Wang et al. BMC Pharmacology 2012, 12:2
`http://www.biomedcentral.com/1471-2210/12/2
`
`RESEARCH ARTICLE
`Open Access
`Potency, selectivity and prolonged binding of
`saxagliptin to DPP4: maintenance of DPP4
`inhibition by saxagliptin in vitro and ex vivo when
`compared to a rapidly-dissociating DPP4 inhibitor
`Aiying Wang1, Charles Dorso1, Lisa Kopcho2, Gregory Locke2, Robert Langish3, Eric Harstad4, Petia Shipkova3,
`Jovita Marcinkeviciene2, Lawrence Hamann5 and Mark S Kirby1*
`
`Abstract
`
`Background: Dipeptidylpeptidase 4 (DPP4) inhibitors have clinical benefit in patients with type 2 diabetes mellitus
`by increasing levels of glucose-lowering incretin hormones, such as glucagon-like peptide -1 (GLP-1), a peptide
`with a short half life that is secreted for approximately 1 hour following a meal. Since drugs with prolonged
`binding to their target have been shown to maximize pharmacodynamic effects while minimizing drug levels, we
`developed a time-dependent inhibitor that has a half-life for dissociation from DPP4 close to the duration of the
`first phase of GLP-1 release.
`Results: Saxagliptin and its active metabolite (5-hydroxysaxagliptin) are potent inhibitors of human DPP4 with
`prolonged dissociation from its active site (Ki = 1.3 nM and 2.6 nM, t1/2 = 50 and 23 minutes respectively at 37°C).
`In comparison, both vildagliptin (3.5 minutes) and sitagliptin ( < 2 minutes) rapidly dissociated from DPP4 at 37°C.
`Saxagliptin and 5-hydroxysaxagliptin are selective for inhibition of DPP4 versus other DPP family members and a
`large panel of other proteases, and have similar potency and efficacy across multiple species.
`Inhibition of plasma DPP activity is used as a biomarker in animal models and clinical trials. However, most DPP4
`inhibitors are competitive with substrate and rapidly dissociate from DPP4; therefore, the type of substrate, volume
`of addition and final concentration of substrate in these assays can change measured inhibition. We show that
`unlike a rapidly dissociating DPP4 inhibitor, inhibition of plasma DPP activity by saxagliptin and 5-
`hydroxysaxagliptin in an ex vivo assay was not dependent on substrate concentration when substrate was added
`rapidly because saxagliptin and 5-hydroxysaxagliptin dissociate slowly from DPP4, once bound. We also show that
`substrate concentration was important for rapidly dissociating DPP4 inhibitors.
`Conclusions: Saxagliptin and its active metabolite are potent, selective inhibitors of DPP4, with prolonged
`dissociation from its active site. They also demonstrate prolonged inhibition of plasma DPP4 ex vivo in animal
`models, which implies that saxagliptin and 5-hydroxysaxagliptin would continue to inhibit DPP4 during rapid
`increases in substrates in vivo.
`
`Background
`Diabetes is a worldwide epidemic, with the World Health
`organization estimating that more than 220 million people
`have diabetes worldwide http://www.who.int/mediacentre/
`factsheets/fs312/en/index.html, with greater than 90% of
`
`* Correspondence: mark.kirby@bms.com
`1Departments of Metabolic Diseases - Diabetes, Pennington NJ 08534, USA
`Full list of author information is available at the end of the article
`
`those having type 2 diabetes mellitus (T2DM). T2DM is
`thought to develop as a combination of insulin resistance
`and pancreatic b-cell failure [1]. Therefore, identification
`of novel treatments that would increase pancreatic insulin
`secretion while protecting pancreatic b-cells are of great
`interest.
`Incretin hormones, such as glucagon-like peptide-1
`(GLP-1), are secreted from cells in the gastrointestinal
`(GI) tract into the circulation in response to nutrient
`
`© 2012 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
`Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
`any medium, provided the original work is properly cited.
`
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`Wang et al. BMC Pharmacology 2012, 12:2
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`
`Page 2 of 11
`
`the
`absorption. They are a major component of
`mechanism regulating post-prandial insulin secretion
`when it is needed following meals [2]. Incretins account
`for up to 60% of the post-prandial insulin secretion in
`healthy individuals, but the incretin response is impaired
`in T2DM [3]. Incretin effects do not lead to insulin
`release per se, but potentiate the physiological release of
`insulin from the pancreas in response to increases in
`plasma glucose. Since GLP-1 has been shown to have
`the major incretin effect on glucose homeostasis in
`patients with type 2 diabetes [4], much work has been
`done to understand the effects of this incretin hormone
`on normal and pathophysiological glucose homeostasis.
