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Biomedical subjects

J F Smyth

Publications and source records attributed to J F Smyth.

At least 73 records · Page 4Linked to original sources
PubMed1995

Characterization of the deamidase enzyme responsible for the metabolism of the anticancer peptide: H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH2.

H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH2, a broad spectrum neuropeptide growth factor antagonist (antagonist G), is soon to enter a phase I clinical trial for the treatment of small-cell lung cancer (SCLC). The pre-clinical pharmacology of this peptide has revealed that its metabolism proceeds from the C-terminus via deamidation. In this study the class of enzyme responsible for the degradation of antagonist G has been characterized. Tissue distribution studies on the enzyme have shown it to be very widespread with high specific activity being detected in the spleen, kidney, H69 SCLC xenograft and liver (12.64, 9.58, 8.00 and 6.94 nmols G/mg protein/hr, respectively). HPLC gel filtration indicated that the G-deamidase enzyme had an apparent molecular mass of 81 kDa. The sub-cellular distribution of the enzyme using differential centrifugation indicates that it is largely soluble with > 85% of the activity located in the cytosolic fraction. The distribution of activity towards antagonist G closely resembles that of esterase and acid carboxypeptidase activity, two activities, along with deamidase activity, known to be possessed by serine carboxypeptidases. Studies using a range of protease inhibitors showed clear inhibition of metabolism by phenylmethylsulphonylfluoride and benzyloxycarbonylphenylalanine chloromethylketone, indicating that the enzyme is a chymotrypsin-like serine carboxypeptidase. This knowledge of the enzyme will be invaluable in the further development of antagonist G and similar compounds. Moreover, the widespread distribution of this enzyme together with its broad specificity for C-terminal group suggests that it should be given serious consideration when designing C-terminally modified peptide drugs.

Amino Acid Sequence↗
PubMed1995

Biochemistry of topoisomerase I and II inhibition by anthracenyl-amino acid conjugates.

Mono-conjugation of an anthraquinone nucleus with a range of naturally occurring amino acids chemically modified at their C-terminus has been adopted as a synthetic approach in the rational design of novel topoisomerase (topo) inhibitors. The biochemistry of topo I and II inhibition has been investigated for a series of 16 new compounds (NU/ICRF 500-515) from which structure-activity relationships have been investigated. Only three compounds could be demonstrated to bind to DNA: two serine derivatives (NU/ICRFs 500 and 506) and an arginine derivative (NU/ICRF 510). In decatenation and relaxation assays with purified enzyme, several compounds were shown to be potent catalytic inhibitors of topo II (100% inhibition at 5 micrograms/mL (10-15 microM) or less) without stabilizing cleavable complex formation. These included the three DNA binding species (of which NU/ICRF 506 was the most active) and a dihydroxyphenylalanine analogue (NU/ICRF 513). Both NU/ICRFs 500 and 506 were further shown to antagonize DNA cleavage induced by amsacrine. Only NU/ICRF 506 unequivocally inhibited the catalytic activity of topo I without induction of DNA cleavage, and was the only combined topo I and II catalytic inhibitor. One compound, NU/ICRF 505 (tyrosine conjugate), stabilized topo I cleavable complexes without inhibiting the catalytic activity of topo I and II. Modifications to the structure of NU/ICRF 505 revealed that the presence of an unhindered hydroxyl on the tyrosine ring and a more hydrophobic ethyl ester at the amino acid C-terminal were both essential, suggesting a highly specific interaction between drug, enzyme and DNA in the ternary complex. Molecular modelling studies suggested that the observed differences in topo inhibition are a consequence of major conformational alterations brought about by small changes in the amino acid substituent, and confirmed a rigid structural requirement for the induction of topo I cleavage, in addition to a less rigid structural requirement for topo II inhibition. A strong correlation was observed between topo inhibition and in vitro cytotoxicity against the human ovarian cancer cell line A2780 (IC50 range 3.4-11.6 microM), suggesting a mechanism of cell kill, at least in part, involving topo inhibition.

