Indomethacin Concentrations Research Articles

Relationship of thrombin-stimulated arachidonic acid release and metabolism to mitogenesis and phosphatidylinositol synthesis.

Thrombin and certain prostaglandins are both capable of stimulating the proliferation of cultured cells. Since thrombin stimulates the release and metabolism of arachidonic acid, the precursor of prostaglandins, we examined the relationship between this release and metabolism and the stimulation of cell division in cultured fibroblasts. We also examined the role of prostaglandin synthesis in thrombin-stimulated phosphatidylinositol synthesis. The data in this report demonstrate that the release and metabolism of arachidonic acid are not necessary for thrombin-stimulated cell division. The presence of a low concentration of chymotrypsin prevented thrombin-stimulated arachidonic acid release and metabolism without affecting the stimulation of cell division. Furthermore, thrombin-stimulated cell division occurred in the presence of indomethacin concentrations that prevented cyclooxygenase-mediated metabolism of arachidonic acid. The following experiments showed that thrombin-stimulated phosphatidylinositol synthesis was brought about by a cyclooxygenase-mediated metabolite(s) of arachidonic acid. Indomethacin inhibited the cyclooxygenase-mediated metabolism of arachidonic acid without affecting the thrombin-stimulated release of arachidonic acid. Indomethacin also inhibited thrombin-stimulated phosphatidylinositol synthesis. The dose dependence of this inhibition paralleled the inhibition by indomethacin of cyclooxygenase-mediated metabolism of arachidonic acid. In addition, prostaglandin F2 alpha stimulated phosphatidylinositol synthesis in the presence of indomethacin concentrations which prevented thrombin-stimulated phosphatidylinositol synthesis.

A high performance liquid chromatography method for the analysis of total and free indomethacin in serum.

A simple rapid and sensitive HPLC technique for the analysis of total and free plasma indomethacin concentrations in neonates is described. The advantages of the technique are small sample volumes (50 microL) enabling the use of capillary samples, direct extraction, low limits of detection (20 ng/mL) and good precision (CV less than 7% between-batch). Indomethacin and the internal standard (flufenamic acid) eluted within 12 min and the peaks were unaffected by indomethacin metabolites or by a number of commonly used drugs. The technique is suitable for the investigation into the pharmacokinetics of total and free indomethacin, in neonates with patent ductus arteriosus, and could be used to define a therapeutic window for free indomethacin levels to enable therapeutic drug monitoring to occur in this condition.

Prediction of plasma concentration profile during single and repeated skin applications of indomethacin and its calcium salt ointments.

To determine the plasma concentrations and to clarify pharmacokinetic behavior from a pharmaceutical application viewpoint, indomethacin (IND) and its calcium salt (IND-Ca) in the form of gel ointments were applied singly and repeatedly to rat abdominal skin under occlusions. In order to describe the plasma IND concentration profile during repeated percutaneous administration, a highly simple pharmacokinetic model including a first-order absorption process with a lag time was applied. IND and IND-Ca were readily absorbed with a short lag time from IND-Ca ointment (1% as IND, 0.14 g/cm2, 3.0 x 1.2 cm area) containing Azone. Even when the applied dose of IND-Ca ointment was decreased to one-fourth (1.0 x 1.0 cm area), the plasma IND concentrations were maintained above the effective levels for 24h. The use of the pharmacokinetic model was justified by good agreement with observed data obtained after repeated percutaneous application of ointments. Thus, the plasma IND concentration-time courses during repeated percutaneous administration can be predicted by using the model.

Effects of estradiol and indomethacin on the contractile activity of the isolated rat uterus

The effects of estradiol (E2) on uterine contractility have been studied by continuous monitoring of the contraction of the whole horn and simultaneous determination of the production of prostaglandins (PG). In 70-minute experiments, uterine horns of ovariectomized rats released more prostaglandin E (PGE2) into the bath medium at the beginning (35 pg/min/mg) than at the end of incubation (4pg/min/mg). However, the decrease in the contraction frequency (CF) was not as large (15%). Estradiol treatment decreased the uterine production of PGE2 and, at low Ca concentrations in the bath medium, it completely abolished spontaneous contractions.Prostaglandin synthesis in the whole organ of ovariectomized rat was inhibited by indomethacin. Spontaneous contractions decreased only at indomethacin concentrations 10 fold higher than those needed to obtain 90% inhibition of prostaglandin synthesis. The effect of high concentrations of indomethacin (10 −5 M) on the CF was calcium-dependent and not related to the inhibition of PG synthesis.The decrease in the CF during hormonal treatment does not seem to be correlated with the decrease in PGE2 production.

