Markers of intestinally-derived lipoproteins: application to studies of altered diet and meal fatty acid compositions

BACKGROUND AND AIM: The atherogenic potential of dietary derived lipids, chylomicrons (CM) and their remnants (CMr) is now becoming more widely recognised. To investigate factors effecting levels of CM and CMr and their importance in coronary heart disease risk it is essential to use a specific method of quantification. Two studies were carried out to investigate: (i) effects of increased daily intake of long chain n-3 polyunsaturated fatty acid (LC n-3 PUFA), and (ii) effects of increasing meal monounsaturated fatty acid (MUFA) content on the postprandial response of intestinally-derived lipoproteins. The contribution of the intestinally-derived lipoproteins to total lipaemia was assessed by triacylglycerol-rich lipoprotein (TRL) apolipoprotein B-48 (apo B-48) and retinyl ester (RE) concentrations. METHODS AND RESULTS: In a randomised controlled crossover trial (placebo vs LC n-3 PUFA) a mean daily intake of 1.4 g/day of LC n-3 PUFA failed to reduce fasting and postprandial triacylglycerol (TAG) response in 9 healthy male volunteers. Although the pattern and nature of the apo B-48 response was consistent with the TAG response following the two diets, the postprandial RE response differed on the LC n-3 PUFA diet with a lower early RE response and a delayed and more marked increase in RE in the late postprandial period compared with the control diet, but the differences did not reach levels of statistical significance. In the meal study there was no effect of MUFA/SFA content on the total lipaemic response to the meals nor on the contribution of intestinally derived lipoproteins evaluated as TAG, apo B-48 and RE responses in the TRL fraction. In both studies, the RE and apo B-48 measurements provided broadly similar information with respect to lack of effects of dietary or meal fatty acid composition and the presence of single or multiple peak responses. However the apo B-48 and RE measurements differed with respect to the timing of their peak response times, with a delayed RE peak, relalive to apo B-48, of approximately 2-3 hours for the LC n-3 PUFA diet (p = 0.002) study and 1-1.5 hours for the meal MUFA/SFA study. CONCLUSIONS: It was concluded that there are limitations of using RE as a specific CM marker, apo B-48 quantitation was found to be a more appropriate method for CM and CMr quantitation. However it was still considered of value to measure RE as it provided additional information regarding the incorporation of other constituents into the CM particle.

Synthesis and glycosidase inhibitory profiles of functionalised morpholines and oxazepanes

In this work libraries of morpholines and oxazepanes have been prepared via the reductive amination reaction between dialdehydes, derived from carbohydrates, and a range of amines. In this way, functionalised morpholines and oxazepanes have been prepared that include N-alkylated derivatives, disaccharide analogues, and ester containing derivatives. The abilities of these functionalised morpholines and oxazepanes to inhibit a broad panel of glycosidase enzymes, that are associated with a range of diseases, have been probed and in this way new inhibitors of a range of glycosidases, but particularly β-d-galactosidase derived from Bovine kidney, have been discovered. N-Alkyl morpholines demonstrated the best inhibition profiles for this enzyme and derivatives (15a)–(15d) acted as non-competitive inhibitors with IC50 values of 55.1–88.6 μM. Within this study, some preliminary structure–activity relationships are proposed, and it is demonstrated that N-substituted morpholines display better inhibitory profiles for the enzymes analysed than any of the N-substituted oxazepanes.

Steric stabilisation of the P_P bond in a bulky tetraorganodiphosphine: synthesis, characterisation and X-ray structure determination of tetrakis(2,4,6-triisopropylphenyl)diphosphine

We report the synthesis and characterisation of tetrakis(2,4,6-triisopropylphenyl)diphosphine. Synthesis is effected by the treatment of PCl3 with an excess of 2,4,6-triisopropylphenyllithium (or the equivalent Grignard reagent) in 70% yield. While under normal circumstances the triarylphosphine would be expected, excessive bulk prevents this, and the resulting diphosphine is, unusually, stable to PP cleavage by further organolithium moieties. The compound is stable, both thermally (m.p. 185°C) and to air and water in the solid state, although conversion to the equivalent diorganophosphinate ester is effected by boiling ethanolic solutions in air. Crystallisation from hexane/ethanol afforded pale yellow crystals of X-ray quality. The molecule is characterised by m.p., IR, NMR, elemental analysis (C, H, P) and MS. The X-ray structure shows an antiperiplanar conformation with a PP separation of 2.2461(16) Å. Comparisons are made with other diphosphines, the title compound being only the fourth simple diphosphine to be structurally characterised.

