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Biomedical subjects

J E Taylor

Publications and source records attributed to J E Taylor.

At least 19 recordsLinked to original sources
PubMed1997

Simultaneous determination of lamotrigine and its glucuronide and methylated metabolites in human plasma by automated sequential trace enrichment of dialysates and gradient high-performance liquid chromatography.

The use of the system, automated sequential trace enrichment of dialysates (ASTED), to prepare plasma samples for the estimation of lamotrigine, its glucuronide and methylated metabolites in plasma prior to gradient high-performance liquid chromatography (HPLC) is described. Using this technique the procedure was observed to be specific for all three compounds and linear over the range 0.04 to 10 microg/ml for lamotrigine and the glucuronide metabolite and 2 to 500 ng/ml for the methylated metabolite. The within-run precision (C.V.) at four different supplemented plasma lamotrigine concentrations of 0.04, 0.10, 2.5 and 10.0 microg/ml was 6.21, 5.17, 1.29 and 0.73%, respectively, and the between-run precision (C.V.) estimated to be 13.49, 6.08, 1.95 and 1.78%, respectively. The overall accuracy (% bias) of the procedure was estimated to be 12.50, 0.00, 2.80 and 1.80%, respectively. The glucuronide and methylated metabolites in plasma showed similar assay performance.

Anticonvulsants↗
PubMed1997

Development of an assay for the simultaneous determination of sildenafil (Viagra) and its metabolite (UK-103,320) using automated sequential trace enrichment of dialysates and high-performance liquid chromatography.

The development of the ASTED (automated sequential trace enrichment of dialysates) system to prepare plasma samples prior to high-performance liquid chromatography (HPLC) of sildenafil and its demethylated metabolite (UK-103,320) is described. Investigations to elucidate potential pitfalls of the ASTED on-line sample preparation system prior to separation of the analytes by HPLC are presented. The procedure is shown to be selective for sildenafil and UK-103,320, and linear over the range 1.00-250 ng/ml. The intra-batch imprecision (C.V.) of the method at plasma analyte concentrations of 1.00, 5.00, 50.0 and 200 ng/ml was 11.2, 3.10, 1.50, and 1.30%, respectively, and the corresponding inter-batch imprecision was estimated to be 13.5, 7.09, 3.69, and 5.43%. At these plasma analyte concentrations the overall inaccuracy (% bias) of the procedure ranged from 3.6 to 8.4%. The method showed similar assay performance for the estimation of the metabolite, UK-103,320. The application of the assay to a pharmacokinetic investigation during a clinical study is presented.

Chromatography, High Pressure Liquid↗
PubMed1997

Somatostatin receptor (SSTR) subtype-selective analogues differentially suppress in vitro growth hormone and prolactin in human pituitary adenomas. Novel potential therapy for functional pituitary tumors.

Previously, we have shown somatostatin receptor (SSTR) subtype-specific regulation of growth hormone (GH), thyroid-stimulating hormone, and prolactin (PRL) secretion in human fetal pituitary cultures, where GH and thyroid-stimulating hormone are mediated by both SSTR2 and SSTR5, whereas SSTR2 preferentially mediates PRL secretion. We now tested SSTR subtype-selective analogues in primary human GH- and PRL-secreting pituitary adenoma cultures. Analogue affinities determined by membrane radioligand binding in cells stably expressing human SSTR forms were either SSTR2 or SSTR5-selective. Analogues preferential either for SSTR2, including octreotide, lanreotide, and novel compounds with improved affinity for SSTR2, or new SSTR5-selective compounds suppressed GH in tumor cell cultures (up to 44% of control; P < 0.0005). However, novel analogues from both groups were 30-40% more potent than octreotide and lanreotide in suppressing GH (P < 0.05). Heterologous analogue combinations containing both SSTR2- and SSTR5-selective compounds were more potent in decreasing GH than analogues used alone (P < 0.05), or than combinations of compounds specific for the same receptor subtype (P < 0.005). In contrast, SSTR2-selective analogues did not suppress PRL release from six cultured prolactinomas studied. However, new SSTR5-selective analogues suppressed in vitro PRL secretion (30-40%; P < 0.05) in four of six prolactinomas. These results suggest that both SSTR2 and SSTR5 are involved in GH regulation in somatotroph adenoma cells, whereas SSTR5 exclusively regulates PRL secretion from prolactinoma cells. Thus, somatostatin analogues with improved selective binding affinity for these receptor subtypes may be effective in the treatment of either GH- or PRL-secreting adenomas.

