mGluR5 (Ki = 1.7 nM)

Potency and selectivity in functional assays [1]
ABP688 was originally shown to be a potent antagonist of quisqualate-induced phosphoinositol (PI) accumulation in L(tk-) cells expressing recombinant human mGluR5 (hmGluR5), with an IC50 value of 2.4 nM (95% CI: 0.5–12 nM) (Fig. 2A). In the same preparation, ABP688 completely inhibited glutamate-induced calcium release with an IC50 value of 2.3 nM (95% CI: 2.1–2.5) nM (data not shown) and had no effect, up to 10 μM, on ATP-induced PI accumulation via stimulation of endogenously expressed purinergic receptors in hmGluR5-expressing cells (Fig. 2A), or on quisqualate-induced PI accumulation in hmGluR1-expressing cells. ABP688 had no effect on basal PI levels in hmGluR5- or hmGluR1-expressing cells (Fig. 2B).

---

[3H]-M-MPEP displacement in membranes of hmGluR5-expressing cells [1]
The affinity of ABP688 for the allosteric binding site in the transmembrane domain of mGluR522 was determined in a radioligand competition assay using [3H]-M-MPEP.9 In membranes prepared from L(tk-) cells expressing recombinant human mGluR5 (hmGluR5),23 ABP688 fully displaced the binding of [3H]-M-MPEP in a concentration-dependent manner (Fig. 3) with an IC50 value of 7.0 nM (95% CI: 6.1–8.1), or a corresponding Ki of 3.5 (3.1, 4.0) nM. The Hill coefficient was −0.92 ± 0.10 (±95% CI).

---

Receptor binding panel [1]
The in vitro binding affinities of ABP688 for a variety of CNS GPCR receptors, ion channels or transporters were determined according to standard filtration methods.24 ABP688, at concentrations of 1 and 10 μM, did not bind to any of the tested receptors or transporters, confirming the high degree of selectivity of ABP688 (details are available as Supplementary information).

---

Binding characteristics of [3H]ABP688 in vitro [1]
The characteristics of the binding of [3H]ABP688 to native mGlu5 receptor were determined in membranes prepared from rat whole brain tissue. The observed association and dissociation time courses were well described by mono-exponential models, the respective rate constants were kobs = 0.103 ± 0.006 (n = 3) and k−1 = 0.075 ± 0.006 min−1(n = 5). The calculated association rate constant k1 was 0.07 nM−1 min−1. From these values, an equilibrium binding constant Kd of 1.07 nM was calculated. The specific binding of [3H]ABP688 was saturable and adequately described by a single-binding site model, with Bmax and Kd values of 0.87 ± 0.096 pmol/mg protein and 2.3 ± 0.34 (n = 4) nM, respectively. (Fig. 4A–C).[1]
In a further series of experiments, we utilized [3H]ABP688 to determine the affinities of ABP688, MPEP, M-MPEP as well as CPCCOEt, an mGluR1 antagonist, to membrane preparations of hmGluR5-expressing cells and rat whole brain tissue. In both preparations, ABP688, MPEP, and M-MPEP fully displaced [3H]ABP688 in a concentration-dependent manner with Hill coefficients near negative unity, while CPCCOEt had no appreciable effect on [3H]ABP688 binding up to 10 μM (Fig. 5 and Table 1, for respective affinities). The solvent, DMSO, had no effect on binding (not shown). The near-equality of the respective binding affinities of APB688, MPEP, and MPEP to hmGluR5 and rat brain membranes indicated that there are no apparent species differences between recombinant human and native rat receptors in vitro.

Autoradiography in rat brain slices [1]
In vitro autoradiography is an established method to determine the binding characteristics of a radioligand and the distribution of the labeled sites. In rat brain slices, incubation with 5 nM (70 Ci/mmol) of [3H]ABP688 gave rise to a regionally differentiated binding pattern (Fig. 6A, left panels). The binding was displaceable by MPEP (10 μM), and the remaining (non-specific) binding was found to be nearly indistinguishable from background (Fig. 6).[1]
High-to-medium specific binding was observed (in descending rank order) in nucleus accumbens, caudate putamen, hippocampus, olfactory tubercle, amygdala, and occipital and frontal cortices; low-to-near background binding was found in thalamus, hypothalamus, midbrain, pons-medulla, and cerebellum (Fig. 7). This pattern is consistent with autoradiographic results obtained with [3H]methoxy-PyEP,10 as well as mGluR5 expression patterns obtained with immunocytochemistry using mGluR5-specific antibodies or mGluR5 mRNA distribution as determined with in situ hybridization. Taken together, our present findings of a good (qualitative) correlation between [3H]ABP688 binding and mGluR5 expression, in combination with low unspecific binding in native brain tissue, encouraged further profiling in vivo.

