## Studies on the **in vitro** Metabolism of Deltamethrin by Rat and Human Tissue Preparations Brian G. Lake LFR Molecular Sciences and Centre

for Toxicology, University of Surrey

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## Outline of Presentation
• Purpose • Study design • Results • Follow- up - hepatocyte and liver microsome/cytosol studies • Implications of Results **Acknowledgements.** Miyoung Yoon ( Hamner Institutes) and Brian Houston (University of Manchester) Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 2

# Purpose

• PBPK modelling needs intrinsic clearance (CLint) values for human tissues and Km and Vmax values for rat tissues • Compare current findings with literature reports

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## Determination of Rates of DLM Metabolism by Rat and Human Liver Preparations

• Phase I metabolism of DLM and other pyrethroids is by cytochrome P450 (CYP) and/or carboxylesterase (CES) enzymes ● 

### Species studied

– Human • Liver microsomes (CYP and CES) • Cytosol (CES)



### – Rat • Liver microsomes, cytosol and plasma (CES enzymes).

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## Determination of Rates of DLM Metabolism by Rat and Human Liver Preparations

• DLM metabolism by substrate depletion

– Monitoring loss of parent pyrethroid from tissue incubations at various times. Determine intrinsic clearance (CLint) values at low (e.g.1 μM) substrate concentration.

– Dissolve substrate in 50% acetonitrile (1% solvent).

– Perform incubations in glass vials to avoid binding to plastics. Stop with acetonitrile containing bifenthrin internal standard.

– Analyse pyrethroids by Waters Acquity UPLC linked to Waters Xevo TQ mass spectrometer (UPLC- MS- MS). Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 5

### DLM Metabolism with Rat and Human Liver Microsomes and Cytosol - Results
• Species differences confirmed

– Human liver - mainly by CES

– Rat liver –both CYP and CES 

### • CES are present in liver microsomes and cytosol

– Determination of rates of metabolism with microsomes alone will underestimate actual hepatic clearance. 

### • Nonspecific binding to liver microsomes and cytosol.

– Needs to be taken into account to obtain correct kinetic data – need to determine fraction unbound (fu)

– Allowing for nonspecific binding results in higher CLint values and different Km values compared to previous data. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 6

### Comparison of EPA and LFR Data on DLM Metabolism in Rat and Human Liver Microsomes

• Godin et al. (2006) utilised rat and human liver microsomal protein concentrations of 1 mg protein/ml incubation mixture. • Reported scaled CLint values expressed as ml/min/kg body weight converted to ml/min/mg protein using scaling factors (liver microsomal protein content and liver weight per kg body weight) reported by Godin et al . (2006). • LFR studies performed with pooled male SpragueDawley rat and commercially obtained human (150 donor pool) liver microsomes. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 7

### DLM Metabolism in Rat and Human Liver Microsomes: Comparison of EPA and LFR Data Liver microsomes Apparent CLint (ml/min/mg protein EPA LFR Rat 0.077 0.040 (52%) Human 0.077 0.174 (226%) Determined at a microsomal protein concentration of 1.0 mg/ml in the presence of NADPH (i.e. CYP and CES metabolism) with a DLM substrate concentration of 1 μM. **Note:** EPA data of Godin et al. (2006) obtained with Long- Evans and not with Sprague- Dawley rats and with different human liver microsomal preparations. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 8

## Issues Concerning Kinetics of DLM Metabolism in Rat Liver

• Corrected CLint values for DLM metabolism by rat liver microsomes and cytosol are higher than those previously reported values predicted by the current Hamner DLM PBPK models – need to reconcile • Hypothesis: Rate limiting step in the clearance by metabolism of DLM in vivo may be the rate of uptake into hepatocytes.

– Example: Saquinavir the rate of metabolism in microsomes at low substrate concentrations is greater than the rate of metabolism in hepatocytes (Parker and Houston (2008) Drug Metab Dispos 36 , 1375- 1384). Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 9

### Studies to Resolve Issues Concerning Kinetics of DLM Metabolism in Rat Liver

• Obtained cryopreserved adult (3 month old) male Sprague- Dawley rat hepatocytes from Triangle Research Labs ( USA) . • Compared rates of DLM metabolism in hepatocytes with two preparations of 90 day old male SD rat liver microsome and cytosol fractions . Rat livers supplied by Charles River from both USA (designated CRL) and previously from UK (designated LFR). • Performed hepatocyte incubations in glass vials using an Eppendorf ThermoMixer C shaker/incubator.

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### Studies to Resolve Issues Concerning Kinetics of DLM Metabolism in Rat Liver

• Studies conducted at a substrate concentration of 1 μM over a range of rat hepatocyte concentrations.

