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Effect of single and repeated doses of a new nitroderivative of acetylsalicylic acid on platelet TXA2 production in rats
ELSEVIER
PII S~24-3205(96)~~-0
Life sdcnces, Vol. 58, No. 11,pp. PL 207-210,19% cqyfight 0 1996Ekcvicr ScienceInc. Printedin the USA. AU ri@s rcscmd OOB-32ns/% smx t .oo
PhTARhfACOLOGY LETTERS AcceleratedCommunicadon
EFFECT OF SINGLE AND REPEATED DOSES OF A NEW NITRODERIVATIVEOF ACETYLSALICYLIC ACID ON PLATELET TXA2 PRODUCTIONIN RATS
Laura Cuzzolin, Alessandra Adami, Maurizio Degan*, Federica Crivellente, Stefano Bonapace*, Piero Minuz* and Giuseppina Benoni Institutes of Pharmacology and Internal Medicine*, University of Verona, Verona, Italy (SubmittedNovember9,1995;acceptedNovember27,1995; received in final form January3, 1596)
Abstract: The effects of a new ASA-nitroderivative compound, NCX 4016 (ASA-NO2), on platelet TXA2 synthesis after single and repeated doses in the rat were investigated. Compared to ASA, cumulative doses of ASA-NO;! showed similar inhibitory effects on platelet TXA2 synthesis and signific~t increases in ni~~ni~a~ plasma con~R~ations 1 h after the last drug administration: 24 h later nitrite/nitrate plasma levels returned to the control values, while serum TXA2 concentrations did not change. A time-course study after a single dose of ASA-NO2 showed a significant inhibition of platelet TXA2 production also 24 h after drug administration and a signific~t increase in ni~ite/ni~ate plasma levels until 10 h. KeyWord: thromboxane
A2 production, awtykakylic
acid-nitroderivative
Introduction A recent approach to reduce the ~s~ointestinal toxicity due to non-steroidal ~tiin~ammatory drugs (NSAID,) is the linking of an NO-pleasing moiety to these compounds (1). In p~limin~ studies (2) it has heen suggested that NO-derivatives of NSAID, may also have anti-aggregating effects on platelets mediated by NO release. Acetylsalicylic acid (ASA) is effective as antithrombotic agent acting as inhibitor of platelet aggregation by blocking platelet cyclooxygenase {COX) (3). In the development of the research on the ph~ac~hemis~ of the NO-releasing NSAID,, we have become interested in a new ASA-nitroderivativecompound, 2 acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester NCX 4016 (a white powder, M.W.=335.3). We investigate the effect of this new ASA-ni~de~vative (ASA-No on platelet ~~rn~xane A2 (TXA2) production. After single and repeated doses in the rat, we performed time-course experiments by measuring serum concentrations of TXB2, the stable non-enzymatic metabolite of TXA2, as an index of the thromboxane synthesis capacity via the constitutive isoform COX-1. Moreover, we evaluated the NO release from this molecule by determining ni~te/ni~ate plasma concentrations. Corresponding author: Dr. Laura Cuzzolin, Institute of Pharmacology, University of Verona, Policlinico Borgo Roma, 37 134 Verona, Italy. Tel.: ~5/8098~9 FAX: 045/581111
PI208
Acetylsalicylic Acid and Platelet m
Vol. 58, No. 11, 19%
Animal treatment Female Sprague-Dawley rats (Charles River, Italy) (6 animals per group), weighing 220-240 g, were treated with ASA or ASA-NO2 initially dissolved in 0.1 ml dimethylsulfoxide, then suspended in carboxymethylcellulose 0.5% and administered orally in a volume of 1 ml 100 g-l body weight: the drugs were administered in equimolar doses. For acute experiment, the dosage chosen was 35 mg kg-l for ASA and 65 mg kg-l for ASA-N02: the animals were fasted overnight and s crified 1, 3, 6, 10 and 24 h after drugs administration. For chronic experiment, ASA 15 mg kg- f and ASA-NO2 28 mg kg-l were administered for 9 days in a single daily dose. At the end of the chronic experiment, 1 