Effects of chemical sympathectomy in neonatal and adult mice on C-1300 neuroblastoma tumor growth and catecholamine content.

The in situ C-1300 murine neuroblastoma (MNB) tumor model was used to investigate the influence of 6-hydroxydopamine (6HD)-induced sympathectomy on tumor growth and catecholamine concentration. One week (adult) and 3 weeks (neonatal) after sympathectomy, mice were implanted with 10(6) disaggregated MNB cells. The time interval between implantation of MNB cells and detection of palpable tumor (tumor onset time), transverse tumor diameter, tumor weight, tumor weight to body weight ratio, and tumor catecholamine concentration were determined. Sympathectomy following 6HD administration was confirmed by analysis of catecholamine concentrations in the heart and spleen by high-pressure liquid chromatography. Treatment of adult animals with 6HD reduced the mean heart and spleen norepinephrine (NE) concentrations to less than 20% of controls (vehicle treated). Neonatal sympathectomy decreased the average heart and spleen NE concentrations to less than 10% of comparable control mice. Whole brain NE and dopamine concentrations were not altered by treatment with 6HD in either age group. Tumor onset time following implantation of MNB cells was significantly increased in animals sympathectomized as either neonates or as adults. In contrast, MNB tumor growth rate following tumor onset was significantly inhibited in animals sympathectomized as neonates but not as adults. The catecholamine concentrations of tumors removed from control and sympathectomized mice 8 days after tumor onset were determined. Tumor NE and dopamine concentrations were increased 9.09 +/- 2.8- (SE) and 7.03 +/- 1.8-fold, respectively, in mice sympathectomized as neonates. There were no significant differences in the NE and dopamine concentrations of tumors obtained from sympathectomized and control adult mice. Pretreatment with desmethylimipramine prior to 6HD administration prevented destruction of sympathetic neurons, inhibition of tumor growth rate, and the increase in tumor catecholamine concentration observed in neonatally sympathectomized mice. These data suggest that the influence of chemical sympathectomy on MNB tumor growth and biochemical differentiation, as defined by catecholamine content, are age dependent.

Influence of host age and tumor load on the growth and catecholamine content of C-1300 murine neuroblastoma in situ.

The influence of host age and tumor load on survival time, tumor growth parameters and biochemical differentiation, as characterized by tumor catecholamine content, were investigated. A/J mice were implanted with tumor loads of 10(4), 10(5) and 10(6) disaggregated C-1300 murine neuroblastoma (MNB) cells at 1, 7, 14, 21 and 56 days of age. Studies performed in mice between 7 and 56 days of age demonstrated that MNB tumorigenicity, tumor growth rate, host survival and catecholamine content were independent of host age and tumor load whereas, tumor onset time was influenced by both factors. In contrast to older animals, tumor onset time and catecholamine content were decreased and tumor growth rate increased in 1-day-old mice. This difference may be due to the presence of endogenous growth factor(s) that modulate cell proliferation in the immediate post-natal period.

Efficacy in sheep and pharmacokinetics in cattle that led to the selection of eprinomectin as a topical endectocide for cattle.

Eprinomectin (MK-397 or 4"-epi-acetylamino-4"-deoxy-avermectin B1) is a novel avermectin selected for development as a topical endectocide for all cattle, including lactating cows. The initial efficacy assessments were made in sheep to identify subclasses of the avermectin/milbemycins that possessed inherent activity against a spectrum of nematode parasites. This included examination of several hundred analogs each given orally to a single sheep experimentally infected with a range of parasitic nematodes. Representatives of several subclasses, most notably the 4"-epi-amino avermectin B1 subclass, were identified as possessing potent, broad-spectrum activity against the endoparasites, whereas subclasses such as those with a variety of synthetic substitutions at C-4a or oximes at C-5 were among the least potent. Eprinomectin, a member of the 4"-epi-amino subclass, possessed potent activity against the range of nematodes when tested at 0.025 mg kg-1 per os. Milk and plasma concentration profiles were also made for these and other selected avermectin/milbemycins following topical administration to lactating dairy cattle. The molecular structure of each compound had a significant effect on the milk to plasma ratio, but the ratio of each was constant over time, implying an equilibrium between the 2 compartments. Compounds that were saturated at the C-22,23 bond had milk to plasma ratios > or = 1.0, whereas those unsaturated at this bond were generally < or = 1.0. The milk to plasma ratio of eprinomectin was < or = 0.2. Therefore, not only is eprinomectin the most potent broad-spectrum avermectin/milbemycin identified to date, but it also possesses one of the lowest milk partitioning coefficients in this class of antiparasitics.

