On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins.

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE.

Pharmaceutical development of a parenteral lyophilized formulation of the investigational polymer-conjugated platinum anticancer agent AP 5280.

AP 5280 is a novel polymer-conjugated platinum anticancer agent showing promising in vitro and in vivo activity against solid tumors. The aim of this study was to develop a parenteral pharmaceutical dosage form for phase I clinical trials. AP 5280 drug substance was characterized by using a wide range of analytical techniques and showed excellent solubility in water.

On-line coupling of size-exclusion chromatography and capillary zone electrophoresis via a reversed-phase C18 trapping column for the determination of peptides in biological samples.

Since biologically active peptides usually exhibit their effects in low concentrations, the development of sensitive analytical methods has become a challenge. In this paper, a multidimensional system is presented, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on a 4 mm x 3 mm ID reversed-phase C18 (RP18) column with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE). The system has been tested with mixtures of six enkephalins and albumin, mimicking biological matrices such as plasma and cerebrospinal fluid.

On-line coupling of size exclusion and capillary zone electrophoresis via a reversed-phase C18 trapping column for the analysis of structurally related enkephalins in cerebrospinal fluid.

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebrospinal fluid (CSF) and tissues. The present study shows the feasibility of a recently developed system, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on an RP18 microcolumn with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE) for the quantification of structural-related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. After SEC separation of the enkephalins from large proteins present in CSF a heart-cut of 200 nuL, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution of the enkephalins with 80% acetonitrile, a fraction of the eluate is electrokinetically injected into the CZE system, where stacking and separation is achieved.

Development of a LC method for pharmaceutical quality control of the antimetastatic ruthenium complex NAMI-A.

Imidazolium trans-tetrachloro(dimethylsulfoxide)imidazoleruthenium(III) (NAMI-A) is a novel ruthenium complex with selective activity against metastases currently in Phase I clinical trials in the Netherlands. Pharmaceutical quality control of NAMI-A drug substance and lyophilized product warranted the development of an assay for determination and quantification of NAMI-A and degradation products. A high performance liquid chromatography (HPLC) method was developed, consisting of a C18 column with 0.

Pharmaceutical development of a parenteral lyophilized formulation of the antimetastatic ruthenium complex NAMI-A.

This paper describes the development of a stable pharmaceutical dosage form for NAMI-A, a novel antimetastatic ruthenium complex, for Phase I testing. NAMI-A drug substance was characterized using several spectrometric and chromatographic techniques. In preformulation studies, it was found that NAMI-A in aqueous solution was not stable enough to allow sterilization by moist heat.

A kinetic study of the chemical stability of the antimetastatic ruthenium complex NAMI-A.

NAMI-A is a novel ruthenium complex with selective activity against cancer metastases currently in Phase I clinical trials in The Netherlands. The chemical stability of this new agent was investigated utilizing a stability-indicating reversed-phase high performance liquid chromatographic assay with ultraviolet detection and ultraviolet/visible light spectrophotometry. The degradation kinetics of NAMI-A were studied as a function of pH, buffer composition, and temperature.

On-line coupling of SEC and RP-LC for the determination of structurally related enkephalins in cerebrospinal fluid.

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebro-spinal fluid (CSF) and tissues. The present study shows the feasibility of the recently reported on-line coupled size exclusion chromatography (SEC) and reversed phase liquid chromatography (RP-LC) separation system for the quantitation of structural related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. The degradation of the peptides, caused by endogenous peptidases in the matrix, could sufficiently be inhibited with imipramine HCL to allow an assay with satisfactory linearity and intraday (0.

Photostability profiles of the experimental antimetastatic ruthenium complex NAMI-A.

NAMI-A is a novel ruthenium complex with selective activity against metastases currently in Phase I clinical trials in The Netherlands. The photostability of this new agent in solid state and in solution has been investigated utilizing a stability-indicating reversed-phase high performance liquid chromatographic (HPLC) assay and ultraviolet/visible (UV/VIS) light spectrophotometry. In solid state, NAMI-A proved to be photostable.

