Tumorigenesis in mice with a fusion of the leukaemia oncogene Mll and the bacterial lacZ gene.

Many different chromosomal translocations occur in man at chromosome 11q23 in acute leukaemias. Molecular analyses revealed that the MLL gene (also called ALL-1, HRX or HTRX) is broken by the translocations, causing fusion with genes from other chromosomes. The diversity of MLL fusion partners poses a dilemma about the function of the fusion proteins in tumour development. The consequence of MLL truncation and fusion has been analysed by joining exon 8 of Mll with the bacterial lacZ gene using homologous recombination in mouse embryonic stem cells. We show that this fusion is sufficient to cause embryonic stem cell-derived acute leukaemias in chimeric mice, and these tumours occur with long latency compared with those found in MLL-Af9 chimeric mice. These findings indicate that an MLL fusion protein can contribute to tumorigenesis, even if the fusion partner has no known pathogenic role. Thus, truncation and fusion of MLL can be sufficient for tumorigenesis, regardless of the fusion partner.

Does the site of intestinal delivery of oleic acid alter the ileal brake response?

Previous work has demonstrated that high doses of oleic acid can activate the ileal brake but the importance of site of delivery has yet to be investigated. The objective of this study was to use modified release capsules to release oleic acid in different regions of the intestine. When tested by in vitro dissolution in pH 6.8 phosphate buffer, one batch released the contents almost immediately, another after around 30 min and the last batch after around 60-70 min. The effect of oleic acid release site on the ileal brake was assessed by the measurement of transit time of radiolabelled non disintegrating tablets by gamma scintigraphy. The results demonstrated that the transit of tablets could be slowed down by oleic acid and therefore it appears the ileal brake can be activated along the entirety of the small intestine.

The mll-AF9 gene fusion in mice controls myeloproliferation and specifies acute myeloid leukaemogenesis.

The MLL gene from human chromosome 11q23 is involved in >30 different chromosomal translocations resulting in a plethora of different MLL fusion proteins. Each of these tends to associate with a specific leukaemia type, for example, MLL-AF9 is found mainly in acute myeloid leukaemia. We have studied the role of the Mll-AF9 gene fusion made in mouse embryonic stem cells by an homologous recombination knock-in. Acute leukaemias developed in heterozygous mice carrying this fusion as well as in chimeric mice. As with human chromosomal translocation t(9;11), the majority of cases were acute myeloid leukaemias (AMLs) involving immature myeloblasts, but a minority were acute lymphoblastic leukaemia. The AMLs were preceded by effects on haematopoietic differentiation involving a myeloproliferation resulting in accumulation of Mac-1/Gr-1 double-positive mature myeloid cells in bone marrow as early as 6 days after birth. Therefore, non-malignant expansion of myeloid precursors is the first stage of Mll-AF9-mediated leukaemia followed by accumulation of malignant cells in bone marrow and other tissues. Thus, the late onset of overt tumours suggests that secondary tumorigenic mutations are necessary for malignancy associated with MLL-AF9 gene fusion and that myeloproliferation provides the pool of cells in which such events can occur.

The effect of oleic acid on the human ileal brake and its implications for small intestinal transit of tablet formulations.

PURPOSE: A human volunteer study was carried out to investigate whether activation of the ileal brake mechanism affects the transit of tablets through the small intestine. METHODS: Oleic acid, which has previously been shown to activate the brake, was delivered to the small intestine in a modified release capsule at doses of 300 mg, 600 mg and 1200 mg. The effect of the oleic acid was determined by measuring the transit of two sets of radiolabelled tablets by gamma scintigraphy. One set of tablets was dosed with the capsule and the other one hour later. RESULTS: The results show that in the majority of the volunteers small intestinal residence time was greater with the oleic acid than control. The effect was most pronounced in the tablets given concomitantly with the capsule and with the higher doses of oleic acid. CONCLUSIONS: The ileal brake, activated by oleic acid, can slow the transit of tablets through the small intestine.

Is the pig a good animal model for studying the human ileal brake?

This study was designed to investigate the existence of an ileal brake mechanism in the pig model. The test substances used (oleic acid, deoxycholic acid, taurocholic acid) had all been previously shown to affect the ileal brake mechanism in other species including man. The substances were infused directly into the terminal ileum of surgically modified pigs, 45 min after the pigs had ingested a meal containing a drug marker. The marker used was sulfasalazine, which is cleaved to form a metabolite, sulfapyridine, when it reaches the colon. The subsequent HPLC analysis of collected blood samples allowed the appearance of sulfapyridine in the plasma and hence the arrival of sulfasalazine in the colon to be determined. Any differences in transit between control and test could be evaluated from a profile of plasma concentrations and corresponding values of AUC. The findings from this study show that the various substances did not affect transit of a test meal in the pig and suggest that it is not possible to use this pig model to make predictions about the human ileal brake.

Rheology and filling characteristics of particulate dispersions in polymer melt formulations for liquid fill hard gelatin capsules.

The rheology and capsule filling properties of molten excipients, Dynafill, Dynasan-114. Lutrol-F68, and polyethylene glycols (PEG) 6000, 8000, 10,000, and 20,000 have been investigated. Lactose (alpha-monohydrate) was selected as a model particulate solid with low solubility in PEG in order to investigate the effects of disperse phase particle size, concentration, and PEG molecular weight on rheology and capsule filling properties of these systems. All excipients behaved as Newtonian fluids between 65 and 90 degrees C, which was chosen as a possible temperature range for liquid filling of hard gelatin capsules. The excipients, apart from Dynasan-114 and PEG 20,000, showed satisfactory capsule filling properties at 70 degrees C using a semi-automatic filling machine. Dynasan-114 (viscosity = 0.012 Pa.s at 70 degrees C) leaked from the seals between the hopper and pump of the filling machine, whereas PEG 20,000 (viscosity = 24 Pa.s at 70 degrees C) showed bridging of the molten polymer between successive capsule bodies during the filling process. The effect of disperse phase (lactose) particle size and concentration, and continuous phase (PEG) molecular weight on the apparent viscosity and filling properties of the non-Newtonian dispersions were investigated at 70 degrees C. Satisfactory filling of the dispersions was achieved at 70 degrees C up to a limiting concentration of disperse phase which was dependent upon disperse phase particle size and continuous phase molecular weight, and corresponded to a pronounced increase in apparent viscosity of the dispersion.