`Following its secretion, dipeptidylpeptidase-4 (DPP4)
`rapidly metabolizes the intact form of GLP-1 (GLP-17-36)
`to inactive GLP-19-36 with a half-life of 1 to 2 minutes in
`vivo [5]. Therefore, two approaches have been taken to
`increase activity of the incretin axis, parenteral adminis-
`tration of DPP4-resistant GLP-1 analogues or oral
`administration of DPP4 inhibitors. DPP4 inhibitors have
`minimal risk of hypoglycemia because they enhance glu-
`cose-dependent insulin secretion and glucagon reduction.
`They are also weight neutral; i.e., they do not promote
`weight gain that is typically seen with many other anti-
`diabetic agents. DPP4 inhibitors are also effective in com-
`bination with several other diabetes drug classes [6-8].
`Finally, data from animal models indicate that GLP-1 is a
`trophic factor for b-cells, and potentiating endogenous
`incretins with DPP4 inhibitors does increase b-cell func-
`tion and number, thereby contributing to improvement
`of b-cell function over the long-term [9].
`There are many examples of enzyme inhibitors display-
`ing time-dependence (e.g. [10,11]), with several becoming
`marketed drugs, including members of the DPP4 inhibi-
`tor class [12-14]. In many cases, prolonged pharmacody-
`namic effects on the target enzyme (when compared to
`the pharmacokinetics of the drug) confers an advantage
`over rapidly dissociating compounds, because time-
`dependent drugs typically require lower plasma levels
`and reduced drug peak-to-trough ratios, reducing the
`risk of off-target toxicity [11,15]. In humans, peak GLP-1
`secretion occurs during the first phase of secretion,
`which occurs rapidly following a meal, giving a 2- to 3-
`fold increase that lasts 30 to 60 minutes [3]. This can be
`followed by a prolonged phase that gives a small increase
`in GLP-1 levels above fasting levels for up to 2 hours
`[reviewed in [16]. Therefore, we hypothesized that if a
`time-dependent inhibitor has a half-life for dissociation
`close to the duration of the first phase of GLP-1 secre-
`tion, the majority of the enzyme-inhibitor complex
`would not dissociate during the release of GLP-1 and this
`would maximize the compound’s beneficial effects while
`minimizing plasma drug levels. DPP4 also has many
`other substrates in vitro, although only a few have been
`
`shown to be cleaved by DPP4 in vivo (reviewed in [17]).
`Therefore, it would be ideal if binding did not extend
`past the duration of first phase GLP-1 secretion, such
`that the inhibitor activity would follow its pharmacoki-
`netics for inhibition of cleavage of other substrates of
`DPP4, should any such substrates have more prolonged
`in vivo half-lives relative to GLP-1.
`Here we describe the inhibitory properties of saxaglip-
`tin and its 5-hydroxy metabolite, which are both slow
`binding DPP4 inhibitors with extended off-rates from
`DPP4 at 37°C, similar to the duration of the first phase of
`release of GLP-1 in vivo. We also use the ex vivo plasma
`DPP assays that are used in preclinical animal models
`and the clinic as a biomarker for efficacy, to demonstrate
`how slow binding compounds such as saxagliptin differ
`from rapidly dissociating DPP4 inhibitors. We show that
`saxagliptin does not have a dilution artifact or a large
`dependence on the pseudo-substrate used in the assay,
`unlike rapidly dissociating DPP4 inhibitors, and we dis-
`cuss the significance of these findings.
`
`Results
`Saxagliptin is a potent inhibitor of human DPP4 in vitro
`irrespective of substrate
`We measured the IC50 for inhibition of substrates across a
`range of substrate concentrations (10 μM to 1000 μM,
`dependent on substrate) that straddled the Km for the
`pseudo-substrate gly-pro-pNA (Km 180 ± 8 μM, kcat 40 ±
`9 s-1, room temp, n = 3), and GLP-1 (Km 24 ± 16 μM,
`kcat 2.9 ± 0.9 s-1; room temp, n = 5), then calculated the
`Ki for inhibition of cleavage by each substrate (Table 1).
