To determine stability in plasma, GHP-88309 was incubated in triplicate in pooled human or CD-1 mouse plasma (BioIVT) in glass tubes in a 37°C shaker-incubator (150 rpm). Procaine and benfluorex served as positive controls and were analyzed in parallel. Aliquots were taken after 0, 5, 15, 30, 60 and 120 minute incubation time, mixed with 400 μL of internal standard in acidified (0.1% (v/v) formic acid) acetonitrile solution, clarified by centrifugation and supernatants analyzed by LC-MS/MS. To determine stability in liver microsomes, GHP-88309 was incubated in triplicate in glass tubes in 50 mM Tris-HCl (pH 7.5) buffer with 8 mM MgCl2, 25 μg/mL alamethicin, Phase I and Phase II cofactors (NADPH regenerating system (NRS) and 2 mM UDPGA, respectively) and 0.5 mg total protein pooled mixed gender human liver microsomes (BioIVT) or pooled CD-1 mouse liver microsomes (Xenotech) in a 37°C shaker incubator (150 rpm). Verapamil served as Phase I positive control, negative controls lacked cofactors and contained 2% (w/v) NaHCO3 in water instead of NRS. Aliquots were taken after 0, 15, 15, 30, 60 and 120 minutes and processed as outlined above.
Cd 1 mouse liver microsomes
Manufactured by Xenotech
CD-1 mouse liver microsomes are a preparation of subcellular fractions derived from the livers of CD-1 strain mice. They contain the endoplasmic reticulum and associated enzymes, including cytochrome P450 enzymes, which are involved in the metabolism of various compounds.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using cd 1 mouse liver microsomes
1
Stability of GHP-88309 in Plasma and Microsomes
2
Metabolic Stability Assay in Liver Microsomes
3
Stability of GHP-88309 in Plasma and Microsomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine stability in plasma, GHP-88309 was incubated in triplicate in pooled human or CD-1 mouse plasma (BioIVT) in glass tubes in a 37°C shaker-incubator (150 rpm). Procaine and benfluorex served as positive controls and were analyzed in parallel. Aliquots were taken after 0, 5, 15, 30, 60 and 120 minute incubation time, mixed with 400 μL of internal standard in acidified (0.1% (v/v) formic acid) acetonitrile solution, clarified by centrifugation and supernatants analyzed by LC-MS/MS. To determine stability in liver microsomes, GHP-88309 was incubated in triplicate in glass tubes in 50 mM Tris-HCl (pH 7.5) buffer with 8 mM MgCl2, 25 μg/mL alamethicin, Phase I and Phase II cofactors (NADPH regenerating system (NRS) and 2 mM UDPGA, respectively) and 0.5 mg total protein pooled mixed gender human liver microsomes (BioIVT) or pooled CD-1 mouse liver microsomes (Xenotech) in a 37°C shaker incubator (150 rpm). Verapamil served as Phase I positive control, negative controls lacked cofactors and contained 2% (w/v) NaHCO3 in water instead of NRS. Aliquots were taken after 0, 15, 15, 30, 60 and 120 minutes and processed as outlined above.
4
In Vitro Microsomal Clearance Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Test compound (0.5 μM) was incubated with female CD1 mouse
liver microsomes (Xenotech LLC; 0.5 mg/mL 50 mM potassium phosphate
buffer, pH 7.4) and the reaction started with addition of excess NADPH
(8 mg/mL 50 mM potassium phosphate buffer, pH 7.4). Immediately, at
time zero, then at 3, 6, 9, 15, and 30 min, an aliquot (50 μL)
of the incubation mixture was removed and mixed with acetonitrile
(100 μL) to stop the reaction. Internal standard was added to
all samples, the samples centrifuged to sediment precipitated protein,
and the plates then sealed prior to UPLCMSMS analysis using a Quattro
Premier XE (Waters Corporation, USA). XLfit (IDBS, UK) was used to
calculate the exponential decay, and consequently the rate constant
(k) from the ratio of peak area of test compound
to internal standard at each time point. The rate of intrinsic clearance
(Cli) of each test compound was then calculated using the
equation Cli (mL/min/g liver) = k × V × microsomal protein yield, where V (mL/mg protein) is the incubation volume/mg protein added and microsomal
protein yield is taken as 52.5 mg protein/g liver. Verapamil (0.5
μM) was used as a positive control to confirm acceptable assay
performance.
liver microsomes (Xenotech LLC; 0.5 mg/mL 50 mM potassium phosphate
buffer, pH 7.4) and the reaction started with addition of excess NADPH
(8 mg/mL 50 mM potassium phosphate buffer, pH 7.4). Immediately, at
time zero, then at 3, 6, 9, 15, and 30 min, an aliquot (50 μL)
of the incubation mixture was removed and mixed with acetonitrile
(100 μL) to stop the reaction. Internal standard was added to
all samples, the samples centrifuged to sediment precipitated protein,
and the plates then sealed prior to UPLCMSMS analysis using a Quattro
Premier XE (Waters Corporation, USA). XLfit (IDBS, UK) was used to
calculate the exponential decay, and consequently the rate constant
(k) from the ratio of peak area of test compound
to internal standard at each time point. The rate of intrinsic clearance
(Cli) of each test compound was then calculated using the
equation Cli (mL/min/g liver) = k × V × microsomal protein yield, where V (mL/mg protein) is the incubation volume/mg protein added and microsomal
protein yield is taken as 52.5 mg protein/g liver. Verapamil (0.5
μM) was used as a positive control to confirm acceptable assay
performance.
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