Sangeeta Tanna

Facilitating pharmacokinetic studies in children: a new use of dried blood spots.

Pharmacokinetic data are used to develop dosing regimens for medicines. The dose regimens of many drugs administered to children have historically been based on pharmacokinetic data generated in adults. The ‘adult’ dose was simply adjusted to the childs body weight or surface area. This practice is potentially unsafe and not acceptable to drug regulatory agencies. Obtaining pharmacokinetic data in children is beset with ethical issues and technical challenges as pharmacokinetic studies require repeated measurement of drug levels in blood. Dried blood spot (DBS) samples used in conjunction with population pharmacokinetic modelling techniques is one potential method for performing pharmacokinetic studies in children. In this article, we review the DBS technique for performing pharmacokinetic studies and highlight issues that still need to be addressed to establish DBS as a method for performing pharmacokinetic studies in children.

In vivo study of a polymeric glucose-sensitive insulin delivery system using a rat model†

This study assesses the feasibility of an intraperitoneal (IP) implantable closed-loop insulin delivery device in rats, that delivers insulin via a glucose-sensitive material such that blood glucose (BG) levels are adjusted automatically to within normal tolerances. A gateway layer of this gel governs the output of insulin from an insulin reservoir device for IP implant. The performance of the system was compared over time in diabetic rats with a control system using oral glucose challenges and daily assessments of BG and body weight. The automated response of the active system was quantified using IP multiple dose injection (MDI) results in the same rat model. Successful control was found for the device containing active gel when assessed daily and when challenged with large glucose doses. This was not found when comparing an inactive gel analog as a control. The regimen was quantified by comparison with the informative MDI study. The device was well tolerated and might operate to further advantage when vascular omentum grows into the perforated front of the device. The successful device must have been outputting approximately 0.5 U/kg/h basal with 2 U/kg boosts in order to match the demand of the challenges. However, the device eventually exhausts and a refill mechanism needs to be devised in future models.

Dexamethasone quantification in dried blood spot samples using LC–MS: The potential for application to neonatal pharmacokinetic studies

A high-performance liquid chromatography (LC-MS) method has been developed and validated for the determination of dexamethasone in dried blood spot (DBS) samples. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30μl blood spots on specimen collection cards. An 8mm disc was cut from the DBS sample and extracted using a combination of methanol: water (70:30, v/v) containing the internal standard, triamcinolone acetonide. Extracts were centrifuged and chromatographic separation was achieved using a Zorbax Eclipse Plus C18 column using gradient elution with a mobile phase of acetonitrile and water with formic acid at a flow rate of 0.2ml/min. LC-MS detection was conducted with single ion monitoring using target ions at m/z 393.1 for dexamethasone and 435.1 for the internal standard. The developed method was linear within the tested calibration range of 15-800ng/ml. The overall extraction recovery of dexamethasone from DBS samples was 99.3% (94.3-105.7%). The accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤15% at all concentrations. Factors with potential to affect drug quantification measurements such as blood haematocrit, the volume of blood applied onto the collection card and spotting device were investigated. Although a haematocrit related effect was apparent, the assay accuracy and precision values remained within the 15% variability limit with fluctuations in haematocrit of ±5%. Variations in the volume of blood spotted did not appear to affect the performance of the developed assay. Similar observations were made regarding the spotting device used. The methodology has been applied to determine levels of dexamethasone in DBS samples collected from premature neonates. The measured concentrations were successfully evaluated using a simple 1-compartment pharmacokinetic model. Requiring only a microvolume (30μl) blood sample for analysis, the developed assay is particularly suited to pharmacokinetic studies involving paediatric populations.