`Each of the DPP4 inhibitors tested were equipotent
`inhibitors of GLP-1 and gly-pro-pNA, as expected for
`inhibitors that are competitive with substrate and bind
`in the active site of DPP4. Therefore, we used gly-pro-
`pNA as a substrate in subsequent experiments. Saxaglip-
`tin was approximately 10-fold more potent than vilda-
`gliptin or sitagliptin under these conditions at room
`temperature.
`
`Potency and selectivity of saxagliptin and 5-
`hydroxysaxagliptin for human enzymes in vitro at 37°C
`Routine screening was performed at room temperature.
`However, as DPP4 inhibition in vivo occurs at 37°C, we
`measured the potency and selectivity of DPP4 inhibitors
`in vitro at that temperature (Table 2). The Km and
`turnover rate of gly-pro-pNA pseudo-substrate for
`DPP4 increased (Km = 209 ± 18 μM; kcat = 67 ± 4 s-1,
`n = 3), as did the Ki values for inhibition of DPP4 by
`DPP4 inhibitors (Table 2). Saxagliptin was 10-fold more
`potent than either vildagliptin or sitagliptin at 37°C. Sax-
`agliptin generates an active metabolite in vivo [18], 5-
`hydroxysaxagliptin;
`it was 2-fold less potent than
`saxagliptin.
`
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`Wang et al. BMC Pharmacology 2012, 12:2
`http://www.biomedcentral.com/1471-2210/12/2
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`Page 3 of 11
`
`Table 1 Inhibition of isolated cloned human DPP4 at
`room temperature
`compound
`Saxagliptin
`Sitagliptin
`Vildagliptin
`
`gly-pro-pNA Ki (nM)
`0.45 ± 0.1 (5)
`8 ± 1 (5)***
`7 ± 2 (5)***
`
`GLP-1 Ki (nM)
`0.41 ± 0.1 (7)
`2.5 ± 0.7 (4) ***
`1.5 ± 0.5 (4) ***
`
`mean ± standard deviation (number of independent experiments). ***P < 0.001
`versus saxagliptin
`
`The gly-pro-pNA pseudo-substrate is not specific to
`DPP4 and is also cleaved by other enzymes, including
`DPP8 (Km = 792 ± 60 μM; kcat = 5.1 ± 0.4 s-1, n = 3) and
`DPP9 (Km = 221 ± 27 μM; kcat = 3.7 ± 0.7 s-1, n = 3),
`although the cleavage rate of both enzymes is 20-fold
`lower than DPP4. Therefore, we also used this substrate to
`investigate inhibition of DPP8 and DPP9 by DPP4 inhibi-
`tors. Saxagliptin is approximately 400-fold selective and
`75-fold selective for DPP4 versus DPP8 and DPP9, respec-
`tively, with the 5-hydroxymetabolite having approximately
`twice the selectivity (DPP8 approximately 950-fold and
`DPP9 approximately 160-fold). In comparison, vildagliptin
`had 400-fold selectivity for DPP8 and 20-fold selectivity
`for DPP9, while sitagliptin had 1900-fold selectivity for
`DPP8 and 3000-fold selectivity for DPP9.
`Saxagliptin and 5-hydroxysaxagliptin were tested against
`multiple other enzymes (at room temperature). Both com-
`pounds had > 1000-fold selectivity against FAP, and >
`6000-fold selectivity against DPP2 and all other proteases
`tested: these included neutral endopeptidase, angiotensin
`converting enzyme, aminopeptidase P, prolidase, prolyl
`carboxypeptidase, activated protein C, chymotrypsin, fac-
`tor IXa, Factor VIIa, Factor Xa, Factor XIa, factor XIIa,
`plasma kallikrein, plasmin, thrombin, tissue kallikrein, tis-
`sue plasminogen activator, trypsin and urokinase (data not
`shown). They also had > 10,000-fold selectivity against a
`panel of 39 unrelated proteins that included 15 G-protein
`coupled receptors, 4 nuclear hormone receptors, 6 ion
`channels, 4 other enzymes and 10 transporters (data not
`shown).
`
`Potency and selectivity of saxagliptin and
`5-hydroxysaxagliptin for inhibition of cynomolgus
`monkey DPP enzymes in vitro at 37°C
`The potency and selectivity for all 4 compounds for
`inhibition of cynomolgus monkey (rhesus monkey has
`
`the same DPP4 DNA sequence) DPP4, DPP8 and DPP9
`is shown in Table 3 and is very similar to that found
`versus the human enzymes.
