Introduction

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Opiate alkaloids such as meperidine and morphine are extensively used for pain relief during labor and delivery. Their use, however, is associated with a number of adverse effects in the neonate because of their rapid transfer across the placenta to the fetus. The distribution of compounds across the placenta is dependent on molecular size and lipophilicity, and all available opiate alkaloids are small and highly lipophilic. The goal of our research group has been to develop novel opioid drugs with limited transfer across the placenta.

The physicochemical characteristics of a peptide drug may be advantageous in the design of an opioid drug for obstetrical use. The incorporation of D-amino acids in endogenous opioid peptides protects them from certain peptidase activity and significantly increases their stability in vitro, but no information exists on their disposition in vivo. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is an endogenous opioid peptide found in amphibian skin that naturally contains a D-amino acid and has been shown to possess many opioid properties (Broccardo et al., 1981; Montecucchi et al., 1981). In vitro degradation studies revealed that dermorphin is hydrolyzed at the Gly-Tyr bond, resulting in dermorphin(1-4) being the major metabolite (Sasaki et al., 1985; Scalia et al., 1986). A synthetic analog of dermorphin(1-4), DALDA (Tyr-D-Arg-Phe-Lys-NH₂), has been found to be a potent and highly selective agonist for the mu opioid receptor (Schiller et al., 1989). In addition, DALDA is unique in that it has a 3+ charge at physiological pH and is therefore very polar and has very limited distribution across the blood-brain barrier (Samii et al., 1994). It can therefore be expected that the distribution of DALDA across the placenta would also be highly restricted, which would make this peptide a promising candidate for use as an obstetrical analgesic.

The objective of this study was to determine the pharmacokinetics of DALDA in nonpregnant and pregnant sheep. We recently developed a specific and sensitive method for the quantification of DALDA in ovine plasma samples (Grigoriants et al., 1997). Here, we present evidence that supports the highly restricted transfer of DALDA across the placenta.

	Materials and Methods

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Animal preparation. Chronic indwelling catheters were surgically placed in four nonpregnant ewes and four pregnant ewes (gestational age, 115-120 days; term, 145 days) as described by Szeto et al. (1990). One polyvinyl catheter was inserted into the femoral artery and advanced to the distal aorta for blood sampling and another was advanced into the inferior vena cava via the femoral vein for drug infusion. In pregnant animals, a fetal hindlimb was exposed via a hysterotomy incision, and chronic indwelling catheters were also placed in the fetal distal aorta and inferior vena cava. Guidelines approved by the Institution for the Care and Use of Animals at Cornell University Medical College were followed for all surgical procedures and experimental protocols.

Compounds. DALDA was prepared by solid-phase synthesis according to a protocol described earlier (Schiller et al., 1989). The synthesis of the deuterated DALDA analog [H-Tyr-D-Arg-Phe(d₅)-Lys-NH₂] was based on the same protocol, except Boc-Phe(d₅)-OH was used in place of Boc-Phe-OH. Pentadeuterophenylalanine was purchased from C/D/N Isotope (Vaudreuil, Quebec, Canada) and converted to Boc-Phe(d₅)-OH through reaction with di-tert-butyldicarbonate. The deuterated peptide was purified by semipreparative reversed-phase HPLC as described previously (Schiller et al., 1989). The purity of DALDA and d₅-DALDA was verified by fast atom bombardment-MS, and the precise amino acid sequence was confirmed by tandem MS.

Experimental protocol. Experiments were performed a minimum of 5 days after surgery to ensure complete recovery from surgical stress. The ewe was placed in a mobile cart with free access to food and water. All studies commenced at 8:00 a.m. with a minimum of 2.5 hr to allow for acclimation of the animal to the study conditions. DALDA (supplied by Dr. Peter Schiller and the National Institute on Drug Abuse) was administered as a constant-rate intravenous infusion (0.6 mg/kg/hr for 4 hr) to the sheep, and blood samples were collected at 0, 1, 2, 3, 3.5, 4, 4.25, 4.5, 5, 6, 7, 12 and 24 hr. Blood samples were collected from the fetus at 0, 3, 3.5, 4, 5 and 6 hr. All blood samples were collected into chilled borosilicate glass tubes containing EDTA and centrifuged, and the plasma was stored in glass containers with Teflon-lined caps and frozen at 80°C until they were shipped overnight to Dr. Dominic Desiderio.

Quantitative analysis of DALDA. DALDA was quantified by using reversed-phase HPLC and MS detection. Details of the quantitative method have been published (Grigoriants et al., 1997) and are presented here briefly. Plasma samples were deproteinated and eluted through a solid-phase extraction cartridge (Sep-Pak C18; Millipore, Milford, MA) with CH₃CN. An internal standard, the deuterated DALDA analog [Tyr-D-Arg-Phe(d₅)-Lys-NH₂], was added to each plasma sample before deproteinization. The filtered plasma sample was chromatographed on an RP-analytical column (Delta Pak, 5 µm, C18, 100Å, 150 × 3.9 mm; Waters, Milford, MA) at a flow rate of 1.5 ml/min, and UV absorption was monitored at 200 nm (Varian Assoc., Walnut Creek, CA). One-minute fractions were collected, and each fraction was lyophilized for MS analysis (AutoSpecQ tandem mass spectrometer; Micromass, Altrincham, UK). Continuous flow-LSIMS was used to quantify DALDA. The (M+H)⁺ ion current for DALDA at m/z 612 was compared with the ion current from d₅-DALDA at m/z 617. The limit of sensitivity of this method is 50 ng/ml DALDA.

