PHARMACODYNAMICS AND DRUG ACTION Tolerance to nicotinic acid flushing The mechanism of tolerance to nicotinic acid flushing was determined in subjects during a 5-day course of treatment. Objective measures of skin blood flow were used to confirm the development of tolerance. Plasma levels of nicotinic acid showed marked intraindividual variability but were not decreased with the development of tolerance. However plasma levels of 9 - a 11-P prostaglandin F,, a stable metabolite of prostaglandin D,, became undetectable in most subjects with the development of tolerance. Thus tolerance is not associated with decreased levels of nicotinic acid or development of tolerance to the prostaglandin mediator, but with decreased levels of the mediator. (CLINPHARMACOL THER1991;50:66-70.)
Ralph H. Stem, PhD, MD, J. David Spence, MD, David J. Freeman, PhD, and Anwar Parbtani, PhD London, Ontario, Canada Nicotinic acid is a remarkably effective hypolipidemic agent, lowering low-density lipoprotein cholesterol, triglycerides, and lipoprotein(a) while raising high-density lipoprotein cholesterol. However, it is under-utilized because of its reputation for adverse effects. The most common, known as flushing, consists of skin erythema caused by increased blood flow and a burning sensation. The response is similar to a sunbum but lasts only an hour. Since its introduction as a hypolipidemic agent in 1955, clinical observers have agreed that tolerance to flushing develops quickly in most individuals. In spite of this, this harmless side effect remains the greatest impediment to the prescription of this agent. The biology of flushing is incompletely characterized. Ingestion of pharmacologic doses of nicotinic acid have recently been shown to produce 400- to 800-fold increases in plasma levels of 9-a 11-P prostaglandin F,, a stable metabolite of prostaglandin D,.' However, the cellular origin and target of this mediator are unknown. Tolerance to nicotinic acid flushing may result from alterations in nic-
From the Department of Medicine, University of Western Ontario. Supported by the Department of Medicine, Victoria Hospital and Hypertension Research Unit, Victoria Hospital. Received for publication Jan. 28, 1991; accepted March 24, 1991. Reprint requests: Ralph Stem, PhD, MD, Hypertension Research Unit D-213, Victoria Hospital/South Street Campus, London, Ontario N6A 4G.5, Canada. 1311129829
otinic acid metabolism, decreased release of prostaglandin D,, or development of tolerance to prostaglandin D,. This study was designed to measure the development of tolerance by using objective measures of skin blood flow and relating the skin blood flow changes to plasma nicotinic acid and prostaglandin levels.
METHODS Clinical investigations. The protocol was approved by the Human Subjects Research Board of the University of Western Ontario, and informed consent was obtained from participants. Healthy men who were taking no medications were recruited, and each subject took a 500 mg nicotinic acid tablet three times a day for a total of 13 doses. The first, seventh, and thirteenth dose were taken in the clinical research unit. Subjects reported at 8 AM while fasting for at least 8 hours. Each subject was placed in bed with his head elevated and an intravenous catheter was inserted. A YSI series 400 attachable surface temperature probe (Yellow Springs. Instrument Co., Yellow Springs, Ohio) was taped 1 cm below the outer canthal margin of the left eye, and the laser Doppler probe of a Medpacific LD5000 capillary perfusion monitor (Medpacific Corporation, Seattle, Wash.) was attached 1 cm below the outer canthal margin of the right eye. Oral temperature was measured with a basal temperature thermometer. After acclimatization to room temperature, which was maintained at 20" to 22" C, a nic-
VOLUME 50 NUMBER I
otinic acid tablet with 300 ml water at room temperature was given at 9 AM. Room, oral, and facial temperatures, as well as laser Doppler readings, were measured every 10 minutes during the first hour and every 15 minutes for the next 45 minutes; additional readings were taken during maximal flushing. Blood was collected into two 10 ml EDTA-anticoagulated tubes and stored on ice. Plasma was removed after centrifugation at lOOOg for 15 minutes at 4" C and stored at -60" C until assay. Nicotinic acid and nicotinuric acid measurements. Nicotinic acid and nicotinuric acid were measured in plasma by ion-pairing chromatography on Waters equipment (Waters Associates, Inc., Milford, Mass.). Plasma was thawed and 0.05 ml placed in microcentrifuge tubes. After addition of 0.02 ml of 20 pglml isonicotinic acid internal standard, 1.5 ml acetone was