Objectives To provide a comprehensive understanding of the competing thermodynamic and kinetic factors governing the crystallization of various hydrate systems. The ultimate goal is to utilize this understanding to improve the control over the unit operations involving hydrate formation, as well as to optimize the bioavailability of a given drug product. Key findings The thermodynamic and kinetic factors that govern hydrate crystallization are introduced and the current status of the endeavour to gain a mechanistic understanding of the phenomena that occur during the crystallization of different hydrate systems is discussed. The importance of hydrate investigation in the pharmaceutical field is exemplified by examining two specific hydrate systems: the polymorphic hydrate system and hydrates of pharmaceutical salts. Summary This review identifies the factors that are of critical importance in the investigation of anhydrate/hydrate systems. This knowledge can be used to control the phase transformation during pharmaceutical processing and storage, as well as in building a desired functionality for the final formulation.

Cyclooxygenase-2 catalyses the biosynthesis of prostaglandins from arachidonic acid but also the biosynthesis of prostaglandin glycerol esters (PG-Gs) from 2-arachidonoylglycerol. Previous studies identified PG-Gs as signalling molecules involved in inflammation. Thus, the glyceryl ester of prostaglandin E2, PGE2-G, mobilizes Ca2+ and activates protein kinase C and ERK, suggesting the involvement of a G protein-coupled receptor (GPCR). To identify the endogenous receptor for PGE2-G, we performed a subtractive screening approach where mRNA from PGE2-G response-positive and -negative cell lines was subjected to transcriptome-wide RNA sequencing analysis. We found several GPCRs that are only expressed in the PGE2-G responder cell lines. Using a set of functional readouts in heterologous and endogenous expression systems, we identified the UDP receptor P2Y6 as the specific target of PGE2-G. We show that PGE2-G and UDP are both agonists at P2Y6, but they activate the receptor with extremely different EC50 values of ~1 pM and ~50 nM, respectively. The identification of the PGE2-G/P2Y6 pair uncovers the signalling mode of PG-Gs as previously under-appreciated products of cyclooxygenase-2.

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Calcitonin gene-related peptide (CGRP) is contained within and secreted by nerves and neuroepithelial bodies in the airway epithelium. To determine whether CGRP is mitogenic for airway epithelial cells, tracheal epithelial cells isolated from 26 guinea pigs were grown in primary culture for 2 days. Subconfluent cells were exposed to 10(-13) to 10(-9) M CGRP for 4 h and then returned to CGRP-free medium. Proliferation was quantified by direct cell count and by measurement of fractional labeling with the thymidine analog, bromodeoxyuridine (BrdU). CGRP exposure increased both cell number (53,980 +/- 9,870 cells after 10(-9) M CGRP versus 33,910 +/- 5,150 cells after control, P < 0.05) and fractional BrdU labeling (12.9 +/- 2.2% after 10(-11) M CGRP versus 3.9 +/- 0.9%, control; P < 0.01, n = 9) at 24 h after exposure. The mitogenic effect of CGRP persisted at least 3 days after exposure. CGRP-induced proliferation was attenuated by co-incubation with the CGRP receptor antagonist, hCGRP-(8-37). These data demonstrate that CGRP causes proliferation of guinea pig tracheal epithelial cells in primary culture through stimulation of a specific receptor, and suggest a role for this neuropeptide in regulating airway epithelial cell growth.