Amino Acids↗
PubMed1995

Processing of the neuropeptide growth factor antagonist [Arg6, D-Trp7.9, NmePhe8]-substance P (6-11) by a small cell lung cancer cell line (H69).

[Arg6, D-Trp7.9, NmePhe8]-substance P (6-11) (antagonist G) is a broad spectrum neuropeptide growth factor antagonist about to enter clinical trials as an anticancer drug. Its fate has been studied after incubation with two densities (5 x 10(4) cells/mL and 1 x 10(6) cells/mL) of the H69 small cell lung cancer cell line for up to 7 days at a concentration of 20 microM, corresponding to the IC50 for growth inhibition. HPLC analyses were conducted on cell pellets and media and in controls consisting of cell free media and water. Over 7 days in media containing cells a 70.4% reduction in parent peptide concentration occurred at the high density and a 44.1% reduction at low density. Despite this, there was a steady elevation in peptide associated with cells reaching a 189% increase by day 7. Oxidation of G at the C-terminal methionine residue occurred in all media studied indicative of a chemical process. The two major active metabolites of antagonist G (deamidated G and G minus Met11) were detected only in media in the presence of cells. These accumulated with time in media and cells together with oxidized products. These results reveal complex cellular pharmacology for antagonist G where H69 cells are increasingly exposed to 4 different peptide products rather than 1.

Antineoplastic Agents↗
PubMed1995

Accumulation of anthracenyl-amino acid topoisomerase I and II inhibitors in drug-sensitive and drug-resistant human ovarian cancer cell lines determined by high-performance liquid chromatography.

Anthracenyl amino acid/dipeptide conjugates (AADC) represent novel structures rationally designed for their DNA-binding properties. A high-performance liquid chromatography method is described for simultaneous determination of five compounds that exhibit novel mechanisms of action as topoisomerase I and II inhibitors. The method uses an Apex ODS-2 column and a mobile phase of 0.25 M ammonium acetate/trifluoroacetic acid (pH 3) in methanol with gradient elution. Selective detection is achieved by monitoring at 545 nm, with limits of detection ranging between 2 and 4 ng on the column. AADC are recovered from cell sonicates by solid-phase extraction using C2 cartridges, with extraction efficiencies ranging from 84% to 95%. Drug uptake studies were performed with three active compounds in the human ovarian cancer cell line A2780 and its multi-drug-resistant counterpart 2780AD. Marked differences were observed in the pattern of cellular accumulation produced by each compound. NU/ICRF 505 (tyrosine derivative) was taken up most avidly, reaching plateau levels of 4000 pmol/10(6) cells after 2 h, with no difference being apparent between A2780 and 2780AD. NU/ICRF 510 (arginine derivative) accumulated slowly in A2780, failing to achieve an equilibrium after 4 h, and appeared to be completely excluded from 2780AD. NU/ICRF 500 (serine derivative) was most rapidly taken up by A2780, producing a plateau of 800 pmol/10(6) cells after only 30 min with approximately 3-fold less accumulation in 2780AD. These results are correlated to the chemosensitivity of the two cell lines to the three compounds.

Amino Acids↗
PubMed1995

Conversion of mitomycin C to 2,7-diaminomitosene and 10-decarbamoyl 2,7-diaminomitosene in tumour tissue in vivo.