hour and 24 h after the last drug administration the rats were sacrified. Sodium pentobarbitone (60 mg kg-l body weight i.p) was used as anaesthetic and then blood samples were obtained by intracardiac puncture. Assay of serum 7XB2 Concentrations were determined in diluted samples by radioimmunoassay using commercial antisera (Biomol-Plymouth, PA, USA), according to the method described by Patron0 et al. (4), partially modified: two-ml aliquots of whole blood were immediately transferred into glass tubes containing thrombin (0.2 U/ml) and allowed to clot at 37 C” for 60 min (the addition of thrombin was made to reduce the variability in TXA2 generation by platelets); serum was separated by centrifugation (10 min at 2000 rpm) and kept at -20 C” until assayed for TXB2. Qluntitation of nitritellrtitrate Nitrite was quantitated calorimetrically after reaction with the Griess reagent (5); nitrate reductase induced in E.coli ATCC 25922 (Difco) by strict anaerobic culture was used to convert nitrate in nitrite according to a modification of the method described by Bartholomew (6). Chemicals ASA was purchased from Sigma Chemical Co. (S.Louis, MO); ASA-NO2 was synthesized and kindly supplied by Nicox (London, U.K.). Statistical analysis The statistical analysis of the data was performed by one-way analysis of variance followed by Student’s t test. PcO.05 or less was considered as indicative of a significant difference. Results and Discussion As shown in Figure 1, ASA 35 mg kg-l induced a significant reduction in platelet-derived TXB2 (97%) during the whole experimental period; on the contrary, in the same experimental conditions ASA-NO2 induced a 47, 78, 76, 73 and 87% reduction of serum TXB2 at 1, 3, 6, 10 and 24 h Espectively. These results suggest that ASA-NO2 does not exert the same inhibitory activity on platelet COX compared to ASA after 1 h, while at the other examined times the values were similar to those observed with ASA. These data indicate that a metabolism of the nitroderivative is required: 1 h is a too short time for the release of NO and the biologically active molecule of ASA. About the nitrite/nitrate plasma levels, we observed the higher concentrations between 6 and 10 h, while after 24 h the values were similar to the controls (Figure 2). When administered for 9 days, lower doses of both ASA and ASA-NO2 almost completely inhibited platelet TXA2 synthesis (99.7 and 94% respectively) 1 h after the last drug administration; these data were confirmed 24 h later, proving that TXB2 product cannot recover until new platelets enter in circulation (Figure. 3). A significant increase in nitrite/nitrate plasma concentrations was observed only 1 h after the last ASA-NO2 treatment, but not when ASA was given; 24 h later, the values returned to basal concentrations (Figure 4). As previously described with other NSAID-nitro-derivatives (1). ASA-NO2 appears to protect gastric mucosa from possible damage induced by the inhibition of prostanoid synthesis (7) also after repeated administrations of the drug.
Vol. 58, No. 11,19%
Acetykakylic Acid and Platelet ‘MA,
PL209
t-
3
6
ASA-NO2
24
10
Time (h)
Fig. 1 Time-course of serum TXB2 concentrations after a single dose of ASA or ASA-N02. The values are expressed as mean&E. (n=6). * ~~0.01 and ** ~~0.005 significance compared to controls.
--t
80 %
controls
70 --
6
3
I
I
10
24
Time (h)
Fig. 2. Time-course of nitrite/nitrate plasma levels after a single dose of ASA or ASA-N02. The values are expressed as mean+S.E. (n=6). * ~~0.05 and ** p
Controls
ASA Ih
ASA-NO2 24h
Fig. 3. Serum TXB2 concentrations lh and 24 h after multiple doses of ASA or ASA-N02. are expressed as mean&S.E. (n=6). * pcO.001 significance compared to controls.
