Synthesis and secretion of thrombospondin by cultured human endothelial cells.

Thrombospondin, the major glycoprotein released from alpha-granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160 kilodalton subunits and apparently functions as a platelet lectin. Because cultured human umbilical vein endothelial cells synthesize and secrete a glycoprotein (GP-160) which is a disulfide-bonded multimer of 160 kdalton subunits, the possibility that GP-160 is thrombospondin was investigated. Tritiated GP-160 could be immunoisolated from [3H]leucine-labeled endothelial cell postculture medium using a rabbit antiserum to human platelet thrombospondin. Thrombospondin and GP-160 comigrated in two different two-dimensional electrophoretic systems. Both proteins are disulfide-bonded trimers of acidic 160-kdalton subunits. A competitive radioimmunoassay for binding of 125I-thrombospondin to the rabbit antibodies indicated that 49 micrograms of thrombospondin antigen per 10(6) confluent endothelial cells accumulated in postculture medium over 24 h. Thus, endothelial cells secrete large amounts of a glycoprotein that is identical or very similar to platelet thrombospondin.

Effect of recombinant human tumor necrosis factor on the induction of murine macrophage tumoricidal activity.

The ability of recombinant human tumor necrosis factor (rH-TNF) alone or in combination with lymphokines (LK) to induce the in vitro activation of murine macrophages was evaluated. The treatment of C57BL/6 mouse resident peritoneal exudate cells (PEC) with rH-TNF and LK was found to induce the activation of macrophages to a tumoricidal state against P815 mastocytoma cells. Neither rH-TNF nor LK alone induced macrophage cytotoxic activity. Furthermore, the macrophage activation seen was not due to small amounts of contaminating lipopolysaccharide. The TNF plus LK-mediated macrophage activation could be totally ablated by rabbit antiserum to murine gamma-interferon, thus suggesting a role for gamma-interferon in this system. Since adherent cells (greater than or equal to 95% macrophages) only marginally responded to stimulation with rH-TNF plus LK and the addition of nonadherent PEC caused a marked augmentation of rH-TNF plus LK-mediated macrophage activation, the involvement of nonadherent PEC was suggested. In addition, using antibodies and complement to deplete subsets of cells from the nonadherent PEC, the requirement for cells bearing Thy 1.2 and asialo GM1 surface markers was demonstrated. These results suggest that TNF may play an autocrine regulatory role in concert with lymphokines in macrophage-mediated host defense against malignant neoplasia.

Effect of anti-thrombospondin antibodies on the hemagglutination activities of the endogenous platelet lectin and thrombospondin.

The proposal that thrombospondin is the endogenous platelet lectin was evaluated using antisera and monoclonal antibodies to thrombospondin. The platelet-bound hemagglutinin activity of human platelets stimulated with A23187 was inhibited by rabbit anti-thrombospondin sera and by a monoclonal anti-thrombospondin IgG. A second monoclonal IgG did not inhibit platelet-bound agglutinin activity. Preparations of purified platelet thrombospondin differed in their hemagglutination activities. The hemagglutination activity of an active preparation of thrombospondin was inhibited by the monoclonal antibody that inhibited platelet-bound lectin activity. The hemagglutination activity of an almost inactive preparation of thrombospondin was enhanced by the anti-thrombospondin monoclonal antibody that did not block platelet-bound lectin activity. The results demonstrate that expression of the platelet-bound form of the endogenous lectin is thrombospondin-dependent and suggest that thrombospondin must become part of a larger complex, either by binding to the platelet surface or by becoming aggregated in solution, before hemagglutination activity can be expressed.

Comparison of tebufelone distribution in rat blood plasma and inflamed and noninflamed tissues following peroral and intravenous administration.

