Determination of mismatched donor HLA in kidney transplant recipients with unknown donor HLA phenotypes.

OBJECTIVE: To determine donor human leukocyte antigen (HLA) from renal allograft biopsies in transplant recipients whose donor HLA phenotype is not known. METHODS: Renal allograft biopsies were obtained from seven renal transplant recipients when indicated for allograft dysfunction or proteinuria. DNA was extracted fresh from allograft specimens, and HLA typing was performed with polymerase chain reaction-specific sequence primers (PCR-SSP) and polymerase chain reaction-sequence-specific oligonucleotides (PCR-SSO). RESULTS: HLA typing of the seven renal allograft biopsies was composed of both recipient and donor HLA phenotypes, allowing the determination of the donor HLA and the degree of HLA mismatching. CONCLUSIONS: Deducing mismatched donor HLA antigens in renal allograft recipients enables detection of donor-specific antibodies, and the management of humoral rejection, and enables more appropriate selection of a donor organ should future retransplantation be required.

Supercritical fluid chromatography and steady-state recycling: phase appropriate technologies for the resolutions of pharmaceutical intermediates in the early drug development stage.

The use of phase appropriate technologies is critical for efficiently moving drug candidates forward in the early stages of drug discovery and development. Phase appropriate purification technology develops the analytical method and subsequently scales up the method and turns the sample around quickly (Kennedy et al., J Chromatogr A 2004; 1046:55). In this article, separation results and conditions from supercritical fluid chromatography (SFC), high-performance liquid chromatography (HPLC), and steady-state recycling (SSR) for the resolutions of three pharmaceutical intermediates in the early stage of the drug development are discussed. The first study used SFC and SSR to separate an impurity for a Good Manufacturing Practice (GMP) campaign. The analytical method development and scale-up conditions are discussed. Productivity, solvent usage, and sample solubility under SFC and SSR conditions are also compared. The second study compared SFC to batch HPLC in separating a diastereomer. Due to higher separation efficiency, SFC was able to resolute multiple peaks. The third study involved the addition of dichloromethane as a co-solvent in SFC purification--improving sample selectivity and solubility. From the separation results of these purifications, SFC and SSR are clearly phase appropriate technologies in the early drug development stage.

Rapid and high throughput separation technologies--steady state recycling and supercritical fluid chromatography for chiral resolution of pharmaceutical intermediates.

The SSR and SFC techniques were used for the enantiomeric resolution of three pharmaceutical intermediates at various sample scales. The separation conditions, the sample purities and yields, the productivities and the solvent consumptions were discussed in three case studies in this paper. In case (I), the SSR process was used for a low selectivity resolution of 2.0 kg of pharmaceutical intermediate. By using this separation process, a productivity of 750 g racemate/kg stationary phase/day was achieved, while solvent usage was minimized ( approximately 200 l/kg racemate). Case (II) pertained to the effectiveness of the SSR process. Productivity using SSR techniques increased by a factor of 4.5, while solvent usage decreased by a factor of 4.1 when compared to the productivity and solvent usage of batch HPLC. Case (III) compared SFC purification to HPLC purification. The SFC process was more effective in terms of an increase in productivity and a reduction in solvent usage. Based on these results, it appears that SSR and SFC are very useful choices at the early stage of the drug development for a high throughput and a rapid turn around of samples.

Chromatographic resolution of atropisomers for toxicity and biotransformation studies in pharmaceutical research.

Atropisomerism can be a complex concept for those who have not encountered it before. This paper discusses the experiments for identification, isolation, thermal stability, toxicity and biotransformation of various species. The identified atropisomers are a series of rotational hindered biaryl, rotational hindered amide, ring flip, and macrocycles atropisomers identified using supercritical fluid chromatography (SFC) and high performance liquid chromatography (HPLC). These technologies offered the advantage of separating various atropoenantiomers, atropdiastereomers and mixed atropisomers with other forms of stereoisomers in both analytical and preparative scales. With ultra-performance convergence chromatography (UPC(2)), the detection of N-oxide atropisomer metabolites can be obtained at very low level thus enabling the observation of conversion in human plasma possible. As the resolution of atropisomers are related to the energy barriers on the rotational axis, a calculated computational protocol was developed to predict the formation. A threshold of 10kcal/mol was established for possible detection of the atropisomers' existence with chromatographic technologies at room temperature or above. The atropisomer with higher energy barrier (>20kcal/mol) were isolated via preparative chromatography and the isolates studied in vitro and in vivo for evaluation of their stability in human plasma. The detailed analytical method development to analyze the biotransformation of the atropisomers in human plasma are also discussed in this paper.

Resolution of a racemic pharmaceutical intermediate. A comparison of preparative HPLC, steady state recycling, and simulated moving bed.

Preparative HPLC and simulated moving bed (SMB) chromatography were used to resolve significant quantities of a racemic pharmaceutical intermediate. In addition, smaller scale studies using closed-loop recycling and steady state recycling (SSR) were performed so that a meaningful comparison of all these techniques could be made using the same real world separation. A highly optimized, six-column SMB process was clearly the superior technique and was used for the process-scale (247 kg of racemate) resolution. At the more moderate lab-scale (33 kg of racemate and 19 kg of racemate), a frequently used but less optimized eight-column SMB process was used. It was found that SSR was comparable to the lab-scale SMB process in productivity and solvent consumption. Thus, it appears that SSR can be a useful choice at such moderate scales. Finally, at moderate scales when neither SSR nor SMB is available, it was found that acceptable results were obtained with both closed-loop recycling and with a two-step preparative process.

