Retinoic acid alters epithelial differentiation during palatogenesis.

Retinoids are teratogenic in humans and animals, producing a syndrome of craniofacial malformations that includes cleft palate. This study investigates the mechanism through which retinoic acid induces cleft palate. Murine palatogenesis after exposure to retinoic acid in utero is compared to normal development and to alterations observed after exposure in organ culture to retinoic acid or epidermal growth factor (EGF). Human embryonic palatal shelves were placed in the organ culture system and the responses to retinoic acid and EGF were compared to those of the murine palatal shelves. Growth factors play a role in normal development and are found in the embryonic palate. In other cell culture systems, retinoids alter the expression of EGF receptors. Our results suggest that in the medial epithelial cells of the palate, retinoic acid sustains the expression of the EGF receptor and the binding of EGF at a time when the expression in control medial cells has declined, and these control cells subsequently undergo programmed cell death. The continued DNA synthesis, proliferation, survival, and shift in phenotype of the medial cells is believed to interfere with the adhesion and fusion of opposing palatal shelves, resulting in cleft palate.

Effects of retinoic acid on embryonic development of mice in culture.

The effects of all-trans-retinoic acid (RA) (tretinoin) on the craniofacial development of mouse embryos were examined using whole embryo culture. In day 8 embryos cultured for 48 h, embryonic growth was inhibited concentration-dependently by all-trans-RA treatment. Most of the treated embryos exhibited hypoplasia of the primary palatal processes and a reduction in the development of the first visceral arches. In day 10 embryos cultured for 48 h, although embryonic growth was not inhibited at any concentrations of all-trans-RA, median cleft lip (93%), hypoplasia of the primary palatal processes (37%) and limb reduction deformities (48%) occurred commonly. Furthermore, RA treatment greatly reduced the size of the secondary palatal processes. The incorporation of 3H-thymidine in the treated maxillary processes was decreased to 65% of the control value at 1.0 x 10(-7) M all-trans-RA. These findings indicate that all-trans-RA is teratogenic in mouse whole embryo culture.

Influence of retinoids on sister chromatid exchanges and chromosomes in cultured human embryonic palatal mesenchymal cells.

The cytogenetic effects of retinoids and their metabolites on human embryonic palatal mesenchymal (HEPM) cells were investigated in cell culture. Treatment with 13-cis, 4-oxo-13-cis-, and all-trans-retinoic acids did not induce chromosomal structural aberrations even at high concentrations in the presence or absence of metabolic activation system. The frequencies of sister chromatid exchanges (SCE) tended to decrease in HEPM cells treated with retinoids in the absence of S-9. 4-Oxo-13-cis-retinoic acid, which is one metabolite of 13-cis-retinoic acid, significantly decreased the SCE frequencies. The mitotic index decreased with increasing concentration of retinoic acids, indicating that retinoids inhibit cell proliferation in HEPM cells. We concluded that retinoids inhibit cell proliferation in HEPM cells in culture without causing DNA or chromosome damage.

Effect of retinoids on proliferation of human embryonic palatal mesenchymal cells in culture.

The cytokinetics and growth effects of retionic acid (RA) (13-cis-RA, and all-trans-RA) were determined in human embryonic palatal mesenchymal (HEPM) cells using cell culture and sister chromatid differential staining. The lowest concentration tested that showed growth inhibition was 1.0 X 10(-4)M for 13-cis-RA, and 6.8 X 10(-5)M for all-trans-RA after 40 h of treatment. When HEPM cells were grown in the presence of BrdU (5-bromodeoxyuridine) for 44 h, approximately 70% of the control cells were in metaphase in the 3rd replication cycle (M3). The percentage of cells in M3 decreased in the presence of RA with or without a metabolic activation system (S-9), thus indicating a longer cell proliferation time for the RA-treated cells. The estimate of cell proliferation time in 13-cis-RA-treated cells (1.0 X 10(-4)M) was approximately 25 h at the 2nd cell cycle compared with 20 h in the control cells. When HEPM cells were exposed for 16 h to [3H]-thymidine, labeling indices in 13-cis-RA-treated cells were significantly reduced even at 1.0 X 10(-6)M. Both 13-cis-RA and all-trans-RA caused concentration-dependent decreases in [3H]-thymidine incorporation into HEPM cells. These results suggest that retinoic acids or their major metabolites interfere with DNA synthesis and decrease the proliferation of HEPM cells.

