A comparison of the pharmacokinetics and safety of enzalutamide in subjects with hepatic impairment and matched healthy subjects.

WHAT IS KNOWN AND OBJECTIVE: Enzalutamide is an androgen receptor inhibitor approved for treatment of metastatic castration-resistant prostate cancer. Enzalutamide is highly protein bound and eliminated primarily by hepatic metabolism; therefore, it is important to understand whether enzalutamide pharmacokinetics is altered by hepatic impairment. METHODS: Pharmacokinetic data were obtained from two non-randomized, open-label, single-dose, phase 1 studies conducted in patients with mild (Child-Pugh class A, n = 6) or moderate (Child-Pugh class B, n = 8) hepatic impairment (NCT01901133) or severe (Child-Pugh class C, n = 8) hepatic impairment (NCT02138162) and their corresponding matched healthy controls; data from both studies are presented here. Subjects with hepatic impairment had liver cirrhosis (n = 19) or chronic hepatitis (n = 3). All subjects received a single oral dose of 160 mg enzalutamide under fasting conditions, with blood samples collected predose and up to 49 days post-dose. RESULTS AND DISCUSSION: Exposure to enzalutamide active moieties, based on the area under the curve of the sum of enzalutamide and N-desmethyl enzalutamide (an active metabolite with similar potency to enzalutamide), increased by 13%, 18% and 4% in subjects with mild, moderate and severe hepatic impairment, respectively, relative to matched controls. Compared with healthy controls, the mean maximum plasma concentration for enzalutamide active moieties was 24% higher in subjects with mild hepatic impairment and 11% and 41% lower in subjects with moderate and severe hepatic impairment, respectively. Enzalutamide was generally well tolerated, with no clinically significant trends in abnormal laboratory findings, vital signs or electrocardiograms. WHAT IS NEW AND CONCLUSIONS: No major differences in single-dose pharmacokinetics were observed in subjects with hepatic impairment vs. matched healthy controls. Therefore, these studies indicate that no initial dose adjustment is necessary when administering enzalutamide to patients with hepatic impairment.

Cyclin D1 overexpression in a model of human breast premalignancy: preferential stimulation of anchorage-independent but not anchorage-dependent growth is associated with increased cdk2 activity.

Cyclin D1 is frequently overexpressed in human breast ductal carcinoma in situ (DCIS) specimens, which confer a high risk for the development of infiltrating ductal carcinoma. If causally involved in the genesis of human breast malignancy, cyclin D1 may represent an interesting target for chemopreventive approaches, as it sits at the junction of many growth factor and hormonal pathways. We have used the MCF-10A human breast cell line, derived from a mastectomy containing a low risk premalignant lesion, as a model system. Three cyclin D1 transfectants exhibited physiologically relevant levels of transgene overexpression, but no coordinate overexpression of other cell cycle related genes. Proliferation assays, flow cytometry, and cdk enzymatic assays of anchorage-dependent proliferation indicated only a minimal and transient effect of cyclin D1. In contrast, cyclin D1 overexpression significantly stimulated anchorage-independent colonization in soft agar or methylcellulose, accompanied by greater Gl-S progression. The cdk4 activity of the control- and cyclin D1 transfectants in colonization assays was comparable, but the cdk2 activity was higher in the latter. Injection of control- and cyclin D1 transfected MCF-10A cells in matrigel into nude mice failed to produce tumors within 1.5 years. The data suggest that cyclin D1 overexpression is an early feature of breast neoplastic progression, and can contribute to cancer development through the promotion of colonization.

Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific.

Thyroid hormone (tri-iodo-thyronine, T3) exerts transcriptional effects on target genes in responsive cells. These effects are determined by DNA/protein interactions governed by the type of T3 receptors (TRs) in the cell. As TRs show tissue and developmental variations, regulation is best addressed in an integrated in vivo model. We examined TR subtype effects on thyrotropin-releasing hormone (TRH) transcription and on the pituitary/thyroid axis end point: thyroid hormone secretion. Polyethylenimine served to transfect a TRH-luciferase construct containing 554 bp of the rat TRH promoter into the hypothalami of newborn mice. Transcription from the TRH promoter was regulated in a physiologically faithful manner, being significantly increased in hypothyroidism and decreased in T3-treated animals. Moreover, when various ligand binding forms of mouse or chicken TRbeta and TRalpha were expressed with TRH-luciferase, all forms of TRbeta gave T3-dependent regulation of TRH transcription, whereas transcription was T3 insensitive with each TRalpha tested. Moreover, chicken TRalpha increased TRH transcription sixfold, whereas mouse TRalpha decreased transcription. These transcriptional effects had correlated physiological consequences: expression of the chicken TRalpha in the hypothalamus of newborn mice raised circulating T4 levels by fourfold, whereas mouse TRalpha had opposite effects. Thus, TR subtypes have distinct, physiologically relevant effects on TRH transcription.