The Delivery of Insulin from Aqueous and Non-Aqueous Reservoirs Governed by a Glucose Sensitive Gel Membrane

A self regulating delivery device, responsive to glucose, has been shown to operate successfully in vitro. This comprises a gel membrane which determines the delivery rate of insulin from a reservoir. The gel consists of a synthetic polysucrose and the lectin, concanavalin A. The mechanism is one of displacement of the branched polysaccharide from the lectin receptors by incoming glucose. The gel loses its high viscosity as a result but reforms on removal of glucose, thus providing the switch controlling the drug diffusion rate. The drug does not require to be chemically modified and thus the device is adaptable to other anti-hyperglycaemic drugs. However, results here indicate that the molecular weight of the solute may be an important parameter. Others include path length, gel formulation and temperature. It had been hypothesised that the reversal might be improved by the use of a non-aqueous reservoir of insulin. However, with the use of insulin, the switching off was found to be superior to that found with other test solutes used in previous studies, irrespective of the reservoir solvent. The advantages in the use of the non-aqueous system include, however, more reproducibility in the magnitude of response and a reduced temperature sensitivity.

Analytical methods used in conjunction with dried blood spots.

This review covers the wide range of different methods that have been used for the microanalysis of dried blood spots (DBS) from the ones reported in 1914 to the sophisticated processes used, for example, in newborn screening, new drug discovery research and application of PCR based DNA studies of HIV in countries with limited infrastructure. The value of the dried blood spot as a sampling tool and the many advantages it offers in combination with suitable analytical methods is discussed. Current developments in both automating the DBS extraction process and the potential for direct analysis of the sample without prior extraction are reviewed. Possible future directions in the analytical methods used for DBS samples are also highlighted.

Quantitative determination of atenolol in dried blood spot samples by LC–HRMS: A potential method for assessing medication adherence

The use of blood spot collection cards was investigated as a means of obtaining small volume samples for the quantification of therapeutic drugs for assessing medication adherence. A liquid chromatography-high resolution TOF mass spectrometry (LC-HRMS) method, based on the measurement at the accurate mass to charge ratio of the target analyte, was used to ensure specificity for atenolol in the dried blood spot (DBS) samples. A working method was developed and validated. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30 μl blood spots on specimen collection cards. A 5mm disc was cut from the dried blood spot and extracted using methanol:water (60:40, v/v) containing the internal standard, atenolol-d(7). Extracts were vortexed, sonicated and then centrifuged. Gradient chromatographic elution was achieved using an Ascentis Express C18 100mm×2.1mm column and a mobile phase flow rate of 0.2 ml/min and the column oven temperature at 30 °C. MS detection was carried out in electrospray positive ion mode for target ions at accurate mass m/z 267.1703 for atenolol and 274.2143 for the IS. Drug extraction efficiency from spiked blood spots was demonstrated to be 96±5% and the drug was stable in DBS for at least 10 weeks. The developed LC-HRMS method was linear within the tested calibration range of 25-1500 ng/ml and validation showed the accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤ 5% at all concentrations with a limit of quantification of 25 ng/ml. Factors with potential to affect drug quantification measurements such as the matrix effects, volume of blood applied onto the collection card and effect of different sampling cards were investigated. The developed LC-HRMS method was applied to blood spots on sampling card taken from adult healthy volunteers previously administered a 50mg atenolol tablet and a DBS concentration-time profile was obtained for atenolol. Requiring only a micro volume (30 μl) blood sample for analysis, the developed DBS based assay has the potential to assess patient adherence to atenolol.

Insulin Delivery Governed by Covalently Modified Lectin-Glycogen Gels Sensitive to Glucose

A glucose‐sensitive gel formulation containing concanavalin A and glycogen has been reported previously. Precipitation resulting from the addition of concanavalin A to glycogen has been documented, but the formation of glucose‐sensitive gels based on lectin‐glycogen interactions is novel and used here in our studies. An improved in‐vitro self‐regulating drug‐delivery system, using covalently modified glucose‐sensitive gels based on concanavalin A and a polysaccharide displacement mechanism, is described. The successful use of the covalently modified gels addresses a problem identified previously where significant leaching of the mitogenic lectin from the gel membranes of non‐coupled gels was encountered.

Bisoprolol, ramipril and simvastatin determination in dried blood spot samples using LC-HRMS for assessing medication adherence.

