Human bioavailability of propranolol from a matrix-in-cylinder system with a HPMC-Gelucire core.

The bioavailability of propranolol from a matrix-in-cylinder system for sustained drug delivery, consisting of a hot-melt extruded ethylcellulose pipe surrounding a drug-containing HPMC-Gelucire 44/14 core, was determined. An oral dose of 80 mg propranolol hydrochloride was administered to healthy volunteers (n = 10) in a randomized cross-over study design either as a commercial pellet formulation (Inderal retard mitis) or as a matrix-in-cylinder system. The influence of concomitant food intake on drug release from the matrix-in-cylinder system was also studied. During the first 10 h after administration, the matrix-in-cylinder system resulted in similar plasma levels as the reference formulation Inderal. The concomitant intake of a high-fat, high-calorie breakfast did not cause dose-dumping. Between 10 h and 24 h after administration of the matrix-in-cylinder system, a remarkable increase of the propranolol plasma levels was noticed (compared to Inderal). This effect was even more pronounced under fed conditions. The matrix-in-cylinder system had a relative bioavailability of 156% (fasted conditions) and 222% (fed conditions) compared to the marketed reference product. In order to elucidate the origin of this increased bioavailability, Caco-2 experiments and dog lymph studies were performed. However, none of these experiments was able to provide a conclusive answer.

Multidrug resistance-associated protein 2 (MRP2) affects hepatobiliary elimination but not the intestinal disposition of tenofovir disoproxil fumarate and its metabolites.

The role of multidrug resistance-associated protein 2 (MRP2) on the intestinal disposition and hepatobiliary elimination of tenofovir disoproxil fumarate (DF) and its metabolites [tenofovir (mono)ester and tenofovir] was studied in the Caco-2 system, Ussing chambers and rat in-situ efflux experiments. In the Caco-2 model and Ussing chambers, no statistically significant differences in transport could be observed when the MRP inhibitor probenecid was included. In Ussing chambers, transport was also similar when using intestinal tissue from MRP2-deficient rats. After intravenous administration of tenofovir DF, the excretion of tenofovir [(mono)ester] in bile was significantly decreased in MRP2-deficient rats and in rats treated with probenecid. The area under the blood concentration-time curve was increased in MRP2-deficient rats [1.0+/-0.1 and 0.36+/-0.03 microM.min-1 for tenofovir and tenofovir (mono)ester, respectively] and rats treated with probenecid (1.42+/-0.04 and 0.36+/-0.02 microM.min-1) compared with control rats (0.64+/-0.05 and 0.15+/-0.06 microM.min-1). The appearance of tenofovir [(mono)ester] in intestinal perfusate was similar in control rats upon co-administering probenecid or when using MRP2-deficient rats. In conclusion, MRP2 appeared to have no modulatory effect on the intestinal disposition of tenofovir and tenofovir (mono)ester. However, inhibition (probenecid) or the total absence of MRP2 (MRP2-deficient rats) significantly reduced hepatobiliary elimination, which was accompanied by increased systemic exposure.

Involvement of a palindromic chromosome 22-specific low-copy repeat in a constitutional t(X; 22)(q27;q11).

Segmental duplications or low-copy repeats (LCRs) on chromosome 22q11 have been implicated in several chromosomal rearrangements. The presence of AT-rich regions in these duplications may lead to the formation of hairpin structures, which facilitate chromosomal rearrangement. Here we report the involvement of such a low-copy repeat in a t(X;22) associated with a neural tube defect. Molecular analysis of the chromosomal breakpoints revealed that the chromosome 22 breakpoint maps in the palindromic non-AT-rich NF1-like region of low-copy repeat B (LCR-B). No palindromic region was encountered near the breakpoint on chromosome X. Our findings confirm that there is no single mechanism leading to translocations with chromosome 22q11 involvement. Because LCR-B does not contain genes involved in neural tube development, we believe that the gene responsible for the observed phenotype is most likely localized on chromosome X.

First experience with an automatic sensing algorithm in single-lead VDD stimulation.

