COX-1 (IC50 = 9 nM); COX-2 (IC50 = 6.3 μM)

In a concentration-dependent manner, the pre-dilution of COX-1 and SC-560 prevents arachidonic acid from being converted to PGE2. Compared to COX-1, SC-560 has an IC50 of 6.3 μM against COX-2, which is about 1,000 times greater[1]. SC-560 shows dose- and time-dependent suppression of the development of HCC cells. In soft agar, SC-560 also prevents colony formation and stimulates dose-dependent HCC cell proliferation. Furthermore, in a manner that is dependent on both dose and time, SC-560 activates caspases 3 and 7 and decreases the amounts of the anti-tumor cell growth sudan proteins Svivin and XIAP [2].

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Two isoforms of cyclooxygenase (COX) are known, and to date most studies have implicated COX-2 in the development and progression of various human cancers. Increasing evidence suggests that COX-1 may also play a similar role. Indeed, we have recently observed that the dual COX-1/COX-2 inhibitor indomethacin induces apoptosis in human hepatocellular carcinoma (HCC) cell lines more effectively than the selective COX-2 inhibitors, possibly implicating COX-1 in HCC. In this study we investigated the expression of COX-1 in non-tumor and malignant human liver tissues, as well as the effects of the highly selective COX-1 inhibitor SC-560 on cell growth and apoptosis in human HCC cell lines. Expression of COX-1 was detected in nearly all the samples assayed, although with a high variability between non-tumoral (NT) and malignant tissues. The percentage of COX-1 positive cells was significantly higher in the NT tissues than in the tumors (p<0.0001). In well-differentiated HCC COX-1 expression was significantly higher than in the poorly-differentiated tissues (p<0.05). SC-560 showed a dose- and time-dependent inhibitory effect on HCC cell growth. The combination of the COX-1 inhibitor with nimesulide and CAY10404, two selective COX-2 inhibitors, resulted in additive effects on cell growth inhibition. SC-560 also inhibited colony formation in soft agar and induced apoptosis in HCC cells in a dose-dependent manner. Moreover, SC-560 decreased the levels of the anti-apoptotic proteins survivin and XIAP and activated caspase-3 and -7 in a dose- and time-dependent fashion. In conclusion, we report for the first time that the selective COX-1 inhibitor SC-560 exhibits anti-tumor and apoptotic effects in human HCC cells. Overall, our previous and present results suggest that both COX-1 and COX-2 inhibitors may have potential therapeutic implications in HCC patients[2].

Ionophore-stimulated TxB2 production was totally suppressed by injury of 10 or 30 mg/kg SC-560 1 hour prior to test, suggesting that SC-560 has biolesion availability and inhibits COX-1 in vivo [1]. In the scaffold tissue, SC-560 was extensively dispersed, and CL was in close proximity to the hepatic venous flow. The medication shows nephrotoxicity, limited bioavailability, and formulation dependence of less than 15% following epidermal formulation [3].

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After an iv dose (10 mg/kg) of SC-560, serum AUC, t(1/2), CL and Vd were 9704 +/- 4038 ng h/mL, 5.4 +/- 0.8 h, 1.15 +/- 0.46 L/h/kg and 9.1 +/- 4.6 L/kg (mean +/- SD, n = 5), respectively. Oral administration of 10 mg/kg SC-560-PEG and MC (n=5 rats) yielded serum AUC, C max, t (max )and t (1/2) of 1203.4 +/- 130.3 and 523 +/- 208 ng h/mL, 218.5 +/- 86.9 and 119.8 +/- 15.5 ng/mL, 1.00 +/- 1.8 and 2.0+/- 0 h, 3.7 +/- 1.6 and 2.7 +/- 1.7 h (mean +/- SD, n = 5), respectively. A single oral dose 10 mg/kg of SC-560 in PEG resulted in an increase in NAG excretion in urine and a reduction in 0-24 h urinary sodium, potassium, and chloride excretion. Conclusions: SC-560 extensively distributes into rat tissues, and has a CL approaching hepatic plasma flow. The drug displays low <15% and formulation dependent bioavailability after oral administration and demonstrates kidney toxicity.[3]

The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H2, the precursor of PGs and thromboxane. These lipid mediators play important roles in inflammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inflammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inflammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50 = 0.009 microM; COX-2 IC50 = 6.3 microM). SC-560 inhibited COX-1-derived platelet thromboxane B2, gastric PGE2, and dermal PGE2 production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inflammation or hyperalgesia at doses that markedly inhibited in vivo COX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inflammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal fluid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inflammatory pain that is mediated at least in part by COX-2[1].