EGF receptor expression in the developing tooth is altered by exogenous retinoic acid and EGF.

Retinoic acid (RA) is a teratogen producing a variety of malformations including clefts of the secondary palate and malformations of the teeth. Recent studies in our laboratory investigating the effects of RA and EGF on the medial epithelium of the palatal shelf have also provided some information on the effects of these agents on the development of the tooth. In the control, toothbud epithelial cells expressed the EGF receptor, bound EGF, and proliferated. The majority of bud stage teeth exposed to RA either in vivo or in organ culture did not detectably bind EGF, express the EGF receptor, or proliferate in the epithelial or mesenchymal components. Toothbuds exposed to exogenous EGF in organ culture also did not bind EGF, the receptor, or proliferate. EGF has been reported to inhibit morphogenesis of toothbuds in culture and when EGF was given to neonates the size of the tooth was reduced. Regulation of EGF receptor expression appears to be important in the development of the toothbud and exposure to RA or EGF disrupts this process and could contribute to the decreased tooth size, agenesis, and malformations observed in the teeth. While limited in scope, these experiments present previously unreported effects of RA and EGF on the toothbud epithelium. These observations should be of interest to those studying tooth development, and warrant further and more detailed studies.

Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture.

Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [125I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [3H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [3H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.

Retinoic acid alters EGF receptor expression during palatogenesis.

Various growth factors are necessary for normal embryonic development and EGF receptors are present in developing palatal shelves of embryonic/fetal mice at least from day 12 of gestation. The medial epithelium of the palatal shelf undergoes a series of developmental events which do not occur in the oral and nasal epithelia. In utero and in organ culture, the control palatal medial epithelium shows a developmental decline in EGF receptors, demonstrated both by a decrease in the binding of antibody to EGF receptors and a decrease in the binding of 125I-EGF; decreases which are not observed in cells of the adjacent oral or nasal epithelium. During this period, medial cells cease DNA synthesis and undergo programmed cell death. Medial epithelial cells exposed to all-trans-retinoic acid continue to express EGF receptors, bind EGF, proliferate, fail to undergo programmed cell death and exhibit a morphology typical of nasal cells. The data suggest that this disturbance by retinoic acid of EGF receptor localization and subsequent alterations in differentiation of the epithelial cells plays a role in the retinoic-acid-mediated induction of cleft palate.

Alterations in craniofacial growth induced by isotretinoin (13-cis-retinoic acid) in mouse whole embryo and primary mesenchymal cell culture.

Recent evidence has demonstrated that 13-cis-retinoic acid (13-cis-RA, or isotretinoin) is responsible for various craniofacial malformations in the rodent and human embryo. Our studies have been directed toward understanding this effect using mouse whole embryo and primary cell cultures. In whole embryo culture, 13-cis-RA caused significant overall embryonic growth retardation, especially in the primary and secondary palatal processes. In embryos explanted on day 10 of gestation and exposed for 24 or 48 hr, the mesenchyme beneath the epithelium of the nasal and maxillary processes contained pyknotic nuclei as well as a dramatically reduced number of nuclei incorporating 3H-thymidine. The secondary palatal processes and the roof of the oral-nasal cavity had fewer mesenchymal cells than control embryos. The incorporation of 3H-thymidine into TCA-insoluble macromolecules was 30% less in the retinoid-treated heads. In primary cell cultures from day-12 mouse secondary palatal mesenchyme, subsequent cell growth was decreased at concentrations of 13-cis-RA greater than 1 X 10(-5) M. After a 40-hr treatment period, labeling indices in retinoid-treated cells were significantly lower than control values (25% compared with 40%). Retinoic acid also caused a significant, concentration-dependent decrease in 3H-thymidine incorporation. The inhibitory effect of 13-cis-RA on proliferation of oral-nasal mesenchymal cells appears to be related to the production of craniofacial malformations.