UNLABELLED: An "Autosensing" algorithm available in SSI(R) and DDR(R) pacemakers automatically adapts the device's sensitivity to changing intracardiac signals. The atrial sensing function of this algorithm was tested for the first time with a VDD pacing system in which large variations of the atrial signal may occur because the atrial electrodes float in the atrial blood pool. METHODS: 15 patients with a VDD pacing system were studied (Unity 292-07, lead 425; Sulzer Intermedics). The atrial sensing threshold was measured, and the atrial sensitivity was programmed with a 2:1 safety margin. The autosensing algorithm and sensitivity profile were temporarily activated, and an ambulatory ECG with continuous marker annotation was recorded. All patients underwent a 30-minute daily life activities protocol. A beat-to-beat analysis of the ambulatory ECG was correlated with the changes in atrial sensitivity. RESULTS: The algorithm changed the baseline sensitivity from 0.57 +/- 0.23 mV during the test to 0.39 +/- 0.20 mV after the final rest period (P < 0.05). During the test 12.6 +/- 10.2 adaptations of the sensitivity occurred (range 0-33). In eight patients atrial undersensing occurred in 4.4% +/- 7.5% of the cycles (4-458 unsensed P waves). In these patients, the algorithm continuously adjusted the sensitivity towards more sensitive values, operating 19.1 +/- 18.3 changes compared with 5.4 +/- 7.3 changes in patients without undersensing (P = 0.009). Oversensing did not occur. CONCLUSION: The autosensing algorithm effectively optimized atrial sensitivity in VDD pacing. In patients with atrial undersensing the algorithm continuously remained near the most sensitive settings, thus reacting as intended. A faster sensitivity adjustment of the system would be desirable.

LPP, the preferred fusion partner gene of HMGIC in lipomas, is a novel member of the LIM protein gene family.

A major cytogenetic subgroup of lipomas is characterized by recurrent chromosome aberrations, mainly translocations, that involve chromosome segment 12q13-q15. Multiple chromosomes have been found as the translocation partners of chromosome 12 but 3q27-q28 is preferentially involved. In previous studies, it has been shown that the high mobility group (HMG) protein gene HMGIC at 12q15 is consistently rearranged as a consequence of these translocations. Here, we report the identification and characterization of the chromosome 3-derived translocation partner gene, which we have designated LPP (lipoma preferred partner gene). Using 3'-RACE analysis of HMGIC fusion transcripts in lipoma cell line Li-501/SV40, ectopic genetic sequences were obtained, which by CASH (chromosome assignment using somatic cell hybrids) and FISH (fluorescence in situ hybridization) analysis were found to originate from chromosome segment 3q27-q28. In Northern blot analysis, an mRNA of over 10 kb was detected by these ectopic sequences in a variety of human tissues but not in brain and peripheral blood leukocytes. Upon partial cDNA cloning, features of the genetic organization of LPP were established. The gene was found to span a genomic region of over 400 kb. Nucleotide sequence analysis of a composite cDNA of LPP revealed an open reading frame of 1836 nucleotides encoding a proline-rich protein containing a leucine-zipper motif in its amino-terminal region and three LIM domains in its carboxy-terminal region. The LPP-encoded protein should be classified as a novel member of the group 3 proteins of the LIM protein gene family. Using reverse transcriptase combined with polymerase chain reactions in the analysis of a number of lipoma cell lines and primary lipomas, it appeared that LPP is frequently rearranged also in cases without a cytogenetically detectable involvement of 3q27-q28. Two alternative HMGIC/LPP hybrid transcripts have been detected; the difference between them is mainly the presence of either two or three LIM domains in the predicted HMGI-C/LPP fusion proteins.

A 6-Mb yeast artificial chromosome contig and long-range physical map encompassing the region on chromosome 12q15 frequently rearranged in a variety of benign solid tumors.

