Dose-finding study of weekly docetaxel, epirubicin and capecitabine, as first-line treatment in advanced breast cancer.

BACKGROUND: Combinations of anthracycline, taxane and fluoropyrimidine are highly active in advanced breast cancer (ABC). In a phase II study of epirubicin 50 mg/m(2), docetaxel 75 mg/m(2), and infusional 5-FU 200 mg/m(2)/day, we found dose-limiting neutropenia and frequent central venous catheter complications. An alternative approach has been tested using weekly fractionation of docetaxel, and oral capecitabine. METHODS: Initially, six women with ABC were treated with epirubicin 60 mg/m(2) day 1, docetaxel 25 mg/m(2) days 1,8,15, and capecitabine 1,000 mg/m(2) twice daily days 1-14, every 21 days. Six further patients received the above with capecitabine escalated to 1,500 mg/m(2) RESULTS: Four DLTs occurred in six patients at the second dose level (febrile neutropenia in 2). There were frequent dose delays/reductions, and fatigue, nausea/vomiting, and diarrhoea were common. Overall, six of ten assessable patients achieved a partial response. CONCLUSIONS: An active regimen, but significant haematological toxicity precluded dose further escalation.

Benzodiazepine site pharmacokinetic/pharmacodynamic quantification in man: direct measurement of drug occupancy and effects on the human brain in vivo.

To date, the study of the relationship between drug occupancy and action in the brain has had to rely on the use of either animal models or of indirect kinetic measures in man, e.g. serum concentrations of unbound drug (as a measure of "free" drug in brain). We describe the first set of experiments which directly measure agonist-induced changes in both pharmacodynamic effects and pharmacokinetic parameters simultaneously and which demonstrate the feasibility of these studies in man. Five healthy volunteers each had two PET scans using [11C]flumazenil (a radiolabelled benzodiazepine site antagonist) as part of a study investigating kinetic models and the relationship between occupancy and effect of benzodiazepine site ligands. In both studies the [11C]flumazenil was displaced from the brain by infusion of midazolam administered i.v. 30 min into the scan. In one study a higher dose of midazolam was administered than in the other (range 12.5-50 micrograms/kg). Time-activity curves of the concentration of radioligand were derived in 17 different brain regions using a stereotactic automatic method of region selection. We demonstrated that there are significant differences in an index of occupancy, induced by the two different doses of midazolam, both across brain regions and within subjects. There was a significant correlation between measured occupancy index change and pharmacodynamic effects as measured by the peak change in beta 1 spectral power on EEG. There was no significant correlation between dose administered and EEG changes; plasma concentrations of midazolam were correlated with the occupancy index and with the EEG measures. In addition, we have demonstrated that a non-regional total index of brain occupancy can be obtained by analysing the non-tomographic data obtained with the PET scanner (total radioactivity counts head curve) and that this index shows significant correlations both with the dose administered and with the pharmacodynamic measure. This last finding validates the use of other non-tomographic counting techniques (Malizia et al., 1995a) where an index of displacement can be obtained after the administration of less than 1% of the dose of radiation needed for a PET study. These studies are likely to be useful in human psychopharmacology, in particular in the assessment of tolerance and of putative changes in benzodiazepine sensitivity in anxiety disorders. The same principles can be applied to other ligand studies and will be useful to validate current PK/PD models.

Multiple TCR V beta usage by infiltrates of young NOD mouse islets of Langerhans. A polymerase chain reaction analysis.

Because a restricted repertoire of T-cell receptor (TCR) V beta gene expression has been reported in other autoimmune diseases, the possibility of similarly restricted V beta gene expression by T-cell infiltrates of NOD mouse islets was examined. With isolated islets from 4- to 12-wk-old NOD mice, a prospective polymerase chain reaction analysis with 18 V beta-specific oligonucleotide primers was performed on the noncloned and unexpanded islet-infiltrating T cells. The methodology used permitted the detection of a minimum of 50 T cells. In contrast to the restricted TCR V beta gene usage reported for other autoimmune diseases, infiltrates of even the youngest mice were characterized by expression of multiple V beta gene segments.

A B-cell-specific nuclear protein that binds to DNA sites 5' to immunoglobulin S alpha tandem repeats is regulated during differentiation.

Immunoglobulin heavy-chain switching is effected by recombination events between sites associated with tandemly repeated switch sequences located 5' to immunoglobulin heavy-chain genes. Using the band mobility shift assay, we have identified two distinct sites 5' to the alpha heavy-chain switch sequence with affinity for a single B-cell-specific DNA-binding protein, S alpha-BP. S alpha-BP was present in nuclear extracts from pre-B and B cells but was not detected in extracts from plasmacytomas, B-cell hybridomas, T-cell lymphomas, or a macrophage cell line. It was also not detectable in other nonlymphoid cells tested. Evidence suggests there are S alpha-BP-binding sites near other immunoglobulin switch sequences. As with the S alpha sites, these sites appear to be distinct from the consensus tandem repeats characteristic of immunoglobulin switch sequences. The possible functions of S alpha-BP on contacting its binding sites are discussed in the context of immunoglobulin heavy-chain switch recombination.

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.

Evidence for haploid expression of mouse testicular genes.

Hybridization of RNA blots of total testicular RNA from prepuberal and sexually mature CD1 mice with several mouse testicular cDNA probes reveals that the mRNA encoding the two mouse protamines, an actin sequence of 1.5 kb, and a post-meiotically expressed 620 nucleotide mRNA are first detected in the testes of mice 22 days of age. These experiments and other studies analysing RNA preparations from isolated populations of testicular cell types with cDNA probes [1, 2] demonstrate that haploid gene expression occurs in the mammalian testis.

Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.

The tetracycline resistance determinants of RP1 and Tn1721: nucleotide sequence analysis.

Nucleotide sequences of the homologous tetracycline resistance (tet) determinants of plasmid RP1 and transposon Tn1721 have been determined. Two open reading frames of divergent polarity have been assigned to a regulatory gene (tetR) and a gene encoding a resistance protein (tetA). The intercistronic region contains appropriate regulatory and transcription signals. The tetR gene can code for a protein of 216 amino acids (deduced mol.wt. 23,288) and the tetA gene for a protein of 399 amino acids (deduced mol. wt. 42,205). Based on the deduced amino acid sequence, the tetA proteins of RP1/Tn1721 are 78% homologous with that of pBR322 and 45% homologous with that of Tn10. We conclude that a single tetA gene mediates resistance in each of these tet determinants.