Teratogenicity of benzoic acid derivatives of retinoic acid in cultured mouse embryos.

Isotretinoin (13-cis-RA) is a human teratogen and mouse embryos exposed to 13-cis-RA in vivo exhibit many of the same defects as humans. Early postimplantation mouse embryos exposed to 13-cis-RA in culture exhibit developmental alterations of the visceral arches, similar to those seen after in vivo exposure. Certain benzoic acid derivatives of retinoic acid have been shown to possess activity equal to or greater than retinoic acid in several in vitro systems. This study examines the teratogenic effects of some of these retinoids on mouse embryos in vitro. Day 8 CD-1 mouse embryos were cultured for 48 hours in the presence of these benzoic acid derivatives. With the exception of Ro-15-0778, all compounds produced visceral arch malformations similar to those seen in embryos exposed to 13-cis-RA, but at dramatically different effective concentrations. Extremely low concentrations of the retinoic acid-related compounds tested appear to have detrimental effects on embryonic development and these compounds may be poor candidates for therapeutic use.

Expression of rat transforming growth factor alpha mRNA during development occurs predominantly in the maternal decidua.

Previous studies have shown that transforming growth factor alpha is expressed during rodent development. To establish the site(s) of transforming growth factor alpha mRNA expression during rat embryogensis, we performed in situ hybridization and Northern blot analyses on samples of embryonic and maternal tissues at various gestational ages. Our results indicate that the high levels of transforming growth factor alpha mRNA that are observed during early development are the result of expression in the maternal decidua and not in the embryo. Decidual expression appears to be induced after implantation, peaks at day 8, and then slowly declines through day 15 at which time the decidua is being resorbed. Expression of transforming growth factor alpha mRNA is highest in that region of the decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and other maternal tissues. The developmentally regulated expression of transforming growth factor alpha mRNA in the decidua, together with the presence of epidermal growth factor receptors in this tissue, suggests that transforming growth factor alpha stimulates proliferation locally through an autocrine mechanism. Since epidermal growth factor receptors are present in the embryo and placenta, transforming growth factor alpha produced in the decidua may also act on these tissues through paracrine or endocrine mechanisms.

TCDD alters the extracellular matrix and basal lamina of the fetal mouse kidney.

The teratogenic effects of the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have previously been studied in several species, and hydronephrosis has been reported to be a frequent abnormality in near-term fetuses. C57BL/6N female mice, given 12 micrograms/kg TCDD, P.O., on day 10 of gestation were killed on days 14, 15, and 16; fetal kidneys were collected and prepared for either immunofluorescent localization of several extracellular matrix components (ECM) or transmission electron microscopy (TEM). The TCDD-treated and control kidneys showed the same pattern of staining for fibronectin, but TCDD-treated kidneys displayed a diminished overall intensity. The intensity of laminin and type IV collagen immunofluorescence also appeared to be decreased, and deviations in the pattern of antibody binding were detected for differentiating TCDD-treated nephrons. Binding of the laminin antibody to the basal lamina was decreased in the parietal layer of Bowman's capsules in more advanced stages of differentiation. TEM analysis focused on the basal lamina of the tubules and Bowman's capsule. In TCDD-exposed kidneys, ECM components adjacent to differentiating nephrons were less abundant, and the basal lamina of the developing Bowman's capsules had a diminished lamina densa. The earliest nephrons to develop display these defects and comprise the first functional filtration units of the metanephric kidney. These ultrastructural changes noted in TCDD-exposed nephrons may promote proteinuria, a condition normally observed in the developing kidney when the filtration barrier is immature.