Therapeutic and cytotoxic effects of the novel antipsoriasis codrug, naproxyl-dithranol, on HaCaT cells

A novel topical codrug, naproxyl–dithranol (Nap-DTH), in which dithranol and naproxen are linked via an ester in a 1:1 ratio to form a single chemical entity, was synthesized. The antiproliferative, anti-inflammatory and toxic effects of Nap-DTH were assessed, at the cellular level, using various in vitro methods. Cultured HaCaT keratinocytes were treated with Nap-DTH, and the cellular effects were compared with those of the parent compounds, individually and as a 1:1 mixture of naproxen:dithranol to mimic 1:1 in situ liberation from Nap-DTH. The results demonstrate that Nap-DTH did not modify proliferation and only exhibited slight toxic effects after 24 h at concentrations >21 μM. At a lower concentration (3.4 μM), Nap-DTH did not alter cell proliferation or inflammation, which suggests that the codrug is therapeutically inert. Relating to this, the 1:1 mixture of naproxen:dithranol exhibited the lowest toxic effect and the highest antiproliferative effect on HaCaT keratinocytes compared to dithranol at the same concentration. Moreover, the 1:1 mixture exhibited a reduced inflammatory effect compared to dithranol alone, as reflected by the upregulation of cyclooxygenase-2 by 45% and 136%, respectively. In spite of the 1:1 mixture showing a greater downregulation of Ki-67 and a 2-fold reduction of proliferating cell nuclear antigen (both cellular markers of proliferation) than dithranol, dithranol showed a much greater induction of cleaved caspase-3 protein expression (upregulated by 287%, compared to 85% for the 1:1 mixture). This suggests that when dithranol was administered with naproxen, inhibition of cell growth plays a more important role in the antiproliferation effects than the induction of apoptotic cell death. These results confirm that the codrug would lead to a better therapeutic profile and fewer adverse effects compared to its parent compounds.

Towards the total synthesis of mycaperoxide B: probing biosynthetic rationale

Studies towards the biomimetic synthesis of mycaperoxide B (1) are described. We have established the synthesis of four diastereoisomers of mycaperoxide B methyl ester (1a) by employing a Michael addition across an α,β-unsaturated ester precursor 2 as the key step. This result strongly suggestsstereocontrol in the addition of the hydroperoxide functionality to the E double bond and discloses the importance of choosing the correct geometry of the α,β-unsaturated double bond when attempting to synthesise mycaperoxide B. Four diastereoisomeric tetrahydrofurans derived from an intramolecular rearrangement of the 1,2-dioxolane enolate 12 were also isolated and characterised.

Distribution of esterase activity in porcine ear skin, and the effects of freezing and heat separation

Porcine ear skin is widely used to study skin permeation and absorption of ester compounds, whose permeation and absorption profiles may be directly influenced by in situ skin esterase activity. Importantly, esterase distribution and activity in porcine ear skin following common protocols of skin handling and storage have not been characterised. Thus, we have compared the distribution and hydrolytic activity of esterases in freshly excised, frozen, heated and explanted porcine ear skin. Using an esterase staining kit, esterase activity was found to be localised in the stratum corneum and viable epidermis. Under frozen storage and a common heating protocol of epidermal sheet separation, esterase staining in the skin visibly diminished. This was confirmed by a quantitative assay using HPLC to monitor the hydrolysis of aspirin, in freshly excised, frozen or heated porcine ear skin. Compared to vehicle-only control, the rate of aspirin hydrolysis was approximately three-fold higher in the presence of freshly excised skin, but no different in the presence of frozen or heated skin. Therefore, frozen and heat-separated porcine ear skin should not be used to study the permeation of ester-containing permeants, in particular co-drugs and pro-drugs, whose hydrolysis or degradation can be modulated by skin esterases.

Possible role for K+ in endothelium-derived hyperpolarizing factor–linked dilatation in rat middle cerebral artery

Background and Purpose— Endothelium-derived hyperpolarizing factor (EDHF) and K+ are vasodilators in the cerebral circulation. Recently, K+ has been suggested to contribute to EDHF-mediated responses in peripheral vessels. The EDHF response to the protease-activated receptor 2 ligand SLIGRL was characterized in cerebral arteries and used to assess whether K+ contributes as an EDHF. Methods— Rat middle cerebral arteries were mounted in either a wire or pressure myograph. Concentration-response curves to SLIGRL and K+ were constructed in the presence and absence of a variety of blocking agents. In some experiments, changes in tension and smooth muscle cell membrane potential were recorded simultaneously. Results— SLIGRL (0.02 to 20 μmol/L) stimulated concentration and endothelium-dependent relaxation. In the presence of NG-nitro-L-arginine methyl ester, relaxation to SLIGRL was associated with hyperpolarization and sensitivity to a specific inhibitor of IKCa, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (1μmol/L), reflecting activation of EDHF. Combined inhibition of KIR with Ba2+ (30μmol/L) and Na+/K+-ATPase with ouabain (1 μmol/L) markedly attenuated the relaxation to EDHF. Raising extracellular [K+] to 15 mmol/L also stimulated smooth muscle relaxation and hyperpolarization, which was also attenuated by combined application of Ba2+ and ouabain. Conclusions— SLIGRL evokes EDHF-mediated relaxation in the rat middle cerebral artery, underpinned by hyperpolarization of the smooth muscle. The profile of blockade of EDHF-mediated hyperpolarization and relaxation supports a pivotal role for IKCa channels. Furthermore, similar inhibition of responses to EDHF and exogenous K+ with Ba2+ and ouabain suggests that K+ may contribute as an EDHF in the middle cerebral artery.