Facile separation of sulfonamides from their degradates by liquid--liquid extraction.

Regulation of acidity for protonation of the free N4-amine can provide for the selective liquid--liquid extraction isolation of a sulfonamide from its degradation products. This principle is applied for the stability-indicating determination of sulfacetamide in the presence of sulfanilamide, sulfaquinoxaline in feed, and sulfabromomethazine in dosage forms. In solution, sulfabromomethazine can exhibit photodecomposition to sulfamethazine. The mean relative errors of the these methods and the precision, represented by relative standard deviations, are each typically less than 2%.

Fluorometric determination of thiazole-containing compounds.

Fluorescence spectroscopy was applied to the development of sensitive analytical methods for the determination of thiazole and several congeners that contain substituted thiazole rings. Treatment to yield thionine, previously used spectrophotometrically to measure thiazole and fluorometrically only for sulfur determinations in inorganic systems, is further characterized and illustrated with the determination of the antibiotic thiopeptin. This method is selective for submicrogram quantities of thiazole rings in the presence of fused-ring derivatives and reduced analogs. It has a precision of +/- 2% RSD (n = 11) at the 15-ng/ml thiazole concentration level with a signal-to-noise ratio of 3:1. For thiopeptin, this method has an accuracy of 5% mean relative error (n = 8) over the 5--20-ppm range in medicated feed.

Difference spectrophotometric determination of p-hydroxybenzoic acid in presence of its esters.

A difference spectrophotometric analytical method was developed for the selective determination of p-hydroxybenzoic acid in the presence of its alkyl esters without prior separation. Based on the spectral shift to a shorter wavelength accompanyint carboxyl dissociation, the procedure measures as little as 2% of this acid in mixtures with the alkyl ester preservatives and has an accuracy of 2% mean relative error over the 0.16-12.0 microgram of p-hydroxybenzoic acid/ml range.

Liquid chromatographic determination of ivermectin in feed.

An analytical method for determining ivermectin in feed at 0.50-3 ppm is presented. The method is based on liquid chromatographic measurement after sample preparation by adsorption chromatography on alumina and solid-phase extraction. Two complete, final, finished medicated feeds and the corresponding control feeds used in their preparation were analyzed. Recoveries from feeds fortified at 50-150% of the 2 ppm ivermectin use concentration also were determined. Mean recoveries from replicate analyses ranged from 90 to 100%, and coefficients of variation (CVs) were less than 4.5%. No significant interferences were found in control feeds. The pooled distribution of individual analytical results (n = 100) gave a mean recovery of 100%, a recovery range of 90-111%, and an overall CV of 5.5%. Resolution of the total variance into its 2 components gave a within-laboratory CV of 4.1% and a between-laboratory CV of 3.4%. There was no significant difference in recoveries among laboratories, days, concentrations, and feed base or between fortified and medicated feeds (P > 0.2).

Pharmacokinetic studies of ivermectin: effects of formulation.

Studies are reported which describe the effects of formulation, animal species, and route of administration on the pharmacokinetics of ivermectin. Biological half-life t1/2 increases in the order: swine (0.5 day) less than dogs (1.8 day) less than cattle approximately equal to sheep (2.8 day). Formulation modifications, based upon the solubility properties of the drug, have been directed towards the development of a nonaqueous injectable formulation for cattle and an aqueous vehicle for horses. Bioavailability following subcutaneous injection in cattle can be regulated by control of injection solvent composition: a vehicle composed of a mixed aqueous-organic solvent exhibits pharmacokinetic properties (i.e., Cp, t1/2, AUC, and F) intermediate between those furnished by an aqueous formulation and via a purely nonaqueous solvent. The longer apparent biological half-life from this latter vehicle (t1/2 = 8.3 days) confirms that a slow absorption process dominates the pharmacokinetics in the nonaqueous injectable product to produce an effective controlled-release formulation. These bioavailability results illustrate the increase in the concentration of an organic solvent and a concomitant decrease in surfactant concentration in a micellar aqueous system for prolonged drug delivery via injection.

Liquid chromatographic method for determination of arprinocid in feed: collaborative study.