In this study we investigated the expression of COX-1 in non-tumor and malignant human liver tissues, as well as the effects of the highly selective COX-1 inhibitor SC-560 on cell growth and apoptosis in human HCC cell lines. Expression of COX-1 was detected in nearly all the samples assayed, although with a high variability between non-tumoral (NT) and malignant tissues. The percentage of COX-1 positive cells was significantly higher in the NT tissues than in the tumors (p<0.0001). In well-differentiated HCC COX-1 expression was significantly higher than in the poorly-differentiated tissues (p<0.05). SC-560 showed a dose- and time-dependent inhibitory effect on HCC cell growth. The combination of the COX-1 inhibitor with nimesulide and CAY10404, two selective COX-2 inhibitors, resulted in additive effects on cell growth inhibition. SC-560 also inhibited colony formation in soft agar and induced apoptosis in HCC cells in a dose-dependent manner. Moreover, SC-560 decreased the levels of the anti-apoptotic proteins survivin and XIAP and activated caspase-3 and -7 in a dose- and time-dependent fashion. In conclusion, we report for the first time that the selective COX-1 inhibitor SC-560 exhibits anti-tumor and apoptotic effects in human HCC cells. Overall, our previous and present results suggest that both COX-1 and COX-2 inhibitors may have potential therapeutic implications in HCC patients[1].

The pharmacokinetics of SC-560 was studied in Sprague-Dawley rats (n = 5 per group) after a single intravenous (i.v.) and oral dose (10 mg/kg) in polyethylene glycol (PEG) 600 and a single oral dose (10 mg/kg) in 1% methylcellulose (MC). Serial blood samples were collected via a catheter inserted in the right jugular vein and serum samples were analysed for SC-560 using reverse phase HPLC. After oral administration of SC-560 in PEG, urine was also collected for 24 h and analysed for urinary sodium, chloride, and potassium as well as NAG.[3]

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Dosing and Sampling Collection [3]
On the morning of study, SC-560 was weighed and dissolved in PEG 600 or suspended in 1% methylcellulose (MC) (~0.5 mL). The formulations were used for dosing rats orally, via gavage needle, or intravenously (iv). Rats received 10 mg/kg (N = 5) of SC-560 (PEG) iv. Five rats received oral doses of 10 mg/kg of SC-560 in PEG or MC. Saline was used to flush the cannulae immediately after injection of the drug (~0.25 mL) and after each collection of blood. Blood (250 m L) was collected pre-dose through the catheter and at 2, 10 min, and 0.5, 1, 2, 4, 8, 12, and 24 h after iv dosing and at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after oral dosing. In a separate study, five rats received ~0.5 mL of vehicle or 10 mg/kg of SC-560 in PEG orally and urine was collected for 24 hours and volume measured. All specimens were kept at -70°C until analysis. Urine was thawed at room temperature and vortexed for 30 seconds and a sample of 0.05 mL was used in each analysis according to manufacturer's instructions. 3-Cresolsulfonphthalenyl-N-acetyl-b-D-glucosaminide, sodium salt is hydrolysed by NAG with the release of 3-cresolsulfonphthalein, sodium salt (3-cresol purple), which is measured photometrically at 580 nm.