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor gamma-chain (FcR gamma-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca(++) elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase C gamma 2 (PLC gamma 2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca(++)](i), during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR gamma-chain during differentiation of megakaryocytes. (Blood. 2000;96:2740-2745)

Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease-activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2-mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR2-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and hypotonic solutions. PAR2-agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cbeta and protein kinases A, C and D inhibited PAR2-induced sensitization of TRPV4 Ca2+ signals and currents. 4alphaPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4alphaPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.

Janus PEG-based dendrimers for use in combination therapy: controlled multi-drug loading and sequential release

The increasing use of drug combinations to treat disease states, such as cancer, calls for improved delivery systems that are able to deliver multiple agents. Herein, we report a series of novel Janus dendrimers with potential for use in combination therapy. Different generations (first and second) of PEG-based dendrons containing two different “model drugs”, benzyl alcohol (BA) and 3-phenylpropionic acid (PPA), were synthesized. BA and PPA were attached via two different linkers (carbonate and ester, respectively) to promote differential drug release. The four dendrons were coupled together via (3 + 2) cycloaddition chemistries to afford four Janus dendrimers, which contained varying amounts and different ratios of BA and PPA, namely, (BA)2-G1-G1-(PPA)2, (BA)4-G2-G1-(PPA)2, (BA)2-G1-G2-(PPA)4, and (BA)4-G2-G2-(PPA)4. Release studies in plasma showed that the dendrimers provided sequential release of the two model drugs, with BA being released faster than PPA from all of the dendrons. The different dendrimers allowed delivery of increasing amounts (0.15–0.30 mM) and in exact molecular ratios (1:2; 2:1; 1:2; 2:2) of the two model drug compounds. The dendrimers were noncytotoxic (100% viability at 1 mg/mL) toward human umbilical vein endothelial cells (HUVEC) and nontoxic toward red blood cells, as confirmed by hemolysis studies. These studies demonstrate that these Janus PEG-based dendrimers offer great potential for the delivery of drugs via combination therapy.

Design, synthesis and in vitro degradation of a novel co-drug for the treatment of psoriasis

Psoriasis is a common, chronic and relapsing inflammatory skin disease. It affects approximately 2% of the western population and has no cure. Combination therapy for psoriasis often proves more efficacious and better tolerated than monotherapy with a single drug. Combination therapy could be administered in the form of a co-drug, where two or more therapeutic compounds active against the same condition are linked by a cleavable covalent bond. Similar to the pro-drug approach, the liberation of parent moieties post-administration, by enzymatic and/or chemical mechanisms, is a pre-requisite for effective treatment. In this study, a series of co-drugs incorporating dithranol in combination with one of several non-steroidal anti-inflammatory drugs, both useful for the treatment of psoriasis, were designed, synthesized and evaluated. An ester co-drug comprising dithranol and naproxen in a 1:1 stoichiometric ratio was determined to possess the optimal physicochemical properties for topical delivery. The co-drug was fully hydrolyzed in vitro by porcine liver esterase within four hours. When incubated with homogenized porcine skin, 9.5% of the parent compounds were liberated after 24 h, suggesting in situ esterase-mediated cleavage of the co-drug would occur within the skin. The kinetics of the reaction revealed first order kinetics, Vmax = 10.3 μM/min and Km = 65.1 μM. The co-drug contains a modified dithranol chromophore that was just 37% of the absorbance of dithranol at 375 nm and suggests reduced skin/clothes staining. Overall, these findings suggest that the dithranol-naproxen co-drug offers an attractive, novel approach for the treatment of psoriasis.