The values
PI+=210
Acetylsalicylic Acid and PlateletTXAa
Controls
Vol. 58,No. 11,1%
ASA-NO2
ASA lh
24h
Fig. 4. Nitrite/nitrate plasma levels lh and 24 h after multiple doses of ASA or ASA-NO2. The values are expressed as mean2S.E. * p
PII S~24-3205(96)~~-0
Life sdcnces, Vol. 58, No. 11,pp. PL 207-210,19% cqyfight 0 1996Ekcvicr ScienceInc. Printedin the USA. AU ri@s rcscmd OOB-32ns/% smx t .oo
PhTARhfACOLOGY LETTERS AcceleratedCommunicadon
EFFECT OF SINGLE AND REPEATED DOSES OF A NEW NITRODERIVATIVEOF ACETYLSALICYLIC ACID ON PLATELET TXA2 PRODUCTIONIN RATS
Laura Cuzzolin, Alessandra Adami, Maurizio Degan*, Federica Crivellente, Stefano Bonapace*, Piero Minuz* and Giuseppina Benoni Institutes of Pharmacology and Internal Medicine*, University of Verona, Verona, Italy (SubmittedNovember9,1995;acceptedNovember27,1995; received in final form January3, 1596)
Abstract: The effects of a new ASA-nitroderivative compound, NCX 4016 (ASA-NO2), on platelet TXA2 synthesis after single and repeated doses in the rat were investigated. Compared to ASA, cumulative doses of ASA-NO;! showed similar inhibitory effects on platelet TXA2 synthesis and signific~t increases in ni~~ni~a~ plasma con~R~ations 1 h after the last drug administration: 24 h later nitrite/nitrate plasma levels returned to the control values, while serum TXA2 concentrations did not change. A time-course study after a single dose of ASA-NO2 showed a significant inhibition of platelet TXA2 production also 24 h after drug administration and a signific~t increase in ni~ite/ni~ate plasma levels until 10 h. KeyWord: thromboxane
A2 production, awtykakylic
acid-nitroderivative
Introduction A recent approach to reduce the ~s~ointestinal toxicity due to non-steroidal ~tiin~ammatory drugs (NSAID,) is the linking of an NO-pleasing moiety to these compounds (1). In p~limin~ studies (2) it has heen suggested that NO-derivatives of NSAID, may also have anti-aggregating effects on platelets mediated by NO release. Acetylsalicylic acid (ASA) is effective as antithrombotic agent acting as inhibitor of platelet aggregation by blocking platelet cyclooxygenase {COX) (3). In the development of the research on the ph~ac~hemis~ of the NO-releasing NSAID,, we have become interested in a new ASA-nitroderivativecompound, 2 acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester NCX 4016 (a white powder, M.W.=335.3). We investigate the effect of this new ASA-ni~de~vative (ASA-No on platelet ~~rn~xane A2 (TXA2) production. After single and repeated doses in the rat, we performed time-course experiments by measuring serum concentrations of TXB2, the stable non-enzymatic metabolite of TXA2, as an index of the thromboxane synthesis capacity via the constitutive isoform COX-1. Moreover, we evaluated the NO release from this molecule by determining ni~te/ni~ate plasma concentrations. Corresponding author: Dr. Laura Cuzzolin, Institute of Pharmacology, University of Verona, Policlinico Borgo Roma, 37 134 Verona, Italy. Tel.: ~5/8098~9 FAX: 045/581111
PI208
Acetylsalicylic Acid and Platelet m
Vol. 58, No. 11, 19%
Animal treatment Female Sprague-Dawley rats (Charles River, Italy) (6 animals per group), weighing 220-240 g, were treated with ASA or ASA-NO2 initially dissolved in 0.1 ml dimethylsulfoxide, then suspended in carboxymethylcellulose 0.5% and administered orally in a volume of 1 ml 100 g-l body weight: the drugs were administered in equimolar doses. For acute experiment, the dosage chosen was 35 mg kg-l for ASA and 65 mg kg-l for ASA-N02: the animals were fasted overnight and s crified 1, 3, 6, 10 and 24 h after drugs administration. For chronic experiment, ASA 15 mg kg- f and ASA-NO2 28 mg kg-l were administered for 9 days in a single daily dose. At the end of the chronic experiment, 1 hour and 24 h after the last drug administration the rats were sacrified. Sodium pentobarbitone (60 mg kg-l body weight i.p) was used as anaesthetic and then blood samples were obtained by intracardiac puncture. Assay of serum 7XB2 Concentrations were determined in diluted samples by radioimmunoassay using commercial antisera (Biomol-Plymouth, PA, USA), according to the method described by Patron0 et al. (4), partially modified: two-ml aliquots of whole blood were immediately transferred into glass tubes containing thrombin (0.2 U/ml) and allowed to clot at 37 C” for 60 min (the addition of thrombin was made to reduce the variability in TXA2 generation by platelets); serum was separated by centrifugation (10 min at 2000 rpm) and kept at -20 C” until assayed for TXB2. Qluntitation of nitritellrtitrate Nitrite was quantitated calorimetrically after reaction with the Griess reagent (5); nitrate reductase induced in E.coli ATCC 25922 (Difco) by strict anaerobic culture was used to convert nitrate in nitrite according to a modification of the method described by Bartholomew (6). Chemicals ASA was purchased from Sigma Chemical Co. (S.Louis, MO); ASA-NO2 was synthesized and kindly supplied by Nicox (London, U.K.). Statistical analysis The statistical analysis of the data was performed by one-way analysis of variance followed by Student’s t test. PcO.05 or less was considered as indicative of a significant difference. Results and Discussion As shown in Figure 1, ASA 35 mg kg-l induced a significant reduction in platelet-derived TXB2 (97%) during the whole experimental period; on the contrary, in the same experimental conditions ASA-NO2 induced a 47, 78, 76, 73 and 87% reduction of serum TXB2 at 1, 3, 6, 10 and 24 h Espectively. These results suggest that ASA-NO2 does not exert the same inhibitory activity on platelet COX compared to ASA after 1 h, while at the other examined times the values were similar to those observed with ASA. These data indicate that a metabolism of the nitroderivative is required: 1 h is a too short time for the release of NO and the biologically active molecule of ASA. About the nitrite/nitrate plasma levels, we observed the higher concentrations between 6 and 10 h, while after 24 h the values were similar to the controls (Figure 2). When administered for 9 days, lower doses of both ASA and ASA-NO2 almost completely inhibited platelet TXA2 synthesis (99.7 and 94% respectively) 1 h after the last drug administration; these data were confirmed 24 h later, proving that TXB2 product cannot recover until new platelets enter in circulation (Figure. 3). A significant increase in nitrite/nitrate plasma concentrations was observed only 1 h after the last ASA-NO2 treatment, but not when ASA was given; 24 h later, the values returned to basal concentrations (Figure 4). As previously described with other NSAID-nitro-derivatives (1). ASA-NO2 appears to protect gastric mucosa from possible damage induced by the inhibition of prostanoid synthesis (7) also after repeated administrations of the drug.
Vol. 58, No. 11,19%
Acetykakylic Acid and Platelet ‘MA,
PL209
t-
3
6
ASA-NO2
24
10
Time (h)
Fig. 1 Time-course of serum TXB2 concentrations after a single dose of ASA or ASA-N02. The values are expressed as mean&E. (n=6). * ~~0.01 and ** ~~0.005 significance compared to controls.
--t
80 %
controls
70 --
6
3
I
I
10
24
Time (h)
Fig. 2. Time-course of nitrite/nitrate plasma levels after a single dose of ASA or ASA-N02. The values are expressed as mean+S.E. (n=6). * ~~0.05 and ** p
Controls
ASA Ih
ASA-NO2 24h
Fig. 3. Serum TXB2 concentrations lh and 24 h after multiple doses of ASA or ASA-N02. are expressed as mean&S.E. (n=6). * pcO.001 significance compared to controls.
The values
PI+=210
Acetylsalicylic Acid and PlateletTXAa
Controls
Vol. 58,No. 11,1%
ASA-NO2
ASA lh
24h
Fig. 4. Nitrite/nitrate plasma levels lh and 24 h after multiple doses of ASA or ASA-NO2. The values are expressed as mean2S.E. * p