In vivo disposition of dermorphin analog (DALDA) in nonpregnant and pregnant sheep.

Although synthetic opioid peptide analogs have been used extensively to study the functional roles of opioid receptors, little is known about their in vivo disposition. Our goal was to develop novel opioid drugs with limited transfer across the placenta. DALDA (Tyr-D-Arg-Phe-Lys-NH2) is a potent and highly selective mu agonist that is quite polar because of its 3+ charge at physiological pH. It can therefore be expected that the distribution of DALDA across the placenta would be highly restricted. In this study, we determined the pharmacokinetics and placental transfer of DALDA after systemic administration in sheep. DALDA was infused intravenously to four nonpregnant and four pregnant sheep at a dose of 0.6 mg/kg/hr for 4 hr. Steady state plasma levels of DALDA were 5436 +/- 464 ng/ml in nonpregnant sheep and 5214 +/- 661 ng/ml in pregnant sheep. A one-compartment open model provided an excellent fit for nonpregnant and pregnant plasma data. The apparent volume of distribution was estimated to be 45.6 +/- 4.4 and 59.2 +/- 7.9 ml/kg in nonpregnant and pregnant animals, respectively. There was no difference in the elimination half-life of DALDA in nonpregnant (1.4 +/- 0.1 hr) and pregnant (1.7 +/- 0.2 hr) animals, and clearance was also similar in nonpregnant (23.1 +/- 1.7 ml/kg/hr) and pregnant (23.7 +/- 1.3 ml/kg/hr) animals. These data suggest that the distribution of DALDA is restricted to plasma volume and that its disposition is not altered in pregnancy. DALDA was not detected in any of the fetal plasma samples (< 50 ng/ml), indicating that fetal plasma concentration is < 1% of maternal concentration. The highly restricted placental distribution of DALDA suggests that it may be a promising opioid drug for obstetrical use.

Mass spectrometric quantification of the mu opioid receptor agonist Tyr-D-Arg-Phe-Lys-NH2 (DALDA) in high-performance liquid chromatography-purified ovine plasma.

The mu opioid receptor agonist Tyr-D-Arg-Phe-Lys-Amide (D-Arg2-Lys4-Dermorphin(1-4)amide=DALDA) was infused continuously for 2 h into sheep. The presence of DALDA in ovine plasma was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) in plasma samples that were obtained at different times during and following that infusion. A stable isotope-incorporated internal standard, deuterated DALDA (d5-DALDA), was used for the MS quantification of DALDA via the protonated molecule ion, (M+H)+, of DALDA and of d5-DALDA. Time-course data (microg DALDA ml(-1) plasma vs. time) were obtained. Tandem MS (MS-MS) provided the product-ion spectrum of the (M+H)+ ion of DALDA in one of the samples to confirm the amino acid sequence of DALDA.

beta-endorphin1-31 in the rat pituitary.

Mass spectrometry was used to determine the molecular mass of rat pituitary beta-endorphin1-31 (BErat, 1-31). The measured molecular mass (3435 +/- 1 Da, n = 5) of endogenous BErat, 1-31 differed from the molecular mass of commercially available synthetic BErat, 1-31 (3465 +/- 1 Da, n = 9), but corresponded to the molecular mass of synthetic BEbovine, 1-31 (3436 +/- 3 Da, n = 3). Based on the combination of these ESIMS molecular mass measurements, HPLC retention time data, LSIMS measurement of the molecular mass of selected tryptic fragments, and consideration of codon sequences, we suggest that the amino-acid sequence of endogenous BErat, 1-31 differs from the DNA-deduced sequence of BErat, 1-31, and that endogenous BErat, 1-31 contains Ala instead of Val in position 26.

Alteration of opioid peptide concentrations in the rat pituitary following survivable closed head injury.

Concentration changes of methionine enkephalin-like immunoreactivity (ME-li) and beta-endorphin-like immunoreactivity (BE-li) in the rat pituitary following diffuse brain injury were studied. Closed head injury was induced by a weight-drop trauma device (450 g x 2 m). The level of closed head injury used in this study altered the pituitary opioid peptide concentrations. The level of ME-li did not change in the experimental groups 3 hours, 10 hours, 24 hours, and 3 days after the trauma, but significantly increased by 34% 10 days after the trauma. BE-li remained constant 3 hours and 10 hours following the injury, increased by 48% at 24 hours, and remained at this level for 10 days after the trauma (44% at 3 days, and 40% at 10 days). The levels of ME-li and BE-li in the control sham-operated rats did not change during these times. The present measurements of BE-li and ME-li in the pituitary indicate that the opioid peptides that derive from two different neuropeptidergic systems, proopiomelanocortin (POMC) and preproenkephalin A, respectively, may participate in the pathophysiology of a closed head injury.