[3H]-M-MPEP competition assays [1]
Radioligand displacement assays utilizing [3H]-M-MPEP were carried out essentially as described previously.22 Details are available in the Supplementary information.

---

[3H]ABP688 binding assays [1]
Reactions were performed in 96-well microtiter plates in a final assay volume of 200 μl per well. The assay mixtures contained membranes, suspended in assay buffer composed of Na–Hepes, (30 mM), NaCl (110 mM), MgCl2 (1.2 mM), KCl (5 mM), CaCl2·2 H2O (2.5 mM) at pH 8.0 [3H]ABP688, and other reagents were added at the concentrations given below. Samples were incubated at 37 °C (duration given below) and then filtered through glass-fiber filters (Unifilter-96 GF/C plate) utilizing a 96-well plate filtration unit. The filters were rinsed five times with cold assay buffer and dried before the addition of a scintillation fluid (Ultimate Gold XR, 100 μl per well). The assay plates were then shaken for 2–3 h and subsequently counted in a liquid scintillation counter. Individual determinations were performed in triplicate.

---

Kinetic studies [1]
To assess the time course of association, mixtures of [3H]ABP688 (0.4 nM) and membranes (ca. 20 μg/well, hmGluR5 40 μg/well) were incubated at 37 °C from 1 to 90 min prior to filtration. The time course of dissociation was assessed in membranes previously equilibrated with [3H]ABP688 (0.4 nM) for 60 min, after which 10 μM M-MPEP was added 1–90 min prior to filtration. A mono-exponential model was used to derive the observed association (kobs) and dissociation (k−1) rate constants. The association rate (k1) was calculated according to k1 = (kobs − k−1)/[L], where [L] was the added radioligand concentration.

---

Saturation binding and displacement assays [1]
Saturation binding was assessed by adding various concentrations of [3H]ABP688 (0.2–50 nM) to the membranes (rat 40 μg/well, hmGluR5 15 μg/well). Samples were left to equilibrate at 37 °C for 60 min prior to filtration. Non-specific binding of [3H]ABP688 was defined as the radioactivity of samples incubated in the presence of an excess of M-MPEP (10 μM). Samples for displacement studies contained a fixed concentration of [3H]ABP688 (1.5 nM) and other ligands at desired final concentrations. The data from the kinetic and equilibrium binding experiments were analyzed by the appropriate non-linear curve fitting procedures using GraphPad Prism 3.03 software package. Inhibition constants were calculated according to the Cheng–Prussoff relation.

---

DMPK studies [1]
In vitro blood distribution and protein binding [1]
Fresh heparinized human and rat blood was used for blood distribution studies and to obtain plasma.

---

Blood distribution [1]
Rat and human blood was spiked with [3H]ABP688 to 5–500,000 pg/mL; blood cells and plasma were separated by centrifugation (1500g, 10 min, 37 °C). Blood distribution was measured at 37 °C with total radioactivity being quantified in both plasma and blood; hematocrite values were determined using microhematocrite capillaries (13,000g, 5 min, n = 3). The fraction of compound in plasma (fP) was calculated as fP (%) = (Cp/Cb) × (1-H) × 100, where Cb is the concentration in blood, Cp is the concentration in plasma, and H is the hematocrite value.

---

Plasma protein binding [1]
Plasma was spiked with [3H]ABP688 to 50–500,000 pg/mL and submitted to ultrafiltration at 37 °C with a Centrifree® device (molecular cut-off of 30 kDa). The total radioactivity was determined in the ultrafiltrate by liquid scintillation (Cu, concentration of unbound compound) and in the sample introduced into the reservoir before ultrafiltration (Cp, total plasma concentration of compound). The unbound (fu) fraction in plasma was calculated as fu (%) = Cu/Cp × 100.

