Understand the physicochemical properties of your compound by using our pKa and log P service.
The determination of pKa and log P is offered within Cyprotex's in vitro experimental services. Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.
pKa affects solubility, permeability, log D and oral absorption by modulating the distribution of neutral and charged species.
1Di L and Kerns EH. (2003) Current Opinion in Chemical Biology 7; 402-408
|Method||Fast UV titration for pKa
UV-metric titration for pKa
Potentiometric (pH-metric) titration for pKa and log P
|Test Article Requirements per Titration||3-5 µL of 10mM stock solution (UV-metric)
1 mg solid compound (pH-metric)
|Partition Solvent used for log P||n-Octanol (others available on request)|
log P (optional)
Calculated log D at pH7.4 (based on pKa and log P)
pKa measurements are determined using the SiriusT3 instrument from Sirius-Analytical using either a UV-metric or pH-metric approach.
Why is the pKa important?
The pKa of a molecule predicts the degree of ionization the molecule will have at a particular pH.
Most drugs are weak acids or weak bases and exist in solution as an equilibrium between unionized and ionized forms. The ionization potential of a compound affects the distribution of the chemical in solution and affects the availability of the chemical to enter into physical, chemical and biological reactions. According to the pH partition hypothesis, only unionized nonpolar drugs penetrate the cell membrane, and at equilibrium, the concentrations of the unionized species are equal on both sides. The pKa of a compound influences properties such as logD and solubility as well as the absorption, distribution, metabolism, elimination and potency of a compound. pKa can be used in conjunction with other in vitro parameters to predict the pharmacokinetics of a compound using the simulation software, Cloe® PK.
How does pKa affect other pharmacokinetic parameters?
Solubility – Acidic compounds tend to be more soluble at high pH values, and basic compounds tend to be more soluble at low pH values.
Permeability – Acidic compounds tend to be less permeable at high pH and basic compounds tend to be less permeable at low pH.
Metabolism – Electrostatic interactions are determined by the pKa of a compound. These interactions can affect binding of the compound to the active sites of enzymes. For example, nitrogen containing bases where the basic nitrogen is 5-7Å from the site of metabolism has been shown to be important in the metabolism of compounds by CYP2D62.
Protein binding – Binding of drugs to plasma proteins tends to be by hydrophobic and electrostatic interactions. Typically, acidic compounds with moderate lipophilicity are more likely to bind to serum albumin whereas basic compounds with moderate lipophilicity are more likely to bind to α1-acid glycoprotein3.
Excretion – Urinary pH is an important factor in the excretion of a drug. For example, acidic drugs are ionized at alkaline urinary pH and basic drugs are ionized at acidic urinary pH. Only unionized compounds in the tubular fluid will be reabsorbed by passive diffusion4.
How does lipophilicity influence pharmacokinetic properties of a drug?
LogP (partition coefficient) is a measure of the lipophilicity of a compound.
Lipophilicity is a key determinant of the pharmacokinetic behavior of drugs. It can influence distribution into tissues, absorption and the binding characteristics of a drug, as well as being an important factor in determining the solubility of a compound.
How much compound will I need to measure pKa and log P?
Although smaller weights are generally required for pKa and log P assays on the SiriusT3, we normally ask customers to send at least 5 mg of solid compound per parameter measured. If only pKa is required, and if it can be measured by a UV-metric technique, then aliquots of sample in stock solution (e.g., 10 mM in DMSO) can be used.
What are the limits to the pKa and log P you can measure?
pH-metric pKa: The standard range is 2.5 to 11.5. Lower or higher pKas can be measured if the sample is sufficiently soluble.
Fast UV pKa: 2.0 to 12.0
UV-metric pKa: 1.0 to 13.0
pH-metric log P: -0.5 to +5.5. The higher limit may be reduced for bases with pKas below 4 or acids with pKa above 10.
Samples must be in solution during pKa measurement. Turbidity is monitored throughout pH-metric pKa titrations to give warning of precipitation. If the compound is insoluble then it may be necessary to perform the measurements either at a lower concentration or in the presence of a co-solvent. Knowledge of the aqueous solubility of the sample helps in designing the experiment and avoids unnecessary use of the compound in repeat experiments. Information regarding compatible organic solvents is also useful.
How do you measure the pKa values of poorly soluble samples?
pKa of poorly soluble samples is determined by measuring psKa values in water-solvent mixtures, and extrapolating to aqueous conditions. Three titrations are done consecutively in the same vial. Yasuda-Shedlovsky is the standard extrapolation method, in which the X-axis plots the inverse of the dielectric constant of the water-solvent mixture at the experimental percentage of solvent. This example shows pKa of Fluoxetine determined from three UV-metric titrations in methanol-water. The pKa is equivalent to the intercept at 0% solvent minus log[H2O] (1.75 for pure water), providing a result of 9.88 for this sample.
Do I need to provide the structure of the compound?
Providing the structure is preferable as it helps design the experiment (i.e., pH range and direction of the titrations and weight of compound). It also helps in the data refinement process and interpretation of the data. If the structure is not provided then information on functional groups is required.
Why do you need to measure the pKa of my compound before you can determine the log P?
When samples are titrated in the two-phase water-octanol system on the SiriusT3, the data generated comprises a set of pH readings vs. volumes of titrant. The log P is determined after calculating each pH value to simulate those measured. One of the parameters required in these calculations is the pKa value.
1 Di L and Kerns EH. (2003) Current Opinion in Chemical Biology 7; 402-408
2 Lewis DF. (2000) Biochem Pharmacol 60; 293-306
3 Urien S et al. (2001) In Pharmacokinetic optimisation in drug research Ed. Testa et al 189-197
4 Timbrell JA. (1991) In Principles of Biochemical Toxicology 65-67