Ozonolysis of methyl oleate monolayers at the air–water interface: oxidation kinetics, reaction products and atmospheric implications

Ozonolysis of methyl oleate monolayers at the air–water interface results in surprisingly rapid loss of material through cleavage of the C[double bond, length as m-dash]C bond and evaporation/dissolution of reaction products. We determine using neutron reflectometry a rate coefficient of (5.7 ± 0.9) × 10−10 cm2 molecule−1 s−1 and an uptake coefficient of [similar]3 × 10−5 for the oxidation of a methyl ester monolayer: the atmospheric lifetime is [similar]10 min. We obtained direct experimental evidence that <2% of organic material remains at the surface on atmospheric timescales. Therefore known long atmospheric residence times of unsaturated fatty acids suggest that these molecules cannot be present at the interface throughout their ageing cycle, i.e. the reported atmospheric longevity is likely to be attributed to presence in the bulk and viscosity-limited reactive loss. Possible reaction products were characterized by ellipsometry and uncertainties in the atmospheric fate of organic surfactants such as oleic acid and its methyl ester are discussed. Our results suggest that a minor change to the structure of the molecule (fatty acid vs. its methyl ester) considerably impacts on reactivity and fate of the organic film.

Phospholipid-based microemulsions suitable for use in foods

The preparation of nonaqueous microemulsions using food-acceptable components is reported. The effect of oil on the formation of microemulsions stabilized by lecithin (Epikuron 200) and containing propylene glycol as immiscible solvent was investigated. When the triglycerides were used as oil, three types of phase behavior were noted, namely, a two-phase cloudy region (occurring at low lecithin concentrations), a liquid crystalline (LC) phase (occurring at high surfactant and low oil concentrations), and a clear monophasic microemulsion region. The extent of this clear one-phase region was found to be dependent upon the molecular volume of the oil being solubilized. Large molecular volume oils, such as soybean and sunflower oils, produced a small microemulsion region, whereas the smallest molecular volume triglyceride, tributyrin, produced a large, clear monophasic region. Use of the ethyl ester, ethyl oleate, as oil produced a clear, monophasic region of a size comparable to that seen with tributyrin. Substitution of some of the propylene glycol with water greatly reduced the extent of the clear one-phase region and increased the extent of the liquid crystalline region. In contrast, ethanol enhanced the clear, monophasic region by decreasing the LC phase. Replacement of some of the lecithin with the micelle-forming nonionic surfactant Tween 80 to produce mixed lecithin/Tween 80 mixtures of weight ratios (Km) 1:2 and 1:3 did not significantly alter the phase behavior, although there was a marginal increase in the area of the two-phase, cloudy region of the phase diagram. The use of the lower phosphatidylcholine content lecithin, Epikuron 170, in place of Epikuron 200 resulted in a reduction in the LC region for all of the systems investigated. In conclusion, these studies show that it is possible to prepare one-phase, clear lecithin-based microemulsions over a wide range of compositions using components that are food-acceptable.

Cocrystalline copolyimides of poly(ethylene 2,6-naphthalate)

Copolycondensation of N,N′-bis(2-hydroxyethyl)-biphenyl-3,4,3′,4′-tetracarboxylic diimide (5–25 mol %) with bis(2-hydroxyethyl)-2,6-naphthalate affords a series of cocrystalline, poly(ethylene 2,6-naphthalate) (PEN)-based poly(ester imide)s. The glass transition temperature rises with the level of comonomer, from 118 °C for PEN itself to 148 °C for the 25% diimide copolymer. X-ray powder and fiber diffraction studies show that, when 5 mol % or more of diimide is present, the α-PEN crystal structure is replaced by a new crystalline phase arising from isomorphic substitution of biphenyldiimide for PEN residues in the polymer crystal lattice. This new phase is provisionally identified as monoclinic, C2/m, with two chains per unit cell, a = 10.56, b = 6.74, c = 13.25 Å, and β = 143.0°.

Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults

Objective The colonic microbiota ferment dietary fibres, producing short chain fatty acids. Recent evidence suggests that the short chain fatty acid propionate may play an important role in appetite regulation. We hypothesised that colonic delivery of propionate would increase peptide YY (PYY) and glucagon like peptide-1 (GLP-1) secretion in humans, and reduce energy intake and weight gain in overweight adults. Design To investigate whether propionate promotes PYY and GLP-1 secretion, a primary cultured human colonic cell model was developed. To deliver propionate specifically to the colon, we developed a novel inulin-propionate ester. An acute randomised, controlled cross-over study was used to assess the effects of this inulin-propionate ester on energy intake and plasma PYY and GLP-1 concentrations. The long-term effects of inulin-propionate ester on weight gain were subsequently assessed in a randomised, controlled 24-week study involving 60 overweight adults. Results Propionate significantly stimulated the release of PYY and GLP-1 from human colonic cells. Acute ingestion of 10 g inulin-propionate ester significantly increased postprandial plasma PYY and GLP-1 and reduced energy intake. Over 24 weeks, 10 g/day inulin-propionate ester supplementation significantly reduced weight gain, intra-abdominal adipose tissue distribution, intrahepatocellular lipid content and prevented the deterioration in insulin sensitivity observed in the inulin-control group. Conclusions These data demonstrate for the first time that increasing colonic propionate prevents weight gain in overweight adult humans