Pharmacokinetic analyses. Plasma levels of DALDA during and after the 4-hr infusion of DALDA were analyzed using both model-dependent and -independent methods with nonlinear regression (WINNONLIN). A one-compartment open model with zero-order input and first-order elimination provided an excellent fit for nonpregnant and pregnant plasma data. The derived pharmacokinetic constants were used to calculate the apparent volume of V_d, t_1/2 and CL. The plasma data were also analyzed using a model-independent approach with area analysis (Perrier and Mayersohn, 1982). Rather than ascribing the data to a specific model, the model-independent approach assumes only that all dispositional processes may be described by first-order kinetics, with elimination occurring from the rapidly equilibrating compartment. The parameters of V_d, CL and MRT are determined from the total AUC and AUMC.

	Results

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DALDA was infused into four nonpregnant and four pregnant sheep at a dosage of 0.6 mg/kg/hr for 4 hr. This infusion rate was selected on the basis of preliminary studies using 0.3 to 0.9 mg/kg/hr for 2 to 4 hr. From these preliminary studies, it was apparent that a 4-hr infusion was necessary for steady-state kinetics; all subsequent studies were therefore carried out using 4-hr infusions. Figures 1 and 2 illustrate the mean plasma DALDA levels in nonpregnant and pregnant sheep, respectively. Steady-state levels were achieved by the end of the 4-hr infusion, with no statistical difference between plasma DALDA levels at 3.5 and 4 hr (paired t test, P > .05). There was no significant difference between the plasma concentration at the end of infusion in nonpregnant (5436 ± 464 ng/ml) and pregnant (5214 ± 661 ng/ml) animals. Samples obtained at 24 hr did not contain measurable levels of DALDA. DALDA was not detected in any of the fetal plasma samples. Because the limit of sensitivity of the analytical method is 50 ng/ml, the ratio of fetal-to-maternal plasma concentration is <0.01.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Plasma concentration of DALDA in nonpregnant sheep. DALDA was infused at a constant rate of 0.6 mg/kg/hr for 4 hr. Data are presented as mean ± S.E.M. (n = 4).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Plasma concentration of DALDA in pregnant sheep. DALDA was infused at a constant rate of 0.6 mg/kg/hr for 4 hr. Data are presented as mean ± S.E.M. (n = 4).

Individual plasma data from nonpregnant and pregnant sheep were analyzed using compartmental and noncompartmental methods. Compartmental analysis revealed that a one-compartment open model provided an excellent fit for both nonpregnant and pregnant animals, with the correlation between observed and predicted values ranging from .92 to .99. Figures 3 and 4 are the observed and predicted values for a representative nonpregnant and pregnant animal, respectively. The calculated parameters for nonpregnant and pregnant animals are summarized in tables 1 and 2, respectively. The coefficient of variation was <10% for all parameter estimates. There was no significant difference in any of the pharmacokinetic parameters between nonpregnant and pregnant animals.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Observed () and predicted () plasma concentration of DALDA in one representative nonpregnant sheep. DALDA was infused at a constant rate of 0.6 mg/kg/hr for 4 hr. Plasma DALDA concentrations were fitted to a one-compartment open model.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Observed () and predicted () plasma concentration of DALDA in one representative pregnant sheep. DALDA was infused at a constant rate of 0.6 mg/kg/hr for 4 hr. Plasma DALDA concentrations were fitted to a one-compartment open model.

The results of the noncompartmental analyses are summarized in tables 3 and 4. The calculated values for V_d, MRT and CL are quite similar to those estimated from the compartmental analysis. The data also show no significant difference between the disposition of DALDA in nonpregnant and pregnant animals.

	Discussion

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Although synthetic opioid peptide analogs have been used extensively to study the functional roles of opioid receptor subtypes, there is no information available on the pharmacokinetics of these peptide analogs in vivo. These data represent the first attempt in understanding the in vivo disposition of synthetic opioid peptide analogs, and this is the first time that detailed pharmacokinetic analyses have been performed using a highly specific method for peptide quantitation in biofluids. The endogenous opioid peptides are rapidly degraded in plasma, and the t_1/2 of met-enkephalin was reported to be 2 min in plasma in vitro (Hambrook et al., 1976). Substitution with D-amino acids significantly increases peptide stability and has been the rationale behind the synthesis of a large number of peptide analogs (D-Ala²) based on the enkephalin structure. However, pharmacokinetic data are not available for even the most widely used analogs, such as DAMGO and DPDPE. Using radioimmunoassay, the t_1/2 of DAMME was determined to be 52 min in sheep (Bolton et al., 1982). The disadvantage with radioimmunoassay, however, is the lack of specificity for the parent peptide, and the antibody may cross-react with degradative fragments. Other in vitro degradation studies used HPLC, but there always is the problem with biofluid samples of coeluting peaks. To the best of our knowledge, this is the first study in which plasma peptide levels were quantified using HPLC and MS. MS optimizes the molecular specificity of the detection of DALDA because the protonated molecule ion, (M+H)⁺, of DALDA at m/z 612 is monitored. Furthermore, an internal standard, the deuterated DALDA analog, was added to each ovine plasma sample before deproteinization. In addition to MS analysis, tandem MS/MS was used to confirm the amino acid sequence of DALDA in selected samples.