An HPLC method for determining arprinocid [9-(2-chloro-6-fluorophenyl)methyl-9H-purin-6-amine] in feed was evaluated in an interlaboratory collaborative study. The samples were prepared in pairs from feeds obtained from 2 commercial feed manufacturers to cover the concentration range 0.0050-0.0070% arprinocid and were distributed to 14 laboratories. Each collaborator was requested to perform one determination on each of 6 samples. In this analytical procedure, the drug is extracted into CHCl3 and, after appropriate sample preparation by liquid-liquid partitioning, determined using a silica column and ultraviolet detection. The means of the analyses reported by the collaborators ranged from 97 to 104% of the true concentration of arprinocid and were not significantly different (P greater than 0.1) from the true values. The average coefficient of variation was 6.8%. The precision standard deviations of the 3 unit blocks (sr) were each less than 0.0004% arprinocid, and the F-test demonstrated that systematic error (sb) did not make a statistically significant contribution (P greater than 0.1) to the standard deviation of the data (sd). This method has been adopted official first action.

Nerve growth factor stimulation of arachidonic acid release from PC12 cells: independence from phosphoinositide turnover.

The effect of nerve growth factor on the metabolism of arachidonic acid and the hydrolysis of phosphatidylinositol in PC12 cells was examined. Addition of nerve growth factor to PC12 cells isotopically labeled with [3H]arachidonic acid caused an increased release of radioactivity. In a similar manner, treatment of PC12 cells prelabeled with [3H]inositol increased inositol monophosphate accumulation in the presence of LiCl. Stimulation of [3H]arachidonic acid release by nerve growth factor was concentration dependent, attaining a maximum at 0.5 nM. Concentrations of nerve growth factor above 0.5 nM caused less than maximal stimulation. In contrast, nerve growth factor-stimulated accumulation of [3H]inositol monophosphate exhibited a sigmoidal dose-response curve with an apparent maximum at 8 nM. Increased accumulation of [3H]inositol monophosphate could be detected as early as 60 s after nerve growth factor addition, whereas nerve growth factor-stimulated release of [3H]arachidonic acid was not observed until 5 min after nerve growth factor treatment. The nerve growth factor-stimulated release of [3H]arachidonic acid was independent of extracellular calcium concentration. Increased [3H]inositol monophosphate accumulation elicited by nerve growth factor was dependent on the presence of extracellular calcium. These results suggest that the increased metabolism of arachidonic acid and the enhanced hydrolysis of phosphatidylinositol are separately regulated by nerve growth factor.

Effect of nerve growth factor on C-1300 murine neuroblastoma tumor growth and catecholamine content in neonatally sympathectomized mice.

The in situ C-1300 murine neuroblastoma (MNB) tumor model was used to investigate the influence of exogenously administered nerve growth factor (NGF) on tumor growth and tissue catecholamine concentration in mice sympathectomized with 6-hydroxy-dopamine (6-OHDA) on postnatal days 4-10. Mice were implanted with 1 x 10(6) disaggregated MNB cells 3 days after termination of 6-OHDA administration. NGF (12-15 micrograms/mouse/day) treatment was initiated at the time of MNB cell implantation and continued until sacrifice of the animal. The time interval between tumor cell implantation and detection of palpable tumor (tumor onset time), transverse tumor diameter, tumor weight, tumor weight to body weight ratio, and tumor catecholamine concentration were determined. Neonatal sympathectomy caused a decrease in myocardial norepinephrine concentration of 88% compared with vehicle-treated animals as well as a significant reduction in total body and organ weight. Average body, brain, heart, and spleen weights were decreased 31%, 16%, 25%, and 42%, respectively, below control values. The daily injection of NGF, from the time of MNB tumor implantation to sacrifice, did not prevent these effects of chemical sympathectomy from being expressed. Tumor onset time following implantation of MNB cells was significantly increased in neonatally sympathectomized mice and was not altered by treatment with NGF. In contrast, the decrease in MNB tumor growth rate observed in sympathectomized mice was reversed by administration of NGF. Mean tumor weight and mean tumor to body weight ratio were 89% and 115% of comparable control values, respectively, in sympathectomized mice receiving exogenous NGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Symptomatic myelolipoma of the adrenal: report of a case with computed tomographic evaluation.

The authors offer what may be only the second histologically verified case of myelolipoma studied by computed tomography. The CT appearance is described.