1021 REDUCTION OF CORD BLOOD POLYMORPHONUCLEAR (PMN) LEUKOCYTE CHEMOTAXIS BY INDOMETHACIN

Protracted exposure to high oxygen tensions has been associated in preterm neonates with egress of leukocytes and alveolar macrophages into pulmonary interstitium and effluent obtained by bronchoalyeolar lavage. Pulmonary effluent serine elastase concentrations are increased in infants exposed to FiO2, >.4 Serum indomethacin concentrations of 10−5 to 10−7 molar have been associated with closure of the patent ductus arteriosus. We hypothesized.that indomethacin, a potent anti-inflammatory agent, might alter leukocyte chemotaxis in infants. Using modified Boyden chambers human term cord blood PMN leukocyte chemotaxis was altered (F=6.68,p=0.025) with linear attenuation (r= -0.77) to zymogen activated human serum. We speculate that indomethacin treatment of neonates may reduce oxygen induced inflammation through reducing leukocyte induced cytotoxic parenchymal lung damage and airways inflammation in addition to closing the patent ductus arteriosus. (HD 16292)

Interferon Induction by Viruses. XIV. Development of Interferon Inducibility and Its Inhibition in Chick Embryo Cells "Aged"In Vitro

Studies with a number of viruses revealed a time-dependent acquisition of interferon (IFN) inducibility in primary chick embryo cells as they "aged" in vitro for 2-12 days at a confluent cell density without a medium change. The time-course for the development of IFN inducibility was established by generating and analyzing a family of dose (multiplicity)-response (IFN yield) curves, using Newcastle disease virus (NDV, strain LaSota) as the inducer. Cells produced little or no IFN for the first 4-5 days. Between 5 and 6 days the cells gradually developed the capacity to respond to NDV (and other viruses). Maximal yields of IFN were inducible by day 10. This time-dependent development of IFN inducibility was abrogated almost completely when "aging" was carried out in the presence of drugs that inhibited the synthesis of cyclic derivatives of C20 oxygenated unsaturated fatty acids, i.e., inhibitors of prostaglandin/leukotriene synthesis and the arachidonic acid cascade. Of the prostaglandin synthesis inhibitors, indomethacin was particularly effective. Cells treated on day 0 with 10 micrograms/ml of indomethacin produced 100- to 1000-fold less IFN than controls when induced on day 8. To prevent maximally the development of IFN inducibility, indomethacin must be added within the first 2 days of seeding. After about 2 days, the cells begin to escape the action of the drug. Indomethacin added at the time of induction had no effect on the yield of IFN. IFN inducibility was partially restored when indomethacin was removed during the aging process. "Aging" chick cells in low concentrations of cycloheximide (0.5 micrograms/ml) produced results comparable to incubation with indomethacin. Neither reagent had any marked effect on the rate of total protein or RNA synthesis, nor did their action prevent the induction of stress (heat shock) proteins. Cells "aged" in indomethacin were threefold less efficient in responding to the action of IFN, whereas aging in cycloheximide had no effect on IFN's action. Plaque formation on drug-treated cells was normal for viruses that were poor inducers of IFN. However, both the plaquing efficiency and plaque size of Sindbis virus (an excellent IFN inducer) were enhanced markedly on cells treated with indomethacin or low concentrations of cycloheximide during the aging process. These data implicate a family of fatty acid derivatives of arachadonic acid, including prostaglandins and leukotrienes, in the development of IFN inducibility in primary chick embryo cells "aged" in vitro.