TCDD-induced hyperplasia of the ureteral epithelium produces hydronephrosis in murine fetuses.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has been recognized as a kidney and palate teratogen for many years. The etiology of the kidney abnormality has not been revealed, and there is some confusion about the exact nature of the defect. This study examines C57BL/6N fetal mouse kidneys from day 14 of gestation through day 17. Pregnant females received a single dose of 0 or 12 micrograms TCDD/kg by gavage on day 10 pregnancy. Fetal urinary systems were examined on days 14, 15, 16 (a.m.), 16 (p.m.), and 17 (p.m.). The patency of the ureteric lumen was examined by injection of dye into the bladder. TCDD treatment did not delay or prevent breakdown of the ureteric membrane between days 15 and 16. On days 16 through 17, the ureteric lumina of TCDD-exposed fetuses were narrow and tortuous when compared to the control lumens. Sections of ureter were observed by light microscopy. On day 15 the lumina of TCDD-exposed ureters were occluded by epithelial cells. As a result of hyperplasia of the ureteric luminal epithelium, hydroureter and hydronephrosis became pronounced by day 17. We conclude that the kidney abnormality induced by TCDD is true hydronephrosis, which is defined as the accumulation of urine in the kidney due to obstructed outflow.

Evaluation of murine placental degradation and transfer of [125I]iodo-epidermal growth factor.

Since epidermal growth factor (EGF) has been postulated to play a role in embryonic and fetal growth, a study was undertaken to assess the placental degradation and transfer of maternally administered EGF. Before iodination, mouse EGF was purified to homogeneity by reversed-phase HPLC. Approximately 5 ng [125I]iodo-EGF (approximately 10(6) cpm) were injected iv into day 10, day 13, or day 17 pregnant CD-1 mice; radioactivity in plasma, placentas, and conceptuses was measured up to 2 h after injection. The time course analysis revealed an initial rapid decline in total plasma radioactivity followed by an increase that was maximal by approximately 30 min. Gel filtration (G-15, G-50) chromatography of plasma revealed that by 5 min, radioactivity was associated with free 125I and with material much larger than EGF. No apparent degradation of [125I]iodo-EGF occurred after direct incubation with maternal plasma. Placental radioactivity had an initial phase of decay between 1 and 5 min followed by an increase that became maximal between 30 and 60 min. Extracts of placentas made with 4 M urea in 0.2 M Tris-HCl, pH 8.0, and taken 1-30 min after injection revealed radioactivity coeluting predominantly with [125I]iodo-EGF at 1 min but shifting to mostly free 125I by 30 min. Uptake of radioactivity by conceptuses was not evident until about 15 min, and only free 125I was detected in extracts; the same results were obtained when 5 micrograms unlabeled EGF were injected simultaneously with [125I]iodo-EGF. Incubation of placental mince with [125I]iodo-EGF yielded [125I]MIT as the apparent major radioactive degradation product. Formation of [125I]MIT in vitro was both time- and temperature-dependent. At 37 C, marked formation of [125I]MIT was observed; at 22 C, only a negligible amount was formed after incubation of mince with [125I]iodo-EGF for 60 min. Incubation of [125I]iodo-EGF with kidney mince yielded predominantly free 125I. When tissues were incubated directly with [125I]MIT, kidney tissue deiodinated the [125I]MIT, but placental tissue did not. When [125I]iodo-EGF was incubated for up to 2 h with kidney tissue in the presence of excess MIT (unlabeled), the major degradation product eluted as [125I]MIT, instead of free 125I. These findings suggest that the mouse placenta can readily bind and then degrade [125I]iodo-EGF to constituent amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Retinoids and epidermal growth factor alter embryonic mouse palatal epithelial and mesenchymal cell differentiation in organ culture.

