A microarray minisequencing system for pharmacogenetic profiling of antihypertensive drug response

We aimed to develop a microarray genotyping system for multiplex analysis of a panel of single nucleotide polymorphisms (SNPs) in genes encoding proteins involved in blood pressure regulation, and to apply this system in a pilot study demonstrating its feasibility in the pharmacogenetics of hypertension. A panel of 74 SNPs in 25 genes involved in blood pressure regulation was selected from the SNP databases, and genotyped in DNA samples of 97 hypertensive patients. The patients had been randomized to double-blind treatment with either the angiotensin II type 1 receptor blocker irbesartan or the beta 1-adrenergic receptor blocker atenolol. Genotyping was performed using a microarray based DNA polymerase assisted 'minisequencing' single nucleotide primer extension assay with fluorescence detection. The observed genotypes were related to the blood pressure reduction using stepwise multiple regression analysis. The allele frequencies of the selected SNPs were determined in the Swedish population. The established microarray-based genotyping system was validated and allowed unequivocal multiplex genotyping of the panel of 74 SNPs in every patient. Almost 7200 SNP genotypes were generated in the study. Profiles of four or five SNP-genotypes that may be useful as predictors of blood pressure reduction after antihypertensive treatment were identified. Our results highlight the potential of microarray-based technology for SNP genotyping in pharmacogenetics.     

Influence of age on the pharmacokinetics and pharmacodynamics of ximelagatran,an oral direct thrombin inhibitor

OBJECTIVE: To investigate the influence of age on the pharmacokinetics and pharmacodynamics of ximelagatran. STUDY DESIGN: This was an open-label, randomised, 3 x 3 crossover study with 4 study days, separated by washout periods of 7 days. SUBJECTS: Subjects comprised 6 healthy young men (aged 20-27 years) and 12 healthy older men and women (aged 56-70 years). METHODS: All subjects received a 2mg intravenous infusion of melagatran over 10 minutes followed, in randomised sequence, by a 20 mg immediate-release tablet of ximelagatran with breakfast, a 20 mg immediate-release tablet of ximelagatran while fasting, and a 7.5 mg subcutaneous injection of ximelagatran. The primary variables were the plasma concentration of melagatran, the active form of ximelagatran, and the activated partial thromboplastin time (APTT), an ex vivo coagulation time measurement used to demonstrate inhibition of thrombin. RESULTS: After oral and subcutaneous administration, ximelagatran was rapidly absorbed and biotransformed to melagatran, its active form and the dominant compound in plasma. The metabolite pattern in plasma and urine was similar in young and older subjects after both oral and subcutaneous administration of ximelagatran clearance of melagatran was correlated with renal function, resulting in about 40% (after intravenous melagatran) to 60% (after oral and subcutaneous ximelagatran) higher melagatran exposure in the older than in the young subjects. Renal clearance of melagatran, was 7.7 L/h and 4.9 L/h in the younger and older subjects, respectively. The interindividual variability inn the area under the melagatran plasma concentration-time curve was low following all regimens (coefficient variation 12-25%). The mean bioavailability of melagatran in young and older subjects was approximately 18 and 12% , respectively, following oral administration of ximalagratan, and 38 and 45%, respectively, following subcutaneous administration of ximelagatran. The bioavailability of melagatran following oral administration of ximelagatran was unaffected by whether subjects were fed or fasting, although the plasma concentration of melagatran peaked about 1 hour later under fed than fasting conditions, due to gastric emptying of the immediate-release tablet formulation used. The APTT as prolonged with increasing melagatran plasma concentration-effect relationship was independent of age. CONCLUSIONS: There were no age-dependent differences in the absorption and biotransformation of ximelagatran, and the observed differences in exposure to melagatran can be explained by differences in renal function between the young and older subjects.     

An example of optimal phase II design for exposure response modelling

This paper presents an example of how optimal design methodology was used to help design a phase II clinical study. The planned analysis would relate the clinical endpoint to exposure (measured via the area under the curve (AUC)), rather than dose. Optimal design methodology was used to compare a number of candidate phase II designs, and an algorithm for finding optimal designs was employed. The sigmoidal E_max with baseline (E₀) model was used to relate the clinical endpoint to individual subject AUCs, and the primary metrics were D optimality and the standard error (SE) of the AUC required to yield a clinically relevant change in the clinical endpoint. The performance of the candidate designs were compared across four different ‘true’ exposure response relationships (determined from the analysis of an earlier proof of concept (PoC) study). The results suggested the total sample size should be increased from the planned 540 individuals, and that the optimal design with 700 individuals would be equivalent to 812 individuals with the reference design (a 16% gain). The performance with this design was considered acceptable, although all designs performed poorly if the true exposure response relationship was very flat. This work allowed a prospective assessment of the likely performance and precision from the exposure response modelling prior to the start of the phase II study, and hence allowed the design to be revised to ensure the subsequent analysis would be of most value.     