Enterotoxin-induced fluid accumulation during experimental salmonellosis and cholera: involvement of prostaglandin synthesis by intestinal cells

Challenge of rabbit intestinal loops with Salmonella typhimurium or Vibrio cholerae resulted in significant elevation of mucosal cyclic adenosine monophosphate (cAMP) and prostaglandin concentrations. This effect could be reproduced in vitro by exposing either isolated epithelial cells from normal rabbits or Chinese hamster ovary cells to purified cholera toxin or cell-free lysates of Salmonella. Indomethacin treatment of animals challenged with Salmonella resulted in less fluid accumulation, in addition to lower concentrations of intestinal cAMP and prostaglandins, compared to that of similarly changed loops in normal animals. Likewise, intestinal loop challenge of indomethacin-treated rabbits with V. cholerae or purified cholera toxin also resulted in decreased fluid secretion and diminished levels of tissue cAMP and prostaglandins. Intestinal loop tissue from normal animals challenged with V. cholerae displayed 10-fold higher levels of prostaglandins than tissue from uninfected animals. Prostaglandin levels in Salmonella-infected intestinal loops were similarly elevated, but by only 2–3 fold. Chinese hamster ovary cell monolayers were simultaneously exposed to either 10 ng of cholera toxin or 20 μl of Salmonella lysate and varying concentrations of indomethacin ranging from 1 ng/ml to 10 μg/ml. Indomethacin decreased both cAMP and prostaglandin levels in Chinese hamster ovary cells. At high indomethacin concentrations, the cells lost their ability to respond to stimulation with either cholera toxin or Salmonella lysate, while retaining greater than 95% viability as determined by trypan blue exclusion. The prostaglandind and cAMP content of epithelial cells from Salmonella-challenged loops was increased in crypt epithelial cell fractions. Prostaglandin concentrations were elevated in isolated intestinal epithelial cells exposed to purified cholera toxin in vitro. These data indicate that prostaglandins synthesized by the epithelial cells are involved in the pathogenesis of both experimental cholera and salmonellosis. The data are consistent with an enterotoxin-mediated mechanism for both diarrheal diseases and argue against the role of inflammatory cells as the source of elevated cAMP and prostaglandins appearing in the intestinal mucosa during experimental salmonellosis.

Effects of prostaglandin E 2, indomethacin, trifluoperazine and drugs affecting the cytoskeleton on collagenase production by cultured adherent rheumatoid synovial cells

Cultured adherent rheumatoid synovial cells with fibroblast properties release large amounts of collagenase and prostaglandin E 2 (PGE 2) into the medium. With age in culture and passage of the cells, the levels of collagenase and PGE 2 decrease, but can be increased by a factor (MCF; mononuclear cell factor) released by cultured human blood monocyte-macrophages. The magnitude of the stimulation varies with different synovial cell strains. To determine some of the mechanisms which regulate the collagenase response, synovial cells were exposed to a cyclooxygenase inhibitor (indomethacin) and substances which alter the cytoskeleton (cytochalasin B or colchicine) or interact with Ca 2+ · calmodulin (trifluoperazine). The collagenase response was retained even when PGE 2 synthesis was totally blocked with indomethacin. The collagenase response, however, was blunted at high indomethacin concentrations (> 10 μM) and paradoxically augmented at lower indomethacin concentrations (0.001 (μM). In some synovial cell strains, the blunting effect of 10 μM indomethacin was reversed by the addition of low concentrations of exogenous PGE 2 (10 ng/ml). Preincubation of synovial cells for 1 or 24 hr with colchicine or cytochalasin B (1–10 μM) resulted in an augmented collagenase and PGE 2 response to MCF. Cells preincubated or incubated with 1–50 μM trifluoperazine, a phenothiazine, also augmented collagenase stimulation by MCF, but, in contrast to colchicine or cytochalasin B, trifluoperazine suppressed the PGE 2 response. Thus, although PGE 2 and collagenase production by synovial cells may be dissociated, altering ambient PGE 2 levels affected basal collagenase production and modulated the collagenase response to MCF.

Intravenous indomethacin for patent ductus arteriosus.

Intravenous indomethacin was given to 36 neonates with a patent ductus arteriosus who where receiving ventilatory support for respiratory distress syndrome. Permanent closure of the ductus arteriosus occurred in 21 (58%) infants and in this group the mean 24 hour plasma indomethacin concentration was 0.37 micrograms/ml. Partial success was achieved in 6 (17%) infants (mean 24 hour indomethacin concentration 0.34 micrograms/ml) but in 9 patients (mean 24 hour indomethacin concentration 0.29 micrograms/ml) there was no clinical change. Although the mean 24 hour indomethacin concentration was lower in the group with no clinical change, this was not statistically significant. Five of the 21 patients in whom there was permanent closure of the ductus required more than one dose of indomethacin. The possible effects of birthweight and age at indomethacin treatment were difficult to separate because of the high negative correlation between these two variables. The chance of closure was enhanced significantly if the patient had either a birthweight of at least 1 kg or the age at indomethacin treatment did not exceed 10 days, or both. Six hour but not 24 hour indomethacin concentrations were higher in patients with a high birthweight treated at an early age.

