Log D_7.4 shake flask assay

Log D shake flask assay protocol

Method Miniaturised shake flask method Partition Solvent n-Octanol Ratio of Buffer:Octanol 50:1, 5:1, 1:2 (v/v) Positive Control Compounds Acebutolol, Ketoconazole Compound Requirements 1 mg solid compound in >10 mL vial Analysis Method LC-MS/MS quantification Data Delivery Log D7.4

Increasing lipophilicity of a compound series generally increases permeability, protein binding and volume of distribution, and decreases solubility and renal extraction2.

Data from the Cloe Screen log D Shake Flask assay

The log D_7.4 values generated by the Cloe Screen shake flask method are consistent with data generated from the GLpKa (calculated from log P and pK_a), which is considered to be the gold standard for these measurements.

Figure 1

Data generated for acebutolol and ketoconazole in the Cloe Screen
Log D_7.4 Shake Flask assay.

The graph illustrates reproducibility of the Cloe Screen Log D_7.4 Shake Flask assay over 17 consecutive experiments for the two control compounds, acebutolol and ketoconazole.

 

Figure 2

Comparison of Cloe Screen Log D_7.4 Shake Flask data with Log D_7.4 calculated from the GLpKa.

 

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Questions and answers on log D_7.4 shake flask

Please provide an overview of the Cloe Screen Log D_7.4 Shake Flask assay.

Log D (distribution co-efficient) is used as a measure of lipophilicity. One of the most common methods for determining this parameter is by measuring the partition of a compound between an organic solvent (typically octanol) and aqueous buffer.

Cloe Screen LogD_7.4 uses the octanol/buffer shake flask method for determining lipophilicity. Octanol is added to a solution of the test compound prepared in buffer at pH 7.4. Three different ratios of octanol to buffer are prepared. The system is mixed to allow distribution of the compound between the two phases. After separation, compound is quantified in the aqueous phase by LC-MS/MS and the following equation is used to calculate the logD.

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 do I interpret the data from the logD assay?

An optimal range for lipophilicity tends to be if the compound has a logD value between 0 and 3. Typically, these compounds have a good balance between solubility and permeability and this range tends to be optimal for oral absorption and cell membrane permeation. The optimal logD for blood brain barrier permeation is approximately 2. Hydrophilic compounds (logD <0) typically are highly soluble but exhibit low permeability across the gastrointestinal tract or blood brain barrier. Highly lipophilic compounds (logD > 5) exhibit problems with metabolic instability, high plasma protein binding and low solubility which leads to variable and poor oral absorption¹.

Why do you prepare three different ratios of octanol/buffer?

By varying the volume ratios of octanol and aqueous, a wider range of lipophilicity should be covered.  Results may be obtained from all three vials or just one, depending on the degree of lipophilicity and analytical sensitivity of the compound.

The vial containing a high octanol volume is more accurate for compounds which have low logD, whereas for compounds with high logD a vial with a low volume of octanol is more likely to yield a more accurate result. This is due to the differences in the distribution of the compound between the two phases.

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