---

In vitro biotransformation and metabolites [1]
Liver microsomes [1]
Liver microsomal pools were used. [3H]ABP688 (0.92 μmol/L) was incubated for 40 min at 37 °C with liver microsomes (0.2 mg protein/mL) in 0.1 M sodium phosphate buffer, pH 7.4, containing 4 mM UDPGA, 1 mM β-NADPH, 5 mM MgCl2, and 12 μg/mL. Individual samples were analyzed on-line by HPLC with radioactivity detection for depletion of unchanged compound as well as formation of metabolites; intrinsic clearance was calculated for each species from the kinetic data of parent compound.

Phosphoinositol accumulation assay [1]
Measurements of phosphoinositide hydrolysis in l-hmGlu5a or CHO-hmGlu1b cells were carried out by determination of inositol monophosphate accumulation in the presence of lithium essentially as published previously. Details are available in the Supplementary Information.

---

Membrane preparation [1]
Membranes of hmGluR5-expressing cells were custom prepared by CMT, according to a previously established protocol. Rat brain membranes were obtained from ABS. All cell membranes used for the receptor binding panel were obtained commercially from Perkin-Elmer or Euroscreen. Details are available in the Supplementary information.

Brain slice autoradiography [1]
Male Sprague–Dawley rats (RA238; 190–220 g) were individually housed in macrolon Type II cages. The animal room was temperature-controlled and equipped with artificial illumination (6:00–18:00 h, lights on). The animals had free access to water and food, ad libitum. Animals were killed by decapitation, the brains were removed and immediately frozen on dry ice and kept at −80 °C. Sagittal brain slices (10 μm) for ex vivo receptor autoradiography were cut from frozen brains with a microtome-cryostat and thaw-mounted on silane-coated microscope slides. Receptor autoradiography was performed according to the following procedure: after 30 min of pre-incubation at room temperature in KRH buffer (Krebs–Ringer Hepes buffer) containing 20 mM Hepes (2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid), pH 7.4, 118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, and 10 mM NaOH, the sections were incubated for 15 min at room temperature in KRH buffer, supplemented with 0.05 mg/mL BSA (bovine serum albumin) and 5 nM [3H]ABP688 (70 Ci/mmol). Non-specific binding was determined in a set of adjacent slides by incubation in the presence of 10 μM MPEP. The washing of labeled sections was carried out as follows: three 20-min washes in the ice-cold KRH buffer (without ligand), 20 s in ice-cold 10 times diluted KRH buffer, and a brief dipping in ice-cold distilled water to remove salts. Finally, the sections were dried under a stream of cold air. Autoradiograms were generated by apposing the labeled tissues to BioMax MR Films at 4 °C for 6 weeks. Sections were finally counterstained with 0.5% Cresyl violet and nuclei localized according to Paxinos and Watson.31 Data from binding were analyzed by optic densitometry of BioMax MR Films using the computerized image analysis system. For a given labeled region, the optic density (OD) corresponding to the total binding and non-specific binding was measured.

---

Disposition of [3H]ABP688 in raty [1]
Male Wistar rats, 220–250 g, were used. The injection solution consisted of 28 μg [3H]ABP688 dissolved in 7.0 mL EtOH/glucose 5% (1:99 v/v). The dose was injected (1 mL/kg) into the surgically exposed femoral vein under slight isoflurane anesthesia. The rats (n = 3 per sampling time) were sacrificed by isoflurane inhalation, heparinized blood was collected and centrifuged to obtain plasma; tissues (lung, heart, liver, kidney, fat, muscle, skin, and brain) were dissected. The radioactivity of all samples was measured by liquid scintillation counting. The concentration of unchanged [3H]ABP688 in plasma and tissue homogenate was determined by LC-RID with 5 μg of non-labeled ABP688 as internal standard: [3H]ABP688 was separated from metabolites and endogenous compounds on a LC-18 column (Supelcosil 5 μm, 4.6 × 150 mm, 40 °C, 10 mM NH4OAc–acetonitrile (45:55 v/v), 1.0 mL/min, UV-detection, λ = 312 nm).The peak corresponding to [3H]ABP688 was collected and radioactivity quantified. Finally, the concentration of [3H]ABP688 was calculated from the ratio of radioactivity in the eluate fraction and the area of the UV peak of non-radiolabeled ABP688.