The progress of mitomycin C (MMC) bioreduction was studied in vivo in the rat Sp 107 mammary carcinoma after intra-tumoural injection of either 100 micrograms or 1 mg. 2,7-Diaminomitosene (2,7-DM) was utilised as a primary bioreductive metabolite and 10-decarbamoyl 2,7-diaminomitosene (DC 2,7-DM) served as a secondary bioreductive metabolite, both of which were measured by high-performance liquid chromatography. 2,7-DM and DC 2,7-DM were produced rapidly, achieving close to their maximal concentrations at the earliest time point studied [5 min]. 2,7-DM was cleared rapidly from the tumour with apparent half-lives of 5 and 35 min after the low and high drug doses, respectively. DC 2,7-DM had a longer apparent half-life of 130 min at the higher dose but, as compared with 2,7-DM, was only a minor metabolite [the area under the curve (AUC) of 2,7-DM was 5.6-fold that of DC 2,7-DM]. At the lower drug dose, DC 2,7-DM was not detectable. Rapid formation and disappearance of bioreductive metabolites of MMC may account for the failure of previous studies to detect these products in vivo.

Animals↗
PubMed1995

Cytogenetic evaluation of the mechanism of cell death induced by the novel anthracenyl-amino acid topoisomerase II catalytic inhibitor NU/ICRF 500.

Anthracenyl-amino acid/dipeptides are novel topoisomerase (topo) inhibitors which can be actively cytotoxic in the low microM range. The present studies have been performed to determine whether cells treated with the topo II catalytic inhibitor NU/ICRF 500 (serine derivative) would manifest cytogenetic lesions consistent with its proposed mechanism of enzyme inhibition. Three other compounds were included for comparison: NU/ICRF 505 (tyrosine) which stabilises topo I cleavable complexes, NU/ICRF 602 (gly-gly) a non-cytotoxic catalytic inhibitor of topo I and II and NU/ICRF 502 (alanine) a non-cytotoxic non-topo inhibitor. Chromosomal damage was measured using the micronucleus test. NU/ICRF 500 (7.5-30 microM) induced an increase in CREST negative micronuclei (11-15 per 500 cells) in human lymphocytes (HL) and blocked the traverse of HL through the cell cycle, with cells accumulating in G2/M at 15 microM drug and G1/S at 30 microM drug. NU/ICRF 502 was without effect in the micronucleus test. NU/ICRF 500 and 602 (90-150 microM) caused no block in passage of synchronised metaphase Chinese hamster ovary cells through mitosis whereas NU/ICRF 505 produced a significant delay. DNA measurements of post-mitotic cells revealed that after NU/ICRF 500 treatment nuclei had a 4C DNA content, indicative of a lack of chromosomal segregation. Normal (2C) DNA content was observed with NU/ICRF 505 and 602. Overall, the data for NU/ICRF 500 are consistent with the cytogenetic modifications expected after catalytic inhibition of topo II and suggest that cell death may be mediated, at least in part, through this mechanism.

Animals↗
PubMed1995

Metabolism of the anticancer peptide H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH2.

H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH2 (Antagonist G) will be the first broad-spectrum neuropeptide antagonist to enter a phase I clinical trial. Its in vitro and in vivo metabolism has been extensively characterized. The major metabolites were identified and their structures elucidated by mass spectroscopy and amino acid analysis. Metabolism occurred almost exclusively at the C-terminus and was arrested by phenylmethylsulfonylfluoride, a known serine-protease inhibitor. Biological characterization of the metabolites demonstrated that the degradation of Antagonist G produces metabolites that retain neuropeptide antagonist properties.

3T3 Cells↗
PubMed1995

Chromosome 11 allele imbalance and clinicopathological correlates in ovarian tumours.

Allele imbalance on chromosome 11 loci in ovarian cancer is a frequent event, suggesting the presence of tumour-suppressor genes for ovarian carcinogenesis on this chromosome. Ten highly polymorphic (CA) repeat microsatellites were used to determine allele imbalance in 60 primary ovarian tumours, including 47 epithelial ovarian cancers (EOCs). Forty EOCs (85%) showed allele imbalance at one or more loci, and in 39 of these (83%) the data suggested subchromosomal deletions: eight of 11p only; six of 11q only; and 25 of both 11p and 11q. Three consensus regions of deletion were indicated at 11p15.5-p15.3, 11q12-q22 and 11q23.3-q24.1. Allele imbalance at the 11q subtelomeric region (D11S912) correlated significantly with adverse survival, while imbalance at 11q14.3 and retention of heterozygosity at 11q22 (close to the site of the progesterone receptor gene) were associated with favourable clinicopathological features. The findings allow development of a preliminary model for the molecular evolution of epithelial ovarian cancer.

