Aldose reductase inhibitors and nanodelivery of diabetic therapeutics.

Nanotechnology is a rapidly emerging drug-delivery system that makes possible the controlled release of small molecules, and nanodelivery of therapeutic molecules using nanoparticles or nanogels represents a major improvement for more focused delivery of such therapeutic molecules. The delivery of insulin for the control of diabetes mellitus (DM) and aldose reductase inhibitor (ARI) for diabetic complications may provide better treatment of diabetes. A structural overview of aldose reductase including computational docking experiments with HAR-1, various ARIs, aldose-keto reductase, and nanodelivery of insulin, ARI's, and drug molecules are described.

Choline acetyltransferase, glutamate decarboxylase, tyrosine hydroxylase, calcitonin gene-related peptide and opioid peptides coexist in lateral efferent neurons of rat and guinea-pig.

The lateral efferent (olivocochlear) innervation of the cochlea originates in the brainstem lateral superior olive. It is likely to use acetylcholine, gamma-aminobutyric acid, dopamine and various neuropeptides as neurotransmitters and/or neuromodulators. In order to determine the different coexistence patterns of these molecules in lateral efferent perikarya, we have used double and triple immunofluorescence co-localization techniques to colocalize choline acetyltransferase, glutamate decarboxylase, tyrosine hydroxylase, calcitonin gene-related peptide and enkephalins in single sections of the lateral superior olive. We also used a non-radioactive in situ hybridization technique onto serial sections of this nucleus to confirm the immunofluorescence co-localization data at the mRNA level. Whatever the pair or triplet of primary antibodies tested was, a high ratio of coexistence was observed in the immunofluorescence experiments. In triple co-localization experiments, 90-93% of the choline acetyltransferase-like immunoreactive neurons were also immunoreactive to the two other antigens investigated. The in situ hybridization co-localization data, based on the use of biotin-labelled oligoprobes, qualitatively confirmed these immunofluorescence data. In conclusion, it can be postulated that acetylcholine, gamma-aminobutyric acid, dopamine, calcitonin gene-related peptide, enkephalins and dynorphins (whose coexistence with choline acetyltransferase and enkephalins has been previously described immunocytochemically) coexist in lateral efferent neurons. Based on these results, it is tempting to propose the lateral efferent innervation as a useful model with which the functional implications of the coexistence of neurotransmitters/neuromodulators can be investigated in vivo.

Effects of L-690,488, a prodrug of the bisphosphonate inositol monophosphatase inhibitor L-690,330, on phosphatidylinositol cycle markers.

In order to enhance the entry into cells of L-690,330, a bisphosphonate inhibitor of inositol monophosphatase (IMPase; a key, enzyme in the phosphatidylinositol (Pl) cell signaling pathway), the tetrapivaloyloxymethyl ester prodrug, L-690,488 [tetrapivaloyloxymethyl 1-(4-hydroxyphenoxy)ethane-1,1-bisphosphonate], was synthesized. The effects of L-690,488 were studied in cholinergically (carbachol)-stimulated rat cortical slices and Chinese hamster ovary cells stably transfected with the human muscarinic m1 receptor (m1 CHO cells). The accumulation of [3H]inositol monophosphates or [3H]cytidine monophosphorylphosphatidate ([3H]CMP-PA) after [3H]inositol or [3H]cytidine prelabeling, respectively, and inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate mass were measured. In rat cortical slices and m1 CHO cells, the maximum response and time course of accumulation of [3H]inositol monophosphates for L-690,488 and lithium were similar. However, the concentrations of L-690,488 required to produce these effects (EC50 values of 3.7 +/- 0.9 and 1.0 +/- 0.2 microM in cortical slices and m1 CHO cells, respectively) were much lower than with lithium (0.3-1.5 mM). Likewise, the time course and maximum accumulation of [3H] CMP-PA in L-690,488-treated m1 CHO cells was similar to lithium but L-690,488 was again much more potent (EC50 values = 3.5 +/- 0.3 microM and 0.52 +/- 0.03 mM for L-690,488 and lithium, respectively). In addition, L-690,488 attenuated the carbachol-induced elevation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in m1 CHO cells, an effect reported previously with lithium. These results are all consistent with L-690,488 and lithium both depleting intracellular inositol as a consequence of inhibition of IMPase. That these effects of L-690,488 on the PI cycle are indeed due to inositol depletion is shown by the observation that the effects of L-690,488 on CMP-PA accumulation could be overcome by addition of exogenous myo-inositol (EC50 = 1.7 +/- 0.5 mM). These data show that inhibition of IMPase produces effects on the PI cycle comparable to lithium. As a corollary, the effects of lithium on the PI cycle are therefore consistent with its major mechanism of action being inhibition of IMPase.

Characterization of the effects of lithium on phosphatidylinositol (PI) cycle activity in human muscarinic m1 receptor-transfected CHO cells.

1. The effects of lithium on [3H]-inositol and [3H]-cytidine incorporation into [3H]-inositol monophosphates ([3H]-InsP1) and [3H]-cytidine monophosphorylphosphatidate ([3H]-CMP-PA), respectively, and inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) mass were studied in carbachol-stimulated human m1 muscarinic receptor-transfected Chinese hamster ovary cells (m1 CHO cells). 2. Lithium alone (10 mM) had no appreciable effects on any of the four parameters measured; it was only in carbachol-stimulated cells that the effects of lithium became apparent. 3. In the presence of carbachol (1 mM), lithium (10 mM) caused a relatively rapid (within 5 min) accumulation of [3H]-InsP1 and [3H]-CMP-PA which continued up to about 20-30 min, after which accumulation slowed down. On the other hand, the elevation in InsP3 and InsP4 levels produced by carbachol was not altered by lithium in the short-term and only at later times (> 20-30 min) was the response attenuated, with InsP3 and InsP4 levels approaching basal. 4. The effects of lithium on carbachol-stimulated [3H]-InsP1 and [3H]-CMP-PA accumulation and the attenuation of the carbachol-induced elevation of InsP3 and InsP4 were all dose-dependent, with EC50s in the region of 1 mM. 5. The lithium-induced effects on [3H]-CMP-PA and InsP3 and InsP4 in carbachol-stimulated cells could be reversed, in a dose-dependent manner, by preincubation with exogenous myo-inositol (EC50 = 2-3 mM) but not by the inactive analogue scyllo-inositol, indicating that these effects occur as a consequence of depletion of inositol. 6. The temporal effects of lithium are consistent with lithium inhibiting inositol monophosphatase,causing accumulation of InsP1, resulting in lower free inositol levels. This leads to accumulation of CMP-PA and reduced PI synthesis which, once agonist-linked membrane inositol phospholipids are depleted, produces attenuated InsP3 and InsP4 responses.7. These results in ml CHO cells support the hypothesis that lithium affects the PI cycle cell signalling pathway by depletion of inositol due to inhibition of inositol monophosphatase.