`Similar data were also obtained for mouse and rat
`enzymes (data not shown). Therefore, we confirmed
`that the potency and specificity of saxagliptin and its 5-
`hydroxymetabolite were similar across species in vitro.
`We did not investigate the effects of DPP4 inhibitors on
`other peptidases from other species because no effect of
`saxagliptin and 5-hydroxysaxagliptin were seen on the
`human proteins tested.
`
`Saxagliptin and 5-hydroxysaxagliptin are long-acting
`DPP4 inhibitors in vitro
`During the course of initial experiments, we noticed that
`there was time dependence to inhibition of DPP4 by
`some DPP4 inhibitors. In order to determine time-
`dependence, we preincubated DPP4 inhibitors with
`DPP4 and measured the rate of dissociation of DPP4
`inhibitors from DPP4 using an ‘infinite dilution’ method.
`The data in table 4 show that saxagliptin and 5-hydro-
`xysaxagliptin have slow binding when tested at 37°C,
`with t1/2 for dissociation of 50 minutes and 23 minutes,
`respectively. While vildagliptin shows some evidence of
`slow binding (t1/2 = 3.5 minutes), this was much less
`pronounced. Sitagliptin showed no time dependence
`(within the limitations of the experimental protocol at <
`2 minutes). The time dependence was only found for
`inhibition of DPP4 and was not seen during experiments
`investigating the inhibition of DPP8 and DPP9; there-
`fore, these prolonged effects would only relate to inhibi-
`tion of cleavage of DPP4 substrates by DPP4.
`
`Saxagliptin does not have a dilution artifact in plasma
`DPP assays in vitro
`The ex vivo assay measuring inhibition of plasma DPP4
`activity has been used as a key biomarker assay for
`DPP4 inhibitor assessment by multiple groups in both
`animal models and in the clinic. Given that the duration
`of the ex vivo assay is typically between 10 and 20 min-
`utes, there would be no dilution artifact predicted for ex
`vivo determination of inhibition from saxagliptin dosed
`animals, because negligible dissociation of saxagliptin
`from DPP4 would occur over the time frame of the
`experiment (Table 4 and the rate of dissociation is
`
`Table 2 Inhibition of isolated, cloned human DPP4, DPP8 and DPP9 at 37°C
`DPP4 Ki (nM)
`DPP8 Ki (nM)
`1.3 ± 0.3 (12)
`508 ± 174 (13)
`2.6 ± 1.0 (12)***
`2495 ± 727 (14)*
`13 ± 2.8 (12)***
`5218 ± 2319 (14)***
`18 ± 1.6 (12)***
`33780 ± 5532 (12)***
`
`Saxagliptin
`5-hydroxysaxagliptin
`Vildagliptin
`Sitagliptin
`
`mean ± standard deviation (number of independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 versus saxagliptin
`
`DPP9 Ki (nM)
`98 ± 44 (11)
`423 ± 64 (12)
`258 ± 93 (12)
`55142 ± 19414 (11)***
`
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`Wang et al. BMC Pharmacology 2012, 12:2
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`Table 3 Inhibition of isolated, cloned cynomolgus monkey DPP4, DPP8 and DPP9 at 37°C
`DPP4 Ki (nM)
`DPP8 Ki (nM)
`1.1 ± 0.2 (14)
`390 ± 82 (6)
`2.9 ± 1.1 (13)***
`2061 ± 658 (6)***
`6.8 ± 2.0 (14)***
`3692 ± 917 (7)***
`15.6 ± 3.6 (14)***
`21949 ± 17461 (6)***
`
`Saxagliptin
`5-hydroxysaxagliptin
`Vildagliptin
`Sitagliptin
`
`mean ± standard deviation (number of independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 versus saxagliptin
`
`DPP9 Ki (nM)
`61 ± 5 (6)
`323 ± 60 (6)*
`125 ± 39 (7)***
`65757 ± 7966 (6)***
`
`decreased with lower temperature). To confirm this for
`saxagliptin, we took naive human plasma and compared
`samples with just the addition of compound to plasma
`only, to samples where compound was added to both
`plasma and buffer (such that total concentration of drug
`was kept constant during the ‘dilution step’).