Stability and compatibility of the investigational polymer-conjugated platinum anticancer agent AP 5280 in infusion systems and its hemolytic potential.

AP 5280 is a novel polymer-conjugated platinum anticancer agent currently undergoing phase I clinical trials. It is pharmaceutically formulated as a lyophilized product containing 200 mg platinum per dosage unit. The aim of this study was to determine the reconstitution and dilution fluid of choice, and to investigate the stability and compatibility of AP 5280 in solution under different storage conditions and with several container materials.

Itraconazole solution: summary of pharmacokinetic features and review of activity in the treatment of fluconazole-resistant oral candidosis in HIV-infected persons.

The clinical pharmacology of itraconazole is presented in relation to its use in the treatment of fluconazole-resistant oropharyngeal candidosis. The oral solution is a new formulation of itraconazole in which itraconazole is solubilised with the use of cyclodextrin. This formulation has a higher bioavailability and leads to higher local concentrations in the oral cavity which are advantages over the solid capsule formulation.

Development of a lyophilized parenteral pharmaceutical formulation of the investigational polypeptide marine anticancer agent kahalalide F.

Kahalalide F is a novel antitumor agent isolated from the marine mollusk Elysia rufescens; it has shown highly selective in vitro activity against androgen-independent prostate tumors. The purpose of this study was to develop a stable parenteral formulation of kahalalide F to be used in early clinical trials. Solubility and stability of kahalalide F were studied as a function of polysorbate 80 (0.

Differential expression of protein kinase C betaI (PKCbetaI) but not PKCalpha and PKCbetaII in the suprachiasmatic nucleus of selected house mouse lines, and the relationship to arginine-vasopressin.

The functional significance of the suprachiasmatic nucleus (SCN) in circadian rhythm control of mammals has been well documented. The role of protein phosphorylation mediated by protein kinase C (PKC), however, is not well known. We report the immunocytochemical localization of three Ca(2+)-dependent PKC isoforms (alpha, betaI, betaII) within the SCN of selected house mouse lines that differ in behavioral circadian rhythm parameters.

Compatibility and stability of the investigational polypeptide marine anticancer agent kahalalide F in infusion devices.

Kahalalide F is a novel marine-derived antitumor agent isolated from the marine mollusk Elysia rufescens, an organism living in the seas near Hawaii. The compound has shown highly selective in vitro activity against prostate tumors and phase I trials in patients with androgen independent prostate tumors incorporating a daily times five and weekly schedule have been initiated. Kahalalide F is pharmaceutically formulated as a lyophilized product containing 150 microg active substance per dosage unit.

Capillary electrophoretic bioanalysis of therapeutically active peptides with UV and mass spectrometric detection after on-capillary preconcentration.

An earlier developed capillary electrophoresis (CE) system with an on-capillary adsorptive phase is investigated for its suitability to quantitate low concentrations of angiotensin II and gonadorelin in plasma. An off-line solid-phase extraction is used for sample preparation. The on-line preconcentration CE system allows multiple capillary volumes of sample solution to be injected, increasing the concentration sensitivity of CE with 3-4 orders of magnitude.

Robust and cost-effective capillary electrophoresis-mass spectrometry interfaces suitable for combination with on-line analyte preconcentration.

This paper describes several successful cost-effective attempts to couple capillary electrophoresis (CE) and mass spectrometry (MS) without make-up flow or nebulizing gas. An in-depth analysis of several interfaces using conductive spray tips was performed as well as an easy-to-prepare T-junction with direct electrode contact, the latter being the most robust interface. No coating is necessary and the spray voltage is applied through a gold wire positioned at the gap between the separation and spray capillaries.

On-line sample preconcentration in capillary electrophoresis, focused on the determination of proteins and peptides.

