Drug development
No standard like a gold standard Invitrogen™ hepatic biology products and services
Drug development
Invitrogen hepatic biology products for drug metabolism and safety
GIBCO® quality—every step of the way
Physiologically relevant in vitro systems, such as primary hepatocytes and liver subcellular fractions, can be used to address a wide array of research ques-
As hepatic scientists in a company that also offers in vitro ADME screening and development services, we deeply understand the importance
tions related to in vivo applications, including those related to xenobiotic metabolism, drug–drug interactions, and cytotoxicity:
of a high quality, reliable supply chain and stringent characterization methods. We offer:
Cytochrome P450 (P450) inhibition and enzyme induction
A robust, extensive tissue procurement network
Metabolic profiling and stability
Carefully honed isolation techniques
Transporter applications
Rigorous quality control standards
At Invitrogen, we strive to offer you industry-leading hepatic products that provide physiologically relevant results, so you can make the right decisions
Ongoing scientific research and development
in relation to your compounds. We are a team of hepatic scientists helping fellow scientists—supporting IND and NDA submissions with our products
Hepatic product specialists to lend technical support
and services, and participating in the advancement of enzyme induction, P450 inhibition, hepatic transport and other metabolism-related research fields. Supplying you with the right tools and technical support is our goal.
Ordering and technical support for ADME/Tox products and services We have dedicated staff to support our line of ADME/Tox products and services—from pre-purchase inquiries, to shipment of your order, though
Complete selection of
hepatic cell products:
Cryopreserved primary hepatocytes Freshly isolated primary hepatocytes (Americas and Europe) Hepatic microsomes Hepatic S9 and cytosol fractions Culture medium Our comprehensive offering includes hepatocytes and subcellular fractions isolated from human, rat, mouse, dog, rabbit, nonhuman primate, trout, and
troubleshooting of experiments. Contact Invitrogen’s ADME/Tox Product Support to: Place and track orders Get assistance with protocols Select hepatic products or particular lots
Americas, Middle East, and Asia Pacific (excluding Japan)
Europe
Orders can be placed through Invitrogen or CellzDirect.
Orders can be placed with your local Invitrogen office.
Email:
[email protected]
Email:
Phone (toll):
+1 919 237 4679 (Toll)
Phone: +44 (0) 141 814 5900
other species upon request. Our products are prequalified for particular research applications, and most are supplied as pooled or single donors, often in
Phone (U.S. toll free): +1 866 952 3559 (U.S. Toll-free)
large lot sizes to facilitate multiple and multi-site experiments.
Fax (toll):
+1 919 237 4600 (Toll)
Fax (U.S. toll free):
+1 866 902 3559 (U.S. Toll-free)
Fax:
[email protected] +44 (0) 141 626 6954
Japan Please direct order inquiries to the Invitrogen Japan office. Email:
[email protected]
Phone: +81 3 5566 6160 Fax:
+81 3 5566 6502
Address: 4-5-4 Hatchobori, Chuo-ku Tokyo 1040032 Japan
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Drug development
Applications table Application
Purpose
FDA guidance*
Plateable hepatocytes
Suspension hepatocytes
Liver microsomes
Liver S9 fractions
Other
Cat. No.
Drug development research (ADME, DMPK, toxicology) Enzyme induction
Enzyme inhibition
Hepatotoxicity
Determine if a compound has the potential to induce hepatic enzymes
Yes
Determine if a compound has the potential to inhibit hepatic enzymes
Yes
Determine if a compound and its metabolites have the potential to be hepatotoxic
+++ ++
+++
Yes
+++
+++
Metabolic profiling
Determine which enzymes metabolize a compound
Metabolic stability
Measure the disappearance of a compound in the presence of metabolizing enzymes
Yes
++
Reaction phenotyping (metabolic identification)
Identify the metabolites formed from a compound when exposed to metabolizing enzymes
Yes
++
Yes
+++
+++
+
+++
+++
+
Recombinant P450 enzymes
+++
Transporter uptake
Determine if a compound has the potential to inhibit or induce liver transporter uptake
No
+++
+++
Transporter efflux
Determine if a compound has the potential to inhibit or induce liver basolateral transporter efflux
No
+++
No
+++
ABC Vesicles, ABC Membranes
HMCPIS and HMCPIT, GIBCO® Human Cryopreserved Plateable Hepatocytes, Induction Qualified Also available: Fresh plateable human hepatocytes Cryopreserved and fresh plateable animal hepatocytes HMMCPL, GIBCO® Human Pooled Microsomes Also available: Cryopreserved suspension hepatocytes HMCPMS and HMCPML, GIBCO® Human Cryopreserved Plateable Hepatocytes, Metabolism Qualified Also available: Fresh and cryopreserved human and animal hepatocytes HMMCPL, GIBCO® Human Pooled Microsomes Also available: Animal microsomes Human and animal, fresh and cryopreserved hepatocytes Human and animal S9 HMMCPL, GIBCO® Human Pooled Microsomes Also available: Animal microsomes Human and animal, fresh and cryopreserved hepatocytes Human and animal S9 HMMCSD, GIBCO® Human Microsomes, Single Donor Also available: VIVID® recombinant P450s HMCSTS and HMCSTL, GIBCO® Human Cryopreserved Suspension Hepatocytes, Transporter Qualified Also available: Human Plateable Hepatocytes, Transporter Qualified Animal hepatocytes HMCPTS and HMCPTL, GIBCO® Human Cryopreserved Plateable Hepatocytes, Transporter Qualified Also available: GenoMembrane™ ABC inside-out vesicles and membranes Animal plateable hepatocytes