added, followed by mixing and standing at room temperature for 10 minutes. Precipitated protein was removed by centrifugation in a microcentrifuge (2 minutes at 13,000 rpm). Supernatant was poured into a 12 X 75 mm glass tube and dried down at 50" C under nitrogen. Samples were resuspended in 0.1 ml water and assayed by HPLC. Mobile phase was 5 mmol/L sodium phosphate, 0.25% dibutylamine, 5% methanol, pH 5.0. The column was a 7.5 x 0.32 cm 5 p Spherisorb C, with a 2 X 0.1 cm 5 p silica guard column (Phase Separations Ltd., Deeside, United Kingdom). Chromatography was performed at 400 C at a flow rate of 0.5 ml/ min with monitoring at 254 nm. Reported values have been calculated by use of peak areas and internal standardization. Samples from each patient were processed and chromatographed together, and quality control samples were interspersed with patient samples. The standard curves for nicotinic acid and nicotinuric acid had correlations of 0.9997 and 0.99992, respectively. Coefficient of variation for measurements of nicotinic acid at 8 mg/L was 2% and nicotinuric acid at 2 mg/L was 8%. The sample preparation was considerably abbreviated from previously used methods to allow for the large number of samples. Artifactual peaks from volatile components of some brands of acetone were observed but could be avoided by use of BDH Omnisolv acetone (BDH Inc., Toronto, Ontario, Canada) and thorough drying of acetone supernatants. In addition, the processed samples were not as pure as those of others. This resulted in the final sample resuspension being cloudy in appearance and a progressive decrease in retention times with multiple injections. Run times were also extended to allow late eluting peaks from
Tole~anceto nicotinic acid 67 the previous injection from interfering with chromatograms. Columns were cleaned with 100% methanol after a run of 25 samples. 9-a 11-P Prostaglandin F, measurements. A commercial radioimmunoassay kit (Amersham TRK 930, Amersham Canada Limited, Oakville, Ontario, Canada) was used to assay content of 0.1 ml plasma from the first and thirteenth dose of nicotinic acid. The manufacturer's instructions were followed except that instead of pouring off the entire 0.8 ml antigenantibody supernatant after charcoal absorption of unbound antigen, only 0.4 ml were pipetted off and then mixed with 5.0 ml ACS-I1 (Amersham Corp) scintillation fluid. Disintegrations per minute obtained from measurements of quenching by external standardization were used and data analyzed after linearization by logit transformation.* The 3 to 200 pg standard curve had a correlation of 0.996. Results are presented as picograms 9-a I 1-p prostaglandin F, per 0.1 ml plasma; however, the antiserum was reported to have
Ralph H. Stem, PhD, MD, J. David Spence, MD, David J. Freeman, PhD, and Anwar Parbtani, PhD London, Ontario, Canada Nicotinic acid is a remarkably effective hypolipidemic agent, lowering low-density lipoprotein cholesterol, triglycerides, and lipoprotein(a) while raising high-density lipoprotein cholesterol. However, it is under-utilized because of its reputation for adverse effects. The most common, known as flushing, consists of skin erythema caused by increased blood flow and a burning sensation. The response is similar to a sunbum but lasts only an hour. Since its introduction as a hypolipidemic agent in 1955, clinical observers have agreed that tolerance to flushing develops quickly in most individuals. In spite of this, this harmless side effect remains the greatest impediment to the prescription of this agent. The biology of flushing is incompletely characterized. Ingestion of pharmacologic doses of nicotinic acid have recently been shown to produce 400- to 800-fold increases in plasma levels of 9-a 11-P prostaglandin F,, a stable metabolite of prostaglandin D,.' However, the cellular origin and target of this mediator are unknown. Tolerance to nicotinic acid flushing may result from alterations in nic-
From the Department of Medicine, University of Western Ontario. Supported by the Department of Medicine, Victoria Hospital and Hypertension Research Unit, Victoria Hospital. Received for publication Jan. 28, 1991; accepted March 24, 1991. Reprint requests: Ralph Stem, PhD, MD, Hypertension Research Unit D-213, Victoria Hospital/South Street Campus, London, Ontario N6A 4G.5, Canada. 1311129829
otinic acid metabolism, decreased release of prostaglandin D,, or development of tolerance to prostaglandin D,. This study was designed to measure the development of tolerance by using objective measures of skin blood flow and relating the skin blood flow changes to plasma nicotinic acid and prostaglandin levels.
