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:

Non-compartmental analysis was employed in all pharmacokinetic analysis. Mean extrapolated AUC after each iv and oral dose was less than 20%. The mean V d of SC-560 was 9.1 ± 4.6 L/kg after iv administration. The mean t1/2 of SC-560 was ~5 h (Table 1). After oral dosing, of the PEG formulation in each of the rats there were measurable amounts of drug in serum at time (15 min) of the first post-dose blood sample (Fig. 2). The tmax ranged from 0.5 to 4 h, and Cmax ranged from 150 to 316 ng/mL (Fig. 2, Table 1). After oral dosing, of the MC formulation in each of the rats there were measurable amounts of drug in serum at time (15 min) of the first post-dose blood sample (Fig. 2). The mean tmax was 2 h, and Cmax ranged from 102 to 132 ng/mL (Fig. 2, Table 1). Comparing the AUC of the iv dose with that obtained after oral dosing of PEG and MC, the mean oral bioavailability of SC-560 in the rat (Table 1) was estimated to be 15 and 5%, respectively. [3]
SC-560 also produced a significant reduction in urinary sodium, chloride and potassium excretion over 24 h compared to controls (Fig. 4). No significant changes in serum creatinine or BUN concentration were apparent between SC-560 treated and control group.
As suggested by its large Vd (mean 9.1 ± 4.6 L/kg), which greatly exceeds body water composition, SC-560 is highly distributed into the rat tissues. The pharmacokinetic parameters of SC-560 in the rat are similar to celecoxib, which similarly possess a large Vd (11). SC-560 is a very lipophilic drug, and is sparingly soluble in aqueous buffers. This physiochemical property is likely a contributor to its large Vd, as it would promote uptake of drug into tissues of high lipid content, such as adipose and brain.
Causes of low oral bioavailability include high first-pass metabolism by the liver and/or incomplete transfer of drug from the gastrointestinal tract to the portal vein. In the rat, mean hepatic blood flow is 3.3 L/h/kg with a hematocrit of 0.48; this yields a mean hepatic plasma flow of ~1.74 L/h/kg. Therefore, the serum clearance of SC-560 after 10 mg/kg of 1.15 ± 0.46 L/h/kg is approaching the mean hepatic plasma flow in the rat Some involvement of incomplete transfer from the gastrointestinal tract is apparent as SC-560 bioavailability in PEG increased 3 fold compared to the MC suspension.
Previous pharmacodynamic and pharmacological studies of SC-560 in vivo have administered this compound intravenously and orally in various formulations without any knowledge of its pharmacokinetics. The current studies suggest that SC-560 undergoes route of administration and formulation dependent pharmacokinetics.
Furthermore, inflammation may reduce clearance of drugs cleared in the liver and SC-560 administration in animal models of disease that involve the liver (i.e. cirrhosis) may demonstrate altered pharmacokinetics.
In agreement with previous studies, we found that SC-560 is orally active. SC-560 induced a significant increase in NAG activity per total urine volume collected over 24 h. NAG is a lysosomal enzyme found in the renal tubules with a molecular weight of 150,000 daltons and thus its large size prevents it from reaching the nephron lumen because it cannot undergo glomerular filtration. NAG has been suggested to be a specific marker of renal tubular damage. Furthermore, a significant decrease in urinary sodium, potassium, and chloride excretion was evident. The decrease in electrolyte excretion may be related to constitutive COX-1 inhibition in the kidney. Interestingly, treatment with SC-560 did not produce any significant changes in either BUN level or serum creatinine after a single dose. We have found that serum creatinine and BUN are relatively insensitive measures of renal toxicity in the rat after administration of single therapeutic doses of non-steroidal anti-inflammatory drugs and various other COX inhibitors. The only other study on the pharmacodynamic effects of SC-560 on renal function was undertaken in rats with cirrhosis and ascites, which also demonstrated a significant decrease in urinary sodium and a significant reduction in glomerular filtration rate, renal plasma flow, and renal prostaglandin E2 after a 20 mg/kg iv dose. It is noteworthy that in cirrhotic rats the effects of selective COX-1 inhibition on furosemide-induced diuretic and naturetic response with SC-560 were dose-dependent.

In summary, SC-560 is a lipophilic drug characterized by a clearance approaching hepatic plasma flow, a high Vd and a low and formulation dependent bioavailability. A single oral dose of 10 mg/kg demonstrated an ability to reduce urinary electrolytes and induced renal tubular damage, as measured by an increase in NAG. This study is the first report regarding the pharmacokinetics and bioavailability of SC-560 and its effects on NAG excretion and renal electrolytes in animals without disease. Further studies characterizing the pharmacokinetic/pharmacodynamic relationships of selective COX-1 inhibition are currently in progress.[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 is a member of the class of pyrazoles that is 1H-pyrazole which is substituted at positions 1, 3 and 5 by 4-methoxyphenyl, trifluoromethyl and 4-chlorophenyl groups, respectively. Unlike many members of the diaryl heterocycle class of cyclooxygenase (COX) inhibitors, SC-560 is selective for COX-1. It has a role as a cyclooxygenase 1 inhibitor, a non-steroidal anti-inflammatory drug, an apoptosis inducer, an antineoplastic agent and an angiogenesis modulating agent. It is a member of pyrazoles, an organofluorine compound, an aromatic ether and a member of monochlorobenzenes.
5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole has been reported in Alstonia yunnanensis with data available.

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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]

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Purpose: To delineate formulation dependent pharmacokinetics and bioavailability of SC-560, a relatively new cycloooxygenase-1 (COX-1) specific inhibitor, in the rat and examine its influence on the renal tubular enzyme, N-acetyl-beta-D-glucosaminidase (NAG), and urinary electrolytes.
Methods: 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.
Results: 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]

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.)