`Saxagliptin is unaffected by the 3-fold dilution in the
`human plasma assay (Figure 1a) using ala-pro-AFC as sub-
`strate. However, sitagliptin clearly has a dilution artifact
`(Figure 1b). When sitagliptin was added only to the
`plasma, the inhibition curve shifted 3-fold to the right
`compared to when compound was added to both the
`plasma and the dilution buffer (IC50 = 152 ± 41 nM versus
`414 ± 116 nM: mean ± s.d., n = 3), except where there is
`virtually no inhibition or full inhibition, consistent with
`the 3-fold dilution during substrate addition. This is pre-
`sumably due to a new equilibrium being rapidly estab-
`lished following dilution, such that the potency of
`sitagliptin will be underestimated when compound is only
`present in the plasma. Similar data were also obtained
`using both cynomolgus and rhesus monkey plasma (data
`not shown).
`
`Maximum inhibition of plasma DPP4 activity in plasma
`samples by DPP4 inhibitors differs between species
`Untreated human plasma samples gave a plasma DPP
`enzyme activity rate of 5.0 ± 0.6 nmoles/min per ml
`plasma (mean ± s.d., n = 3 independent experiments)
`when ala-pro-AFC was used as substrate. Untreated
`cynomolgus and rhesus monkey plasma DPP4 rates were
`similar to those seen in human, with rates of 5.2 ± 0.3
`and 7.3 ± 0.2 nmoles/min per ml plasma, respectively.
`However, the ability of DPP4 inhibitors to inhibit cleave-
`age of peudo-substrates differs among species. Figure 2
`
`shows that the maximum inhibition of plasma DPP activ-
`ity seen with saxagliptin was approximately 85% in rhesus
`and 80% in cynomolgus monkeys, but > 95% in humans
`(Figure 2). Like human, rodent (mouse and rat) and dog
`plasma DPP is inhibited > 95% by saxagliptin (data not
`shown).
`These effects were shown to be independent of DPP4
`inhibitor and similar data were obtained with gly-pro-
`pNA as the pseudo-substrate (Table 5; Figure 3). Since
`the pseudo-substrates are not specific for DPP4, pre-
`sumably these findings reflect a species difference in the
`relative activity of all the other plasma peptidases that
`cleave these pseudo-substrates.
`The in vitro IC50 for inhibition of human, rhesus
`monkey and cynomolgus monkey plasma DPP activity
`by saxagliptin, vildagliptin and sitagliptin was similar
`across species (Table 5). Therefore, although there were
`different amounts of maximal inhibition among species,
`DPP4 inhibitors have similar potency in rhesus monkey,
`cynomolgus monkey and human plasma for inhibition
`of plasma DPP activity.
`
`Choice of assay affects the IC50 measured for inhibition of
`plasma DPP activity by DPP4 inhibitors at steady-state in
`vitro
`The measured IC50 varies with the ratio of substrate con-
`centration to substrate Km for competitive inhibitors (see
`Methods). Using the assays described here, Km values
`were calculated as 57 ± 13 μM (n = 3 independent experi-
`ments) for the ala-pro-AFC assay, and 180 ± 10 μM (n =
`3) for gly-pro-pNA assay in human plasma. Km values
`were similar in cynomolgus monkey plasma, at 35 ± 6 μM
`(n = 3) for ala-pro-AFC assay and 134 ± 5 μM (n = 3) for
`gly-pro-pNA assay. Since the majority of DPP4 inhibitors
`are competitive with substrate, a difference in substrate
`concentration will affect the measured IC50 of these inhibi-
`tors. In the two pseudo-substrate assays we used to mea-
`sure inhibition of plasma DPP activity, the ratio of Km to
`substrate concentration is approximately 7-fold in the ala-
`pro-AFC assay (370 μM substrate concentration), but is
`only 2-fold in the gly-pro-pNA assay (400 μM substrate
`concentration). Therefore, using gly-pro-pNA as substrate
`would give an apparent increase in potency of DPP4 inhi-
`bitors compared to ala-pro-AFC. Further, the difference in
`temperature (30°C versus room temperature) and pH (7.4
`
`Table 4 On and off rates of DPP4 inhibitors at 37°C
`kon, 105 M-1 s-1
`koff, 10-5 s-1
`Compound
`t1/2 (min.)