Physical properties of autoclaved bone. Torsion test of rabbit diaphyseal bone

Structural properties of autoclaved diaphyseal bone in the rabbit were investigated by torsional test. Heat propagation into the bone was studied by means of thermocouples. The torsional test included 54 pairs of diaphyseal bones. Autoclaving was performed to the same degree of sterilization, although with variations of time and temperature. Standard autoclaving at 121 degrees C for 20 min was found to cause a moderate decrease (23 per cent) in torsional strength. The decrease was more pronounced (35 per cent) for bones autoclaved at 110 degrees C for 255 min and less (9 per cent) for those autoclaved at 131 degrees C for 2 min. Heat propagation into bone during autoclaving proved to be rapid at both 121 degrees C and 131 degrees C, indicating that complete, uniform sterilization of diaphyseal bone may be performed to an accurate, predetermined degree. Diaphyseal bone subjected to standard autoclaving remains mechanically adequate for skeletal substitution. Reimplantation of autoclaved tumorous bone might provide a simple combined means for tumor devitalization and subsequent reconstruction.     

In vitro evaluation of microcrystalline chitosan (MCCh) as gel-forming excipient in matrix granules.

Although much research has been carried out into the effects of chitosan and its chemical properties on drug release, less attention has been paid to the effects of its physical properties. The aim of this study was to characterize microcrystalline chitosan (MCCh) as a gel-forming excipient. Matrix granules containing chitosans of differing physicochemical properties (crystallinity, molecular weight, degree of deacetylation) and ibuprofen or paracetamol as model drugs were prepared. Gel formation by the chitosans in the granules and subsequent effects on drug release were studied at pH 1.2 and pH 5.8. The chitosan granules acted as slow-release formulations in the case of ibuprofen (a class-II drug in the Biopharmaceutics Classification System) but with paracetamol (class-I) no controlled-release formulation could be developed. Microcrystalline grades of chitosan had the most marked retardant effects on drug release, with the efficacy of gel formation by MCCh explaining the results. The kinetic constant for ibuprofen release (at pH 5.8) ranged from 22%.h(-1) (MCCh) to 31%.h(-1) (unmodified chitosan). The release rate was easily controlled by varying the amount or molecular weight of MCCh, and to a lesser extent by the degree of deacetylation. The effects were most pronounced when pH was markedly acidic, suggesting that MCCh granules might be particularly useful in preparing stomach-specific slow-release dosage forms.     

Knowledge, attitudes and practice among nursing staff concerning pressure ulcer prevention and treatment – a survey in a Swedish healthcare setting

The aim of this study was to investigate (i) attitudes among Registered Nurses (RNs) and Nursing Assistants (NAs) regarding pressure ulcer prevention, (ii) knowledge among RNs and NAs of pressure ulcer prevention and treatment, (iii) practice of risk assessment and documentation regarding pressure ulcers among RNs and NAs and (iv) to identify perceived possibilities and barriers in pressure ulcer prevention and treatment. In this cross-sectional study, a total of 230 questionnaires were distributed to an equal number of RNs and NAs in both municipality as well as hospital care settings. The response rate was 67% (n = 154). In general, all respondents displayed good knowledge on prevention and treatment of pressure ulcers and demonstrated a positive attitude towards this area of care. However, answers provided to some questions indicate that recent research findings and guidelines have not succeeded in reaching out to these occupational groups. Furthermore, only 37% (n = 55) of the participants said that they have an agreed strategy for the prevention of pressure ulcers in their unit. These shortcomings may affect the quality of care provided to the patient and lead to pressure ulcers developing as a consequence. Today, evidence-based methods for risk assessment are available but are not adopted and used in practice. The study highlights the need to further reduce the gap between research and practice.     

Diaphyseal reconstruction by autoclaved bone: Reimplantation experiments in rabbits

In 8 adult rabbits, reconstruction of large humeral defects by reimplantation of resected, autoclaved bone supplemented with allogeneic bone matrix was investigated with respect to incorporation (radiography, histology), bone metabolic activity (scintigraphy, autoradiography), and strength (torsional test). Radiography showed that seven out of eight implants were incorporated at 3 months. Scintigraphy and autoradiography disclosed bone metabolic activity in the reconstructions still 8 months postoperatively. Histological investigations evidenced abundant new viable bone that partially had replaced the implants at 8 months. The torsional test disclosed that the strength of the reconstructions was 84 per cent of normal 8 months after reimplantation. Our experiment supports the concept of reimplantation of autoclaved tumorous bone supplemented with allogeneic bone matrix in reconstruction after local resection.