Adenoma↗
PubMed1997

Somatostatin receptor subtype specificity in human fetal pituitary cultures. Differential role of SSTR2 and SSTR5 for growth hormone, thyroid-stimulating hormone, and prolactin regulation.

Somatostatin (SRIF), a hypothalamic inhibitor of pituitary growth hormone (GH) and thyroid-stimulating hormone (TSH) secretion, binds to five distinct receptor (SSTR) subtypes. We therefore tested SSTR subtype-specific SRIF analogs in primary human fetal pituitary cultures (23-25-wk gestation) to elucidate their role in regulating human pituitary function. Using reverse transcription-PCR, mRNA expression of SSTR2 and SSTR5 were detected in fetal pituitary by 25 wk. SRIF analog affinities were determined by membrane radioligand binding in cells stably expressing the human SSTR forms. GH secretion was suppressed equally (40-60%, P < 0.005) by analogs preferential for either SSTR2 (IC50 for receptor binding affinity, 0.19-0.42 nM) or SSTR5 (IC50, 0.37 nM), and compounds with enhanced affinity for SSTR2 were more potent (EC50 for GH suppression, 0.05-0.09 nM) than Lanreotide (EC50, 2.30 nM) and SRIF (EC50, 0.19 nM). Similarly, analogs with high affinity for SSTR2 or SSTR5 decreased TSH secretion (30-40%, P < 0.005). However, prolactin was effectively inhibited only by compounds preferentially bound to SSTR2 (20-30%, P < 0.05). Luteinizing hormone was modestly decreased (15-20%) by SSTR2- or SSTR5-specific analogs. An SSTR5-specific analog also exclusively inhibited GH in acromegalic tumor cells. Thus, SRIF regulation of GH and TSH in primary human fetal pituitary cells is mediated by both SSTR2 and SSTR5, both of which are abundantly expressed by 25 wk. In contrast, suppression of prolactin is mediated mainly by SSTR2. These results indicate that SSTR5 is critical for physiologic regulation of GH and TSH. SRIF analogs with selective affinity for this receptor may therefore be more effective in the treatment of hormone-secreting pituitary adenomas.

Adrenocorticotropic Hormone↗
PubMed1997

Characterization of bombesin binding sites in the rat stomach.

We characterized the bombesin receptor population in the rat stomach and determined the receptor subtype mediating the contractile effect of bombesin in the gastric fundus. Using in vitro receptor autoradiography, we evaluated the ability of the specific gastrin-releasing peptide-preferring receptor antagonist [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester to inhibit binding of 125I-[Tyr4]bombesin to the gastric fundus, corpus and antrum. Binding to these regions was completely inhibited by [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester suggesting that these receptors are the gastrin-releasing peptide-preferring subtype. We found that the rank order of potency for the contractile effect of bombesin, and the related mammalian peptides neuromedin C and neuromedin B, was bombesin > neuromedin C > neuromedin B. [D-F5,Phe6,D-Ala11]bombesin-(6-13) methyl ester was equipotent in antagonizing contractions produced by all three peptides. Furthermore, receptor tachyphylaxis to either neuromedin C or neuromedin B abolished the subsequent contractile response elicited by neuromedin C and neuromedin B, suggesting that one bombesin receptor subtype mediates rat gastric fundal contractions. Together, these results demonstrate that the bombesin receptor subtype in the rat stomach is gastrin-releasing peptide-preferring subtype and that this subtype is responsible for the effects of bombesin-like peptides on fundal smooth muscle contraction.

Animals↗
PubMed1997

Caudal hindbrain neuromedin B-preferring receptors participate in the control of food intake.

Recent studies have identified two subtypes of bombesin (BN) receptors in the rat central nervous system: gastrin releasing-peptide (GRP) preferring and neuromedin B (NMB) preferring. To investigate a role for the NMB-preferring receptor subtype in feeding suppression elicited by fourth ventricular (4V) BN administration, we evaluated the ability of a selective NMB-preferring receptor antagonist, BIM-23127, to block suppression of glucose intake produced by 4V BN (10 pmol). Our results showed that 4V administration of BIM-23127 dose dependently antagonized the suppression of glucose intake produced by 4V BN. In addition, 4V administration of BIM-23127 alone increased glucose intake above that observed in the baseline condition. These results support a role for the NMB-preferring BN receptor subtype in the suppression of intake produced by 4V BN administration and suggest that endogenously released NMB participates in ingestive control.