It is recognized that some acidic nonsteroidal antiinflammatory drugs (NSAIDs) accumulate in the synovial fluids of inflamed joints. The distribution of tebufelone, a member of the di-tert-butylphenol class of NSAIDs, between rat plasma and paw tissues (inflamed and noninflamed) was determined. Tebufelone was administered (doses providing 80% maximal effectiveness) perorally (po, 10 mg/kg) or intravenously (i.v., 0.5 mg/kg) to Sprague-Dawley rats just prior to injection of an inflammatory adjuvant (carrageenan) into one of the hind paws. Levels of the drug were measured in plasma, inflamed paws, and noninflamed paws at prescribed times postdosing by capillary gas chromatography/stable isotope dilution mass spectrometry. Tebufelone levels, as determined from areas under the curves of concentration versus time were sevenfold (po) and 11-fold (i.v.) higher in paw tissue than in plasma. Inflamed and noninflamed tissues have equal affinity for the drug. Calculated terminal-phase half-lives of tebufelone in paw tissue were similar following i.v. and po dosing (approximately 14 h) and equivalent to the plasma half-life of the drug after po dosing (approximately 10 h). The plasma half-life determined following i.v. dosing was considerably lower (2.8 h). The anti-inflammatory activity of tebufelone appears to correlate more closely with tissue distribution profiles than plasma drug levels at the dose levels examined.

Absorption, bioavailability, and pharmacokinetics of tebufelone in the rat.

Tebufelone (NE-11740) is a member of the new di-tert-butylphenol class of anti-inflammatory agents. It exhibits good inhibitory activity against cyclooxygenase and 5-lipoxygenase in vitro. It also shows excellent anti-inflammatory activity and inhibits bone resorption in vivo in the rat adjuvant arthritis model at an oral dose level of 1 to 2 mg/kg. The absorption, bioavailability, and pharmacokinetics of tebufelone were investigated in male Sprague-Dawley rats. Tebufelone labeled with carbon-14 was administered intravenously at doses of 0.5 and 2 mg/kg and perorally at doses of 2 and 10 mg/kg to fasted rats. Plasma samples taken from the rats at timed intervals were analyzed for total radiolabel by scintillation counting and for tebufelone by a mass spectrometric method. Comparison of the total radiolabel and tebufelone areas under the curves (AUCs) of concentration of tebufelone versus time from the 2-mg/kg intravenous and 2-mg/kg oral doses indicates that tebufelone is completely absorbed and 100% bioavailable at this dose level in the rat. The AUCs are a linear function of dose at the 0.5- and 2-mg/kg dose levels, but the AUC of the 10-mg/kg dose exhibits a nonproportional increase, suggesting saturation of elimination processes at this higher dose.

Evaluation of ketorolac concentrations in plasma and gingival crevicular fluid following topical treatment with oral rinses and dentifrices.

Two clinical studies were conducted to determine the relative amounts of ketorolac detectable locally in the gingival crevicular fluid (GCF) and systemically in plasma after oral, topical drug administration. The rinse study compared topical administration of three concentrations of ketorolac tromethamine (0.1%, 0.5%, and 0.01%) in oral rinse formulations administered topically and a perorally administered capsule (10 mg), and the dentifrice study compared two concentrations of ketorolac in dentifrice formulations (0.15% and 1.0%) with a 0.1% oral rinse, all treatments administered topically. The dose-corrected systemic availability of the three oral rinses evaluated in the rinse study relative to the peroral capsule was about 15%. However, the ratios of the observed maximum GCF ketorolac concentration to maximum plasma ketorolac concentration ranged from 22 to 49, compared to less than 1 for the peroral ketorolac capsule. Using this ratio as an estimate of the ability of a treatment to target the drug to the gingival tissue, these data indicate that the ketorolac oral rinses achieved greater delivery of drug to the gingival tissue (presumed site of action for periodontitis) with a lower systemic drug load than peroral administration of a ketorolac capsule. The dose-corrected relative systemic bioavailabilities for the dentifrice treatments with respect to the 0.1% rinse in the dentifrice study were 59.2% and 86.4% for the 1.0% and 0.15% dentifrices, respectively, indicating that significantly less ketorolac was systemically available from the two dentifrices relative to the oral rinse. The relative bioavailabilities of ketorolac in the GCF after dosing with the dentifrice formulations with respect to the rinse were 89.1% for the 1.0% dentifrice and 19.7% for the 0.15% dentifrice. Thus, the 1.0% dentifrice appears to provide statistically equivalent levels of ketorolac to the gingival tissue as the 0.1% oral rinse with significantly less systemic exposure. The T1/2 of ketorolac in the GCF was about 0.5 h for all three treatments, which is significantly less than the plasma half-life of about 5.3 h. These data suggest that GCF levels of ketorolac should remain above the IC50 for PGE2-stimulated IL-1 bone resorption for about 7 h following treatment, assuming continuation of the first-order elimination observed over the first two postdosing hours. We conclude that oral rinses and dentifrices are effective and preferred vehicles for administration of ketorolac for use in treatment of periodontitis.