Cytogenetic analysis of a variety of benign solid tumors, among which uterine leiomyoma, lipoma, pleomorphic salivary gland adenoma, and pulmonary chondroid hamartoma, has indicated that these tumors often display chromosome breakpoints in region q13-q15 of chromosome 12. In previous studies, we have reported that these breakpoints map between locus D12S8 and the CHOP gene, the latter of which has been shown to be consistently rearranged in myxoid liposarcomas with t(12;16)(q13;p11). Here, we report directional chromosome walking studies starting from D12S8 and resulting in the construction of a YAC contig of about 6 Mb. This YAC contig, whose orientation on chromosome 12 was determined by double-color fluorescence in situ hybridization (FISH) analysis, has at least double coverage and consists of 75 overlapping YAC clones, all isolated from CEPH YAC libraries. Their insert sizes were estimated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomal localization and chimerism of the YACs were investigated by FISH analysis. Chimerism of YAC clones was independently determined by restriction mapping. On the basis of YAC end-derived DNA markers and sequence-tagged sites (STSs), with an average spacing of approximately 70 kb, as well as restriction enzyme analysis, a long-range physical map was established for the 6-Mb DNA region of chromosome 12 covered by the YAC contig. Within the YAC contig, the relative positions of various known genes, an expressed sequence-tagged site, and a number of CEPH/Généthon polymorphic markers were determined. The latter data allow full integration of our mapping data with those obtained by CEPH/Généthon as well as those reported at the Second International Workshop on Human Chromosome 12 Mapping. Finally, this YAC contig constitutes the basis for the contstruction of a transcriptional map of this region and is likely to facilitate identification of genes involved in the formation of various benign solid tumor types.

Recurrent rearrangements in the high mobility group protein gene, HMGI-C, in benign mesenchymal tumours.

We recently showed that the 1.7 megabase multiple aberration region (MAR) on human chromosome 12q15 harbours recurrent breakpoints frequently found in a variety of benign solid tumours. We now report a candidate gene within MAR suspected to be of pathogenetical relevance. Using positional cloning, we have identified the high mobility group protein gene HMGI-C within a 175 kilobase segment of MAR and characterized its genomic organization. By FISH analysis, we show the majority of the breakpoints of eight different benign solid tumour types fall within this gene. By Southern blot and 3'-RACE analysis, we demonstrate consistent rearrangements in HMGI-C and/or expression of altered HMGI-C transcripts. These results suggest a link between a member of the HMG gene family and benign solid tumour development.

Identification of the chromosome 12 translocation breakpoint region of a pleomorphic salivary gland adenoma with t(1;12)(p22;q15) as the sole cytogenetic abnormality.

Cell line Ad-312/SV40, which was derived from a primary pleomorphic salivary gland adenoma with t(1;12)(p22;q15), was used in fluorescence in situ hybridization (FISH) analysis to characterize its translocation breakpoint region on chromosome 12. Results of previous studies have indicated that the chromosome 12 breakpoint in Ad-312/SV40 is located proximally to locus D12S8 and distally to the CHOP gene. We here describe two partially overlapping yeast artificial chromosome (YAC) clones, Y4854 (500 kbp) and Y9091 (460 kbp), which we isolated in the context of a chromosome walking project with D12S8 and CHOP as starting points. We present a composite long-range restriction map encompassing the inserts of these two YAC clones and show by FISH analysis that both YACs span the chromosome 12 breakpoint as present in Ad-312/SV40 cells. Subsequently, we have isolated cosmid clones corresponding to various sequence-tagged sites (STSs) mapping within the inserts of these YAC clones. These included cRM51, cRM69, cRM85, cRM90, cRM91, cRM110, and cRM111. In FISH studies, cosmid clones cRM85, cRM90, and cRM111 appeared to map distally to the chromosome 12 breakpoint, whereas cosmid clones cRM51, cRM69, cRM91, and cRM110 were found to map proximally to it. These results assign the chromosome 12 breakpoint in Ad-312/SV40 to a DNA region of less than 165 kbp. FISH evaluation of the chromosome 12 breakpoints in five other pleomorphic salivary gland adenoma cell lines indicated that these are located proximally to the one in Ad-312/SV40, at a distance of more than 0.9 Mbp from STS RM91. These results, while pinpointing a potentially critical region on chromosome 12, also provide evidence for the possible involvement of 12q13-q15 sequences located elsewhere.

Identification, molecular cloning, and characterization of the chromosome 12 breakpoint cluster region of uterine leiomyomas.

