pK_a and log P

pK_a and log P protocol

Method Potentiometric titration Instrument GLpKa (Sirius Analytical Instruments Ltd) Compound Requirements 10 mg solid compound Partition Solvent used for Log P Determination n-Octanol (others available on request) Data Delivery pKa Log P (optional) Standard error Goodness of fit Calculated log D at pH7.4 (based on pKa and log P)

Log P is determined by measuring the pKa in the presence of increasing concentrations of the partition solvent, octanol.

Data from Cloe Select pK_a and log P Determination

pK_a measurements are determined using the GLpKa instrument from Sirius-Analytical.

Figure 1

Titration curves for acebutolol in the absence (Figure 1a) and presence
(Figure 1b) of octanol.

The GLpKa automatically performs pH-metric titrations. A blank acid-base titration is compared to a titration in the presence of the compound. A difference curve is produced from the volume of titrant required to reach a given pH with and without the compound.

 

Figure 2

Bjerrum plot for acebutolol in the presence of increasing concentrations
of octanol.

In this example, increasing the concentration of octanol reduces the pK_a confirming the presence of a basic pK_a for acebutolol.

 

The titration curve is converted to a Bjerrum plot. The axes are reversed and the volume difference is converted to a unit of ñH; the average number of bound protons per molecule of compound. The pK_a is the pH at which the molecule is 50 % protonated. Titrations are performed in the presence of increasing concentrations of the partition solvent (n-octanol). Log P is calculated from shifts in the apparent pK_a that occur in the presence of the partition solvent. From the measured pK_a and log P values, the log D value can be calculated as a function of pH.

back to top

Questions and answers on pK_a and log P determination

The pK_a of a molecule predicts the degree of ionisation the molecule will have at a particular pH. LogP (partition coefficient) is a measure of the lipophilicity of a compound.

pK_a measurements are determined using the GLpKa instrument from Sirius-Analytical. The GLpKa automatically performs pH-metric titrations. A blank acid-base titration is compared to a titration in the presence of the compound. A difference curve is produced from the volume of KOH required to reach a given pH with and without the compound.

The difference curve is converted to a Bjerrum plot. The axes are reversed and the volume difference is converted to units of ñ_H; the average number of bound protons per molecule of compound. The pK_a is the pH at which the molecule is 50 % protonated.

Figure 3

Graphs showing the titration curves with and without sample, the difference curve and the Bjerrum plot.

 

 

Titrations are then performed in the presence of varying amounts of partition solvent. Log P is calculated from shifts in the apparent pK_a(s) that occur in the presence of partition solvent. Typically, n-octanol is used for lipophilicity measurements although alternative solvents may be used if required. From the measured pK_a and logP values, the logD value can be calculated as a function of pH, typically LogD_7.4 is reported with the results.

Why is the pK_a important?

Most drugs are weak acids or weak bases and exist in solution as an equilibrium between unionised and ionised forms. The ionisation 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 unionised nonpolar drugs penetrate the cell membrane, and at equilibrium, the concentrations of the unionised species are equal on both sides. The pK_a of a compound influences properties such as logD and solubility as well as the absorption, distribution, metabolism, elimination and potency of a compound. pK_a 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 pK_a 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 pK_a 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 ionised at alkaline urinary pH and basic drugs are ionised at acidic urinary pH. Only unionised compounds in the tubular fluid will be reabsorbed by passive diffusion⁴.

How does lipophilicity influence pharmacokinetic properties of a drug?

Lipophilicity is a key determinant of the pharmacokinetic behaviour 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 pK_a and LogP?

Approximately 10 - 20 mg of solid compound is required to perform a measurement using the GLpKa. Sufficient sample is required to produce a measurable buffering effect and hence good quality data, particularly if extreme pK_as are being measured (<4 and >10), or the compound has several overlapping pK_as. Concentrations as low as 0.1mM (mol wt 200 g/mol) may be sufficient to measure mid-range pK_as. However, concentrations of at least 2.5mM would be required to reliably measure extreme pK_as. If aqueous solubility is limiting then a co-solvent is used; three different titrations are carried out at different percentages of co-solvent and the 100% aqueous pK_a(s) found by extrapolation.

What are the limits to the pK_a you can measure?

The pH electrode used on the GLpKa can measure the pH in the range 1.2 to 12, any pK_a outside this range can not be measured by this technique.

A turbidity probe is deployed throughout the pK_a titrations to evaluate if precipitation is evident. 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.

What co-solvent systems are supported?

When performing a pK_a in the presence of co-solvent the result is termed a psK_a, where s stands for solvent. The GLpKa uses organic co-solvents mixed with different percentages of ISA (ionic strength adjusted) water to help in the dissolution of the test compound. The presence of the co-solvent inhibits ionisation due to the lower dielectric constants of the system and shifts the pK_a(s) up for acidic pK_a’s and down for basic pK_a’s. Due to the impact on the standard electrode parameters, only solvents supported by the software can be used. The solvents supported are listed in Table 1 along with information on dissolving powers, weight percentages and potential errors associated with each solvent.

Table 1: Table to show the co-solvent systems supported by the GLpKa software with useful information on each system.

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 pK_a of my compound before you can determine the LogP?

The GLpKa uses the shift in the apparent pK_a in the presence of the partition solvent, typically n-octanol, to determine the LogP therefore to assess the shift a measured pK_a values is needed.

Figure 4a shows a typical multiset pK_a difference curve where the blue and red plots represent two separate titrations. Figure 4b shows the same difference curve in the presence of 3 different volumes of n-octanol, showing the shift in pK_a in the presence of 3 different volumes of the partition solvent. The solid dark blue line represents the pK_a curve from figure 4a. The downward shift in the pK_a represents a basic compound. If the logP was measured for an acidic compound the apparent shift would be upwards.

What does the goodness of fit and error values represent?

The goodness of fit represents the quality of the refined result of a single data set. The closer to zero the value the better the data fit is. The goodness of fit is based on a number of parameters as detailed below:

• Experimental uncertainty in the measurement of the pH, related to the absolute accuracy of the pH meter, and the volume of titrant dispensed which is related to the absolute accuracy of the dispenser.
• The difference between pH^calc and pH^obs. A weighting scheme is used which focuses the difference towards the area of the proposed pK_a (or logP) where the titration is buffered by the compound.

Typically, a number of compatible single data sets (N ≥ 2) are used to create a multiset. The goodness of fit of the multiset is based on the goodness of fit of each individual data set. An acceptable goodness of fit for a multiset pK_a or logP refinement would be less than 10.

The error value is an estimate of the precision of the refinement process, and is, in part, based on the goodness of fit. For acceptable data, the error value should be below 0.1.

back to top