Differential effects of 6-hydroxydopamine-induced ablation of peripheral and central adrenergic axons on C-1300 murine neuroblastoma tumor growth and catecholamine concentration in the A/J mouse.

The relationship between 6-hydroxydopamine (6-OHDA)-induced ablation of central and peripheral adrenergic neurons and in situ C-1300 murine neuroblastoma (MNB) tumor growth and catecholamine concentration were investigated. Destruction of central and/or peripheral adrenergic neurons was produced by the intracerebral and/or s.c. administration of 6-OHDA to neonatal A/J mice. Disaggregated MNB cells (1 x 10(6)) were implanted s.c. into each mouse 3 weeks after treatment with 6-OHDA or diluent. Tumor onset time (the time interval between implantation of MNB cells and detection of palpable tumor), tumor weight, tumor weight to body weight ratio, tumor growth rate constant and tissue catecholamine concentrations were determined. Central axotomy caused a significant increase in tumor onset time and decrease in tumor weight when compared to controls. However, neither the tumor weight to body weight ratio or tumor growth rate constant were significantly lowered. In contrast, a reduction in all tumor growth parameters was produced by peripheral axotomy, which differed significantly from centrally axotomized and control animals. The catecholamine concentration of MNB tumors excised from control and 6-OHDA-treated mice 8 days after tumor onset were determined. Norepinephrine and dopamine levels were elevated above controls in MNB tumors obtained from mice that had been either peripherally or peripherally and centrally axotomized; whereas, no change in tumor catecholamine concentrations was noted in centrally axotomized mice. This investigation has demonstrated that ablation of central as well as peripheral adrenergic innervation exerts an inhibitory effect on MNB tumor growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of protein kinase C alpha in nerve growth factor-induced arachidonic acid release from PC12 cells.

Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 microM) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs alpha, delta, epsilon, and zeta. PMA down-regulation depleted PKCs alpha, delta, and epsilon, and partially depleted zeta. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs alpha and beta specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs alpha, beta, and gamma, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC alpha plays a role in NGF-induced AA release.

Determination of ivermectin in medicated swine feeds at the 2 ppm concentration level.

An analytical method has been developed that is applicable to the determination of Ivermectin in medicated feeds at the 2 ppm concentration level. It is based upon liquid chromatographic analysis with a reverse-phase column and ultraviolet detection. After the drug is extracted from the feed into methanol, an analytical sample is prepared by the consecutive use of column chromatography on alumina and solid-phase extraction on Sep-Pak C18 and silica cartridges. This procedure has been applied to the concentration range 0.50-3.0 ppm of Ivermectin in feed with an accuracy of +/- 2% mean relative error and a precision of +/- 2% relative standard deviation at the 2 ppm concentration level.

Evolution of a specific fluorogenic derivatization of ivermectin for bioanalytical applications. A review.

The evolution of the fluorogenic derivatization of ivermectin is traced through a series of continual modifications that have resulted in improvements in speed and sensitivity. Since the original development of this selective analytical technique, the reaction time has been shortened from 24 h at 100 degrees C to < 30 s at room temperature and, through modifications of the derivatization reagent and catalyst, the sensitivity has also been increased 50-fold to 20 pg of analyte with no significant decrease in precision. A procedure is reported, based on the use of fluorescence derivatization, which eliminates the use of solid-phase columns for sample preparation and fluorophore isolation, and is faster and less cumbersome than previous methods. The method was evaluated with cattle and canine plasma samples over the concentration range 1.0-40 ng ml-1 of ivermectin. It has an accuracy of 1.9% (mean relative error) over this concentration range and a precision of 5.6% (RSD) at the 1 ng ml-1 ivermectin concentration level in a 1 ml plasma sample.

High pressure liquid chromatographic determination of arprinocid in feed.

Arprinocid [9 - (2 - chloro - 6 - fluorophenylmethyl)-9H-purin-6-amine] is determined in feed by high pressure liquid chromatography with a silica column and ultraviolet detection. The drug is extracted from the feed into chloroform in the presence of pH 7 phosphate buffer, transferred to 0.1N HCl, and separated from interfering substances by partitioning with hexane. The acidic solution is neutralized, and the analyte is extracted into chloroform for injection into the chromatograph. This procedure has been applied to feeds containing 0.0030--0.0090% arprinocid with a precision of less than 5% relative standard deviation at the 0.0060% formulated concentration level. The results of this chromatographic procedure also correlate with those from a colorimetric analysis.