Warfarin IC50 for hVKORC1 variants determined by cell culture-based and dithiothreitol-driven in vitro assays and compared with patient dosage phenotypes
| hVKORC1 variant . | Warfarin IC50 ± SEM (nM)* . | Variant IC50/wild-type IC50 ratio* . | Mean patient dosage in HDT multiples [drug]† . | Warfarin IC50 by dithiothreitol-driven VKOR assay . |
|---|---|---|---|---|
| Wild-type | 24.7 ± 3.6 | 1.0 ± 0.2 | 1.0 [W, P] (n = 77) | Warfarin-sensitive (by definition) |
| Ala26Pro | 1224 ± 5.2 | 49.6 ± 0.1 | >3.0 [W] (n = 1) | 11.2-Fold increased Ki (IC50)‡ |
| Ala26Thr | 74 ± 7.5 | 3.0 ± 0.2 | >2.0 [P] (n = 1) | Sensitive‡ |
| Leu27Val | 62 ± 10.1 | 2.5 ± 0.2 | >3.0 [F], 1.0 [W] (n = 1)‖ | Sensitive‡ |
| His28Gln | 72 ± 8.2 | 2.9 ± 0.2 | 3.5 [P] (n = 1) | Sensitive‡ |
| Val29Leu | 136 ± 3.3 | 5.5 ± 0.1 | 2.0 [W] (n = 1) | Absence of expression‡/sensitive§ |
| Asp36Gly | 78 ± 6.6 | 3.2 ± 0.2 | 3.0 [W] (n = 1) | Sensitive‡ |
| Asp36Tyr | 93 ± 19.4 | 3.8 ± 0.3 | 1.5-3.5 [W] (n = 10) | Sensitive‡ |
| Val45Ala | 152 ± 4.4 | 6.2 ± 0.1 | >2.0 [W] (n = 1) | Sensitive‡/sensitive§ |
| Ser52Leu | 182 ± 8.9 | 7.4 ± 0.2 | >3.0 [P] (n = 1) | Low VKOR activity, Ki (IC50)determination not possible‡ |
| Ser52Trp | 140 ± 23.0 | 5.7 ± 0.2 | 3.5 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Val54Leu | 112 ± 2.8 | 4.5 ± 0.1 | 1.5-5.5 [W] (n = 2) | 4.6-Fold increased Ki (IC50)‡ |
| Ser56Phe | 167 ± 11.9 | 6.8 ± 0.2 | >5.0 [P] (n = 1) | Sensitive‡ |
| Arg58Gly | 85 ± 7.8 | 3.4 ± 0.2 | 5.0 [W] (n = 1) | Sensitive‡/sensitive§ |
| Trp59Arg | 433 ± 5.0 | 17.5 ± 0.1 | 7.0 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Trp59Cys | 188 ± 8.4 | 7.6 ± 0.2 | >3.5 [P] (n = 1) | Sensitive‡ |
| Trp59Leu | 1858 ± 4.8 | 75.2 ± 0.1 | >5.0 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Val66Gly | 69 ± 5.7 | 2.8 ± 0.2 | 2.5 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Val66Met | 134 ± 7.3 | 5.4 ± 0.2 | 3.0-6.0 [W] (n = 7) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Gly71Ala | 127 ± 6.3 | 5.1 ± 0.2 | >2.0 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Asn77Ser | 131 ± 15.5 | 5.3 ± 0.2 | >3.0 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Asn77Tyr | 96 ± 3.7 | 3.9 ± 0.2 | 3.5 [W] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Ile123Asn | 209 ± 3.7 | 8.5 ± 0.1 | >7.0 [P] (n = 1) | 2.4-Fold increased Ki (IC50)‡ |
| Leu128Arg | 1226 ± 8.4 | 49.7 ± 0.1 | >4.0-7.0 [W] (n = 5) | Low VKOR activity, Ki (IC50) determination not possible‡/sensitive§ |
| Tyr139His | 113 ± 5.2 | 4.6 ± 0.2 | >3.0 [W] (n = 1) | 3.6-Fold increased Ki (IC50)‡ |
| TYA → Leu-Ile-Val | 361 ± 11.1 | 14.6 ± 0.1 | — | — |
| hVKORC1 variant . | Warfarin IC50 ± SEM (nM)* . | Variant IC50/wild-type IC50 ratio* . | Mean patient dosage in HDT multiples [drug]† . | Warfarin IC50 by dithiothreitol-driven VKOR assay . |
|---|---|---|---|---|
| Wild-type | 24.7 ± 3.6 | 1.0 ± 0.2 | 1.0 [W, P] (n = 77) | Warfarin-sensitive (by definition) |
| Ala26Pro | 1224 ± 5.2 | 49.6 ± 0.1 | >3.0 [W] (n = 1) | 11.2-Fold increased Ki (IC50)‡ |
| Ala26Thr | 74 ± 7.5 | 3.0 ± 0.2 | >2.0 [P] (n = 1) | Sensitive‡ |