Recent molecular cytogenetic analysis of uterine leiomyoma cell lines with chromosome 12 aberrations has indicated that their chromosome 12 breakpoints map between linkage locus D12S8 and the CHOP gene. Here, we report fluorescence in situ hybridization (FISH) and molecular cloning studies of the chromosome 12 breakpoints in a panel of seven such uterine leiomyoma cell lines; five with the frequently observed t(12;14)(q15;q24), one with t(12;15)(q15;q24), and one with ins(12;11)(q14;q21qter). Directional chromosome walking studies starting from D12S8 and the CHOP gene resulted in the isolation of a relatively large number of overlapping YAC clones, including Y5355 (465 kbp), Y7673 (360 kbp), and Y9899 (275 kbp). In total, the inserts of these three YAC clones span an 800 kbp long and presumably contiguous stretch of human genomic DNA. All chromosome 12 breakpoints of the uterine leiomyoma cell lines studied were found by FISH analysis to be mapping within a 445 kbp subfragment of this region and, furthermore, to be dispersed over a DNA region which is at least 150 kbp in size. The chromosome 12 breakpoint of t(12;14)(q15;q24) in uterine leiomyoma cell line LM-30.1/SV40 was tentatively mapped within the 60 kbp region between YAC clones Y9899 and Y5355. From this 60 kbp region and close to sequence-tagged site RM99, we isolated probe pRM118-A, which showed in Southern blot analysis that it detected a rearrangement in LM-30.1/SV40 DNA, and generated restriction maps of the normal and rearranged genomic DNA regions detected with this probe. Finally, we molecularly cloned part of one of those rearranged DNA fragments using a vectorette-PCR-based technique and demonstrated that it consisted of 12q13-q15 sequences fused to DNA sequences derived from 14q23-24 and most likely represented the translocation junction on der(14) in LM-30.1/SV40 cells. Our studies strongly suggest that we have identified and isolated the uterine leiomyoma cluster region of chromosome 12 breakpoints, which we designate ULCR12, and molecularly cloned and characterized the der(14) translocation junction in cells derived from a uterine leiomyoma carrying the frequently observed t(12;14)(q15;q24).

Physical mapping of chromosome 12q breakpoints in lipoma, pleomorphic salivary gland adenoma, uterine leiomyoma, and myxoid liposarcoma.

We report here the physical mapping of recurrent chromosome 12q13-q15 breakpoints in cell lines derived from primary myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands. In fluorescence in situ hybridization (FISH) experiments, we first mapped the position of the chromosome 12 translocation breakpoint in uterine leiomyoma cell line LM-30.1/SV40 relative to loci COL2A1, D12S4, D12S17, D12S6, D12S6, D12S19, D12S8, and D12S7. It mapped between linkage probes CRI-C86 (D12S19) and p7G11 (D12S8). We then isolated YAC clones using CRI-C86- and p7G11-derived sequence-tagged sites, constructed corresponding YAC contigs of 310 and 800 kb, respectively, and established long-range physical maps of these. Cosmid clones LLNL12NCO1-98C10 and LLNL12NCO1-113D12 were isolated using STSs within the CRI-C86- and the p7G11-derived YAC contigs, respectively, and a mixture of them was used to routinely study the various tumor cell lines by FISH analysis. The chromosome 12 breakpoints of all tumor cell lines tested mapped between cosmids LLNL12NCO1-98C10 and LLNL12NCO1-113D12. None of the breakpoints appeared to map within any of the isolated YAC clones. Furthermore, FISH analysis using cosmid LLNL12-NCO1-144G3, which maps at the CHOP locus, revealed that the chromosome 12 breakpoints in all cell lines of the three benign solid tumors that were tested were located distal to the chromosome 12 translocation breakpoint with the CHOP gene in myxoid liposarcoma cells with t(12;16). In conclusion, our studies seem to indicate that the chromosome 12 breakpoints of myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands are all clustered within the 7-cM interval between D12S19 and D12S8, with those of the benign solid tumors distal to CHOP. Finally, the MYF5 gene mapped telomeric to LLNL12NCO1-113D12, and the MIP gene mapped centromeric to the chromosome 12 translocation breakpoint in myxoid liposarcoma cells.