Sequence variations in the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene and their relationship with pyrimethamine resistance.

The gene encoding dihydrofolate reductase-thymidylate synthase of the human malaria parasite, Plasmodium vivax, was isolated by polymerase chain reaction from genomic DNA and cloned. The sequences of the dihydrofolate reductase domain of 30 clinical isolates originating from various geographic areas were compared. Interstrain analysis revealed several genotypic variations, including short tandem repeat arrays which produced length polymorphism between different parasite isolates and point mutations in the putative dihydrofolate reductase active site cavity corresponding to those associated with pyrimethamine resistance in P. falciparum and rodent malaria parasites. Amino acid substitutions Ser-->Asn-117 and Ser-->Arg-58 were associated with decreased level of in vitro pyrimethamine sensitivity. These findings suggest that the P. vivax dihydrofolate reductase domain is characterized by polymorphism that has not been observed in P. falciparum and may explain the resistance of some P. vivax isolates to pyrimethamine. Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB databases under the accession numbers X98123 (isolate ARI/Pakistan), AJ003050 (isolate CNC/Thailand), AJ003051 (isolate COU/unknown geographic origin), AJ003052 (isolate DUF/French Guiana), AJ003053 (isolate GRO/Madagascar), AJ003054 (isolate HRT/Comoros Islands), AJ003071 (isolate LFT/Cambodia), AJ003072 (isolate LGF/'India), AJ003073 (isolate MAN/Comoros Islands), AJ003074 (isolate MAT/Surinam), AJ003075 (isolate PHI/Djibouti), AJ003076 (isolate PIT/Madagascar), AJ003077 (isolate YTZ/Indonesia), AJ222630 (isolate Burma-1), AJ222631 (isolate Burma-151), AJ222632 (isolate Burma-5), AJ222633 (isolate Burma-6), AJ222634 (isolate Burma-98).

Analysis of the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene sequence.

A basis for the intrinsic resistance of some Plasmodium vivax isolates to pyrimethamine is suggested following the isolation of the bifunctional gene encoding dihydrofolate reductase-thymidylate synthase (DHFR-TS) of this human malaria parasite. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to an inhibition of DNA replication. The gene was isolated by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotides designed to hybridize on the highly conserved regions of the sequence. The nucleotide sequence was completed by screening P. vivax genomic bank. Sequence analysis revealed an open reading frame (ORF) of 1872 nucleotides encoding a deduced protein of 623 amino acids (aa). Alignment with other malarial DHFR-TS genes showed that a 237-residue DHFR domain and a 286-residue TS domain were separated by a 100-aa linker region. Comparison with other malarial species showed low and essentially no isology in the DHFR and junctional domains, respectively, whereas an extensive isology was observed in the TS domain. The characteristic features of the P. vivax DHFR-TS gene sequence include an insertion of a short repetitive tandem array within the DHFR domain that is absent in another human malaria parasite, P. falciparum, and a GC-biased aa composition, giving rise to highly GC-rich DHFR (50.8%), junctional (58.7%), and TS (40.5%) domains, as compared with other malaria parasites. Analysis of the 5' noncoding region revealed the presence of a putative TATA box at 116 nucleotides upstream of the ATG start codon as well as a putative GC box at -636. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed two residue changes: Ser Arg-58 and Ser Asn-117. These aa residues correspond to codons 59 and 108 in the P. falciparum DHFR active site in which similar aa substitutions (Cys Arg-59 and Ser Asn-108) are associated with pyrimethamine resistance. These findings may explain the intrinsic resistance of some P. vivax isolates to pyrimethamine.

Differential expression of nucleoside diphosphate kinases (NDPK/NM23) during Xenopus early development.

In Xenopus laevis, three nucleoside diphosphate kinase (NDPK) monomers have been described (NDPK X1, X2 and X3) (Ouatas et al., 1997). In eucaryotes, this kinase is known as a hetero- or homohexamer. Here, we examine the distribution of the enzyme and its different subunit mRNAs during oogenesis and early embryogenesis of Xenopus laevis, respectively by immunohistofluorescence and whole-mount in situ hybridization. These analyses show that NDPKs and their mRNAs are differentially distributed throughout the oocyte and early embryos with a high level of transcription in somites and brain. We emphasize two points. First, each mRNA displays a distinct subcellular localization in somites, suggesting a complex regulation of NDPK genes both at the transcriptional and translational level and a possible involvement of NDPK X2 homohexamers in the dorsal muscle differentiation. Second, in oocytes and early embryos, the proteins are mainly localized in the nucleus, suggesting a new mechanism for their nuclear import, since they do not possess any known nuclear import sequences.

T3-dependent physiological regulation of transcription in the Xenopus tadpole brain studied by polyethylenimine based in vivo gene transfer.

