DPP-4 (IC50 = 4 nM)

Dipeptidyl peptidase-4 (DPP-4) is one of the extensively studied novel targets for the type 2 diabetes mellitus (T2DM) strategy that inhibits the DPP-4 action in order to maintain the endogenous glucagon-like peptide (GLP)-1 activity[1].
Trelagliptin has a strong inhibitory effect on DPP-4 that is prepared from Caco-2 cells, with an IC50 value of 5.4 nM. Additionally, trelagliptin inhibits the plasma DPP-4 activity of rats, dogs, and humans with IC50 values of 4.2 nM, 6.2 nM, and 9.7 nM, respectively[2].
Trelagliptin exhibits >10,000-fold selectivity over DPP-2, DPP-8, DPP-9, PEP, and FAPα activities, and it is highly selective for DPP-4, with IC50 values >100,000 nM. Trelagliptin is approximately 4- and 12-fold more potent than sitagliptin and alogliptin in terms of DPP4 selectivity[2].

Trelagliptin (oral gavage; 7 mg/kg; single dose) inhibits DPP-4 activity >80% of the time even after 24 hours in dogs, demonstrating a sustained Parkinson's disease effect[1]. Trelagliptin (oral gavage; 3 mg/kg; single dose; 60 min prior to oral glucose) reduces the AUC_0−120min of 19.3% in ob/ob mice when compared to the vehicle group, greatly improving the glucose tolerance capacity[3]. Trelagliptin (oral gavage; 10 mg/kg; once a week; 8 weeks) significantly lowered fasting blood glucose (FBG) levels; over the course of the treatment period, the average decrease was 16.8% lower than in the control group.Additionally, it raises insulin levels, which in ob/ob mice are raised by 1.7-fold in AUC_0−120min[3].

Enzyme inhibition assays[2]
Human DPP-4 enzyme used in these studies was obtained from several sources. Human DPP-4 partially purified from Caco-2 cells purchased from the ATCC (ATCC No. HTB-37; www.atcc.org), as described previously, was used to confirm trelagliptin inhibitor potency. For comparison among the DPP-4 inhibitors, trelagliptin, alogliptin and sitagliptin, commercially available recombinant human DPP-4 (Abnova, Taiwan) was used. For detailed kinetic studies, recombinant human DPP-4 was cloned, expressed and purified as described previously. In addition, inhibition of plasma DPP-4 activity was determined using plasma samples of humans, dogs, and rats. The DPP-4 related proteases, dipeptidyl peptidase-2 (DPP-2) and prolyl endopeptidase (PEP), were prepared from rat kidney and brain, respectively, according to the method previously reported. Human dipeptidyl peptidase-8 (DPP-8), dipeptidyl peptidase-9 (DPP-9), and fibroblast activation protein α (FAPα) were purified by affinity chromatography from 293-F cells expressing each FLAG-tagged protein.[2]
For detailed kinetic studies, GP-pNA was used as substrate and assays carried out in pH 7.4 buffer containing 20 mmol/L HEPES, 20 mmol/L MgCl2, 0.1 mg/ml bovine serum albumin, and 1% (v/v) DMSO at room temperature. In most cases, DPP-4 enzyme (1 nmol/L final concentration) was added last to initiate the enzymatic reaction, except when measuring the recovery of DPP-4 enzyme activity from a preformed DPP-4-inhibitor complex, in which case enzyme was first pre-incubated with trelagliptin for 70 min before initiating the reaction by dilution 50-fold into a reaction buffer containing a large excess (2 mmol/L, ca. 17x Km) of GP-pNA substrate. All assays were conducted as duplicates in 96-well format with total assay volume of 200 uL and absorbance at 405 nm was measured every 10 seconds to determine the reaction time-course. In most cases, the entire reaction progress curve was analyzed as described below. However, for initial rate studies to establish GP-pNA substrate-competitive inhibition by trelagliptin, only absorbance measurements from the first 40 seconds were used.

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In Vitro Bioassay, Crystal Structure Determination, and Pharmacokinetic Assay in SD Rats[3]
The in vitro DPP-4 inhibition study (at least three independent experiments), binding kinetics study using surface plasmon resonance, the cocrystallization of DPP-4 with compound 5 as well as structure determination, and the pharmacokinetic assay in SD rats were all conducted using the same method of operation reported in our previous work.

DPP-4 activity from Caco-2 cells or plasma was assayed using the chromophoric substrate Gly-Pro-p-nitroaniline (GP-pNA) (0.5 mmol/L final concentration) and carried out in pH 7.5 buffer containing 100 mmol/L Tris-HCl, 1 mg/mL bovine serum albumin, and 0.5 mg/mL CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) for 60 min at 37°C (DPP-4 fraction from Caco-2 cells) or 30°C (plasma). Change in absorbance at 405 nm was measured to determine the reaction rate. Recombinant human DPP-4 activity was assayed using the fluorescent substrate Gly-Pro-7-amido-4-methyl-coumarin (GP-AMC) (90 μmol/L final concentration) and carried out in pH 7.8 buffer containing 25 mmol/L HEPES, 140 mmol/L NaCl, 1 mg/mL bovine serum albumin for 15 min at 37°C. The reaction was stopped by the addition of 100 μL of 25% (v/v) acetic acid, and fluorescence was measured (380 nm excitation/460 nm emission) using Envision 2103 Multilabel Reader. Reaction conditions for measurement of DPP-2, DPP-8, DPP-9, PEP, and FAPα activities are described in Table 1. Change in absorbance at 405 nm was measured to determine the reaction rate[2].

