Target animal safety study of meloxicam administered via transmucosal oral spray (Promist(®) technology) for 6 months in dogs.

This study evaluated the safety of meloxicam administered via transmucosal oral spray (TMOS) at 1, 2, 3 and 5 times the maximum proposed dose for 6 months in dogs. After baseline assessments, 40 Beagles (20 M, 20 F) were randomized to gender-balanced groups administered either water or meloxicam TMOS at 1, 2, 3 and 5 times the maximum proposed dose once daily for 26 weeks. Dogs were subjected to daily food consumption measurements and clinical and dose site observations. Periodic evaluations were made of body weight, physical examination, clinical pathology, urinalysis, buccal mucosal bleeding time (BMBT) and gastroduodenal endoscopy. At study completion, all dogs were subjected to gross necropsy. Histopathology was performed on tissues from dogs in groups 0X and 5X and from selected tissues in other dose groups. Clinical signs of previously reported NSAID-associated gastrointestinal upset were noted with higher frequency in meloxicam-dosed animals than in controls. Despite the presence of statistically significant effects on some clinicopathological variables, no toxicologically relevant dose-associated effects were determined on these or on food consumption, dose site observations, body weight, physical examination, urinalysis, BMBT, endoscopic examination or gross and histopathological examination of necropsy tissues.

Plasma concentrations of immunoreactive relaxin activity and progesterone in the pregnant Asian elephant (Elephas maximus).

Immunoreactive relaxin activity and progesterone concentrations in weekly plasma samples were measured throughout six pregnancies in five Asian elephants (Elephas maximus). One animal aborted, one delivered a stillborn calf at term and the remaining pregnancies concluded normally with living young. Immunoreactive relaxin activity increased by week 20 of pregnancy to reach a mean peak concentrations of > 6 ng/ml in the second trimester. This was followed by a slow decline beginning approximately at 30 weeks before term. A smaller, secondary relaxin rise was observed during the final eight weeks preceding parturition and circulating concentrations remained above pre-pregnancy baseline values for 72 h after birth. In contrast, progesterone concentrations increased immediately after mating and rose to a maximum of > 2 ng/ml by mid-gestation. As with relaxin, progesterone concentrations began to decline gradually approximately 30 weeks before birth. More frequently collected samples before, during and after parturition revealed a decline to pre-pregnancy levels between 2-5 days before parturition, in contrast to elevated relaxin levels (1 ng/ml) during this time. The aborted pregnancy at week 35 was accompanied by a precipitous decline in both immunoreactive relaxin and progesterone concentrations one week before the visible termination of the pregnancy. The delivery of the term stillborn calf occurred five weeks after both immunoreactive relaxin and progesterone concentrations had declined to pre-pregnancy values. Results suggest that immunoreactive relaxin may be important, along with progesterone, in the maintenance of pregnancy in the Asian elephant as well as playing a role in preparation for birth and possibly to indicate foetal well-being.

Alpha 2-macroglobulin conformation determines binding affinity for activin A and plasma clearance of activin A/alpha 2-macroglobulin complex.

Activin A is a member of the transforming growth factor-beta family of growth factors and a potent regulator of cellular activity. A number of binding proteins for activin A have been identified, including alpha 2-macroglobulin (alpha 2M). Alpha 2M has several conformational states that are known to have different growth factor-binding properties. The effect of alpha 2M conformation on activin A binding has not been characterized. The aims of this study were to determine 1) whether activin A binds preferentially to the native (alpha 2M-N) or "activated" (alpha 2M*) conformation of alpha 2M, 2) the affinity of different alpha 2M conformations for activin A, and 3) the fate of activin A complexed with alpha 2M-N or alpha 2M* in vivo. [125I]Activin A associated with alpha 2M in plasma and follicular fluid and with purified alpha 2Ms. In this qualitative assay, more activin A was associated with alpha 2M* than with alpha 2M-N. The affinity of the activin A-alpha 2M interaction was determined. The Kd values for activin A-alpha 2M* and activin A-alpha 2M-N were 190 +/- 30 and 510 +/- 60 nM, respectively. The plasma clearance profiles and tissue distribution of uncomplexed activin A and purified alpha 2M*-activin A complex were determined. Radiolabeled activin A cleared in a biphasic manner, with rapid clearance over the initial 10 min and substantially slower clearance over the subsequent 20 min. During the slow phase of clearance, activin A formed a complex with circulating alpha 2M-N. In contrast, radiolabeled activin A-alpha 2M* complexes were rapidly cleared from plasma with a half-life of approximately 5 min and were specifically targeted to alpha 2M receptors in vivo. These studies reveal that alpha 2M can maintain activin A in the circulation or rapidly target the hormone for plasma clearance depending on the conformational state of the carrier protein in vivo.

