A physicochemical profile is important for predicting drug absorption, disposition and transport. This involves measuringa number of properties which many laboratories are ill-equippedto handle. One solution is to use contract analytical services.
Most pharmaceutical and biotech companies now use combinatorial chemistry to synthesise large numbers of new chemical entities.
New methods have been developed for early-stage pharmacokinetic tests, to help decide which molecules have the properties required to make them efficacious and successful in the marketplace. The highest throughput is attained using computational methods, which predict important pharmacokinetic properties.
High throughput measurements are also important. Measured pKa values aid the interpretation of solubility, lipophilicity and permeability measurements. The likelihood of a molecule being absorbed by passive transport can be successfully assessed.
One important area of chemical analysis is tools for predicting drug absorption, disposition and transport. However, prediction may not be accurate, and there is a need to measure properties. In the fields of logP, solubility and pKa however, some laboratories do not have instruments in situ for their measurement.
The instruments needed for such physicochemical profiling can be purchased from UK-based manufacturer Sirius Analytical, innovators of tools for compound analysis. For those laboratories where justification to purchase such an instrument is difficult to quantify, or who may not have suitably trained staff to carry out the analysis, the company offers contract analytical services for physicochemical profiling.
The analysis is carried out in the UK, and is offered to customers worldwide. PH-metric technology, solution equilibria, UV spectroscopy, chemometrics, lipophilicity, solubility and permeability are particular analytical strengths.
The important properties
Why are pKa, log P, solubility and permeability important in pharmaceutical science anyway (see Glossary section at the end of the article).
Most drug molecules ionise in solution; their pKa value/s denote the pH at which ionisation occurs. Lipophilicity, solubility and permeability are all pKa-dependent for ionisable drugs. pKa is important for understanding how a charged drug interacts with a receptor. When drugs metabolise they form ionised, hydrophilic metabolites, which are excreted at physiological pH. pKa is important in drug formulation for choice of counter-ion and excipient. In addition, pKa must be measured for regulatory compliance.
For their part, partition coefficients are a measurement of lipophilicity. Many drugs have a logP value of between one and four, making them suitable for oral methods of delivery. The term LogD is used for ionisable drugs. Measured logP values are used at several stages during drug design, development and formulation, and to satisfy regulatory requirements. LogP values are also widely used in the science of QSAR.
A measure of solubility required at several stages of drug development. These include during HTS, to screen for very insoluble molecules, and during formulation, to decide optimal drug delivery route and optimise formulation for regulatory purposes.
In terms of permeability, the technique of PAMPA permeability measurement using artificial membranes is currently arousing a lot of interest as a way of assessing how easily molecules will pass through membranes.
A contract analytical service
Sirius offers a contract analytical service that makes use of all these techniques. It is also fully confidential, with experienced operators providing quick turn-round times (714 days for standard analyses) and comprehensive analysis reports.
A recent investment in a larger analytical laboratory has extended the services offered. Many customers often make use of these before purchasing their own instruments.
To ensure reproducibility, all work is carried out under strictly controlled conditions. All calibrations and reagent standardisations are recorded. Assay results are quoted with reference to all relevant calibrations, standardisations, instrument identification, and operator identification. All relevant documentation is available for audit.
The range of techniques offered by the company can be divided into four categories:
* logP: pH-metric, liquid-liquid chromatography.
* pKa: rapid screening, pH-metric, UV spectroscopic.
* Solubility: pH-metric, intrinsic, thermodynamic.
* Permeability: PAMPA.
Neutral and ionic logP is determined using Sirius GLpKa. A sample must contain at least one pKa. Octan-1-ol is the standard partition solvent, with alternative solvents attracting a surcharge. LogP values between -1 and 5 are reported as standard a surcharge applies to values outside this range.
Aqueous pKa(S) is determined using Sirius GLpKa too. A titration range of pH2 to 12 is offered, with pH3 to 11 as the standard working range. Samples must contain one or more groups which ionise within the titration range. CoSolvent assays are performed for samples with a solubility of <0.25mM, with aqueous pKa value(s) derived from a Yasuda-Shedlovsky extrapolation.
Aqueous pKa(s) can also be determined using GLpKa fitted with the Sirius D-PAS spectroscopy module. For this, a sample must contain a UV-active chromophore, no more than 34 bond lengths from the ionisable group. The working range here is from pH1.3 to 12 and sample concentrations in the range 0.5 to 0.005mM are used. Determination of kz (the tautomerisation constant) is also available. CoSolvent assays can be performed for sampleswith low aqueous solubility (<0.005mM), with aqueous pKa value(s) derived from aYasuda-Shedlovsky extrapolation.
Rapid screening for pKa and log D is another service. Here aqueous pKa(s) determined using Sirius ProfilerSGA and Log D (octanol-water) determined using Sirius ProfilerLDA. Rapid screening is achieved on minimal amounts of compound, although a sample must contain a UV active chromophore no more than 3-4 bond lengths from the ionisable group. This service supports solids and solutions, for example 10mM in DMSO and pre-prepared 96-well microtitre plates.
Aqueous intrinsic solubility is determinedpH-metrically using pSOL-3. A sample must contain one or more group which ionises within the titration range and accurate pKa values are required. The titration range is from pH1.7 to 12. CoSolvent titrations are performed for samples with an intrinsic solubility <4ug/mL. Both aqueous and CoSolvent intrinsic solubilities reported.
Aqueous thermodynamic solubility can be determined, but the sample must contain aUV-active chromophore. Solids and solutions are supported, as for the rapid screening service. Assays can be performed at any pH between 3 and 10 (this can be specified at time of order).
Passive permeability through an artificial phospholipid membrane is also possible. The assay is a modified version of the PAMPA approach and the sample must contain a UV-active chromophore. Solids and solutions are again supported, while assays are performed in the same pH range as for aqueous thermodynamic solubility.
To use its analytical testing service, all customers have to do is simply complete the relevant assay questionnaire from the range provided and send it in with the sample. Any unused sample can be returned upon completion of analysis.
Glossary
* pH expresses the degree of acidity of a solution.
* pKa represents the state of ionisation of a substance in solution.
* logP represents the ratio between the concentration of a substance dissolved in oil or fat and the concentration dissolved in water. LogP is used to predict absoption and is pKa dependent.
* The solubility of a substance is the concentration of a substance dissolved in water in a saturated solution. Solubility is used to predict bioavailability and is pKa dependent.
Sirius Analytical Instruments Limited is based in Sussex, England. For more information, tel: +44 (0)1342 820720or email sirius@sirius-analytical.com