A comparison of topical ketorolac, systemic flurbiprofen, and placebo for the inhibition of bone loss in adult periodontitis.

Systemic non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce alveolar bone loss in periodontitis. This study assesses the efficacy of a topical NSAID rinse, containing ketorolac tromethamine as the active agent. Adult periodontitis patients (n = 55) were studied in this 6-month randomized, double blind, parallel, placebo and positive-controlled study. Each patient had a least 3 sites at high risk for bone loss as assessed by low dose bone scan. Groups, balanced for gender, were assigned to one of three regimens: bid ketorolac rinse (0.1%) with placebo capsule; 50 mg bid flurbiprofen capsule (positive control) with placebo rinse; or bid placebo rinse and capsule. Prophylaxes were provided every 3 months. Monthly examinations assessed safety, gingival condition, and gingival crevicular fluid PGE2. Standardized radiographs were taken at baseline and at 3 and 6 months for digital subtraction radiography. A significant loss in bone height was observed during the study period in the placebo group (-0.63 +/- 0.11; P < 0.001), but not in the flurbiprofen (-0.10 +/- 0.12; P = 0.40) or ketorolac rinse (+0.20 +/- 0.11 mm; P = 0.07) groups. Nested ANOVA revealed that ketorolac and flurbiprofen groups had less bone loss (P < 0.01) and reduced gingival crevicular fluid PGE2 levels (P < 0.03) compared to placebo. ANOVA suggests (P = 0.06) that ketorolac rinse preserved more alveolar bone than systemic flurbiprofen at the dose regimens utilized. These data indicate that ketorolac rinse may be beneficial in the treatment of adult periodontitis.

Determination of leukotriene B4 in human plasma by gas chromatography using a mass selective detector and a stable isotope labelled internal standard. Effect of NE-11740 on arachidonic acid metabolism.

A highly selective gas chromatographic method, coupled with selected ion monitoring using a mass selective detector and positive electron ionization, was developed for the determination of leukotriene B4 (LTB4) in human plasma. Plasma was separated from whole human blood via centrifugation, proteins precipitated with acetonitrile and LTB4 recovered (approximately 82.0%) by ethyl acetate extraction. The methyl ester, bis-t-butyldimethylsilyl ether derivative of LTB4 was formed prior to analysis and determined quantitatively using [18O]2-LTB4 as an internal standard. The limit of detection (S/N = 2) was 425 pg on column (m/z 335/339) using a 1-microliter injection. Standard curves were linear over two orders of magnitude with an RSD of less than 5.0% (n = 10). NE-11740, a new anti-inflammatory drug, was shown to inhibit, in a dose-dependent manner (ED50 = 22 microM) ionophore-stimulated LTB4 biosynthesis by human whole blood in vitro.

Pain after periodontal scaling and root planing.

BACKGROUND: Although periodontal scaling and root planing, or SRP, is one of the most common procedures used in dental practice, there is little information available about the degree of postprocedural pain associated with it. The authors undertook this study to document the intensity and duration of pain after SRP with a view toward helping practitioners and their patients manage postprocedural discomfort. METHODS: Using the Heft-Parker self-assessment pain scale, 52 adults with moderate periodontitis evaluated their pain before and after SRP conducted with local anesthetic. RESULTS: After SRP, 28 percent of all patients reported faint-to-weak pain, 18 percent experienced weak-to-mild pain, 28 percent experienced mild-to-moderate pain, 8 percent had moderate-to-strong pain and 8 percent reported strong-to-intense pain. The average time to onset of maximum pain was approximately three hours after SRP, and the average duration of mild or greater pain was about six hours. Upon awakening the morning after SRP, subjects found that pain had returned to pre-SRP levels. Overall, 23 percent of all patients reported self-medicating with analgesics to relieve postprocedural pain. Women self-medicated earlier (P < .05) and more often than men (43 percent vs. 10 percent; P < .05). CONCLUSIONS: Patients experienced significant duration and magnitude of pain after SRP. This pain peaked between two and eight hours after SRP, lasted about six hours, and returned to pre-SRP levels by the morning after the procedure. Almost 25 percent of all patients self-medicated to relieve pain after SRP, and women took analgesic medication earlier and more often than men. CLINICAL IMPLICATIONS: Practitioners should consider using appropriate analgesic drugs to alleviate mild-to-moderate pain after SRP. On the basis of this study, it would appear that an analgesic that has a peak effect two to eight hours after the completion of SRP would be the most appropriate medication. Moreover, it is unlikely that analgesic medication would be needed by most patients beyond the day on which SRP was performed.