The mechanism by which retinoids (RA) induce cleft palate is not known. During normal palatogenesis, the medial epithelia of opposing palatal shelves cease DNA synthesis, come into contact, adhere, and undergo programmed cell death (PCD). In organ cultures of day 12 embryonic mouse palatal shelves, epidermal growth factor (EGF) blocks PCD, and DNA synthesis continues. In the present study, the effects of trans-RA, 13-cis-RA, EGF, and combinations of EGF and RA on surface morphology, DNA synthesis, and cellular ultrastructure are determined for CD-1 embryonic mouse palatal shelves cultured on day 12 of gestation. DNA synthesis in the medial cells was sustained and PCD was blocked by EGF, trans-RA, and 13-cis-RA. Exposure to trans-RA, but not to 1-cis-RA, induced the medial epithelia to undergo hyperplasia, and addition of EGF enhanced the effect. In the presence of RA, particularly trans-RA, medial epithelial cells acquired nasal cell characteristics, and EGF enhanced this effect. Expansion of the mesenchymal extracellular spaces was blocked by trans-RA and to a lesser degree by 13-cis-RA. The RA-induced alterations in normal epithelial and mesenchymal cell differentiation may be relevant to the etiology of RA-induced cleft palate in vivo.

Human embryonic palatal epithelial differentiation is altered by retinoic acid and epidermal growth factor in organ culture.

Reports of adverse human pregnancy outcomes including cleft palate have increased as the clinical use of isotretinoin (13-cis-retinoic acid) and other retinoic acid (RA) derivatives have increased, but the mechanisms by which their effects are exerted are not understood. Research in craniofacial development is generally performed in rodents, and mouse palatal shelves exposed in organ cultures to retinoids and epidermal growth factor (EGF) display altered medial epithelial cell morphology blocking normal union of apposing shelves. In the present study, precontacting human palatal shelves were maintained in organ culture for 2, 3, or 6 days and exposed to labeled thymidine (3H-TdR) during the last 16 hr. Retinoids and EGF were included in the media so that each shelf was exposed to one of the following: control, EGF, trans-RA at 10(-5)M, cis-RA at 10(-7) or 10(-9) M, or RA + EGF. After exposure of cultured human embryonic palatal shelves to 13-cis-RA and trans-RA with or without EGF, medial epithelial cells do not degenerate, cell surface morphology shifts toward a nasal type, glycogen deposits decrease, smooth endoplasmic reticulum (SER) increases, and basal lamina appear altered. In shelves exposed to EGF and trans-RA early in their development, DNA synthesis appears to terminate prematurely as compared to shelves cultured in control media, and this effect is accompanied by excessive mesenchymal extracellular space expansion. Exposure of shelves to EGF alone is sufficient to block degeneration and induce hyperplasia of the medial epithelial cells but does not induce other ultrastructural changes seen with both EGF and RA. The observed alterations in medial cell morphology could interfere with adhesion of the palatal shelves and may play a role in retinoid-induced cleft palate in the human embryo.

Retinoic acid inhibits migration of cranial neural crest cells in the cultured mouse embryo.

Clinical observations have demonstrated that isotretinoin (13-cis-retinoic acid; cis-RA) is a human teratogen causing primarily heart and craniofacial malformations. Isotretinoin exposure to the early postimplantation mouse embryo in culture results in specific defects in craniofacial development that may be due to an interference in the early migration of cranial neural crest (CNC) cells [Goulding and Pratt, 1986]. The present study was designed to test this hypothesis by examining the migration of these cells in whole embryo culture. Day 8 CD-1 mouse embryos were cultured for 6-48 hr in the presence or absence of cis-RA at 2 X 10(-6) to 2 X 10(-5) M. Embryos either were fixed for light microscopy using Nichols' method for localization of CNC cells or were processed for scanning and transmission electron microscopy. At the light microscopic level, CNC cells in the mid-brain region of control embryos had migrated to the region of the first and second visceral arches after 6 hr in culture. Cis-RA interfered with this migration; CNC cells in treated embryos either did not leave the neuroepithelium (NE) or were aggregated near the NE. Autoradiographic studies indicated that cis-RA did not affect the overall viability or DNA synthesis of the CNC cells. However, at the TEM level, there was a dramatic increase in the number of cellular blebs in the CNC cells. Our results demonstrate a direct effect of 13-cis-RA on the CNC cells and suggest that this effect is due to alterations in the cell surface.