ICR ob/ob mice[3]
10 mg/kg
Oral gavage; 10 mg/kg; once a week; 8 weeks

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Effect on DPP-4 Activity in ob/ob Mice[3]
Eight-week-old ob/ob mice (n = 10 in each group, 5 male and 5 female) were randomly assigned to treatment groups. After 2 h of fasting, baseline blood was collected into a tube containing EDTA. Mice were then treated orally with vehicle (0.5% sodium carboxymethyl cellulose, 10 mL/kg), compound 5 (0.3, 1, 3, 1, and 10 mg/kg), omarigliptin (3 mg/kg), or trelagliptin (3 mg/kg). Subsequently, blood per animal was collected at 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 h. All samples were centrifuged at 10 000 rpm for 2 min, and the plasma was harvested. Aliquots of plasma samples were stored at −80 °C until analysis. The measurement of in vivo DPP-4 activity was the same as the method with ICR mice.

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Effect on OGTT in db/db Mice[3]
To examine the effect of compound 5 on blood glucose after an oral glucose challenge in 6 week old db/db mice (n = 10 in each group, 5 male and 5 female), compound 5 (3 and 10 mg/kg), omarigliptin (10 mg/kg), trelagliptin (10 mg/kg), or vehicle (0.5% sodium carboxymethyl cellulose) was orally administered to 6 h-fasted db/db mice 60 min prior to the oral glucose challenge (1.5 g/kg). Blood glucose was estimated using a glucometer at 60 min before the glucose load and 0, 15, 30, 60, 90, and 120 min post-glucose challenge. The AUC for the glucose tolerance test was calculated using the trapezoidal method.

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Long-Term Antidiabetic Effects in db/db Mice[3]
Six-week-old db/db mice were divided into 5 groups (n = 10 in each group, 5 male and 5 female) based on nonfasting blood glucose and 6 h FBG, serum insulin levels, PBW (non-FBW), and 6 h FBW. Lean littermates were used as the lean control. Compound 5 (3 and 10 mg/kg), omarigliptin (10 mg/kg), trelagliptin (10 mg/kg), or vehicle (0.5% sodium carboxymethyl cellulose) was orally administered once weekly for 8 weeks. Nonfasting glucose and FBG, PBW, and 6 h FBW were determined at 7 d intervals. After 7 weeks of treatment, the 6 h-fasted animal was challenged by 1.5 g/kg glucose. Blood glucose was estimated using a glucometer at 0, 15, 30, 60, 90, and 120 min post-glucose challenge. After 8 weeks of treatment, the 6 h-fasted animal was challenged by 1.5 g/kg glucose. Blood samples were collected at 0, 15, 30, and 60 min post-glucose challenge to test plasma insulin levels. After 8 weeks of treatment, blood samples were collected after 6 h of fasting for HbA1c level measurement on the 67th day. The detailed dosing regimen is provided in the Supporting Information (Figure S11).

[1]. Recent approaches to medicinal chemistry and therapeutic potential of dipeptidyl peptidase-4 (DPP-4) inhibitors. Eur J Med Chem. 2014 Mar 3;74:574-605.

[2]. Trelagliptin (SYR-472, Zafatek), Novel Once-Weekly Treatment for Type 2 Diabetes, Inhibits Dipeptidyl Peptidase-4 (DPP-4) via a Non-Covalent Mechanism. PLoS One. 2016 Jun 21;11(6):e0157509.

[3]. Discovery of a Natural-Product-Derived Preclinical Candidate for Once-Weekly Treatment of Type 2 Diabetes. J Med Chem. 2019 Mar 14;62(5):2348-2361.

Trelagliptin is a member of benzenes and a nitrile.
Trelagliptin is under investigation in clinical trial NCT03555591 (Specified Drug-Use Survey of Trelagliptin Tablets "Survey on Long-term Use in Patients With Type 2 Diabetes Mellitus").

C18H20FN5O2

357.38

357.16

C, 60.49; H, 5.64; F, 5.32; N, 19.60; O, 8.95

865759-25-7

Trelagliptin succinate;1029877-94-8

15983988

White to off-white solid powder

1.4±0.1 g/cm3

519.0±60.0 °C at 760 mmHg

267.7±32.9 °C

0.0±1.4 mmHg at 25°C

1.646

1.88

1

6

3

26

657

1

C(C1C=C(F)C=CC=1C#N)N1C(=O)N(C)C(=O)C=C1N1CCC[C@@H](N)C1

IWYJYHUNXVAVAA-OAHLLOKOSA-N

InChI=1S/C18H20FN5O2/c1-22-17(25)8-16(23-6-2-3-15(21)11-23)24(18(22)26)10-13-7-14(19)5-4-12(13)9-20/h4-5,7-8,15H,2-3,6,10-11,21H2,1H3/t15-/m1/s1

2-[[6-[(3R)-3-aminopiperidin-1-yl]-3-methyl-2,4-dioxopyrimidin-1-yl]methyl]-4-fluorobenzonitrile

SYR 472; Trelagliptin; SYR-472; TRELAGLIPTIN; 865759-25-7; Trelagliptin [USAN]; Trelagliptin [USAN:INN]; UNII-Q836OWG55H; Q836OWG55H; Trelagliptin (USAN); SYR472; trade name: Zafatek

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.00 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)