Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration

'Stealth' liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (<1 micromol/kg) and upon repeated administration (time interval between the injections 5 days-4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 micromol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.     

Metabolism and Excretion of Mood Stabilizers and New Anticonvulsants

1. The mood stabilizers lithium, carbamazepine (CBZ), and valproate (VPA), have differing pharmacokinetics, structures, mechanisms of action, efficacy spectra, and adverse effects. Lithium has a low therapeutic index and is renally excreted and hence has renally-mediated but not hepatically-mediated drug–drug interactions.2. CBZ has multiple problematic drug–drug interactions due to its low therapeutic index, metabolism primarily by a single isoform (CYP3A3/4), active epoxide metabolite, susceptibility to CYP3A3/4 or epoxide hydrolase inhibitors, and ability to induce drug metabolism (via both cytochrome P450 oxidation and conjugation). In contrast, VPA has less prominent neurotoxicity and three principal metabolic pathways, rendering it less susceptible to toxicity due to inhibition of its metabolism. However, VPA can increase plasma concentrations of some drugs by inhibiting metabolism and increase free fractions of certain medications by displacing them from plasma proteins.3. Older anticonvulsants such as phenobarbital and phenytoin induce hepatic metabolism, may produce toxicity due to inhibition of their metabolism, and have not gained general acceptance in the treatment of primary psychiatric disorders.4. The newer anticonvulsants felbamate, lamotrigine, topiramate, and tiagabine have different hepatically-mediated drug–drug interactions, while the renally excreted gabapentin lacks hepatic drug–drug interactions but may have reduced bioavailability at higher doses.5. Investigational anticonvulsants such as oxcarbazepine, vigabatrin, and zonisamide appear to have improved pharmacokinetic profiles compared to older agents.6. Thus, several of the newer anticonvulsants lack the problematic drug-drug interactions seen with older agents, and some may even (based on their mechanisms of action and preliminary preclinical and clinical data) ultimately prove to have novel psychotropic effects.     

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.     

Metabolic-flux analysis of Saccharomyces cerevisiae CEN.PK113-7D based on mass isotopomer measurements of (13)C-labeled primary metabolites

Metabolic-flux analyses in microorganisms are increasingly based on (13)C-labeling data. In this paper a new approach for the measurement of (13)C-label distributions is presented: rapid sampling and quenching of microorganisms from a cultivation, followed by extraction and detection by liquid chromatography-mass spectrometry of free intracellular metabolites. This approach allows the direct assessment of mass isotopomer distributions of primary metabolites. The method is applied to the glycolytic and pentose phosphate pathways of Saccharomyces cerevisiae strain CEN.PK113-7D grown in an aerobic, glucose-limited chemostat culture. Detailed investigations of the measured mass isotopomer distributions demonstrate the accuracy and information-richness of the obtained data. The mass fractions are fitted with a cumomer model to yield the metabolic fluxes. It is estimated that 24% of the consumed glucose is catabolized via the pentose phosphate pathway. Furthermore, it is found that turnover of storage carbohydrates occurs. Inclusion of this turnover in the model leads to a large confidence interval of the estimated split ratio.     

Endothelial mediators and communication through vascular gap junctions

Cellular interaction in vessels is achieved by multiple communication pathways, including gap junctions (GJs). They provide intercellular channels, allowing direct interaction of endothelial and smooth muscle cells and the coordination of cellular behaviour along the vessel. The latter is a prerequisite for large flow increases because an adaptation of resistance along the vessel length is required. Longitudinal communication is studied by confined local stimulation of arterioles and the observation of responses at distant locations. Certain vascular stimuli induce local and concomitant remote responses of a similar type, verifying rapid longitudinal conduction of vasomotor signals, most likely changes in membrane potential. This is achieved for dilatory responses via the endothelium, possibly by an endothelium-derived hyperpolarising factor (EDHF) that induces local hyperpolarisation, which is then transferred to remote sites through GJs. In vessels, GJs are composed of different connexins (Cx), but Cx40 is of special importance because its lack impairs longitudinal conduction of vasodilations. Interestingly, Cx40-deficient mice are hypertensive, suggesting that Cx40-dependent coupling is necessary to regulate vascular behaviour and peripheral resistance. While the role of other connexins is less well established, an abundance of data has proven the necessity of GJ communication to coordinate vascular behaviour during blood flow regulation.     