Quantification of hyperbaric oxygen-induced toxicity utilizing a malarial system.

This study was undertaken in recognition of the need to develop quantitative systems to evaluate the toxicity associated with hyperbaric oxygen (HBO) exposure. Malaria-infected (P. berghei berghei) mice were briefly exposed to 100% oxygen at 3 ATA on day 10 of infection. At 25, 48, and 72 h thereafter, the levels of circulating erythrcytes and percent parasitized RBC were monitored and compared to those of infected non-exposed controls. The total erythrocyte counts of the infected HBO-exposed and non-exposed mice did not differ significantly. In contrast, percent parasitized cells in the oxygen-exposed mice were lowered to 55-60% control values at 24, 48, and 72 h. The mechanism of this difference needs further study, but we believe that P. berghei-infected erythrocytes are preferentially hemolyzed as a consequence of HBO exposure. this mode system is useful in the study of HBO-induced toxicity because of its high degree of selectivity and sensitivity and its amenability to strict quantification over a period of at least several days.

Evaluation of Cetylpyridinium Chloride-Containing Mouthwashes Using In Vitro Disk Retention and Ex Vivo Plaque Glycolysis Methods.

The Disk Retention Assay (DRA) is an in vitro method developed to measure the available level of cetylpyridinium chloride (CPC) in mouthwash formulations. This method is based on the binding of the cationic CPC molecule to the anionic surface of a cellulose filter disk. Aqueous CPC solutions demonstrate a linear response (A545) for concentrations up to 0.3%. Higher levels of CPC showed no increased response in the assay. Among common oral product ingredients, at relevant concentrations, surfactants are the primary compounds which inhibit CPC detection and hence, chemical availability. Poloxamer-407 decreased CPC availability to 60% at 0.1%, to 10% at 0.5%, and to 24-33% for 0.2-0.4%. Polysorbate-80 decreased CPC availability to 30% at 0.1% and 6% at 0.25%. A range (4-54%) of available levels of CPC were determined for several commercial products containing 0.045-0.05% nominal levels of CPC indicating significant formulation excipient influence. A plaque glycolysis (PG) assay was used to determine the biological activity of all mouthwash products analyzed by DRA. An experimental series of mouthwash formulations having nominal CPC levels of 0-0.10% demonstrated a good correlation (r2 = 0.955) between the calculated available level of CPC (DRA) and inhibition of plaque glycolysis. The calculated available level of CPC from select commercial mouthwash products, also fit the established correlation with biological activity. The combination of DRA and plaque glycolysis methods are valuable tools which can be used during development to maximize the biological activity of CPC mouthwash formulations prior to clinical evaluation.

Hybrid approach offers option for long term older adult care.

Hospitals in the future will be called on increasingly to administer to the medical needs of an aging population. But what are alternative forms of care beyond nursing homes and what must hospitals do to position themselves with those alternatives? In the following article, the author discusses the advantages of a "hybrid approach" to caring for older patients.

Medication errors: 1977 to 1988. Experience in medical malpractice claims.