Animals↗
PubMed1996

Pharmacological profiles of two bombesin analogues in cells transfected with human neuromedin B receptors.

We examined the effect of two des-Met-bombesin analogues, [(CH3)2CHCO-His-Trp-Ala-Val-D-Ala-His-Leu-NHCH3] (ICI 216140) and [D-Phe6,des-Met14]bombesin(6-14) ethylamide (DPDM-bombesin ethylamide), on neuromedin B-induced Ca2+ and [3H]arachidonate release in BALB 3T3 cells transfected with human neuromedin B receptors. ICI 216140 and DPDM-bombesin ethylamide both stimulated Ca2+ mobilisation in a concentration-dependent manner but were less potent and efficacious than neuromedin B. BIM 23042 [D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH2], a selective neuromedin B antagonist and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P, a broad-spectrum peptide receptor antagonist inhibited neuromedin B-, ICI 216140 and DPDM-bombesin ethylamide-induced Ca2+ release. Pretreatment of cells with either des-Met-bombesin analogue attenuated neuromedin B-induced Ca2+ elevations, suggesting similar agonist-sensitive Ca2+ pools. The pharmacological profiles revealed from the [3H]arachidonate assay were similar, although ICI 216140 was less potent and efficacious than DPDM-bombesin ethylamide. The data suggest that ICI 216140 and DPDM-bombesin ethylamide behave as agonists at the neuromedin B receptor, perhaps as a consequence of neuromedin B receptor overexpression.

3T3 Cells↗
PubMed1996

Somatostatin receptor-binding peptides labeled with technetium-99m: chemistry and initial biological studies.

The synthesis of peptides which possess a high affinity for the somatostatin receptor and contain a chelator for the radionuclide technetium-99m is described. The target compounds were designed such that they would form stable, oxotechnetium(V) chelate complexes in which the Oxorhenium(V) chelate complexes of these peptides were prepared as nonradioactive surrogates for the technetium complexes. Peptide oxorhenium complexes and Tc-99m complexes eluted closely upon HPLC analysis. The receptor-binding affinities of both the free and rhenium-coordinated species were measured in vitro. The binding affinities of the free peptides (Ki's in the 0.25 - 10 nM range) compared favorably with [DTPA]octreotide (Ki = 1.6 nM), which, as the indium-111 complex, is already approved for somatostatin receptor (SSTR)-expressing tumor imaging in the United States and Europe. Furthermore, the rhenium-coordinated peptides had binding affinities which, in many cases, were higher than those of the corresponding free peptides, with several complexes having a Ki's of 0.1 nM. Some of the more potent SSTR-binding peptides were labeled with technetium-99m and assessed in an in vivo study with tumor-bearing rats. The 99m Tc-labeled peptides prepared in this study should be useful as SSTR-expressing tumor-imaging agents due to their high SSTR-binding affinities, ease of preparation, and, because they are low molecular weight peptides, expected pharmacokinetics characterized by rapid tracer excretion from the body resulting in high-contrast images.

Amino Acid Sequence↗
PubMed1996

There are three distinct forms of bombesin. Identification of [Leu13]bombesin, [Phe13]bombesin, and [Ser3,Arg10,Phe13]bombesin in the frog Bombina orientalis.