Inhibition of purified glutathione S-transferases by indomethacin

Soluble rat liver glutathione S-transferases have been purified and a previously undescribed peak was observed. This peak contained glutathione S-transferase activity which was extensively inhibited by indomethacin. Glutathione conjugation of 1-chloro-2,4-dinitrobenzene by this isozyme, designated glutathione S-transferase VII, was inhibited 44 and 68% at indomethacin concentrations of 0.20 and 1.00 μM, respectively. The other six basic glutathione S-transferase isozymes were relatively unaffected by low concentrations of indomethacin. The pharmacological significance of this inhibition by indomethacin is largely dependent on the role of the glutathione S-transferase VII in leukotriene synthesis.

Determinants of Bumetanide Response in the Dog: Effect of Indomethacin

Four male unanesthetized dogs each weighing 22.0–29.0kg received 0.250mg/kg iv of bumetanide before (treatment I) and after (treatment II) indomethacin pretreatment. Lactated Ringer’s solution was administered intravenously throughout both treatments at a flow rate of 2ml/min to avoid fluid and electrolyte depletion. Unchanged bumetanide and indomethacin concentrations were analyzed using high-performance liquid chromatography. Sodium was measured by flame photometry and creatinine by colorimetry. Indomethacin pretreatment did not significantly change the pharmacokinetics of bumetanide, affecting neither the total amount of drug nor time course of drug delivered into the urine. In contrast, indomethacin pretreatment resulted in a dramatic reduction in the 4-hr sodium excretion and urine volume. Therefore, a pharmacokinetic interaction may be eliminated as a possible mechanism for the attenuation, by indomethacin, of the natriuretic and diuretic response of bumetanide. Instead, it appears that indomethacin diminishes the response to bumetanide via prostaglandin inhibition.

In vitro biosynthesis of cyclooxygenase metabolites in ovalbumin-sensitized and control lungs of guinea pigs

Cyclooxygenase metabolism may regulate mediator release in anaphylaxis. The in vitro biosynthesis of cyclooxygenase metabolites was examined, using isolated microsomal membranes from ovalbumin-sensitized and control guinea pig lungs, under conditions in which the substrate concentration ([ 3H]arachidonic acid) was varied (5–168 μM). With microsomes from sensitized lungs, there was stimulation of cyclooxygenase activity at substrate concentrations from 20 to 100 μM, with the major increases in activity occurring in the biosynthesis of thromboxane B 2 and prostaglandin D 2. At substrate concentrations greater than 100 μM, the biosynthesis of thromboxane B 2 and prostaglandin D 2 decreased in microsomes of sensitized lungs to the level of production found in control-lung microsomes. In contrast, prostacyclin production was significantly higher in the control-lung microsomes at the higher substrate concentrations. The ratio of prostacyclin to thromboxane production was lower in sensitized-lung microsomes at all substrate concentrations. No changes in the biosynthesis of prostaglandins F 2α or E 2 were detected between the two groups. Kinetic analysis of the data demonstrated that the cyclooxygenases in control and sensitized-lung microsomes exhibited different apparent K m and V max values. When the enzymes from both the control and the sensitized-lung microsomes were assayed in the presence of indomethacin (1–10 μM), a cyclooxygenase inhibitor, thromboxane synthesis was preferentially inhibited. The microsomal enzymes from control and sensitized lungs showed similar responses to the drug. At the indomethacin concentrations used, no significant inhibition of prostacyclin or prostaglandin production occurred. The results of these in vitro experiments support the hypothesis that net changes in the biosynthesis of cyclooxygenase metabolites may modulate the anaphylactic response of the lungs in sensitized animals.

Variability of serum indomethacin concentrations after oral and intravenous administration to preterm infants.