Non-invasive monitoring of ovarian function in Asian elephants (Elephas maximus) by measurement of urinary 5 beta-pregnanetriol.

The development of an enzymeimmunoassay for 5 beta-pregnanetriol and its use for non-invasive monitoring of reproductive cycles in Asian elephants is described. Gas chromatography-mass spectrometry (GCMS) and high performance liquid chromatography (HPLC) confirmed the presence of 5 beta-pregnane-3 alpha,17 alpha,20 alpha/beta-triols as the two most abundant urinary progesterone metabolites. The assay developed used the antiserum anti-5 beta-pregnane-17 alpha,20 alpha-diol-3 alpha-gamma l glucuronide but was designed to measure the free steroid in urine samples after hydrolysis and extraction. HPLC confirmed the presence of immunoreactive pregnanetriol in urine, but indicated that the measurement was nonspecific. Immunoreactive pregnanetriol concentrations were significantly correlated with the concentrations of both progesterone (r = 0.98, n = 269, P < 0.01) and 17 alpha-hydroxyprogesterone (r = 0.95, n = 205, P < 0.01), the metabolic precursor of pregnanetriol. The mean +/- SEM deviation of cycles as determined by measurements of plasma progesterone, 17 alpha-hydroxyprogesterone and urinary pregnanetriol, respectively, were 15.54 +/- 1.5 (n = 23, where n = number of cycles), 15.21 +/- 1.7 (n = 15) and 15.45 +/- 0.94 weeks (n = 20). These results demonstrate that it is possible to monitor ovarian function in Asian elephants by the measurement of urinary immunoreactive pregnanetriol concentrations.

Altered androstenedione to testosterone ratios and LH concentrations during musth in the captive male Asian elephant (Elephas maximus).

Greater concentrations of androstenedione than testosterone were usually present during periods of non-musth in plasma collected weekly for various periods up to 2 years in 8 male Asian elephants (4-35 years of age). For the 6 males that exhibited musth the androstenedione/testosterone ratio shifted greatly in favour of testosterone. The severity of musth was assessed weekly using a scale of 1 to 5 for each of 8 behavioural traits including urine dribbling, temporal gland secretion and aggression. A significant correlation (P less than 0.05) was noted between plasma testosterone concentrations and the musth score value in 5 of 6 musth episodes. Brief shifts in the ratio of two androgens when testosterone predominated (n = 106) were seen during the non-musth period in 3 of the males studied continuously for 2 years. In 82% of these instances, stimuli of a sexual or aggressive nature had occurred in the preceding 48 h (chi 2, P less than 0.01). A heterologous bovine assay was used to measure LH values in plasma collected every 15 min for 12 h. Increases in testosterone concentrations followed pulsatile increases in plasma LH concentrations during 7 non-musth periods in 4 animals. Apart from pulse frequency, increases in the variables describing pulsatile LH secretion were seen in 2 strong musth and 2 mild musth episodes compared to non-musth values. A strong musth, however, was characterized by a much greater increase in pulsatile testosterone secretion than was a mild musth and which may be a function of the duration of musth.