`37°C
`4.6 ± 0.6
`0.7 ± 0.1
`1.2 ± 0.2
`> 100
`
`23 ± 1
`50 ± 2
`330 ± 30
`> 580
`
`50
`23
`3.5
`< 2
`
`Saxagliptin
`5-hydroxysaxagliptin
`Vildagliptin
`Sitagliptin
`
`mean ± standard error. Standard errors for kon were calculated from
`equations (2), (3) and (4), and for koff are from the fits to equation (5)
`
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`Figure 1 The effect of dilution on the IC50 for inhibition of human plasma DPP activity by saxagliptin (Panel A) and sitagliptin (Panel
`B) using ala-pro-AFC as substrate (activity is measured as arbitrary fluorescence units. Mean ± s.e.m., n = 3 independent experiments, 3
`replicates per experiment).
`
`versus 7.9) of the two assays would also affect the mea-
`sured IC50. Figure 3 shows the inhibition of cynomolgus
`monkey plasma DPP activity in vitro for saxagliptin and
`sitagliptin under pseudo-steady-state conditions. There
`was a small change in IC50 for saxagliptin (2.5 ± 0.2 nM to
`9.8 ± 0.3 nM, P < 0.0001), with a narrow concentration
`range over which a difference would be seen between the
`two assays (1 to 10 nM. Figure 3A). This presumably
`reflects differences in temperature and pH between the
`two assays. However, at concentrations of sitagliptin
`between 5 and > 3000 nM, much more inhibition of DPP
`activity is seen with the gly-pro-pNA assay than with the
`ala-pro-AFC assay (Figure 3B). Further, the IC50 for inhibi-
`tion in the ala-pro-AFC assay was significantly increased
`26-fold compared to the gly-pro-pNA assay, from 17 ± 2
`nM to 440 ± 163 nM (P < 0.0001). Similar data were
`obtained using human plasma (data not shown).
`
`Figure 2 Percent inhibition of plasma DPP activity in human,
`rhesus monkey and cynomolgus monkey plasma by
`saxagliptin using ala-pro-AFC as substrate.
`
`Measurement of plasma DPP activity in ex vivo assays
`The differences in dissociation rate from DPP4 and the
`substrate used have substantial implications for mea-
`surement of activity following dosing in animals and
`humans. Figure 4 shows data from an in vivo study
`where various doses of saxagliptin and sitagliptin were
`given to cynomolgus monkeys and plasma DPP inhibi-
`tion was measured after 24 hours, at trough.
`When the ala-pro-AFC assay was used to measure
`plasma DPP inhibition ex vivo, saxagliptin treatment
`resulted in close to maximal inhibition of the inhibitable
`plasma DPP activity at its highest doses, with the 1, 3
`and 10 mg/kg doses being statistically different from
`vehicle. However, sitagliptin treatment had no effect on
`plasma DPP activity at any of the doses. When the gly-
`pro-pNA assay was run on exactly the same samples,
`similar results were obtained for saxagliptin and there
`was no statistical difference between the data obtained
`with either assay at any dose. In contrast to the ala-pro-
`AFC assay, sitagliptin treatment gave statistically signifi-
`cant inhibition of plasma DPP activity at 3, 10 and 40
`mg/kg doses when compared to vehicle in the gly-pro-
`pNA assay. Further, plasma DPP inhibition in the gly-
`pro-pNA assay at the 10 and 40 mg/kg doses were sta-
`tistically significantly different from those obtained
`using the ala-pro-AFC assay (P = 0.07 for the 3 mg/kg
`dose). However, the highest dose tested still did not give
`maximal inhibition of plasma DPP activity. Therefore,
`choice of assay had significant relevance for the inter-
`pretation of inhibition by sitagliptin in this study.
`
`Discussion
`Saxagliptin (BMS-477118) is a potent inhibitor of DPP4
`that is approximately 10-fold more potent than vilda-
`gliptin or sitagliptin. Saxagliptin also has an active
`
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`Wang et al. BMC Pharmacology 2012, 12:2
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`Page 6 of 11
`
`Table 5 Maximum levels of inhibition of human and monkey plasma DPP activity using ala-pro-AFC as substrate
`Rhesus
`Cyno
`Human
`IC50 (nM)
`IC50 (nM)
`Max % inhib
`IC50 (nM)
`Max % inhib
`Max % inhib
`81 ± 6††
`86 ± 1†††
`2.9 ± 0.3
`saxagliptin
`4.0 ± 0.8
`2.4 ± 0.2
`100 ± 0
`79 ± 6††
`85 ± 1†††
`17 ± 5**
`vildagliptin
`20 ± 7*
`13 ± 4**
`98 ± 1
`80 ± 7††
`87 ± 1†††
`17 ± 2***
`sitagliptin
`22 ± 3***
`17 ± 2***
`98 ± 0
`N = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus saxagliptin. ††P < 0.01, †††P < 0.001 versus human
`
`metabolite, 5-hydroxysaxagliptin, which is present in
`human plasma at levels between 2- and 7-fold higher
`than saxagliptin (Fura 2009). 5-hydroxysaxagliptin is 2-
`fold less potent than saxagliptin, but approximately 5-
`fold more potent than sitagliptin and vildagliptin.