– Assumption: only viable cells (based on Trypan blue exclusion) can metabolise DLM, but nonspecific binding of DLM will occur with both viable and non viable hepatocytes.

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## DLM Metabolism in Rat Hepatocytes: Effect of Cell Concentration

1 μM DLM. Adult male SD rat hepatocytes 0.018- 0.242 million total cells/ml incubation. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 12

### Kinetics of DLM Metabolism in Rat Hepatocytes, Liver Microsomes and Cytosol

• Obtained apparent CLint values for DLM metabolism in rat hepatocytes and liver microsomes and cytosol over a range of protein concentrations. • Calculated unbound CLint and affinity constant ( Ka) with the equation: CLint (apparent) = CLint 1 + Ka.P Where P is incubation cell or protein concentration • Calculated fraction unbound (fu) values with the equation: fu = 1 1 + Ka.P Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 13

### Rates of DLM Metabolism in Rat Hepatocytes and Liver Microsomes and Cytosol

• Compared rates of metabolism of 0.05, 0.1, 0.3 and 1 μM DLM in hepatocytes, microsomes (with NADPH) and cytosol. • Experimentally observed CLint values were corrected by allowing for the fraction unbound (fu), by dividing apparent CLint by the fu value.

– The corrected (unbound) CLint values were scaled to per gram of liver

– The data for liver microsomes and cytosol were summed to compare with hepatocytes. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 14

### Determination of Rates of DLM Metabolism in Rat Hepatocytes and Liver Microsomes and Cytosol Tissue preparation Incubation conditions Affinity constant (Ka) Fraction unbound (fu) Scaling factor per gram of liver* Hepatocytes 0.1 x 106 viable cells/ml 3.8583 0.722 120 million Microsomes 0.25 mg protein/ml 7.7635 0.340 45 mg protein Cytosol 0.4 mg protein/ml 7.6466 0.246 90 mg protein *Data from Houston and Galetin (2008) Curr Drug Metab 9 , 940-951. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 15

### Effect of Substrate Concentration on Rates of DLM Metabolism in Rat Hepatocytes Parameter DLM substrate (μM) 0.05 0.1 0.3 1 Apparent CLint* 0.328 0.291 0.234 0.172 Unbound CLint* 0.454 0.404 0.324 0.239 Scaled CLint** 54.48 48.44 38.93 28.64 Results are mean values (n=3 experiments) *Units ml/min/1 x 106 viable cells. **Units ml/min/gram liver. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 16

### Comparison of Scaled Rates of DLM Metabolism in Rat Hepatocytes with Liver Microsomes and Cytosol Tissue fraction DLM substrate (μM)* 0.05 0.1 0.3 1 Hepatocytes 54.48 48.44 38.93 28.64 CRL microsomes 57.47 52.91 49.12 40.04 CRL cytosol 4.64 8.98 11.25 7.98 CRL sum 62.11 61.89 60.37 48.02 LFR microsomes 59.72 47.78 36.22 27.88 LFR cytosol 10.63 10.05 13.22 7.75 LFR sum 70.34 57.83 49.44 35.63 *Units ml/min/gram liver. Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 17

### Comparison of Scaled Rates of DLM Metabolism in Rat Hepatocytes with Liver Microsomes and Cytosol Tissue fractions DLM substrate (μM) 0.05 0.1 0.3 1 Hepatocytes* 54.48 48.44 38.93 28.64 CRL sum** 1.14 1.28 1.55 1.68 LFR sum** 1.29 1.19 1.27 1.24

*Units ml/min/gram liver. **Fold hepatocytes (i.e. sum of rates liver microsomes and cytosol divided by rate in hepatocytes). Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 18

### Comparison of Scaled Rates of DLM Metabolism in Rat Hepatocytes with Liver Microsomes and Cytosol
• No marked difference between rates of DLM metabolism in rat hepatocytes compared to liver microsomes and cytosol

– Thus rates of DLM metabolism can be estimated from CYP- and CES-dependent activities in liver microsomes and cytosol with no further contribution from other enzymes (e.g. phase II conjugative enzymes) present in intact hepatocytes. 

### • Limitation of uptake into hepatocytes does not provide an explanation for the higher rates of DLM metabolism observed in LFR studies in rat tissue Exclusive Property of the Council for the Advancement of Pyrethroid Human Risk Assessment, LLC (CAPHRA) 19

# Implications of Results

• Reconciliation of differences in metabolic clearance necessitates other explanation and accommodation in the PBPK model

– To be discussed by Dr. Yoon

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