Adenocarcinoma↗
PubMed1995

Pharmacokinetics, metabolism, tissue and tumour distribution of the neuropeptide growth factor antagonist [Arg6, D-Trp7,9, NmePhe8]- substance P(6-11) in nude mice bearing the H69 small-cell lung cancer xenograft.

BACKGROUND: [Arg6, D-Trp7,9, NmePhe8]-Substance P (6-11) (codenamed antagonist G) represents the first board spectrum antagonist of a number of neuropeptides shown to act as growth factors in small-cell lung cancer (SCLC) and is shortly to enter clinical trials. DESIGN: Pharmacokinetics, metabolism, tissue disposition have been studied in mice (nu/nu) bearing the NCI-H69 human SCLC xenograft after systemic drug adminstration at an active dose (45 mg/kg i.p.). RESULTS: The peptide exhibited relatively long half life (28.9 min; clearance 45.6 ml/min/kg) and distributed widely (volume of distribution 1490 ml/kg). Marked accumulation of antagonist G (and its metabolites) was noted in the liver (AUC5278 micrograms/g x min) and to a lesser extent the spleen (AUC 930 micrograms/g x min) but only low levels appeared to cross the blood brain barrier (AUC in brain, 20 micrograms/g x min) or be taken up into the heart (AUC 101 micrograms/g x min). Tumour uptake was intermediate in value out of the 7 tissues studied (AUC 195 micrograms/g x min). Metabolism was restricted almost exclusively to the C terminal of the peptide producing 4 major products: M1, deamidated antagonist G; M2, Harg-DTrp-NmePhe-DTrp-Leu-OH, both of which retain growth factor antagonist activity; M3, a combination of oxidised antagonist G [Met11(O)] and oxidised deamidated antagoinst G; and M4, a combination of H-Arg-DTrp-NmePhe-DTrp-OH and H-DTrp-NmePhe-DTrp-Leu-OH. Extensive biotransformation to predominately M1 and M2 occurred in most tissues including the tumour where the parent peptide accounted for only 48.5% of the total. CONCLUSION: Levels of antagonist G required to produce a small but significant effect on the growth of SCLC cell lines in vitro are in the region of 4-7 microM. Taking into account metabolites, a peak concentration of 4.1 microgram/g (4.3 microM) was achieved in the H69 xenograft. These studies reveal a favourable preclinical pharmacology profile for antagonist G and offer hope that anticancer activity may be achievable in man.

Animals↗
PubMed1995

Effect of human recombinant interferon-alpha on the activity of cis-diamminedichloroplatinum(II) in human non-small cell lung cancer xenografts.

Interferons (IFNs) augment the effect of some antitumor agents, including cis-diamminedichloroplatinum(II) (cDDP), in experimental systems. The effect of human recombinant interferon-alpha 2b (rIFN alpha) on the cDDP-dependent growth delay of a human non-small cell lung cancer established as a xenograft in nude mice (NX002) has been investigated. IFN (10(5) IU/mouse, s.c.) as a single agent had no effect on the growth of the xenograft. cDDP (4.2 mg/kg, i.p.) caused a specific growth delay of 0.42, and this delay was significantly enhanced (to 1.08) by concomitant dosing with the otherwise inactive IFN. Possible mechanisms for this supra-additive relationship between IFN and cDDP have been investigated: increased intratumoral accumulation of platinum was seen at late time points (maximally at 36 hr) during the pharmacokinetic beta-phase of cDDP elimination from the plasma of the nude mice. Tumor:plasma platinum concentration ratios at 36-48 hr indicated significantly increased accumulation of platinum in tumors from IFN-treated mice compared to controls (p < 0.05). Scheduling experiments suggest that this IFN-mediated effect can persist for 4 hr. These differences may account for the enhanced antitumor activity of cDDP when coadministered with IFN.