Amphibian bombesin is the prototypic peptide that defines the bombesin-like peptide family. In this paper we show that in the frog Bombina orientalis, there are actually 3 distinct forms of bombesin, and each of these peptides is an agonist with differing affinities for the known bombesin receptors. Oligonucleotides complementary to the 5'- and 3'-untranslated regions of the bombesin mRNA were used to amplify bombesin-related cDNAs from the skin, brain, and gut of B. orientalis. Three classes of cDNAs were found. One class encoded the previously characterized form of bombesin which has a Leu at position 13 ([Leu13]bombesin). The other two classes, respectively, encoded new bombesin-like peptides which we have designated as [Phe13]bombesin and [Ser3,Arg10,Phe13]bombesin ([SAP]bombesin). The existence of [SAP]bombesin in skin was confirmed by tandem mass spectrometry. Polymerase chain reaction analysis of genomic DNA showed the mRNAs for [Leu13]bombesin, [Phe13]bombesin, and [SAP]bombesin most likely arise from separate genes. Polymerase chain reaction analysis showed different patterns of tissue-specific expression for each form. [Leu13]Bombesin and [SAP]bombesin were predominantly expressed in skin, brain, and gut; [Phe13]bombesin was expressed only in brain, and [Leu13]bombesin predominated in oocytes. [SAP]Bombesin contained a cleavage site between residues 4 and 5, which if used would yield the peptide [SAP]bombesin(5-14) which has the sequence [Gln3,Arg6]neuromedin B. Thus a frog homolog of NMB could derive from the [SAP]bombesin prohormone. [Phe13]Bombesin, [SAP]bombesin, and [SAP]bombesin(5-14) were synthesized and their affinities for the mammalian bombesin-like peptide (GRP and NMB) receptors determined. These peptides acted as agonists for the GRP and NMB receptors, with relative potencies for the GRP receptor of [Leu13]bombesin > [Phe13]bombesin > [SAP]bombesin(5-14) > [SAP]bombesin and for the NMB receptor of [Phe13]bombesin > [SAP]bombesin(5-14) > [Leu13]bombesin > [SAP]bombesin. None of these peptides demonstrated high affinity binding for the BRS-3 receptor. The different receptor affinities and tissue distribution of these peptides suggests distinct physiologic roles and raises the possibility of as yet uncharacterized mammalian homologs of these new amphibian peptides.

Amino Acid Sequence↗
PubMed1996

Receptor-specific somatostatin analogs: correlations with biological activity.

A number of cyclic and linear somatostatin (SRIF) analogs have now been found to have promising levels of selectivity for rodent somatostatin receptors (rsst2,3,5), but not sst1 and sst4. Comparisons between binding affinities for these and transfected human receptors are just beginning to emerge and we present results from a comparison of affinities of several key families of peptides for sst2 present on rat AR42J cells and on cells transfected with human (h)sst2. The typical cyclic octapeptide analogs, octreotide, lanreotide, and RC-160, exhibited similar affinities to SRIF for rsst2, but somewhat lower affinities for the human receptor. Affinities of several analogs for transfected hsst5 were also measured. As with the rat receptor, octreotide-related analogs had low affinity for hsst5. The highly specific rsst5 analog, DC-23-99, was less so for the human receptor; however, a D-Tyr1 version of DC-23-99 had subnanomolar affinity (Ki, 0.68 nmol/L) and high selectivity. A new extended-ring analog, BIM-23268D, showed superior affinity to DC-23-99 and even to SRIF and SRIF-28 for hsst5 (K(i), 0.38 nmol/L), and had the highest sst5/sst2 selectivity ratio of any analog that we have tested thus far.

Amino Acid Sequence↗
PubMed1996

Improved analogs and novel delivery systems for somatostatin octapeptides.

Appropriate N-terminus modification can result in somatostatin (SRIF) octapeptide analogs that are both more potent and more selective in vitro for the human SRIF receptor type 2 (hsst2). In addition, these modifications can improve the duration of action and bioavailability of SRIF analogs following parenteral administration, as shown by both pharmacological and distribution studies in vivo with BIM-23190 and BIM-23197 in the rat.

Amino Acid Sequence↗
PubMed1996

Synthesis, structure, and antagonistic properties of des-Asn29[D-Trp28,32]NPY(27-36).