METHODS Clinical investigations. The protocol was approved by the Human Subjects Research Board of the University of Western Ontario, and informed consent was obtained from participants. Healthy men who were taking no medications were recruited, and each subject took a 500 mg nicotinic acid tablet three times a day for a total of 13 doses. The first, seventh, and thirteenth dose were taken in the clinical research unit. Subjects reported at 8 AM while fasting for at least 8 hours. Each subject was placed in bed with his head elevated and an intravenous catheter was inserted. A YSI series 400 attachable surface temperature probe (Yellow Springs. Instrument Co., Yellow Springs, Ohio) was taped 1 cm below the outer canthal margin of the left eye, and the laser Doppler probe of a Medpacific LD5000 capillary perfusion monitor (Medpacific Corporation, Seattle, Wash.) was attached 1 cm below the outer canthal margin of the right eye. Oral temperature was measured with a basal temperature thermometer. After acclimatization to room temperature, which was maintained at 20" to 22" C, a nic-
VOLUME 50 NUMBER I
otinic acid tablet with 300 ml water at room temperature was given at 9 AM. Room, oral, and facial temperatures, as well as laser Doppler readings, were measured every 10 minutes during the first hour and every 15 minutes for the next 45 minutes; additional readings were taken during maximal flushing. Blood was collected into two 10 ml EDTA-anticoagulated tubes and stored on ice. Plasma was removed after centrifugation at lOOOg for 15 minutes at 4" C and stored at -60" C until assay. Nicotinic acid and nicotinuric acid measurements. Nicotinic acid and nicotinuric acid were measured in plasma by ion-pairing chromatography on Waters equipment (Waters Associates, Inc., Milford, Mass.). Plasma was thawed and 0.05 ml placed in microcentrifuge tubes. After addition of 0.02 ml of 20 pglml isonicotinic acid internal standard, 1.5 ml acetone was added, followed by mixing and standing at room temperature for 10 minutes. Precipitated protein was removed by centrifugation in a microcentrifuge (2 minutes at 13,000 rpm). Supernatant was poured into a 12 X 75 mm glass tube and dried down at 50" C under nitrogen. Samples were resuspended in 0.1 ml water and assayed by HPLC. Mobile phase was 5 mmol/L sodium phosphate, 0.25% dibutylamine, 5% methanol, pH 5.0. The column was a 7.5 x 0.32 cm 5 p Spherisorb C, with a 2 X 0.1 cm 5 p silica guard column (Phase Separations Ltd., Deeside, United Kingdom). Chromatography was performed at 400 C at a flow rate of 0.5 ml/ min with monitoring at 254 nm. Reported values have been calculated by use of peak areas and internal standardization. Samples from each patient were processed and chromatographed together, and quality control samples were interspersed with patient samples. The standard curves for nicotinic acid and nicotinuric acid had correlations of 0.9997 and 0.99992, respectively. Coefficient of variation for measurements of nicotinic acid at 8 mg/L was 2% and nicotinuric acid at 2 mg/L was 8%. The sample preparation was considerably abbreviated from previously used methods to allow for the large number of samples. Artifactual peaks from volatile components of some brands of acetone were observed but could be avoided by use of BDH Omnisolv acetone (BDH Inc., Toronto, Ontario, Canada) and thorough drying of acetone supernatants. In addition, the processed samples were not as pure as those of others. This resulted in the final sample resuspension being cloudy in appearance and a progressive decrease in retention times with multiple injections. Run times were also extended to allow late eluting peaks from
Tole~anceto nicotinic acid 67 the previous injection from interfering with chromatograms. Columns were cleaned with 100% methanol after a run of 25 samples. 9-a 11-P Prostaglandin F, measurements. A commercial radioimmunoassay kit (Amersham TRK 930, Amersham Canada Limited, Oakville, Ontario, Canada) was used to assay content of 0.1 ml plasma from the first and thirteenth dose of nicotinic acid. The manufacturer's instructions were followed except that instead of pouring off the entire 0.8 ml antigenantibody supernatant after charcoal absorption of unbound antigen, only 0.4 ml were pipetted off and then mixed with 5.0 ml ACS-I1 (Amersham Corp) scintillation fluid. Disintegrations per minute obtained from measurements of quenching by external standardization were used and data analyzed after linearization by logit transformation.* The 3 to 200 pg standard curve had a correlation of 0.996. Results are presented as picograms 9-a I 1-p prostaglandin F, per 0.1 ml plasma; however, the antiserum was reported to have