Animals↗
PubMed1995

Identification of anthracenyl-dipeptide conjugates as novel topoisomerase I and II inhibitors and their evaluation as potential anticancer drugs.

As part of an ongoing rational drug design programme aiming to develop monosubstituted anthracenyl-peptides as potential anticancer drugs, three novel dipeptide conjugates have been synthesized and evaluated as inhibitors of topoisomerase (topo) I and II. Each of the three conjugates (designated NU/ICRF 600-602) was shown to inhibit the catalytic activity of both topoisomerase I and II, of which NU/ICRF 602 was the most active [100% inhibition of both enzymes at 5 micrograms/ml (approximately 15 microM) or less]. In a topo I/DNA unwinding assay, none of the compounds bound to DNA, suggesting genuine inhibition of catalytic activity. NU/ICRF 600 stabilized topo I cleavable complexes, although none of the compounds induced topo II-mediated DNA cleavage. Using a panel of Chinese hamster ovary cell lines along with the human ovarian cancer cell line, A2780, none of the three compounds were actively cytotoxic at concentrations < 100 microM. Subsequent drug uptake studies with NU/ICRF 600 and 602, using a method developed to correlate the chemosensitivity of A2780 cells with the uptake of anthracenyl-amino acid conjugates, revealed a lack of cellular uptake for both dipeptide conjugates. The significance of this finding in relation to drug design and the future development of this series of compounds is discussed.

Animals↗
PubMed1995

Expression of the heat shock protein HSP27 in human ovarian cancer.

The relationship of the heat shock protein HSP27 in ovarian cancer to several biological and clinical parameters was investigated in a series of primary tumors and cell lines. Analysis of 72 primary tumors (54 malignant, 5 borderline, and 13 benign neoplasms) indicated that malignant tumors expressed higher HSP27 concentrations than benign tumors (median values, 0.56 versus 0.25 ng/microgram cytosolic protein; P = 0.032). Tumors from patients with advanced stage (stages II, III, or IV) disease contained significantly higher HSP27 concentrations than tumors from stage I patients (P = 0.018), and an HSP27 content >2.0 ng/microgram cytosolic protein was associated with reduced survival (P = 0.03). Tumors that had demonstrated progressive growth after chemotherapy had a significantly higher HSP27 content than tumors that were static or responsive (P = 0.022). These data indicate that HSP27 is associated with more aggressive malignant ovarian disease and with inherent resistance to chemotherapy. Concentrations of HSP27 were also correlated with indicators of estrogen sensitivity. Therefore, the HSP27 concentration correlated with the estrogen receptor (all tumors, P = 0.0014; malignant tumors only, P = 0.047) but not with the progesterone receptor concentration. Analysis of ovarian cancer cell lines in vitro and in vivo indicated that the HSP27 content was higher in cell lines that were estrogen receptor rich and whose growth was modulated by estrogen as compared with those that were not. Additionally, two estrogen receptor-rich ovarian carcinoma lines demonstrated a small but significant decrease in HSP27 levels in response to 17beta-estradiol in culture. These results suggest that HSP27 may help identify tumors responsive to estrogens.

Animals↗
PubMed1995

Loss of heterozygosity at 11q22 correlates with low progesterone receptor content in epithelial ovarian cancer.