The formulation of cationic polymers of polyethylenimine (PEI) with plasmid DNA has been optimized to deliver genes into the Xenopus tadpole brain in vivo. Using intraventricular microinjections of 1 microl (containing 0.5 to 1 microg DNA) we show that the linear, low molecular weight polymer, 22 kDa PEI was significantly more efficient than a branched 25 kDa polymer. Complexes bearing a slightly positive net charge (formed with a ratio of 6 PEI amines per DNA phosphate) provided the best levels of transfection. Transgene expression was DNA-dose dependent and was maintained over 6 days, the time course of the experiment. Spatial distribution was examined using a beta-galactosidase construct and neurones expressing this transgene were found spread throughout the brain. The possibility of using this technique to evaluate physiological regulations was approached by examining the effects of tri-iodothyronine (T3), on transcription from the mammalian TRH and Krox-24 promoter sequences. Adding physiological concentrations of T3 to the aquarium water significantly reduced transcription from the rat TRH promoter whilst the same treatment increased transcription from a mouse Krox-24 -luciferase construct. Thus, PEI-DNA transfection provides a versatile and easily applied method for following physiological regulations at the transcriptional level in the tadpole brain.

Three different genes encode NM23/nucleoside diphosphate kinases in Xenopus laevis.

Nucleoside diphosphate kinases (NDPKs) catalyse the phosphorylation of nucleoside diphosphates. In mammals, the functional enzyme is a hexamer composed of different amounts of two homologous acidic (A) and basic (B) subunits encoded by separate genes. In prokaryotes and invertebrate eukaryotes, only one cytoplasmic enzyme has been isolated. Other genes encoding chloroplastic and mitochondrial forms as well as related proteins have been cloned. Here, we show that in Xenopus laevis, as in mammals, the cytoplasmic NDPK is encoded by several homologous genes. With Xenopus laevis being a pseudotetraploid species, each monomer is encoded by two genes. The amino acid sequences are very similar, and all the differences concern amino acids located at the outer surface of the hexameric enzyme. The Xenopus genes share 82-87% identity with their human counterparts. Interestingly, in vitro, the Xenopus X1 enzyme binds to a specific nuclease hypersensitive element (NHE) of the human c-myc promoter, as does its human counterpart. X1 also binds to a single-stranded (CT)(n) dinucleotide repeat. The NHE is present in the coding strand of a pyrimidine-rich region of the 3' non-coding sequence of the Xenopus NDPK genes. We propose that NDPK is indeed able to bind to its own mRNA and prevent polyadenylation at the normal position. This could provide an autoregulatory translation mechanism. A phylogenetic tree of the vertebrate NDPK sequences supports the idea that in amphibians, as in mammals, gene duplication has resulted in functional diversification.

[Retrospective evaluation of the detection of Toxoplasma gondii by polymerase chain reaction in AIDS patients]. / Evaluation rétrospective de la détection de Toxoplasma gondii par réaction de polymérisation en chaîne chez des patients sidéens.

OBJECTIVES: We retrospectively evaluated routine detection of Toxoplasma gondii by polymerase chain reaction (PCR) amplification of the B1 gene and the TGR 1E sequence in blood and CSF samples from patients with acquired immune deficiency syndrome (AIDS) and suspected toxoplasmosis. METHODS: From January 1993 to February 1994, 93 blood, 33 cerebrospinal fluid (CSF) and 1 aqueous humor samples were obtained from 83 HIV-positive patients with CD4 counts under 200/mm3 and suspected toxoplasmosis. RESULTS: Authentic cerebral toxoplasmosis was confirmed by response to specific treatment in 18/29 patients with typical focal brain lesions. Blood samples were available in 17/18 and CSF in 6. PCR was positive for both B1 and TGR (23.5% sensitivity, 100% specificity) in 4/17 blood samples and for either B1 or TGF (58.9% sensitivity, 72.7% specificity) in 10/18. PCR of CSF was positive in only 1/6 patients with cerebral toxoplasmosis. PCR (TGR 1E only) was positive in 3/11 blood samples and in one CSF sample from patients without cerebral toxoplasmosis. Five out of 21 patients with diffuse neurologic symptoms and presumed HIV encephalitis had positive Toxoplasma detection in blood or CSF. However, no clinical improvement was obtained after specific antitoxoplasmic therapy. Two out of 38 patients with unexplained fever with or without pneumonia had a proven disseminated toxoplasmosis. These two patients had PCR-positive blood samples. One of them was cured by a specific anti-toxoplasmic treatment and the other died two days later. Seven other patients had a positive PCR result in blood or CSF. Three of them improved with specific treatment and 2 died before treatment could be initiated. The one patient with toxoplasmic retinitis was PCR-positive both in blood and aqueous humor. CONCLUSION: PCR detection of Toxoplasma gondii appears to add little to the diagnosis of cerebral toxoplasmosis but could be helpful in the diagnosis of cerebral toxoplasmosis associated with extracerebral reactivation and in disseminated toxoplasmosis.