Blood distribution, plasma protein binding, and in vitro metabolism [1]
The assessments were performed using [3H]ABP688 with the objective of allowing a pharmacokinetic scaling from animal to man.

---

In vitro blood distribution [1]
[3H]ABP688 was stable when incubated in plasma at 37 °C and equilibrated rapidly between plasma and blood cells at 37 °C. The blood distribution was independent of concentration but slightly species-dependent, as illustrated by the fraction present in plasma of 76% in rat and 92% in human. The blood to plasma concentration ratios were 0.74 and 0.57 in rat and human, respectively. The characteristics of the blood distribution of [3H]ABP688, that is, large fraction of the compound present in plasma compartment, independent of concentration, support the use of plasma concentrations to estimate the input function for PET studies.

---

Plasma protein binding [1]
The fraction of [3H]ABP688 bound to plasma proteins was independent of concentration from 50 to 500,000 pg/mL, the average unbound fraction in plasma was higher in rat (6.8%) than in man (4.4%) in accordance with the stronger partitioning into blood cells.

---

Metabolic stability [1]
[3H]ABP688 was incubated with liver microsomes from rat and man. The metabolic stability was assessed by calculation of the intrinsic clearance CLint: the intrinsic clearance was medium to high in rat and human (roughly 150 μL/min/mg). HPLC analysis with radioactivity detection indicated oxidative hydroxylation to be the main metabolic pathway, leading to formation of tritiated water and four mono-hydroxylated metabolites. The exact positions of the hydroxy group could not be determined from the available LC–MS/MS data. No human-specific metabolites were detected when compared to rat.

---

Disposition of [3H]ABP688 in rat following an intravenous microdose injection [1]
The main objective of this pharmacokinetic study was to establish the disposition of ABP688 in plasma and brain of rats.

---

Pharmacokinetics [1]
After a 4 μg/kg [3H]ABP688 intravenous application, plasma concentrations declined rapidly (Fig. 8, left panel). Compartmental analysis according to a bi-exponential model resulted in plasma half-lives of approximately 5 min (t1/2,1) and 1 h (t1/2,2), respectively, where the fraction of AUC associated with the second half-life amounted to 59%. Since [3H]ABP688 was stable when incubated in blood or plasma at 37 °C and only trace amounts of parent compound were detected in excreta, its elimination occurred almost exclusively by metabolism. Indeed, already 5 min. after administration, the fraction unchanged in plasma had decreased to 34% of plasma radioactivity, whereas the AUC of parent drug represented merely 1.4% of the AUC for total radioactivity. The blood clearance value of ABP688 (60 mL/min) was close to the cardiac output in rat indicating an important contribution of extra-hepatic metabolism, possibly located in the lung. The penetration of [3H]ABP688 into the brain was rapid and extensive, and resulted in brain to plasma ratios of up to 20. Total radioactivity and parent compound concentrations in the brain were similar (Fig. 8, right panel), indicating that the proportion of radiolabeled metabolites entering the brain was negligible. The parallel decline of brain and plasma levels, with terminal half lives of 1.1 and 1.0 h, respectively, suggested passive diffusion at the blood–brain barrier.

---

Tissue distribution [1]
The fraction of [3H]ABP688 escaping a rapid elimination was largely distributed into tissues as indicated by the volume of distribution (VSS = 11 L/kg). At 5 min post-dose the highest tissue concentration of [3H]ABP688 (21 pmol/g) was observed in the brain. At this time, the amount of parent drug left in the body represented only 21% of the administered dose, with a considerable fraction (0.8% of dose) present in the brain (Fig. 9). The next highest [3H]ABP688 concentration occurred in fat tissue where the concentration measured between 5 min and 2 h post-dose corresponded to 6–10% of the administered dose. No brain penetration of metabolites and no active transports of ABP688 from and to the brain were observed. These rat results predict a favorable human PK profile for ABP688 as PET ligand.