`The data here shows that temperature and the choice of
`substrate can have significant effects on IC50 values,
`accounting for some of the differences in values typically
`reported (reviewed in [17]). However, Thomas et al. [14]
`reported very high IC50 values for saxagliptin and vilda-
`gliptin in particular. In the case of saxagliptin, the value
`was 50-fold higher than the Ki value reported here and
`50-fold higher than the value reported for their DPP4 inhi-
`bitor, linagliptin. Saxagliptin (Onglyza®) is available com-
`mercially in many world markets at doses of 2.5 mg and
`5 mg [6] and the minimal efficacious dose of linagliptin is
`5 mg [19]. It is difficult to conceive how saxagliptin could
`be 50-fold less potent than linagliptin in vitro, but at least
`equipotent in the clinic; therefore, it seems likely that the
`data reported here are more accurate determinations of
`the activity of saxagliptin and vildagliptin. A known con-
`centration of pure DPP4 is typically used in the majority
`of these in vitro studies (we used 100 pM of isolated
`cloned human DPP4) because a low concentration of
`DPP4 is important for avoiding obtaining artifactually high
`inhibition values. Part of the reason for the discrepancy
`may be that Thomas et al. [14] are the only group to use
`
`“DPP4 extracted from Caco cell membranes”, with DPP4
`purity and concentration undisclosed.
`The increased potency and prolonged binding of saxa-
`gliptin compared to sitagliptin and vildagliptin may be a
`reflection of its strong interactions with both Ser630 and
`Glu205/Glu206, whereas sitagliptin interacts primarily with
`Glu205/Glu206, and vildagliptin with Ser630 [17]. Further,
`we showed that saxagliptin demonstrated slow-onset
`inhibition at room temperature that was partially due to
`a covalent, but reversible enzyme-adduct formation at
`Ser630, but also partially due to another conformational
`change induced by saxagliptin [20]. Both saxagliptin and
`5-hydroxysaxagliptin also have slow binding kinetics at
`37°C and would be expected to have prolonged pharma-
`codynamic effects in vivo.
`The data in Figure 4 shows that saxagliptin bound to
`DPP4 prior to substrate addition remained bound during
`the time course of the ex vivo assay. The half life for disso-
`ciation of saxagliptin from DPP4 (t1/2 of 50 minutes at
`37°C) is similar to the duration of first phase of elevated
`GLP-1 in the plasma following a meal in humans [4]. This
`may be relevant to the cleavage of endogenous substrate(s)
`because saxagliptin (and 5-hydroxysaxagliptin) given
`before a meal will already be bound to DPP4 at its site(s)
`of action. When incretins are released in response to a
`meal, incretins will increase and compete with inhibitors
`with rapid off-rates. However, this would be unlikely to
`
`Figure 3 Inhibition of cynomolgus monkey plasma DPP activity by saxagliptin (Panel A) and sitagliptin (Panel B) using the ala-pro-
`AFC and gly-pro-pNA assays (mean of 3 independent experiments).
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`Figure 4 The effect of pseudo-substrate choice on the measurement of plasma DPP activity ex vivo in cynomolgus monkeys 24 hours
`post dose. **P < 0.01, ***P < 0.001 versus vehicle. †P < 0.05, ††P < 0.01, versus ala-pro-AFC at the same dose. The dashed line represents the
`average maximum inhibition seen (68.9 ± 3.9%, 1 hour post dose at the highest doses tested).
`
`occur for saxagliptin, unless substrate concentrations were
`raised for significantly longer than the duration of the first
`phase of GLP-1 secretion, when saxagliptin would equili-
`brate with substrate as with the other DPP4 inhibitors. In
`clinical practice, DPP4 inhibitors currently have a placebo-
`like side-effect profile in trials up to 2 years in length.
`Therefore, rapidly dissociating DPP4 inhibitors can be
`given at high enough doses that any theoretical advantages
`from extended binding are currently not seen in the clinic.