MIIENJ has paid $30,144,636 in indemnity from 1977 through 1988 for medical malpractice suits arising from medication errors. A review of these files revealed that patients incurred death and serious morbidity, and that several specific behavior errors accounted for the majority of patient injury. Among the damages that occurred during that time period were 88 deaths, 15 patients with profound brain damage, 15 patients who alleged that they had become addicted to medications prescribed for pain, 10 patients who required amputations of limbs, and 12 patients who suffered some degree of hearing loss or decreased visual acuity. The predominant categories where insureds incurred difficulty were in disregarding patient allergies to specific drugs (acetylsalicylic acid (aspirin), penicillin and its derivatives); prescribing drugs without consideration of the patient's medical history; failing to monitor therapy with anticoagulants, nonsteroidal anti-inflammatory drugs, digitalis derivatives, theophylline, and aminoglycoside antibiotics; antibiotic therapy; and errors in the writing of prescriptions. In order to reduce the number of patient injuries and accompanying medical malpractice suits from medication errors, the following suggestions are offered: 1. Heed the patient's warning regarding drug allergies and prescribe a substitute drug, especially if the drug is aspirin or penicillin (and derivatives) or if the patient gives a history of having asthma. If the patient gives a history of aspirin sensitivity, make certain the drug you are prescribing does not contain aspirin as one of its components. Often physicians stated that they were not aware that aspirin was contained in the drug they prescribed for patients with a documented allergy to aspirin.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of tebufelone, a new anti-inflammatory drug, in plasma and tissue using capillary gas chromatography/stable isotope dilution mass spectrometry.

A capillary gas chromatographic/mass spectrometric method for the determination of tebufelone, a new anti-inflammatory drug, in plasma and rat paw tissue is described. Trideuteriated tubufelone was employed as an internal standard and the drug quantified by selected ion monitoring using a mass-selective detector following positive electron ionization (70 eV). Tebufelone was recovered from plasma (greater than 93%) and pulverized paw tissue (greater than 88%) by ethereal extraction. Standard curves were linear between 5 ng ml-1 and 5 micrograms ml-1 with a detection limit (S/N = 2) of 2 ng ml-1. The coefficient of variation for successive injections of standards (200 ng ml-1) or samples (10-1500 ng ml-1) was 7.3% and 8.5%, respectively.

Determination of menthol and menthol glucuronide in human urine by gas chromatography using an enzyme-sensitive internal standard and flame ionization detection.

The rate of peppermint oil absorption and excretion, following peroral administration, was determined by measuring urinary levels of menthol glucuronide. Menthol, a major component of peppermint oil, was liberated from its glucuronide metabolite by treating raw urine with beta-D-glucuronidase (Patella vulgata). Phenyl glucuronide was employed as an enzyme-sensitive internal standard. Menthol and phenol were recovered by ethyl acetate extraction and quantitated by capillary gas chromatography using flame ionization detection. Standard curves were linear between 25 and 250 micrograms/ml with a detection limit (signal-to-noise ratio = 2) of 0.25 microgram/ml. Assay precision was shown to be +/- 1.2% relative standard deviation.

Biochemical changes in developmentally retarded Xenopus laevis larvae. I. The lens crystallin transition.

Premetamorphic tadpoles of Xenopus laevis reared in water containing 0.01% propylthiouracil are developmentally retarded and metamorphosis is prevented. When uncrowded, they continue to grow to a giant size. Moderate crowding leads to a slower rate of growth. Thus morphologically premetamorphic tadpoles were produced with lens diameters appropriate to either normal premetamorphic, climactic or post-metamorphic animals. The lens crystallins of such tadpoles have been separated by immunoelectrophoresis and polyacrylamide gel electrophoresis. The crystallin pattern was correlated with lens diameter rather than with body stage. Giant retarded larvae possessed crystallin patterns identical to those or normal adults. The thyroid antagonist propylthiouracil is therefore unable to prevent the lens crystallin transition, the beginning of which is normally temporally correlated with metamorphosis.

Determination of tebufelone (a new anti-inflammatory drug) strength and stability in bulk drug, dosage formulations and feed admixtures by reversed-phase high-performance liquid chromatography.

A rugged reversed-phase high-performance liquid chromatographic method suitable for the quantitative determination of tebufelone, a new anti-inflammatory drug, in bulk drug, various pharmaceutical formulations and animal feed admixtures is described. Tebufelone was easily separated from synthetic by-products and detected by ultraviolet absorption (280 nm). Standard curves were linear (r2 greater than 0.999) over 2 orders of magnitude with a detection limit of 0.1 microgram/ml at a signal-to-noise ratio of 2 (0.05 ml injected). Recovery of tebufelone from bulk drug and dosage formulations was greater than 99% with a coefficient of variation of 1.8% throughout the range of the standard curve. Recovery of tebufelone from feed admixtures was 96-102% with a less than 5% relative standard deviation at the levels assayed.