Fifteen preterm infants with patent ductus arteriosus and respiratory distress syndrome were given indomethacin (0.2 mg/kg) at 12 h intervals up to three times, either orally or intravenously, in an uncontrolled, non-randomized study. Serum indomethacin concentrations were determined in blood samples taken 12 h after dosing. There was considerable variability in the serum indomethacin concentrations, especially after oral administration, although the mean concentrations after each of the three doses were similar after both oral and intravenous administration. The frequency of closures and transient closures of the ductus arteriosus was also similar for both routes of administration. There was, however, no relation between concentration and effect in individual patients. The sustained exposure to indomethacin which appears to be necessary for ductal closure can sometimes be attained by oral administration.

Serum indomethacin concentrations after intravenous administration to preterm infants with patent ductus arteriosus.

Six preterm infants with PDA received 14 treatments with indomethacin 0.2 mg/kg intravenously. Auscultatory and echocardiographic assessment indicated closure of the duct in 2, partial closure in 2, and no effect in 2 infants. The mean serum concentration of indomethacin was: 15 min after the first injection 1 314 ng/ml, after 1 hour 970 ng/ml, after 6 hours 718 ng/ml, and after 24 hours 388 ng/ml. The mean half-life of indomethacin in the serum was 20 hours (range 9-50 hours). Side effects in all infants were hyponatraemia, decreased urinary output, decreased urinary sodium excretion, and weight gain. One infant had transient thrombocytopenia and gastrointestinal haemorrhage. By intravenous administration of indomethacin in a dose of 0.2 mg/kg to preterm infants a sufficiently high serum concentration is obtained shortly after the injection. To maintain a high serum concentration for a longer period it is recommended to give a second dose of 0.2 mg/kg after 6 hours and if necessary a third dose of 0.1 mg/kg 24 hours after the first dose.

Excretion of indomethacin into saliva following intravenous administration to dogs.

Parotid saliva (Pr) and mandibular-sublingual saliva (MS) were collected separately by means of permanent fistulae in order to investigate the excretion of indomethacin in saliva of dogs receiving a single intravenous dose of 20 mg/kg. Drops of citric acid solution were placed on the tongue to stimulate salivary secretion. The concentrations of indomethacin both in saliva and plasma declined biexponentially with time. There was a good linear relationship between the drug concentration in each saliva and plasma. The Pr and MS levels were 7.4% and 4.4% of the plasma levels, respectively. Indomethacin concentrations in Pr were significantly higher than in MS (p less than 0.05). The roles of salivary pH and salivary protein binding were discussed in respect to the mechanism of salivary excretion of the drug.

The disposition of indomethacin in preterm babies

Limited pharmacokinetic data are available concerning the disposition of indomethacin in preterm infants. Since oral absorption of the drug may be poor or incomplete, the current report provides pharmacokinetic data on 37 premature infants who received indomethacin intravenously. Each of these infants had a hemodynamically significant patent ductus arteriosus. Findings included variable serum indomethacin concentrations from four to 12 hours after a single dose of 0.2 mg/kg. Female preterm infants generally had lower serum drug values at 12 hours and beyond when compared to males. An extrauterine age dependence was found of serum indomethacin levels. Total body clearance, serum half lives and volumes of distribution also bore a direct relationship to extrauterine age. Thus, when indomethacin is administered shortly after birth, one may anticipate a longer duration of action after a single dose and a relatively greater risk of accumulation of the drug when more than one dose is required for treating a duct al patency.

Indomethacin-sensitive suppressor cells regulate the cell-mediated cytotoxic response to SV 40-induced tumor-associated antigens in mice.