`However, the chronic adverse event profiles of current
`DPP4 inhibitors are not yet fully defined and differences
`between DPP4 inhibitors may yet be seen.
`Saxagliptin and 5-hydroxysaxagliptin are selective for
`DPP4 versus DPP8 and DPP9 at 37°C. Neither saxaglip-
`tin nor 5-hydroxysaxagliptin exhibited slow binding
`kinetics to DPP8 or DPP9, so any DPP8/9 related phar-
`macodynamics would closely match their pharmacoki-
`netics. The potential for inhibition of DPP8 and DPP9
`in vivo is difficult to assess in the absence of a known
`physiologically relevant substrate and knowledge of the
`specific tissues and cells where either may play a physio-
`logical role. Further, as DPP8 and DPP9 are cytosolic
`enzymes [21], the cytosolic concentration of saxagliptin
`and 5-hydroxysaxagliptin in those cells would also be
`required to accurately predict the potential for inhibi-
`tion. Since this information is unknown (and to test for
`other off-target issues), extensive toxicity studies are
`typically undertaken in several species and adverse
`events are scrutinized in clinical studies. As discussed
`
`previously (reviewed in [17]), the preponderance of evi-
`dence for saxagliptin would show that there is no toxi-
`city attributable to inhibition of DPP8 or DPP9 at
`clinically relevant doses across species.
`There was no significant species difference for potency
`of inhibition of human and cynomolgus monkey DPP4,
`DPP8, DPP9 or plasma DPP activity for three DPP4 inhi-
`bitors (saxagliptin, vildagliptin or sitagliptin). However,
`there were significant differences in the maximum
`amount of inhibition of plasma DPP activity seen
`between rhesus and cynomolgus monkey plasma (80 to
`85%) and the other species tested (95%). This presumably
`shows a difference between the levels of dipeptidylpepti-
`dases found in the plasma of the two species. The identi-
`ties of the peptidases that underlie this difference are
`unknown and were not a focus of these experiments.
`However, as there was no differentiation between saxa-
`gliptin, vildagliptin and sitagliptin, it is unlikely to be
`attributable to species differences in DPP8 or DPP9
`expression (also consistent with current understanding
`that DPP8 and DPP9 are not secreted or thought to be
`present in plasma [17]).
`Based on the data presented here, interpretation of
`data from ex vivo assays for DPP4 inhibitors should be
`done with caution because the choice of substrate, as
`well as the nature of the ex vivo assay (such as dilution
`artifact), can all affect measured efficacy. The difference
`in plasma DPP inhibition for sitagliptin between these
`two assays (Figure 4) can be explained by sitagliptin’s
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`rapid dissociation and reassociation with the active site
`of DPP4. Due to the difference in the ratio of Km to
`substrate concentration between the two assays, one
`substrate is less effective at competing out the inhibitor
`and the inhibitor appears more potent in that assay.
`Consistent with this conclusion was the in vitro data
`that showed that this issue would most likely manifest
`itself across the range of plasma concentrations typically
`found at trough (24-hour post dose) for sitagliptin in
`cynomolgus monkeys. In contrast, saxagliptin would not
`dissociate appreciably from the active site during the
`time course of the assay and there would be no inhibi-
`tor-substrate competition.
`Krishna et al. [22] recently illustrated the benefits of
`accelerating drug development using biomarkers with
`the example of DPP4 inhibitors and the plasma DPP4
`assay. While this approach can be fundamentally useful,
`some important caveats to this approach have been
`demonstrated here. The pseudo-substrate gly-pro-pNA
`was used in both clinical assays to determine plasma
`DPP inhibition for saxagliptin and sitagliptin. However,
`the conditions are very different: i.e. the substrate con-
`centration is 2000 μM (10-fold the Km), with an 11-fold
`dilution step and 120 minute assay duration in the saxa-
`gliptin assay [23]; the substrate concentration is 400 μM
`(2-fold Km), with a 2.5-fold dilution step and a 14 min-
`ute assay duration in the sitagliptin assay [24]. The data
`presented here show that, given the comparably longer
`duration of the assay, the larger dilution factor and the
`higher substrate concentration in the saxagliptin clinical
`assay, it is not possible to perform a meaningful direct
`comparison between clinical data for the two drugs
`using these different assays. This may explain why
`Krishna et al. [22] reported that they need 80% inhibi-
`tion of plasma DPP4 activity at trough (24-hours post
`dose) to obtain maximal glucos