| Leu27Val | 62 ± 10.1 | 2.5 ± 0.2 | >3.0 [F], 1.0 [W] (n = 1)‖ | Sensitive‡ |
| His28Gln | 72 ± 8.2 | 2.9 ± 0.2 | 3.5 [P] (n = 1) | Sensitive‡ |
| Val29Leu | 136 ± 3.3 | 5.5 ± 0.1 | 2.0 [W] (n = 1) | Absence of expression‡/sensitive§ |
| Asp36Gly | 78 ± 6.6 | 3.2 ± 0.2 | 3.0 [W] (n = 1) | Sensitive‡ |
| Asp36Tyr | 93 ± 19.4 | 3.8 ± 0.3 | 1.5-3.5 [W] (n = 10) | Sensitive‡ |
| Val45Ala | 152 ± 4.4 | 6.2 ± 0.1 | >2.0 [W] (n = 1) | Sensitive‡/sensitive§ |
| Ser52Leu | 182 ± 8.9 | 7.4 ± 0.2 | >3.0 [P] (n = 1) | Low VKOR activity, Ki (IC50)determination not possible‡ |
| Ser52Trp | 140 ± 23.0 | 5.7 ± 0.2 | 3.5 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Val54Leu | 112 ± 2.8 | 4.5 ± 0.1 | 1.5-5.5 [W] (n = 2) | 4.6-Fold increased Ki (IC50)‡ |
| Ser56Phe | 167 ± 11.9 | 6.8 ± 0.2 | >5.0 [P] (n = 1) | Sensitive‡ |
| Arg58Gly | 85 ± 7.8 | 3.4 ± 0.2 | 5.0 [W] (n = 1) | Sensitive‡/sensitive§ |
| Trp59Arg | 433 ± 5.0 | 17.5 ± 0.1 | 7.0 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Trp59Cys | 188 ± 8.4 | 7.6 ± 0.2 | >3.5 [P] (n = 1) | Sensitive‡ |
| Trp59Leu | 1858 ± 4.8 | 75.2 ± 0.1 | >5.0 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Val66Gly | 69 ± 5.7 | 2.8 ± 0.2 | 2.5 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Val66Met | 134 ± 7.3 | 5.4 ± 0.2 | 3.0-6.0 [W] (n = 7) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Gly71Ala | 127 ± 6.3 | 5.1 ± 0.2 | >2.0 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Asn77Ser | 131 ± 15.5 | 5.3 ± 0.2 | >3.0 [P] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Asn77Tyr | 96 ± 3.7 | 3.9 ± 0.2 | 3.5 [W] (n = 1) | Low VKOR activity, Ki (IC50) determination not possible‡ |
| Ile123Asn | 209 ± 3.7 | 8.5 ± 0.1 | >7.0 [P] (n = 1) | 2.4-Fold increased Ki (IC50)‡ |
| Leu128Arg | 1226 ± 8.4 | 49.7 ± 0.1 | >4.0-7.0 [W] (n = 5) | Low VKOR activity, Ki (IC50) determination not possible‡/sensitive§ |
| Tyr139His | 113 ± 5.2 | 4.6 ± 0.2 | >3.0 [W] (n = 1) | 3.6-Fold increased Ki (IC50)‡ |
| TYA → Leu-Ile-Val | 361 ± 11.1 | 14.6 ± 0.1 | — | — |
[P], phenprocoumon; [W], warfarin.
IC50 values determined by the cell-based assay method as cited in Fregin et al18 used for the present study.
Data from Figures 2 and 3 and supplemental Table 3 of Watzka et al. HDT is defined as the high-dosage threshold and is equivalent to the mean patient population dosage divided by the control group (homozygous wild-type VKORC1 alleles with VKORC1:c.-1639GG haplotype) population mean OAC dosage for each drug, where the patient achieved stable anticoagulation with an international normalized ratio within the range 2.0-3.0. n, number of reported patients with mutation.13
Results from Hodroge et al, the study assumes that Ki = IC50.17
results calculated from data in Rost et al1 by methods detailed in Fregin et al.18 ‖patient had cytochrome P450 isoform 2C9*2*3 haplotype, which results in a reduced warfarin dosage requirement to achieve a stable, therapeutic international normalized ratio compared with patients with wild-type cytochrome P450 isoform 2C9*1*1 haplotype.