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ADME PK

Log D7.4 shake flask assay

Use the Log D shake flask assay to determine the lipophilicity of your compound.

Log D7.4 shake flask is one of Cyprotex's in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.

Measurement of Log D by the shake flask method as a measure of lipophilicity

  • 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.
  • Log D (distribution co-efficient) is used as a measure of lipophilicity. Determining the partition of a compound between an organic solvent (typically octanol) and aqueous buffer is one of the most common methods for determining this parameter.
  • Cyprotex’s Log D7.4 assay uses the octanol / buffer shake flask method for determining lipophilicity. LC-MS/MS is used to quantify the samples.
A compound with moderate lipophilicity (Log D 0–3) has a good balance between solubility and permeability and is optimal for oral absorption, cell membrane permeation in cell-based assays, is generally good for BBB penetration (optimal Log D ~2) and has low metabolic liability.

1Di L and Kerns EH. (2003) Current Opinion in Chemical Biology 7; 402-408

Protocol

Log D shake flask assay protocol

Method Octanol:buffer shake flask method
Partition Solvent n-Octanol
Ratio of Buffer:Octanol 2:1 (v:v); other ratios available on request
Control Compounds Tolbutamide, ketoconazole
Analysis Method LC-MS/MS quantification of both phases
Data Delivery Log D7.4

Data

Data from Cyprotex's Log D shake flask assay

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

 
Figure 1
Comparison of Log D7.4 values generated in Cyprotex’s Log D7.4 shake flask assay with Log D7.4 values reported in the literature3.

The graph illustrates good correlation of Cyprotex’s Log D7.4 shake flask data with literature data3.
Figure 2
Inter-assay reproducibility for a range of compounds in Cyprotex’s LogD7.4 shake flask assay.

Cyprotex’s Log D7.4 shake flask assay exhibits good reproducibility for compounds over a range of lipophilicity.

Q&A

Please provide an overview of Cyprotex's Log D7.4 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.

Cyprotex’s Log D7.4 assay uses the octanol/buffer shake flask method for determining lipophilicity. Briefly, octanol (pre-saturated with buffer) is added to the test compound and sonicated. Buffer at pH 7.4 (presaturated with octanol) is then added to the octanol. A ratio of 2:1 v/v buffer to octanol is prepared. The system is mixed to allow distribution of the compound between the two phases. After separation, compound is quantified in the aqueous phase and octanol phase by LC-MS/MS and the following equation is used to calculate the Log D7.4.

Where: Coct = Concentration in octanol sample (corrected for dilution)
Caq = Concentration in aqueous sample (corrected for dilution)

How does lipophilicity influence pharmacokinetic properties of a drug?

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 do I interpret the data from the Log D assay?

An optimal range for lipophilicity tends to be if the compound has a Log D 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 Log D for blood brain barrier permeation is approximately 2. Hydrophilic compounds (Log D < 0) typically are highly soluble but exhibit low permeability across the gastrointestinal tract or blood brain barrier. Highly lipophilic compounds (Log D > 5) exhibit problems with metabolic instability, high plasma protein binding and low solubility which leads to variable and poor oral absorption1. Furthermore, high lipophilicity has been shown to increase the likelihood of adverse outcomes in preclinical toxicology studies4.

What range of Log D7.4 values can you measure using Cyprotex's Log D7.4 shake flask assay?

The range of Log D7.4 values which can be measured using the standard Log D7.4 shake flask assay is between -1 and 5, depending on the range of aqueous concentrations measured. In order to ensure the most appropriate range is used in the assay, it is useful if a predicted Log D value is provided. The volume ratio of buffer:octanol used in the assay can also be altered to provide more accurate results in cases where compounds have predicted Log D values at the upper or lower ends of the scale.

A high octanol volume is more accurate for compounds which have Log D < 0, whereas for compounds with a Log D > 3.5, 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.

References

1Di L and Kerns EH. (2003) Profiling drug-like properties in discovery research. Current Opinion in Chemical Biology 7; 402–408
2Kerns EH and Di L (2003) Pharmaceutical profiling in drug discovery. Drug Discovery Today 8(7); 316-323
3Wenlock MC, Potter T, Barton P, Austin RP. (2011) A method for measuring the lipophilicity of compounds in mixtures of 10. Journal of Molecular Screening 16(3); 348-355
4Hughes J et al., (2008) Physicochemical drug properties associated with in vivo toxicological outcomes. Bioorganic and Medicinal Chemistry Letters, 18; 4872-4875

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