Exposure of 21-day-old Sprague-Dawley rats to hyperoxia (> 95% O2 for 8 days) causes thickening of the airway epithelial and smooth muscle layers. To test the hypothesis that hyperoxic exposure increases airway layer DNA synthesis, we labeled the nuclei of cells undergoing S-phase by administering the thymidine analog bromodeoxyuridine (BrdU). BrdU was administered on days 3 and 4, 5 and 6, or 7 and 8 of air or O2 exposure, and the lungs were harvested immediately thereafter. Histologic sections were stained with an avidin-biotin-immunoperoxidase stain that revealed BrdU incorporation into nuclei, and a hematoxylin counterstain. After 4 days of air or O2 exposure, there was no difference in BrdU fractional labeling between control and hyperoxic animals. Thereafter, fractional BrdU labeling of the small airway (circumference < 1,000 microns) epithelium and smooth muscle layer was significantly increased in O2-exposed animals (P < 0.01, unpaired t test). The fractional labeling of larger, central airway smooth muscle layer cells was also increased after 8 days of O2 exposure (P < 0.01). In another cohort of O2-exposed animals, measurements of airway layer dimensions demonstrated increases in small airway epithelial and smooth muscle layer thickness that paralleled the time course seen for BrdU incorporation. We conclude that O2 exposure of immature rats increases airway epithelial and smooth muscle layer cellular DNA synthesis. These data suggest that hyperplasia of airway epithelial and smooth muscle layer cells may contribute to hyperoxia-induced airway remodeling.

We recently found that exposure of 21-day-old rats to hyperoxia (> 95% O2 for 8 days) significantly increased in vivo airway cholinergic responsiveness and that O2 exposure also increased airway epithelial and smooth muscle layer thicknesses in a separate cohort of animals. There was substantial variation in the magnitude of both the functional and structural responses to hyperoxia. The present study was designed to test whether the magnitude of O2-induced airway remodeling could account for individual differences in airway responsiveness after O2 exposure, as well as for the difference in responsiveness between air- and O2-exposed animals. We assessed in vivo airway responsiveness to aerosolized acetylcholine (ACh) and airway architecture in 14 O2- and 5 air-exposed, immature rats. Total respiratory system resistance was determined using a plethysmographic method. The mean thicknesses and fractional areas of the airway epithelial and smooth muscle layers were determined by contour tracing using a digitizing pad and microcomputer. Both the small (circumference < 1,000 microns) and central (circumference 1,000 to 4,000 microns) airways were studied. For O2-exposed rats, individual values of EC200 ACh correlated negatively with small airway smooth muscle layer thickness (r = -0.59, p < 0.05; ANOVA), small airway smooth muscle layer fractional area (r = -0.75, p < 0.01), small airway epithelial thickness (r = -0.54, p < 0.05), small airway epithelial fractional area (r = -0.69, p < 0.01), and central airway smooth muscle layer thickness (r = -0.53, p < 0.05). When both air- and O2-exposed animals were considered, EC200 ACh correlated negatively with all eight parameters of airway layer thickness and fractional area.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously demonstrated that hyperoxic exposure (> 95% O2 for 8 d) induces airway cholinergic hyperresponsiveness and remodeling in 21-d-old rats. To examine the potential relationship between airway hyperresponsiveness and remodeling in these animals, we exposed rats to air or hyperoxia for 8 d, returned them to air-breathing, and measured airway responsiveness to inhaled acetylcholine (ACh) and layer thicknesses immediately after or 16 or 48 d after cessation of air or O2 exposure. The ACh concentration required to increase resistance by 100% (EC200ACh) was calculated by linear interpolation. Small airway (circumference < 1,000 microns) and medium-sized, conducting airway (1,000 to 3,000 microns) epithelial and smooth muscle layer mean thicknesses and fractional areas (layer area/luminal cross-sectional area) were determined from lung sections by contour tracing using a digitizing pad and computer. As we reported previously, after 8 d of O2 exposure, group mean log EC200ACh was significantly reduced relative to that in control animals (p < 0.001). Similarly, hyperoxic exposure was associated with significant increases in all parameters of airway layer thickness assessed (p < 0.05). However, by 16 d after cessation of O2 exposure, there were no longer statistically significant differences in log EC200ACh, airway layer thickness, or fractional area between control and O2-exposed animals. Further studies, in a second cohort of animals killed 0, 3, 6, 8, or 13 d after cessation of O2 exposure, demonstrated progressive reductions in small airway epithelial and smooth muscle layer thicknesses, confirming that hyperoxia-induced airway remodeling resolves by approximately 2 wk after termination of O2 exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

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