Other research applications (R&D, toxicology, environmental safety) Environmental bioaccumulation
Evaluate bioaccumulation of drugs or chemicals in humans and fish
++
+++
Liver disease research
Improve understanding of how the liver is involved in disease
No
+++
+++
++
siRNA, basic research
Identify the effects of gene suppression on disease
No
+++
TRS9PL, GIBCO® Fish (Rainbow Trout) S9 Fractions Also available: Animal and human, fresh and cryopreserved hepatocytes HMCS1S, HMCS1L, HMCS2S, and HMCS2L, GIBCO® Human Cryopreserved Suspension Hepatocytes, Metabolism Qualified Also available: Animal and human, fresh and cryopreserved, hepatocytes Animal and human microsomes HMCPTS or HMCPTL, GIBCO® Human Cryopreserved Plateable Hepatocytes, Transporter Qualified Also available: Human fresh plateable hepatocytes Animal cryopreserved plateable hepatocytes
* Drug Interaction Studies-Study Design, Data Analysis, and Implications for Dosing and Labeling, Draft Guidance for Industry, US FDA, September 2006
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Drug development
Primary hepatocytes isolated from the liver are effective tools for the in vitro evaluation of metabolism, drug–drug interactions, and hepatotoxicity, as well as transporter assessment. At Invitrogen, our hepatocyte isolationists are extensively trained in proper techniques to ensure optimal cell health. As a result, GIBCO® hepatocytes have high viabilities, in vivo–like enzyme expression levels, and if released as plateable cells, excellent confluencies that contribute to polarization and functioning cell–cell contacts (Figure 1). Extensive selection of lots Viabilities routinely >80%
Metabolic activity testing—ECOD, 7-HCG, 7-HCS; human hepatocytes include CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, FMO Application qualification tests—attachment efficiency, monolayer confluency (Figure 2), fold induction with prototypical inducers, transporter uptake and efflux, in situ intrinsic clearance Additional characterization data—genotyping, optimal seeding densities (Figures 3 and 4), viability stabilities, donor demographics
Lack of monolayer integrity ("unhealthy" morphology)
12000 Testosterone 6β-hydroxylation (pmol/mg/min)
GIBCO® cryopreserved human and animal hepatocytes
rifampicin
Transporter-qualified cryopreserved suspension hepatocytes are suitable for
8000
hepatic uptake studies, which typically measure the rate of appearance of substrate in cells after a relatively short incubation period, in most cases be4000
tween 15 seconds and 3 minutes. Each of our transporter-qualified lots (suspension and plateable) has been functionally tested for the activities of NTCP,
0
Ideal monolayer integrity ("healthy" morphology)
OATP1B3, and OATP transporter pathways using the substrates taurocholate, 100%
50%
33%
25%
Optimal plating density Figure 3. Seeding density effects. Induction response varies based on the plating density. A better response to rifampicin is obtained when the plating density is optimized. CYP3A4 activity was measured by testosterone 6β-hydroxylation.
Characterized for phase I and phase II drug metabolizing enzyme activities
Measurement of transporter uptake using suspension hepatocytes
digoxin, and estradiol 17β glucuronide (E2-17G). They have also been tested for phase I and phase II metabolic activities. Visualization of the bile canalicular networks is attained by fluorescence microscopy using the compound 5-(6)-carboxy-2’,7’-dichlorofluorescein diacetate (CDFDA). CDFDA is a substrate of the MRP2 transporter protein and accumulates in the bile canaliculi
Multiple large lots available—ideal for long-term studies across sites
A
B
Low confluency (90%)
Limited cell–cell contact
Good cell–cell contact
Grainy cytoplasm
Clear cytoplasm
Visible cell stretching
3-dimensional configuration
(e.g., fibroblast-like) Severe cell flattening
* Note: An unhealthy monolayer can demonstrate confluence but lack
Characterization and quality control
Figure 2. Representative images that demonstrate (A) the lack of monolayer integrity and (B) ideal monolayer integrity of cryopreserved human hepatocytes isolated from separate donor tissues and cultured for multiple days.
cell membrane integrity, organelle size, presence of lipid droplets, nucleus size and shape, cytosolic clarity, cell shape, level of cell debris, cell excretion products, cell–cell contacts (plateable cells only) and reestablishment of bile canalicular networks (plateable cells only)
6
integrity due to cell stretching and flattening
of suspension hepatocytes is not affected by cryopreservation (Figure 6).