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Assay [3]
A validated HPLC procedure was used for assay of SC-560 in rat serum. To serum samples (0.1 ml) was added 50 m l of internal standard solution of testosterone 17-propionate, (10 m g/ml) and 1 mL ice-cold acetonitrile. The mixture was vortexed for 1 min, and centrifuged at 15000 rpm at 4°C for 5 min. The supernatant was collected and evaporated to dryness using a Heto Vac concentrator. The residue was reconstituted with 100 ml of 70% methanol (v/v), vortexed for 1 min and centrifuged at 8000 rpm at 4°C for 5 min, and 40 m l of the supernatant was injected onto the column. The HPLC system used was a Shimadzu HPLC, consisting of an LC-10AT VP pump, an SIL-10AF auto injector, an SPD-M10A VP spectrophotometric diodearray detector, and an SCL-10A VP system controller. Data collection and integration were accomplished using Shimadzu EZ Start 7.1.1 SP1.The analytical column used was Beckman ultrasphere octyl column (150 × 2 mm I.D., 5-m particle size) equipped with a pre-column (7.5 × 2 mm I.D., 5 m) of the same packing material. The mobile phase consisted of methanol and water (7:3, v/v), filtered and degassed under reduced pressure, prior to use. Separation was carried out isocratically at ambient temperature (25 ± 1°C), and a flow rate of 0.25 mL/min, with UV detection at 240 nm. For all runs, quality control samples were incorporated to ensure integrity of the results. Standard curves were linear, and bias and precision data were less than 10% at high (5000 ng/mL) and low (20 ng/mL) drug concentrations. The accuracy was estimated based on the mean percentage error of measured concentration to the actual concentration.

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Pharmacokinetic Analysis [3]
The elimination rate constant (λn) was estimated by linear regression of the serum concentrations in the log-linear terminal phase. Peak concentrations of SC-560 in the serum (Cmax ) and the corresponding Tmax were estimated for each rat from the serum concentration profile using WinNonlin® (version 1.0). In order to estimate serum concentrations (C0 ) immediately after injection of iv SC-560, compartmental models were fitted to the serum concentration versus time data using WinNonlin® (version 1.0). The estimated C0 was then used in conjunction with the actual measured serum concentrations to determine the area under the plasma concentration-time curve (AUC). The AUC0∞· was calculated using the combined log-linear trapezoidal rule for data from time of dosing to the last measured concentration, plus the quotient of the last measured concentration divided by λn. Non-compartmental pharmacokinetic methods were used to calculate clearance (CL) and volume of distribution (Vd) after iv dosing. The oral bioavailability (F) was calculated as follows:

All pharmacokinetic analyses were performed using a non-compartmental model. The mean extrapolated AUC was less than 20% after each intravenous and oral administration. Following intravenous administration, the mean volume of distribution (Vd) of SC-560 was 9.1 ± 4.6 L/kg. The mean half-life (t1/2) of SC-560 was approximately 5 hours (Table 1). Following oral administration, PEG formulation drug concentrations were detectable in the serum of each rat 15 minutes (15 min) after the first dose (Figure 2). The time to peak concentration (tmax) ranged from 0.5 to 4 hours, and the peak concentration (Cmax) ranged from 150 to 316 ng/mL (Figure 2, Table 1). Following oral administration, MC formulation drug concentrations were detectable in the serum of each rat 15 minutes (15 min) after the first dose (Figure 2). The mean time to peak concentration (tmax) was 2 hours, and the peak concentration (Cmax) ranged from 102 to 132 ng/mL (Figure 2, Table 1). The mean oral bioavailability of SC-560 in rats was estimated to be 15% and 5% (Table 1) by comparing the AUC of the intravenous dose with the AUC after oral administration of PEG and MC. [3] Compared with the control group, SC-560 also significantly reduced urinary sodium, chloride and potassium excretion over 24 hours (Figure 4). There were no significant changes in serum creatinine or blood urea nitrogen concentrations in the SC-560 treatment group and the control group. Since its volume of distribution (Vd) (mean 9.1 ± 4.6 L/kg) is much larger than that of body fluid composition, it indicates that SC-560 is widely distributed in rat tissues. The pharmacokinetic parameters of SC-560 in rats are similar to those of celecoxib, both of which have large volumes of distribution (Vd) (11). SC-560 is a highly lipophilic drug with low solubility in aqueous buffers. This physicochemical property may be one of the reasons for its large volume of distribution, as it promotes the absorption of the drug by high-fat tissues such as adipose tissue and brain tissue. The low oral bioavailability can be attributed to a high first-pass metabolism rate in the liver and/or incomplete transport of the drug from the gastrointestinal tract to the portal vein. In rats, the mean hepatic blood flow is 3.3 L/h/kg, and the hematocrit is 0.48; the mean hepatic plasma flow is calculated to be approximately 1.74 L/h/kg. Therefore, after administration of SC-560 at a dose of 10 mg/kg to rats, the serum clearance rate was 1.15 ± 0.46 L/h/kg, close to the mean hepatic plasma flow. Since the bioavailability of SC-560 in PEG is 3 times higher than that in MC suspension, it suggests the possibility of incomplete gastrointestinal transport. Previous pharmacodynamic and pharmacological studies of SC-560 involved intravenous and oral administration, but its pharmacokinetics were not well understood. Current research indicates that the pharmacokinetics of SC-560 is related to the route of administration and formulation. Furthermore, inflammation may reduce the clearance of drugs eliminated by the liver, and administration of SC-560 may alter its pharmacokinetics in animal models of liver diseases such as cirrhosis. Consistent with previous studies, we found that SC-560 has oral activity. SC-560 significantly increased the activity of NAG in total 24-hour urine. NAG is a lysosomal enzyme present in the renal tubules with a molecular weight of 150,000 Daltons. Due to its large molecular weight, it cannot pass through glomerular filtration into the renal tubular lumen. NAG is considered a specific marker of renal tubular injury. In addition, the excretion of urinary sodium, potassium, and chloride was also significantly reduced. The decrease in electrolyte excretion may be related to constitutive inhibition of COX-1 in the kidneys. Notably, SC-560 did not cause significant changes in BUN levels or serum creatinine after a single dose. We found that serum creatinine and BUN levels showed relatively low sensitivity to nephrotoxicity after a single therapeutic dose of nonsteroidal anti-inflammatory drugs (NSAIDs) and various other COX inhibitors in rats. Another study on the pharmacodynamic effects of SC-560 on renal function was conducted in cirrhotic rats with ascites, which also showed a significant decrease in urinary sodium, glomerular filtration rate, renal plasma flow, and renal prostaglandin E2 levels after intravenous administration of a 20 mg/kg dose. Notably, in cirrhotic rats, the selective COX-1 inhibitor SC-560 showed a dose-dependent effect on furosemide-induced diuresis and excretion. In summary, SC-560 is a lipophilic drug characterized by clearance close to hepatic plasma flow, a high volume of distribution (Vd), low bioavailability, and formulation dependence. A single oral dose of 10 mg/kg can reduce urinary electrolyte levels and induce renal tubular damage, manifested as elevated NAG levels. This study is the first to report the pharmacokinetics and bioavailability of SC-560, as well as its effects on NAG excretion and renal electrolytes in disease-free animals. Further studies are currently underway to characterize the pharmacokinetic/pharmacodynamic relationship of selective COX-1 inhibitors. [3]

[1]. Pharmacological analysis of cyclooxygenase-1 in inflammation. Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13313-8.

[2]. The selective cyclooxygenase-1 inhibitor SC-560 suppresses cell proliferation and induces apoptosis in human hepatocellular carcinoma cells. Int J Mol Med. 2006 Feb;17(2):245-52.

[3]. Formulation dependent pharmacokinetics, bioavailability and renal toxicity of a selective cyclooxygenase-1 inhibitor SC-560 in the rat. J Pharm Pharm Sci. 2003 May-Aug;6(2):205-10.

SC560 belongs to the pyrazole class of compounds, with the structure 1H-pyrazole, substituted at positions 1, 3, and 5 by 4-methoxyphenyl, trifluoromethyl, and 4-chlorophenyl, respectively. Unlike many diaryl heterocyclic cyclooxygenase (COX) inhibitors, SC-560 is selective for COX-1. It can function as a cyclooxygenase 1 inhibitor, a nonsteroidal anti-inflammatory drug, an apoptosis inducer, an antitumor drug, and an angiogenesis regulator. It belongs to the pyrazole class, organofluorine compounds, aromatic ethers, and monochlorobenzene class of compounds. 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole has been reported in Alstonia yunnanensis, and relevant data are available. Cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG)H2, a precursor of prostaglandins and thromboxanes. These lipid mediators play important roles in inflammation, pain, and normal physiological function. While substantial data suggest that the inducible isoenzyme COX-2 plays a significant role in inflammation and pain, the constitutively expressed isoenzyme COX-1 is also thought to play a role in inflammatory processes. To address the latter from a pharmacological perspective, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50 = 0.009 μM; COX-2 IC50 = 6.3 μM). SC-560 inhibited the production of COX-1-derived platelet thromboxane B2, gastric PGE2, and skin PGE2, indicating oral activity, but did not inhibit COX-2-derived prostaglandins (PGs) in a lipopolysaccharide-induced rat air sac model. In a carrageenan-induced rat paw pad model, therapeutic or prophylactic doses of SC-560 significantly inhibited COX-1 activity in vivo, but did not affect acute inflammation or hyperalgesia. In contrast, the selective COX-2 inhibitor celecoxib exhibited anti-inflammatory and analgesic effects in this model. However, both SC-560 and celecoxib reduced paw PGs to the same level, contrary to expectations. Following carrageenan injection, cerebrospinal fluid prostaglandin (PG) levels increased, and celecoxib significantly reduced these levels, but SC-560 had no effect. These results suggest that, in addition to the effects of peripherally generated PGs, there is an important centrally mediated neural component in inflammatory pain, which is at least partially mediated by COX-2. [1]