This overview highlights the possibilities of on- or in-line preconcentration procedures in combination with a CZE separation, focused on the determination of peptides and proteins. The discussed methods, including sample stacking, field-amplified injection, isotachophoresis, solid phase extraction, membrane preconcentration, electroextraction, supported liquid membranes, hollow fibers, immunoaffinity, and molecularly imprinted polymers technology preconcentration are categorized in electrophoresis-based and chromatography-based preconcentration. The chromatography-based preconcentration is subdivided in low-specificity and high-specificity methods.

In vitro hemolysis and buffer capacity studies with the novel marine anticancer agent kahalalide F and its reconstitution vehicle cremophor EL/ethanol.

An in vitro biocompatibility study was performed with the pharmaceutical formulation of the investigational, marine-derived anticancer agent kahalalide F developed for early clinical studies. The pharmaceutical formulation consists of a lyophilized product containing 150 micrograms kahalalide F, 3 mg citric acid, 3 mg polysorbate 80, and 150 mg of sucrose per dosage unit, to be reconstituted with 3 mL of a mixture composed of Cremophor EL, ethanol, and water (5/5/90% v/v/v), resulting in a solution of pH 3 and to be further diluted in normal saline for infusion. The reconstituted product, infusion solutions, and Cremophor/ethanol (CE) vehicle were tested for hemolytic potential and buffer capacity.

Degradation kinetics of aplidine, a new marine antitumoural cyclic peptide, in aqueous solution.

The degradation kinetics of aplidine were investigated using reversed-phase high-performance liquid chromatography combined with UV detection. Aplidine consists of at least two isomers that undergo interconversion at a low rate. Influences of pH, temperature, buffer ions and ionic strength on the degradation kinetics were studied.

Phase response curve and light-induced fos expression in the suprachiasmatic nucleus and adjacent hypothalamus of Arvicanthis niloticus.

This article describes the phase response curve (PRC), the effect of light on Fos immunoreactivity (Fos-IR) in the suprachiasmatic nucleus (SCN), and the effect of SCN lesions on circadian rhythms in the murid rodent, Arvicanthis niloticus. In this species, all individuals are diurnal when housed without a running wheel, but running in a wheel induces a nocturnal pattern in some individuals. First, the authors characterized the PRC in animals with either the nocturnal or diurnal pattern.

Derivatization trends in capillary electrophoresis.

This survey gives an overview of recent derivatization protocols, starting from 1996, in combination with capillary electrophoresis (CE). Derivatization is mainly used for enhancing the detection sensitivity of CE, especially in combination with laser-induced fluorescence. Derivatization procedures are classified in tables in pre-, on- and postcapillary arrangements and, more specifically, arranged into functional groups being derivatized.

Pharmaceutical development of anticancer agents derived from marine sources.

The marine ecosystem is more and more acknowledged as a source of potential anticancer agents. After the identification of a potential substance several hurdles have to be overcome before a marine candidate can enter the clinic. Amongst these are the establishment of a method which ensures sufficient supply and, which is the focus of this review, the development of a clinically useful pharmaceutical formulation.

Breaking through artificial selection limits of an adaptive behavior in mice and the consequences for correlated responses.

Previous divergent selection for nest-building behavior at 22 +/- 1 degrees C resulted in a 40-fold difference between the high and the low lines in amount of cotton used to build a nest. Correlated responses to selection indicated positive genetic correlations with body weight, nest-building at 4 +/- 1 degrees C, and litter size and negative genetic correlations with food consumption. At generation 46, the replicate high-selected (High 1 x High 2), randomly bred control (Control 1 x Control 2), and low-selected (Low 1 x Low 2) lines were crossed and the F1 showed significant heterosis for nest-building behavior.

Quantitative analysis of pharmaceutically active peptides using on-capillary analyte preconcentration transient isotachophoresis.

An on-capillary adsorptive phase in combination with capillary electrophoresis (CE), frequently referred to as preconcentration CE, for quantitative analysis of low peptide concentrations was developed. The capillary containing the on-line analyte preconcentrator can be constructed within 5 min from commercially available extraction disks. These disks contain poly(styrenedivinylbenzene) adsorbent particles incorporated in a matrix of inert Teflon, creating a mechanically stable sorbent.