AbstractSpecific secondary cytotoxic reactivity (as measured by 51Cr‐release assay) against SV 40‐induced tumor‐associated antigens was generated in vitro in spleen cells of tumor‐free (BALB/c × C57 BL/6)F1 (CBF1) mice immunized against a syngeneic SV 40‐induced tumor of BALB/c origin (mKSA), following in vitro sensitization for 5 days with the relevant antigens in mixed lymphocyte‐tumor cell cultures. In contrast, spleen cells of CBF1 mice bearing the SV 40‐induced tumor demonstrated suppressed specific secondary cytotoxic reactivity following incubation with the corresponding antigens. Spleens from tumor‐bearing mice contained 4 times the number of mononuclear cells and 3 times the percentage of macrophages, as compared to spleens of normal mice. The percentage of B cells was also elevated in spleens of tumor‐bearing mice. There was a slight reduction in the percentage of T cells. The cytotoxic reactivity of spleen cells of tumor‐bearing mice was restored following removal of macrophages by either rayon adherence columns or iron and magnet, or incubation on plastic petri dishes. No such effect was seen with spleen cells of tumor‐free or normal mice. Spleen cells of tumor‐bearing mice inhibited the in vitro generation of secondary cytotoxic reactivity of spleen cells of tumor‐free mice, sensitized in vitro with SV 40‐induced tumor cells by mixing experiments. The suppressor cells were found to be macrophages by the 3 techniques for removal of macrophages described above. The addition of indomethacin (1–10 μg/ml), a noncompetitive irreversible prostaglandin synthesis inhibitor, to cultures of responding spleen cells from tumor‐bearing mice and stimulating SV 40‐induced tumor cells resulted in marked augmentation of spleen cells responsiveness. With higher indomethacin concentrations (100 μg/ml), no enhancement was seen. The augmenting effect was noted only when indomethacin was present during the initial 24 h of the 5‐day culture. Indomethacin at 1–10 μg/ml had no effect on cytotoxic reactivity of spleen cells of tumor‐free mice, whereas at higher concentrations (100 μg/ml) it had a strong suppressive effect. Preincubation of spleen cells of tumor‐bearing mice with indomethacin for 3 days abrogated their ability to suppress the generation of secondary cytotoxic reactivity of spleen cells of tumor‐free mice. It is hypothesized that indomethacin‐sensitive suppressor macrophages regulate the immune responsiveness in tumor‐bearing mice.

The possible role of prostaglandins in mediating immune suppression by nonspecific T suppressor cells

Nonadherent splenic lymphoid cells from tumor-bearing animals exhibit a poor primary in vitro plaque-forming cell (PFC) response to SRBC. This is attributable to the presence, in this population, of a T suppressor cell. This nonadherent population suppressed the in vitro anti-SRBC PFC response of normal spleen cells (NSC). This nonspecific suppressive effect was eliminated by adding indomethacin, a prostaglandin synthetase inhibitor, to the in vitro cultures. These effects were seen at indomethacin concentrations which have no direct effect on the NSC response. When exogenous prostaglandins were added to NSC cultures plus SRBC, prostaglandins PGE 1 and PGE 2 were shown to markedly inhibit the PFC response at a wide range of concentrations, whereas PGA 1, PGF 1α, and PGF 2α were inhibitory only at the highest concentrations tested.

Quantitative gas—liquid chromatographic method for the determination of indomethacin in biolgical fluids

A sensitive and specific gas chromatographic method for the analysis of indomethacin has been developed. After extraction of the drug from blood or urine with ethylene dichloride a derivative is formed with pentafluorobenzyl bromide. 5-Fluoro-indomethacin is used as an internal standard. The overall recovery of indomethacin is 85.1 ± 2.3% and the method can detect 10 ng indomethacin in a 1 ml plasma or urine sample. No interference in the method is seen with some commonly used drugs. The method has been used to measure plasma indomethacin concentrations in 20 patients with rheumatoid arthritis, being treated with 25 mg indomethacin three times daily. The plasma concentration ranged from 168–596 ng/ml.

250 INDOMETHACIN DISPOSITION IN PREMATURE INFANTS: BLEEDING DUE TO PLATELET DYSFUNCTION AFTER SINGLE DOSES OF INDOMETHACIN

Indomethacin failed to produce permanent ductal closure in any of four premature infants with patent ductus arteriosus to whom the drug was given (0.15-0.30 mg/kg). Indomethacin half-lives measured in two premature infants were 21 and 24 hours, much longer than in full-term newborns or adults. Platelet function, as measured by platelet aggregation, was grossly abnormal for two to four days after indomethacin administration, normal values returning only by the ninth and tenth day. Gastrointestinal bleeding and transient renal dysfunction occurred in one infant. Measurement of plasma indomethacin concentrations in sick, low birthweight infants could help to monitor indomethacin dose and dosage interval, thereby preventing drug accumulation and reducing toxicity. Further studies of potential toxicity seem to be indicated before instituting widespread indomethacin administration for ductal closure in premature infants.