Our data show that steady-state plasma levels were achieved with a 4-hr infusion of DALDA to nonpregnant and pregnant sheep and that there was no significant difference in the steady-state plasma levels between pregnant and nonpregnant animals. Model-dependent and -independent methods resulted in similar pharmacokinetic parameters. The results show that the distribution of DALDA is very limited, with an apparent volume of distribution (50 ml/kg) that is approximately equivalent to plasma volume. This small volume of distribution is consistent with the polar character of this highly charged (3+) peptide at physiological pH and is consistent with earlier findings of very restrictive distribution of DALDA across the blood-brain barrier (Samii et al., 1994; Schiller et al., 1990). This apparent volume of distribution is even smaller than the distribution volume reported for -endorphin (180 ml/kg) in humans (Foley et al., 1979), DAMME (190 ml/kg) in sheep (Bolton et al., 1982) and DPDPE (486 ml/kg) in rats (Chen and Pollack, 1996, 1997). In addition to the polar nature of DALDA, the small volume of distribution may also be due to an extensive binding of DALDA to plasma proteins. There is at present no information on the plasma protein binding of DALDA. Studies in rats recently showed that 50% of DPDPE is bound to plasma proteins (Chen and Pollack, 1997).

The structural features of DALDA, with its D-Arg² and Lys⁴-amide, should confer high metabolic stability. Indeed, our results revealed a t_1/2 of 1.6 hr in sheep, which is longer than the t_1/2 of -endorphin in humans (37 min) (Foley et al., 1979) or DAMME in sheep (52 min) (Bolton et al., 1982). More importantly, the t_1/2 of DALDA is considerably longer than that for the opiate alkaloids in the sheep, which is on the order of 20 to 30 min (Szeto et al., 1982, 1978, 1981). These data suggest that the incorporation of the D-Arg² and Lys⁴-amide significantly enhanced the stability of the dermorphin peptide against peptidase activity. The elimination pathway of DALDA in vivo is not known but is likely to be via renal excretion because of its polar character. The plasma CL of DALDA was estimated to be 20 ml/kg/hr, which is much less than the reported CL for -endorphin (192 ml/kg/hr) or DAMME (168 ml/kg/hr) (Bolton et al., 1982; Foley et al., 1979). The slow CL of DALDA can be explained, at least in part, by its very small apparent volume of distribution. In addition, tubular reabsorption may contribute to the recirculation of DALDA into plasma.

The results of this study suggest that the pharmacokinetics of DALDA are not significantly altered in pregnancy. Pregnancy is associated with a number of physiological changes that are expected to affect the disposition of hydrophilic and hydrophobic drugs, including an increase in plasma volume, total body water and fat mass (Reynolds and Knott, 1989). Although the apparent volume of distribution of DALDA was higher in pregnant animals based on compartmental and noncompartmental estimation, the difference did not reach statistical significance. The influence of pregnancy on pharmacokinetics may be more considerable for lipophilic drugs with more extensive distribution. Similarly, neither the t_1/2 nor CL of DALDA was altered in pregnant animals.

As we predicted, the placental transfer of DALDA was highly restricted, with no measurable levels detected in fetal plasma even after 4 hr of drug infusion to the mother. With the limit of sensitivity of our analytical method being 50 ng/ml, our data suggest that <1% of the peptide reaches the fetus. In contrast, the percentages of methadone, meperidine and morphine reaching the ovine fetus are 30%, 15% and 13%, respectively. The negligible distribution of DALDA to the fetus can readily account for the lack of effects on fetal cardiovascular and metabolic function after maternal DALDA administration (Clapp et al., in press).

The very low extent of fetal drug exposure suggests that DALDA would be a very safe drug for use in pregnancy. However, the restricted distribution of DALDA would also mean that it would have difficulty gaining access into the central nervous system. In fact, available data have shown that the distribution of DALDA to the central nervous system is very slow (Samii et al., 1994), and DALDA has only a small effect in the hot-plate test 2 hr after subcutaneous administration to mice (Schiller et al., 1990). A solution to the problem would be to administer DALDA via the intrathecal route. When administered intrathecally to rats with indwelling intrathecal catheters, a single dose of DALDA was found to be highly effective in the tail-flick assay with a duration of action of >4 hr.² The polar character of DALDA, together with its metabolic stability, can account for the extraordinary long duration of antinociception. When administered in the subdural space, its distribution across the dura is likely to be very restricted, and DALDA can therefore remain in cerebrospinal fluid for a long time. Together, these data suggest that DALDA is a most promising drug for regional obstetrical analgesia.