Employment of a noninvasive magnetic method for evaluation of gastrointestinal transit in rats

ABSTRACT: AC Biosusceptometry (ACB) was previously employed towards recording gastrointestinal motility. Our data show a reliable and successful evaluation of gastrointestinal transit of liquid and solid meals in rats, considering the methods scarcity and number of experiments needed to endorsement of drugs and medicinal plants. ACB permits real time and simultaneous experiments using the same animal, preserving the physiological conditions employing both meals with simplicity and accuracy.     

A metabolome study of the steady-state relation between central metabolism, amino acid biosynthesis and penicillin production in Penicillium chrysogenum

The relation between central metabolism and the penicillin biosynthesis pathway in Penicillium chrysogenum was studied by manipulating the steady-state flux in both pathways. A high producing industrial strain was cultivated at a growth rate mu=0.05 h(-1) in glucose-limited chemostat cultures, both under penicillin-G producing and non-producing conditions. Non-producing conditions were accomplished in two ways: (1) by cultivation without addition of the side chain precursor phenylacetic acid and (2) by cultivation of a mutant strain which lost all copies of the gene cluster coding for the penicillin biosynthesis pathway. Manipulation of the fluxes through central metabolism was obtained by cultivation on either glucose or ethanol as sole carbon source. A positive relation was observed between metabolite concentrations and carbon flux in central metabolism. Furthermore, in many cases a positive relation was found between the concentrations of free amino acids and their direct precursors in central metabolism. This corresponds with control of the biosynthesis of these amino acids via feed back inhibition by the end product. With respect to the penicillin production pathway, the flux seems not influenced by two of the three precursor amino acids, namely alphaAAA and valine but is only influenced by cysteine, which requires a large NADPH supply, and the ATP level. An interesting observation is that the absence of penicillin production seems to stimulate storage metabolism (trehalose metabolism). This leads to the final conclusion that the penicillin production flux appears to be mostly influenced by the availability of energy and redox cofactors, where ATP is supposed to exert its influence at ACV-synthetase and NADPH at the cysteine level.     

Lipase catalyzed HEMA initiated ring-opening polymerization: in situ formation of mixed polyester methacrylates by transesterification

2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the enzymatic ring-opening polymerization (ROP) of omega-pentadecalactone (PDL) and epsilon-caprolactone (CL). The lipase B from Candida antarctica was found to catalyze the cleavage of the ester bond in the HEMA end group of the formed polyesters, resulting in two major transesterification processes, methacrylate transfer and polyester transfer. This resulted in a number of different polyester methacrylate structures, such as polymers without, with one, and with two methacrylate end groups. Furthermore, the 1,2-ethanediol moiety (from HEMA) was found in the polyester products as an integral part of HEMA, as an end group (with one hydroxyl group) and incorporated within the polyester (polyester chains acylated on both hydroxyl groups). After 72 h, as a result of the methacrylate transfer, 79% (48%) of the initial amount of the methacrylate moiety (from HEMA) was situated (acylated) on the end hydroxyl group of the PPDL (PCL) polyester. In order to prepare materials for polymer networks, fully dimethacrylated polymers were synthesized in a one-pot procedure by combining HEMA-initiated ROP with end-capping using vinyl methacrylate. The novel PPDL dimethacrylate (>95% incorporated methacrylate end groups) is currently in use for polymer network formation. Our results show that initiators with cleavable ester groups are of limited use to obtain well-defined monomethacrylated macromonomers due to the enzyme-based transesterification processes. On the other hand, when combined with end-capping, well-defined dimethacrylated polymers (PPDL, PCL) were prepared.