50%
Note: For studies using B-CLEAR® technology, please refer to our B-CLEAR® Hepatic Transport Kits. GIBCO® Human Cryopreserved Hepatocytes, TransPlating density * 0.6-0.9 x 106 cellx/mL, 0.5 mL for 24-well plate
Bile canaliculi formation
Figure 1. Confluent, healthy sandwich-cultured human hepatocytes shown after 5 days of culture. The high level of confluency (>90%) and observable canaliculi are signs that this lot is suitable for metabolism, induction, and transporter uptake experiments.
Transporter uptake data have been compared before and after cryopreservation with similar results, suggesting that transporter gene expression
Cuboidal cell structure
Absence of bile canaliculi
10-point cell morphology check backed by photomicrographs—
Monolayer confluency
as the cells polarize and form bile canaliculi over 3–4 days in culture (Figure 5). 100%
porter Qualified, are not prequalified for use with B-CLEAR® reagents and their purchase conveys no rights to practice the B-CLEAR® technology.
Figure 4. General relationship between plating density and monolayer confluence. For human hepatocytes, the optimal plating range to achieve maximal confluence is 0.7–0.9 x 10 6 cells/mL for a 24-well plate.
GIBCO® Human Cryopreserved Hepatocytes, Transporter Qualified Contain functional membrane receptors and transporters Facilitate effective transporter uptake and basolateral efflux (plated) experiments Have stringent release specs: ≥80% viability and ≥80% confluency
Figure 5. Visualization of functional bile canaliculi networks showing the accumulation of 5-(6)-carboxy-2’,7’-dichlorofluorescein (CDF).
(plated cells)
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Drug development
25
fresh cryo
20 15 10 5
A
0
0.5
1
Digoxin accumulation (pmol/mg)
TC accumulation (pmol/mg)
25
2
and dose in triplicate with vehicle (0.1% DMSO), omeprazole (OMP),
fresh cryo
20
phenobarbital (PB) and rifampin (RIF) for 72 hours. Once monolayers are
15
washed, they are incubated with substrates phenacetin, bupropion, and
10
testosterone to determine CYP1A2, CYP2B6 and CYP3A activities, respec-
B
5
tively (Table 2, Figure 7). Fold induction of specific activity is expressed
0 0.5
Time (minutes)
1
2
Time (minutes)
Figure 6. Comparison of human suspension hepatocyte activities before and after cryopreservation. A single lot of human hepatocytes was functionally tested for transporter uptake after 2 hours, post-isolation and subsequent cryopreservation. Average accumulation of two substrates, (A) Na-taurocholate and (B) digoxin, was similar under both conditions.
as the ratio of induced activity to vehicle activity. mRNA content is also determined by TaqMan® qRT-PCR analysis after 48 hours treatment.
GIBCO® Human Cryopreserved Hepatocytes, Plated Metabolism Qualified Useful for the assessment of intrinsic clearance (CLint) in lowturnover compounds Prequalified according to CLint of midazolam, tolbutamide, dextromethorphan ≥80% viability and ≥75% attachment efficiency
Metabolic assay conditions for plated metabolism qualified human hepatocytes
GIBCO® Human Hepatocytes, Induction Qualified
Our plated metabolism-qualified hepatocytes have been tested for the
Prequalified for CYP1A2, CYP2B6 and CYP3A induction
enzymatic functions of CYP3A4, CYP2C9, and CYP2D6 using the proto-
≥80% viability and ≥80% confluency (if plated cells)
typical P450 substrates midazolam, tolbutamide, and dextromethorphan,
Minimum specific activities:
respectively (Figure 8). Using 48-well collagen-coated plates, hepatocytes
• • •
≥10-fold induction of CYP1A2
are allowed to attach prior to incubations in duplicate in serum-free Wil-
≥5-fold induction of CYP2B6
liams Medium E, with reactions stopped with ice-cold acetonitrile at
≥3-fold induction of CYP3A4
time points indicated in Table 3. Well contents are stored at –70°C prior to analysis. The disappearance of parent compound is monitored by LC/
Enzyme induction assay to qualify lots for use in experiments that monitor enzyme induction Our induction-qualified hepatocytes have passed our test for specific activity and mRNA levels in response to prototypical inducers. We culture our cryopreserved human hepatocytes in 24-well collagen-coated plates
MS/MS and intrinsic clearance values determined by linear regression.