The use of dried blood spot (DBS) collection cards was investigated for the quantification of three therapeutic drugs used in cardiovascular therapy for assessing medication adherence. A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed and validated for the determination of bisoprolol, ramipril and simvastatin. Whole blood spiked with target analytes was used to produce 30 μl blood spots on specimen collection cards. An 8mm disc was cut from the dried blood spot and extracted using methanol: water (70:30, v/v) containing the internal standard, atenolol. Extracts were vortexed, sonicated and then centrifuged. Gradient chromatographic elution was achieved using a Zorbax Eclipse C18 HD 100 mm × 2.1 mm, 1.8 μm pore size column and a mobile phase flow rate of 0.6 ml/min and the column oven temperature at 40 °C with a run time of 3 min. MS detection was carried out in electrospray positive ion mode for the three target drugs and for the IS. Drug recoveries from spiked blood spots were ≥ 92% for bisoprolol and ramipril and ~43% for simvastatin and the drugs were stable in DBS for at least 12 weeks. Validation of the LC-HRMS method showed good linearity and the accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤ 15% at all concentrations. Matrix effects and the effects of different volumes of blood applied to the collection card were investigated. The LC-HRMS method successfully identified control volunteers who were known to be either adherent or non-adherent. There were no false positives from volunteers taking other cardiovascular drugs or from volunteers receiving no medication.

Covalent coupling of concanavalin A to a Carbopol 934P and 941P carrier in glucose-sensitive gels for delivery of insulin.

A novel glucose‐sensitive gel formulation, containing concanavalin A and specific polysaccharides, was stabilised via covalent coupling to two structurally different carbomers. The bonding was done to minimise leaching of gel components thereby preventing toxicity and preserving the working mechanism of the gel. Increased gel stability was introduced by covalently bonding amine groups present on the lysine residues of concanavalin A to carboxylic moieties on Carbopol 934P NF and 941P NF using carbodiimide chemistry. The introduction of dextran then produced a glucose‐sensitive formulation that transformed from gel to sol in the presence of free glucose. Rheological examination of glucose‐sensitive gels stabilised in this way and containing varying concentrations of glucose was conducted with a cone and plate viscometer used in continual rotation mode. A decrease in viscosity over the chosen glucose concentration range was exhibited by both carbomer‐stabilised formulations. The subsequent testing of such formulations in in‐vitro diffusion experiments revealed that the leaching of concanavalin A from the covalently coupled gels is restricted significantly with respect to non‐coupled formulations. In addition, insulin delivery in response to glucose in the physiologically relevant glucose concentration range has been demonstrated using the carbomer‐stabilised gels at 37°C. The performance of this self‐regulating drug delivery system has been improved in terms of increased gel stability with reduced component leaching.

A Covalently Stabilised Glucose Responsive Gel Formulation with a Carbopol ® Carrier

A novel glucose-responsive gel formulation was stabilised via covalent coupling to a carbomer resin. The gel formed between the plant lectin, concanavalin A and specific polysaccharides was stabilised to minimise leaching of gel components into the surroundings. This was required to prevent toxicity and to preserve the working mechanism of the formulation. Increased gel stability was introduced by covalently bonding amine groups present on the lysine residues of concanavalin A to carboxylic moieties on Carbopol ® 974P NF using carbodiimide chemistry. The introduction of dextran then produced a glucose-responsive formulation that transformed from gel to sol in the presence of free glucose. The rheological properties and in vitro component and insulin release of the carbomer-stabilised gel were evaluated. A decrease in viscosity over a chosen glucose concentration range was exhibited by the carbomer-based gel. The testing of such a formulation in in vitro diffusion experiments revealed that the leaching of concanavalin A from the covalently coupled gels was restricted significantly with respect to a non-coupled gel. Insulin delivery in response to glucose in the physiologically relevant glucose concentration range was demonstrated using the carbomer-stabilised gel at 37°C. The performance of this novel self-regulating drug delivery system has been improved in terms of increased gel stability with reduced component leaching.