[1]. ABP688, a novel selective and high affinity ligand for the labeling of mGlu5 receptors: identification, in vitro pharmacology, pharmacokinetic and biodistribution studies. Bioorg Med Chem. 2007 Jan 15;15(2):903-14.

Initial efforts to identify a mGluR5-selective PET tracer based on the structural framework of the selective antagonist MPEP failed to produce a valid PET tracer. It is only very recently that a first series of 11C and 18F-derivatives of MTEP and MTEB (2-methyl-4-phenylethynyl-thiazole) has been described, allowing in vivo PET imaging in rhesus monkey.[1]
The aim of our program was to identify a molecule with an improved tracer profile compared to the MPEP series, particularly a higher affinity, selectivity, and lower lipophilicity. Targeted chemical modifications of the original structure allowed the identification of ABP688, a derivative in which the aromatic ring of the MPEP series is replaced by a functionalized cyclohexenone moiety.[1]
These modifications allowed a significant improvement of the physico-chemical properties such as a log P below 4 and a high water solubility (120 mg/L) compared to the previously published ligands bearing a substituted aromatic ring. As a result, ABP688 maintained an excellent selectivity and affinity for the allosteric site of the mGlu5 receptor while significantly improving physicochemical properties. The radiosynthesis was straightforward, with the label introduced in the last step of the synthesis by alkylation of oxime 7 with methyl iodide yielding either [3H]- or [11C]ABP688 in high radiochemical yield and specific activity.[1]
In vitro, ABP688 was found to be a highly potent functional inhibitor of the hmGlu5 receptor and to bind with high affinity to the human mGlu5 receptor (Ki = 3.5 nM). The characterization of the binding of the tritiated analog, [3H]ABP688, showed a highly specific binding to the human (hmGluR5-expressing cells) or rat (whole brain) mGlu5 receptor. The binding was found to be fully displaceable by MPEP and M-MPEP, reversible, and with a very low level of non-specific binding. In membranes from whole rat brain, the saturation binding experiments with [3H]ABP688 revealed a high Bmax value of 0.87 ± 0.1 pmol/mg and a Kd of 1.9 nM. The profiling of ABP688 against a panel of CNS receptors further confirmed an excellent degree of selectivity for mGluR5.Autoradiography experiments in rat brain slices demonstrated that [3H]ABP688 has a high specific binding to brain regions such as the striatum, cortex, and hippocampus with low or no binding to the cerebellum or to mid-brain regions, in line with the known distribution of the mGlu5 receptor.[1]
In rats, despite an extensive plasma protein binding, [3H]ABP688 rapidly and extensively penetrated the blood–brain barrier (BBB) with brain to plasma ratios of up to 20. The maximal brain concentrations were reached 5 min after iv injection. Importantly, despite extensive metabolism, no metabolites which could interfere with the imaging quality were detected in the brain.[1]
In conclusion, the profile of ABP688 supports its selection for radiolabeling with [11C] and further development as a PET tracer, to image mGlu5 receptors in vivo in the brain. The expected clinical benefits of such a tracer are multiple, from example receptor expression studies in patients, to receptor occupancy studies and clinical dose selection for drug candidates acting at mGlu5 receptors.

C15H16N2O

240.30

239.1377

C, 74.87; H, 6.74; N, 11.71; O, 6.69

1224977-89-2

1224977-89-2; 924298-51-1

Light brown to brown solid-liquid Mixture

5.6

0

3

3

18

411

0

CC1=CC=CC(C#CC(CCC/2)=CC2=N\OC)=N1

GOHCTCOGYKAJLZ-UHFFFAOYSA-N

InChI=1S/C19H13ClF3N3O/c1-12-17(8-3-14-9-10-24-18(20)11-14)25-13(2)26(12)15-4-6-16(7-5-15)27-19(21,22)23/h4-7,9-11H,1-2H3

2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4-yl]ethynyl]pyridine

ABP688; ABP 688; CTEP; 871362-31-1; 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine; mGluR5 inhibitor; CTEP (RO4956371); 2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4-yl]ethynyl]pyridine; E3BWG5775S; CHEMBL3410223; ABP-688

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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