Batch and simulated moving bed chromatographic resolution of a pharmaceutical racemate.

The preparative chromatographic resolution of racemates has become over the past few years a standard approach for the generation of enantiomers in pharmaceutical research and development. This paper will discuss the chromatographic resolution of a racemic pharmaceutical intermediate. Initial analytical method development to determine the best preparative conditions will be presented. Batch resolution of kg quantities of racemate followed by the simulated moving bed resolution of tens and hundreds of kg of racemate will also be discussed. Finally the different approaches used for the separation will be compared.

On the method development of immobilized polysaccharide chiral stationary phases in supercritical fluid chromatography using an extended range of modifiers.

Polysaccharide-derived selectors are often used in the separation of enantiomers by supercritical fluid chromatography (SFC). Their recognition patterns are normally investigated with alcohols and acetonitrile as modifiers. The present paper describes the results of a research program designed by Pfizer and Chiral Technologies Inc. to explore the potential of other solvents (i.e. ethyl acetate, tetrahydrofuran, dichloromethane) in SFC by using a series of polysaccharide-derived supports with broad solvent versatility (CHIRALPAK IA, IB, IC, ID, IE and IF). The contribution of such extended solvent range to the overall success rate, as well as to overcome racemization, solubility and stability issues was confirmed by using standard non-proprietary samples and research molecules. Elution patterns with such lower polarity solvents, compared to alcohols, and the role of the different additives were also investigated.

Cardiac ion channel safety profiling on the IonWorks Quattro automated patch clamp system.

The normal electrophysiologic behavior of the heart is determined by the integrated activity of specific cardiac ionic currents. Mutations in genes encoding the molecular components of individual cardiac ion currents have been shown to result in multiple cardiac arrhythmia syndromes. Presently, 12 genes associated with inherited long QT syndrome (LQTS) have been identified, and the most common mutations are in the hKCNQ1 (LQT1, Jervell and Lange-Nielson syndrome), hKCNH2 (LQT2), and hSCN5A (LQT3, Brugada syndrome) genes. Several drugs have been withdrawn from the market or received black box labeling due to clinical cases of QT interval prolongation, ventricular arrhythmias, and sudden death. Other drugs have been denied regulatory approval owing to their potential for QT interval prolongation. Further, off-target activity of drugs on cardiac ion channels has been shown to be associated with increased mortality in patients with underlying cardiovascular diseases. Since clinical arrhythmia risk is a major cause for compound termination, preclinical profiling for off-target cardiac ion channel interactions early in the drug discovery process has become common practice in the pharmaceutical industry. In the present study, we report assay development for three cardiac ion channels (hKCNQ1/minK, hCa(v)1.2, and hNa(v)1.5) on the IonWorks Quattro™ system. We demonstrate that these assays can be used as reliable pharmacological profiling tools for cardiac ion channel inhibition to assess compounds for cardiac liability during drug discovery.

The application of preparative batch HPLC, supercritical fluid chromatography, steady-state recycling, and simulated moving bed for the resolution of a racemic pharmaceutical intermediate.

This article discusses the chromatographic resolution of a racemic pharmaceutical intermediate. Preparative batch high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), steady-state recycling (SSR), and simulated moving bed (SMB) were used to resolve a total of 12.2 kg of a racemic pharmaceutical intermediate. In this study, a first batch of 0.8 kg of racemate was separated on the preparative batch HPLC and SFC, and subsequently another 5.9 kg of racemate was separated on the SSR. Lastly, a third batch of 5.5 kg was separated on the SMB. The separation conditions and results of these techniques are discussed. The productivities and solvent costs of SFC versus HPLC are compared. The productivities and solvent costs of SMB, SSR, and HPLC are also compared. The analytical method development and process optimization of these processes are also discussed in this article.

Saliva and bloodspot cortisol: novel sampling methods to assess hydrocortisone replacement therapy in hypoadrenal patients.

BACKGROUND: In patients with hypoadrenalism, it is often difficult to assess the optimal dose of glucocorticoid replacement. Serial serum cortisol measurements for a cortisol day curve are sometimes used, but this has low acceptability for patients. In this study, we evaluate the reliability of saliva and capillary bloodspot cortisol as alternative methods in assessing cortisol profiles in hypoadrenal patients on hydrocortisone replacement. METHODS: We first examined the correlations between serum cortisol, saliva and bloodspot cortisol in in-hospital patients not on glucocorticoid therapy. We then studied 18 hypoadrenal patients on hydrocortisone therapy and measured their serum, saliva and bloodspot cortisol concurrently at seven different time points in a single day. RESULTS: For in-hospital patients, a significant correlation exists between saliva and serum cortisol (R = 0.7121, P < 0.0001), but there is a stronger correlation between bloodspot and serum cortisol (R = 0.9494, P < 0.0001). The correlations were weaker in hypoadrenal patients on hydrocortisone (saliva and serum cortisol: R = 0.6262, P < 0.001; bloodspot and serum cortisol: R = 0.7871, P < 0.001). When we evaluated each measurement with respect to an arbitrary target range, there was a greater degree of agreement between serum and capillary bloodspot cortisol (85% agreement) than between serum and saliva cortisol (65% agreement) (P < 0.001). CONCLUSION: Bloodspot samplings provide a simple and convenient way for ambulant hypoadrenal patients on hydrocortisone replacement therapy to assess cortisol levels at multiple times in a single day. This may be useful in determining the optimal glucocorticoid dose for hypoadrenal patients.