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Objective: To elucidate the formulation-dependent pharmacokinetics and bioavailability of a novel cyclooxygenase-1 (COX-1) specific inhibitor, SC-560, in rats, and to investigate its effects on renal tubular enzyme N-acetyl-β-D-glucosidase (NAG) and urinary electrolytes.
Methods: In Sprague-Dawley rats (n = 5 per group), SC-560 was administered intravenously (iv) and orally (10 mg/kg) in a solution of polyethylene glycol (PEG) 600, and SC-560 was administered orally (10 mg/kg) in a solution of 1% methylcellulose (MC) to investigate its pharmacokinetics. A series of blood samples were collected by catheter insertion into the right jugular vein, and the concentration of SC-560 in serum was analyzed by reversed-phase high-performance liquid chromatography (HPLC). Following oral administration of SC-560 in polyethylene glycol (PEG), 24-hour urine samples were collected, and urinary sodium, chloride, potassium, and NAG were analyzed. Results: After intravenous administration of SC-560 (10 mg/kg), serum AUC, t(1/2), CL, and Vd were 9704±4038 ng·h/mL, 5.4±0.8 h, 1.15±0.46 L/h/kg, and 9.1±4.6 L/kg, respectively (mean ± standard deviation, n=5). Following oral administration of 10 mg/kg SC-560-PEG and MC (n=5 rats), serum AUC, Cmax, t(max), and t(1/2) were 1203.4±130.3 and 523±208 ng·h/mL, 218.5±86.9 and 119.8±15.5 ng/mL, 1.00±1.8 and 2.0±0 h, and 3.7±1.6 and 2.7±1.7 h, respectively (mean ± standard deviation, n=5). A single oral administration of 10 mg/kg of the polyethylene glycol (PEG) formulation SC-560 increased urinary NAG excretion and decreased urinary sodium, potassium, and chloride excretion over 0–24 hours. Conclusion: SC-560 is widely distributed in rat tissues, and its clearance rate is close to that of hepatic plasma flow. After oral administration, the bioavailability of the drug is less than 15%, which is formulation-dependent, and it exhibits nephrotoxicity. [3]

C17H12CLF3N2O

352.7382

352.059

C, 57.89; H, 3.43; Cl, 10.05; F, 16.16; N, 7.94; O, 4.54

188817-13-2

4306515

White to yellow solid powder

1.3±0.1 g/cm3

440.6±45.0 °C at 760 mmHg

63 °C

220.3±28.7 °C

0.0±1.0 mmHg at 25°C

1.564

6.13

0

5

3

24

407

0

ClC1C([H])=C([H])C(=C([H])C=1[H])C1=C([H])C(C(F)(F)F)=NN1C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H]

PQUGCKBLVKJMNT-UHFFFAOYSA-N

InChI=1S/C17H12ClF3N2O/c1-24-14-8-6-13(7-9-14)23-15(10-16(22-23)17(19,20)21)11-2-4-12(18)5-3-11/h2-10H,1H3

5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole

SC-560; SC 560; SC-560; 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; SC 560; 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)pyrazole; SC560; 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole; 1H-Pyrazole, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-; SC560.

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)

DMSO : ≥ 100 mg/mL (~283.49 mM)

Solubility in Formulation 1: ≥ 3 mg/mL (8.50 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 30.0 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 (7.09 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.)