Figure 7. GIBCO® human cryopreserved hepatocytes tested for fold induction of CYP3A. GIBCO® human cryopreserved hepatocytes are tested for response to prototypical inducers to determine suitable applications. In this example, the fold induction of CYP3A (number shown above the bar line) is calculated, illustrating inherent variability in individual lots of hepatocytes.
Figure 8. Cryopreserved human hepatocytes prequalified for plated metabolism (intrinsic clearance). CLint (µL/1 x 10 6 cells/min) results were midazolam, 14.6; tolbutamide, 1.34; dextromethorphan, 7.20.
GIBCO® Human Cryopreserved Single Donor Hepatocytes, Metabolism Qualified Characterized for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A, ECOD, 7-HCG, and 7-HCS activities (Table 4) High quality lots with batch sizes >500 vials Specialty lots available, including low CYP2D6 metabolizers Our single donor human cryopreserved suspension hepatocytes are ideal for metabolism studies such as metabolic profiling and metabolic stability. As an industry leader in liver tissue sourcing and hepatocyte isolations, Invitrogen offers one of the largest selections of single donor lots (Figure 9).
Table 3. Incubation conditions for CLint in plated cryopreserved human hepatocytes.
Concentration
Incubation time
Midazolam
Substrate
0.50 µM
0, 1, 2, 4, 6, 8 hr
Tolbutamide
1.00 µM
0, 4, 6, 8, 18, 24 hr
Dextromethorphan
1.00 µM
0, 1, 2, 4, 6, 8 hr
Table 2. Substrate probes for the assessment of P450 activity in GIBCO® Human Cryopreserved Plateable Hepatocytes, Induction Qualified.
Enzyme
8
Inducer
Inducer concentration
Substrate
Substrate concentration
Incubation time
Marker metabolite
CYP1A2
Omeprazole
50 µM
Phenacetin
100 µM
15 min
Acetaminophen
CYP2B6
Phenobarbital
1000 µM
Bupropion
500 µM
20 min
Hydroxybupropion
CYP3A
Rifampicin
10 µM
Testosterone
200 µM
14 min
6β-Hydroxytestosterone
Figure 9. Human hepatocytes qualified for suspension metabolism applications.
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Drug development
Table 5. Troubleshooting guide for plateable cryopreserved human hepatocytes, cont’d.
Table 4. Test conditions for determining the metabolic capacity of GIBCO® human cryopreserved suspension hepatocytes.
Enzyme
Substrate
Concentration
Incubation time
Marker metabolite
CYP1A2
Phenacetin
100 µM
15 min
Acetaminophen
CYP2B6
Bupropion
500 µM
20 min
Hydroxybupropion
CYP2C8
Paclitaxel
20 µM
45 min
6α-Hydroxypaclitaxel
CYP2C9
Diclofenac
25 µM
15 min
4’-Hydroxydiclofenac
CYP2C19
(S)-Mephenytoin
250 µM
30 min
4’-Hydroxymephenytoin
CYP2D6
Dextromethorphan
15 µM
15 min
Dextrorphan
CYP3A4
Testosterone
200 µM
14 min
6β-Hydroxytestosterone
Phase I and II
7-Ethoxycoumarin
100 µM
30 min
7-HCG, 7-HCS, 7-HC *
* 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin sulfate, and 7-hydroxycoumarin
Problem Suboptimal monolayer confluency
Possible cause
Seeding density too low Insufficient dispersion of hepatocytes during plating Insufficient plating volume used for well format Low attachment efficiency (see above)
Low cell viability, post-thaw
Dirty monolayers (rounded-up cell clumps or debris on top of monolayer)
Seeding density too high Insufficient dispersion of cells during plating Improper plating volume used for well format
Improper thawing technique Suboptimal thawing medium Rough handling of hepatocytes during counting Improper counting technique Cells sat too long prior to counting or plating
Recommendation
Unexpected cell yield
Improper thawing technique Suboptimal thawing medium Incorrect centrifugation speed Rough handling of hepatocytes during counting Improper counting technique
Low attachment efficiency
Not enough time for cells to attach Poor-quality substratum Hepatocyte lot not characterized as plateable
10
Possible cause
Table 5. Troubleshooting guide for plateable cryopreserved human hepatocytes. Problem
Recommendation
Review Invitrogen’s detailed thawing, plating, and counting protocols Thaw cells