We have previously reported that [D-Trp32]NPY and its centrally truncated analogues such as des-AA7-24[D-Trp5,32,Aoc6]NPY can competitively antagonize NPY effects on rat hypothalamus and Y1 (SK-N-MC AND HEL) cells, respectively. In continuation of this work, we performed structure-activity studies with C-terminal decapeptide sequence keeping D-Trp at position 32 to develop lower molecular weight Y1-selective antagonists. This study led to the development of des-Asn29[D-Trp28,32]NPY(27-36), which bound to both Y1 (SK-N-MC, Ki > or = 10 microM) and Y2 (SK-N-BE2, Ki = 1.01 +/- 0.03 microM) receptors. This peptide did not exhibit any agonist activity at Y1 receptors, and exhibited comparable potencies in antagonizing the effects of NPY on the synthesis of cAMP and mobilization of [Ca2+]i in HEL cells. However, in SK-N-MC cells, it was more potent in antagonizing the mobilization of [Ca2+]i than inhibition of cAMP synthesis. Substitution of Nva for Gln34 to increase the hydrophobicity without altering the carbon skeleton substantially increased Y1 affinity (Ki = 0.33 +/- 0.15 microM) and imparted Y1 selectivity (Ki for Y2 affinity = 3.16 +/- 0.50). Moreover, this peptide exhibited good antagonistic potency in HEL cells. 2D NMR studies of des-Asn29[D-Trp28,32]NPY(27-36) revealed the existence of a fairly stable loop-like structure between residues 27 and 32 and a less stable one between residues 32 and 36. The increased Y1 affinity of des-Asn29[D-Trp28,32,Nva34]NPY(27-36) may be due to the stabilization of the 32-36 loop by Nva34. It appears therefore that stabilization of the loop structures in these peptides should result in the development of more potent Y1 receptor antagonists. Our investigations also suggest that HEL cells express a homogeneous population of NPY Y1 receptors whereas SK-N-MC cells express high- and low-affinity Y1 receptors coupled to Ca2+ and cAMP, respectively.

Animals↗
PubMed1996

Real-time evaluation of somatostatin subtype 2 receptor activity employing the technique of cytosensor microphysiometry.

Five types of somatostatin (SS) receptors (sst1-5) have been cloned and are widely distributed in the central nervous system and variably expressed in target tissues of the periphery. At the cellular level, adenylate cyclase inhibition has been classically described in native and transfected cells expressing sst subtypes. In addition, ion channel modulation (K+, Ca2+), phospholipase C, phospholipase A2, and tyrosine phosphatase activation have also been reported. The present study describes a novel in vitro approach based on quantifying receptor-activated metabolic rate changes to evaluate SS biological activity in cells (CHO-K1) stably expressing the human (h) sst2 receptors. Real-time metabolic rate changes were evaluated by determining the rate of extracellular acidification (microphysiometry). The metabolic rate was transiently and potently (EC50 1 nM) increased in response to natural SS ligands, SS-14 and SS-28. The peak activation time was approximately 2 min. Pharmacological analysis for the sst2 receptor yielded rank order of potency for SS analogues of: MK-678 > BIM-23027 > octreotide > BIM-23014C << L-362,855 > BIM-23052 << BIM-23056. Similar rank orders were obtained from in vitro receptor binding studies in the same cell line. These results demonstrate that microphysiometry is a rapid and valid technique to evaluate the pharmacology SS receptor activation.

Animals↗
PubMed1996

Design of a laser scanner for a digital mammography system.

We have developed a digital readout system for radiographic images using a scanning laser beam. In this system, electrostatic charge images on amorphous selenium (alpha-Se) plates are read out using photo-induced discharge (PID). We discuss the design requirements of a laser scanner for the PID system and describe its construction from commercially available components. The principles demonstrated can be adapted to a variety of digital imaging systems.

Diagnosis, Computer-Assisted↗
PubMed1996

Hindbrain GRP receptor blockade antagonizes feeding suppression by peripherally administered GRP.

Bombesin (BN)-like peptides injected peripherally or centrally suppress food intake in rats. The relationship between the central and peripheral actions of BN is unknown. However, experimental evidence supports a critical role for the caudal hindbrain in mediating the feeding effects of BN. To investigate this relationship further, we examined the ability of fourth ventricular infusion of a specific gastrin-releasing peptide (GRP) antagonist, [D-F5, Phe6, D-Ala11]BN-(6-13) methyl ester (BN-ME), to block suppression of glucose intake (0.5 kcal/ml) produced by intraperitoneal administration of GRP-(18-27) in 5-h food-deprived male Sprague-Dawley rats (n = 10). We found that fourth ventricular administration of 10, 32, and 100 ng BN-ME blocked the suppression of glucose intake produced by peripherally administered 10 nmol/kg GRP-(18-27). The most effective dose of BN-ME (100 ng) blocked the ability of peripheral injection of GRP-(18-27) to inhibit glucose intake but had no effect on intake when given alone. These results demonstrate that the availability of caudal hindbrain GRP receptors is necessary for peripherally administered GRP-(18-27) to reduce food intake in rats.

Animals↗