Forty-seven epithelial ovarian cancers were analyzed for loss of heterozygosity (LOH) at D11S35 (11q22), close to the progesterone receptor (PR) gene, and for tumoral estrogen receptor (ER) and PR content. Thirty-eight of 47 tumors were informative, and, of these, 14 exhibited LOH. There was a significant association (P = 0.014) between D11S35 LOH and low tumoral PR content. For all informative tumors, there was no correlation between ER and PR; however, exclusion of tumors with LOH from the informative series revealed a linear correlation between tumoral ER and PR (P = 0.013), and established ER (P = 0.025) and PR (P = 0.05) content as significant factors in relation to patient survival. Patients with ER-rich tumors with D11S35 LOH had particularly poor survival compared with ER-rich, D11S35 heterozygous, no loss patients (P = 0.014). Analysis of the same tumors using two other microsatellites, D11S935 (11p13) and NM23 (17q22), showed no statistically significant relationships, although there were nonsignificant trends for the correlation of ER and PR expression in informative tumors without allele loss at these loci. We propose that genomic structural alteration at or close to the PR gene locus has biological and clinical sequelae in ovarian cancer.

Chromosomes, Human, Pair 11↗
PubMed1994

Conducting clinical research in the new NHS: the model of cancer. United Kingdom Coordinating Committee on Cancer Research.

The United Kingdom Coordinating Committee on Cancer Research represents the major organizations funding cancer research in the United Kingdom. The deliberations of a working party convened by the committee to evaluate recently expressed concerns that the changes in the NHS threaten research, especially clinical trials to evaluate new treatments, are reported. A survey of contributors to trials coordinated by the committee showed that half are now experiencing difficulties in continuing to participate in clinical trials. The two major problems identified were lack of time and of staff, especially for NHS staff in non-teaching hospitals. Recent changes in junior doctors' hours and proposed reductions in the length of time for training will exacerbate this. It is possible to identify the direct and indirect excess costs of conducting research in the NHS, but currently the mechanism does not exist to designate funds specifically for this purpose. Consultation with the regional directors of research and development confirmed that the service increment for teaching and research is not the solution for this. Proposals are made to secure future clinical research in the NHS, including finance, indemnity, the licensing of new drugs, the greater use of nurse counsellors, and the value of cancer registries.

Clinical Trials as Topic↗
PubMed1994

Encapsulation of mitomycin C in albumin microspheres markedly alters pharmacokinetics, drug quinone reduction in tumour tissue and antitumour activity. Implications for the drugs' in vivo mechanism of action.

Pharmacokinetics and metabolism of mitomycin C (MMC) have been studied in NMRI mice bearing MAC 16 colon adenocarcinoma after direct intratumoural injection of either 500 micrograms free MMC or the same dose incorporated in albumin microspheres. Microspheres produced a tumour pharmacokinetic profile of steady state drug levels, avoiding the much higher early peak (20.5 micrograms/tumour vs 98.9 micrograms/tumour) and lower trough of free MMC, and reducing significantly the levels of drug reaching the systemic circulation (AUC 1.8 micrograms/mL x hr for microspheres vs 6.8 micrograms/mL x hr for free drug). 2,7-Diaminomitosene (2,7-DM), a key intermediate in MMC quinone bioreduction, was used as an indicator of drug metabolic activation in tumour tissue. Peak levels were 10-fold higher (11.2 micrograms/tumour vs 1.1 micrograms/tumour) and area under the curve 5-fold higher after free drug. Even taking into account differences in tumour pharmacokinetic profiles of the parent drug, microspheres actively inhibited 2,7-DM formation 3-fold. However, the microspheres generated a completely different pattern of drug metabolism where four previously uncharacterized mitosane metabolites and elevated levels of cis and trans 1-hydroxy 2,7-diaminomitosene were detected. Despite similar parent drug exposure in tumours, free drug was significantly more active (P < 0.05, Student's t-test) against MAC 16. These results suggest that formation of 2,7-DM correlates more closely with antitumour activity than sustained parent drug levels or appearance of other key metabolites. Potentially, they provide the first direct evidence for an in vivo mechanism of action dependent on bioreductive activation and formation of 2,7-DM.

Adenocarcinoma↗